Manabu Tokeshi - Applications of Microfluidic Systems in Biology and Medicine-Springer (2019)

Bioanalysis
Series Editor: Tuan Vo-Dinh
Manabu Tokeshi Editor
Applications
of Microfluidic
Systems in
Biology and
Medicine
Bioanalysis
Advanced Materials, Methods, and Devices
Volume 7
Series editor
Tuan Vo-Dinh
Fitzpatrick Institute for Photonics
Duke University
Durham, NC, USA
More information about this series at http://www.springer.com/series/8091
Manabu Tokeshi
Editor
Applications of Microfluidic
Systems in Biology and
Medicine
Editor
Manabu Tokeshi
Division of Applied Chemistry, Faculty
of Engineering
Hokkaido University
Sapporo, Japan
ISSN 2364-1118 ISSN 2364-1126 (electronic)

Bioanalysis
ISBN 978-981-13-6228-6 ISBN 978-981-13-6229-3 (eBook)
https://doi.org/10.1007/978-981-13-6229-3
Library of Congress Control Number: 2019935498
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Preface
About 30 years have passed since the concept of μTAS was proposed by Andreas
Manz in 1990. In the mid-1990s, as the human genome project was being promoted,
many researchers were working on the development of microfluidic “electrophoresis”
devices. From around 2000, development of devices integrating chemical analysis,
bioassay, etc. has increased. Then, this research field called μTAS, lab-on-a-chip,
microfluidic device, etc. has greatly expanded and developed. Currently, μTAS field
continues to expand; new concepts such as digital microfluidics, nanofluidics, μPAD
(microfluidic paper-based analytical device), and organ-on-a-chip which were origi-
nally unexpected have been proposed; and not only to analytical chemistry but also to
various fields such as medical diagnosis and biological applications has being carried
out. Especially, medical and biological applications are developing rapidly, and there
are striking things. However, the published scientific papers on these applications are
enormous, and it is hard for experts in this field to read all of them.
This book focuses on state-of-the-art microfluidic research in medical and bio-
logical applications. The top-level researchers in this research field explain carefully
and clearly what can be done by using microfluidic devices. Beginners in the field —
undergraduates, engineers, biologists, medical researchers—will easily learn to
understand microfluidic-based medical and biological applications. Because a wide
range of topics are summarized here, it also helps experts to learn more about fields
outside their own specialties. The book covers many interesting subjects, including
protein separation, protein crystallization, cell separation, single-cell analysis, cell
diagnosis, point-of-care testing, immunoassay, and regenerative medicine. Readers
will be convinced that microfluidic devices have great potential for medical and
biological applications.
I would like to thank Shinichi Koizumi and Asami Komada at Springer for their
help in pulling the book together. The publication of this book would have been
impossible without their help. Finally, I would like to express my sincere thanks to
the authors for all of the time and efforts they spent writing their chapters.
Sapporo, Japan Manabu Tokeshi

August 2018
v
Contents
1 Acoustofluidic Blood Component Sample Preparation

and Processing in Medical Applications . . . . . . . . . . . . . . . . . . . . . 1
Maria Antfolk and Thomas Laurell
2 Microfluidic Technologies and Platforms
for Protein Crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Masatoshi Maeki and Manabu Tokeshi
3 Application of SERS-Based Microfluidics
for In Vitro Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Jinhyeok Jeon, Namhyun Choi, Joung-Il Moon, Hao Chen,
and Jaebum Choo
4 Miniaturized Electrochemical Sensors to Facilitate Liquid
Biopsy for Detection of Circulating Tumor Markers . . . . . . . . . . . . 71
Yi-Ge Zhou, Leyla Kermansha, Libing Zhang,
and Reza M. Mohamadi
5 Spiral Inertial Microfluidics for Cell Separation
and Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Ning Liu, Chayakorn Petchakup, Hui Min Tay, King Ho Holden Li,
and Han Wei Hou
6 Worms on a Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Han-Sheng Chuang, Wen-Hui Wang, and Chang-Shi Chen
7 Microfluidic Devices for Gamete Processing and Analysis,
Fertilization and Embryo Culture and Characterization . . . . . . . . . 197
Séverine Le Gac, Verena Nordhoff, and Bastien Venzac
8 Microfluidic Organs-on-Chips to Reconstitute Cellular
Microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Yu-suke Torisawa
vii
viii Contents
9 In Vitro Tissue Construction for Organ-on-a-Chip

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Yuya Morimoto, Nobuhito Mori, and Shoji Takeuchi
10 Nanobiodevices for Cancer Diagnostics
and Stem Cell Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Daisuke Onoshima, Hiroshi Yukawa, and Yoshinobu Baba
11 Nanopore Device for Single-Molecule Sensing Method
and Its Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Masateru Taniguchi and Takahito Ohshiro
12 Paper Microfluidics for POC Testing
in Low-Resource Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Elain Fu
13 Paper-Based Microfluidics for Point-of-Care
Medical Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Kentaro Yamada and Daniel Citterio
Chapter 1
Acoustofluidic Blood Component Sample
Preparation and Processing in Medical
Applications
Maria Antfolk and Thomas Laurell
Abstract Recent developments of bulk acoustofluidic technology (BAW – bulk

acoustic wave) in biomedical applications is described in this chapter. The basic
principles for setting up an acoustic standing wave in a microchannel in 1 or
2 dimensions in the transversal direction to flow is outlined. BAW acoustofluidics
is a preferred solution as compared to SAW based acoustofluidics due to the
relatively higher acoustic energies that can be accomplished in BAW systems.
This in turn lends BAW technology to perform cell manipulation based handling
in a sufficiently high flow through format that can fulfill many biomedical and
bioanalytical applications. Several unit operations for BAW based cell handling
have today reached a level of maturity where these are being integral components in
cytometry and cell processing instrumentation. Most of these applications are still
realized at an analytical level and have not yet reached process scale or therapeutic
scale throughput. However, intense developments are in progress to also reach into
this domain of larger scale processing since the performance and label free operation
offered from BAW systems would significantly impact current bioprocess industry
and clinical practice. The importance of having full control of the buffer systems
used is discussed since poorly matched buffers/fluids, with respect to the acoustic
properties (acoustic impedance), may significantly impact the processing outcome as
a consequence of acoustically driven fluid relocation. Also, the challenge of manip-
ulating smaller bioparticles, e.g. bacteria, is discussed and strategies to tackle the fact
that the inherent acoustic streaming in acoustic standing wave based microfluidics
may be counteracting the desired alignment of cell/particles defined by the acoustic
standing wave. A focus is put on applications in blood component processing, where
unit operations such as cell separation (WBC, RBC, WBC subpopulations, CTC and
platelets), buffer exchange and concentrating cell samples have become important
modalities in cell based microfluidics. The current state of diagnostic BAW appli-
cations such as blood plasma separation, circulating tumor cell (CTC) isolation,
rapid hematocrit determination and bacteria enrichment/purification in sepsis are
discussed.
M. Antfolk · T. Laurell (*)

Department of Biomedical Engineering, Lund University, Lund, Sweden
e-mail: [email protected]
© Springer Nature Singapore Pte Ltd. 2019 1

M. Tokeshi (ed.), Applications of Microfluidic Systems in Biology and Medicine,
Bioanalysis 7, https://doi.org/10.1007/978-981-13-6229-3_1
2 M. Antfolk and T. Laurell
Keywords Acoustofluidics · Acoustophoresis · Cell separation plasmapheresis ·

CTC · Sepsis · Hematocrit
1.1 Introduction
Acoustofluidics uses ultrasound to separate and handle cells either in continuous

flow, using bulk acoustic waves (BAW) [1–3] or standing surface acoustic waves
(SSAW) [4], or in a batch mode using acoustic trapping. The methods rely to a
majority on the formation of standing acoustic waves to handle and process cells
based on their size, density, and/or compressibility.
Acoustophoresis is most often operated in a label-free mode which is advanta-
geous when isolating cells that are not susceptible to surface marker based separa-
tions, but has also been combined with affinity beads to target specific cell types. In a
continuous flow mode, devices depending on BAW can be operated at relatively
high flow rates compared to other microfluidic methods, and where SSAW devices
have not shown the same throughput. Devices based on SSAW, however, are
fabricated in softer materials such as PDMS, while BAW devices generally rely on
more rigid materials such as silicon or glass in order to efficiently support a standing
wave formation defined solely by the geometry of the microfluidic compartment.
BAW systems are commonly operated by exciting the entire chip structure at an
oscillation frequency that coincides with a fundamental resonance mode of a
microfluidic compartment. By designing the resonance cavity as a microchannel,
particles or cells can be focused in well-defined and reproducible positions/stream-
lines [5]. In a SAW device the microchannel is placed between a pair of IDTs on a
piezoelectric substrate. When actuating the substrate the SAWs propagate in oppo-
site directions on the substrate surface and leak into the liquid of the microchannel.
The interference between the two propagating fields causes pressure fluctuations in
the liquid resulting in the formation of acoustic pressure nodes and antinodes
[6]. Both types of acoustophoresis offer opportunities for a number of different
unit operations such as cell separation, sample concentration, alignment and buffer
exchange (Fig. 1.1).
1.1.1 Acoustofluidics
When a particle is suspended in an ultrasound standing wave field, the particle is

subjected to both a primary and secondary radiation force, as well as a Stokes’ drag
force induced by the acoustic streaming. The primary acoustic radiation force
originates from scattering of the standing wave on a particle and affects the particles
position within the microchannel. The secondary acoustic radiation force is due to
interactions of the scattered waves from two particles and affects the particle-particle
1 Acoustofluidic Blood Component Sample Preparation and Processing in. . . 3
Fig. 1.1 Schematic of different cell handling unit operation achievable with acoustophoresis
relative position. This force is commonly orders of magnitude smaller and is only
significant on very short particle-particle distances, i.e. at very high particle concen-
trations. Most acoustofluidic devices are operated below this critical particle con-
centration where these secondary forces are negligible. The fundamental theory on
acoustic standing wave forces on particles have been further described by King [7],
Yoshioka and Kawashima [8], Gorkov [9] and Nyborg [10], among others.
1.1.1.1 Primary Acoustic Radiation Force
The primary acoustic radiation force (Frad) is responsible for moving the cells within
the microchannel, to the node or anti-node of the standing wave. The expression for
the primary acoustic radiation force for a plane standing acoustic wave, on a
spherical particle with a radius much smaller than the wavelength can be written,
Eq. 1.1:

4π 3 1 2 3
F rad ¼ ∇U rad ¼ ∇ a f 1 κ 0 pin f 2 ρ0 v2in ð1:1Þ
3 2 4
κp
f 1 κ~ ¼ 1 κ~ and κ~ ¼
κ0

2 ρ~ 1 ρp
f 2 ρ~ ¼ and ρ~ ¼
ρþ1
2~ ρ0
Urad denotes the acoustic radiation potential, a the particle radius, f1 and f2 are the
monopole and dipole scattering coefficients respectively, κ0, ρ0, κp and ρp are the
Fig. 1.2 Schematic of

acoustic pressure field (solid
blue line) and acoustic
radiation force (dashed
green line) in the channel
cross-section. Red arrows
indicate direction of Frad for
particles with a positive
acoustic contrast factor, Φ
compressibility and density of the fluid and particle, respectively, hpini and hvini are
the pressure and velocity field time averages. When considering an ideal
one-dimensional standing wave (Fig. 1.2), the expression for the acoustic radiation
force can be simplified to the following [11]; Eq. 1.2.

F zrad ¼ ∂z U rad ¼ 4πϕ κ~; ρ~ ka3 E ac sin ð2kzÞ ð1:2Þ
p2a
E ac ¼
4ρ0 c20

1 5~ρ2
ϕ κ~; ρ~ ¼ κ~
ρþ1
3 2~

where k ¼ 2π/λ, and ϕ κ~; ρ~ is the acoustic contrast factor, Eac the acoustic energy
density, z the position of the particle in the direction of the channel cross-section
(Fig. 1.2), pa the pressure amplitude, ρ0 and c0 the density and the speed of sound in
the medium respectively. It should be noted that Eac is proportional the square of the
voltage applied to the piezo electric element.
The acoustic radiation force-induced movement of cells and particles in a stand-
ing wave is balanced by the Stokes’ drag force such that Frad ¼ Fdrag. When these
forces are balanced the velocity, urad, of the particle can be derived according to
Eq. 1.3, where η is the fluid viscosity.
2Φ 2
urad ¼ a ky E ac sin 2k y y ð1:3Þ
3η
It can be seen that the particle velocity is dependent on the square of the particle
radius and hence acoustophoretic separation of particles of the same physical
properties are strongly dependent of the particle size. Using Eq. 1.3 the unknown
acoustic contrast factor for a particle or cell with a known radius can be estimated by
comparing the voltage needed to translate the particle to the center outlet under
constant flow rate versus the corresponding voltage for a reference particle where
both the contrast factor and radius are known.
Fig. 1.3 Schematic of one-dimensional and two-dimensional acoustophoretic focusing
The sign of the acoustic contrast factor, Φ, depends on the density and compress-
ibility of the particles in relation to the suspending medium, and determines if the
particle will move towards the pressure node or anti-node. This factor is typically
positive for cells suspended in physiological fluids such as plasma, phosphate
buffered saline (PBS), or cell culture medium, and negative for particles such as
air bubbles or oil particles in the same medium. It should however be noted that the
acoustic contrast factor of a cell is dependent of the acoustophysical properties of the
buffer conditions and by designing the suspending medium accordingly a cell can
also display a negative acoustic contrast [12]. The acoustic contrast factor of a cell in
a given medium can be calculated if the density and compressibility of the cell is
known. Although density data many times can be found in the literature, the
compressibility is scarcely found. To alleviate this shortcoming, Cushing et al.
recently presented a method to measure the compressibility for specific cell types
[13], which opens the route to a simplified way to tailor make acoustophoresis
buffers for optimized separation performance.
1.1.1.2 Two-Dimensional Focusing
Early acoustophoresis based particle separation experiments relied on focusing the

particles in one dimension (Fig. 1.3, left) [5, 14]. The parabolic flow prole in the
microchannels will have an impact on the separation performance when operated in
this mode. This is due to the fact that particles suspended in different parts of the
channel will have different retention time in the device and, thus, experience the
acoustic standing wave field increasingly longer when the particles are positioned
nearer the walls or the top and bottom of the channel. A smaller particle initially
positioned closer to the wall and especially top and bottom of the channel may, thus,
be able to move to the same final position as a larger particle initially positioned
closer to the channel center. As the retention time in the force field for the smaller
particle will be longer it will make up for the fact that this particle will experience a
smaller acoustic radiation force and thus move slower.
In order to improve the resolution of the separation, the particles can be
pre-aligned in two dimensions, both horizontally and vertically, to ensure that they
all are positioned in the same flow vector and thereby have the same retention time in
Fig. 1.4 Schematic of the

impact of separation
performance in free flow
acoustophoresis using either
(a) one-dimensional
pre-alignment or (b)
two-dimensional
pre-alignment
the separation microchannel (Fig. 1.3). Particles pre-aligned in 1-D along the
sidewall, Fig. 1.4a, will display a broadened distribution at the outlet depending
on the initial vertical distribution of the particles in the acoustophoresis channel
during one-dimensional separation, whereas particles that are pre-aligned in
two-dimensions before the acoustophoresis separation step, Fig. 1.4b, will display
a minimized dispersion at the separation outlet. Two-dimensional pre-alignment has
been used to improve the sorting efficiency [15–17].
Two-dimensional focusing has also been used to improve the ability to concen-
trate cells and particles in continuous flow using acoustophoresis [18]. The
two-dimensional focus positions the cells in the fastest moving fluid regime when
passing the outlet region. In the outlet region where the channel widens, the channel
width no longer matches the wavelength of the ultrasound. In this region resonances
occur in several directions, creating an spurious acoustic standing wave field that
may divert the particles from their original trajectories to end up in the waste outlet.
Also, acoustically induced vortices appear at the edges of the outlet flow splitter
which may divert one-dimensionally focused particles from the central stream line,
i.e. if the particles move in the slower moving fluid regime past this region they may
spend enough time there to be diverted. If they are, however, two-dimensionally
focused to be positioned in the fast-moving central fluid regime they will pass this
region rapidly and will not divert from their original trajectories.
1.1.1.3 Acoustic Streaming
An acoustic standing wave in a microchannel will not only exercise a primary

radiation force on particles suspended in the acoustic standing wave field, it will
Fig. 1.5 Schematic of Rayleigh streaming in the cross-section of a microchannel actuated at a λ/2
resonance. Lower insert illustrates the boundary driven Schlichting streaming that induce the
Rayleigh streaming rolls
also induce the acoustic streaming, driven by the shear stress near rigid walls in the
acoustic boundary layers of the standing wave [19]. This phenomenon was origi-
nally described by, and named after, Lord Rayleigh to Rayleigh streaming
[20]. Later, the theory has been extended by Schlichting [21], Nyborg [22], Hamilton
[23], and Muller [24], among others.
The Rayleigh streaming seen in acoustofluidic devices is driven by the viscous
dissipation of acoustic energy into the boundary layer of the fluid along the channel
walls. The dissipation in the boundary layer next to the wall is relatively large in
comparison with the bulk dissipation because of the steep velocity gradient that is
formed perpendicular to the channel wall due to the non-slip boundary condition,
dictating that the velocity by the wall decreases to zero. The boundary layer δv in
most acoustofluidic devices is less than 1 μm thick, harboring the steep velocity
gradient. The viscous dissipation results in a momentum flux close to the channel
wall, which in turn results in the formation of boundary layer vortices called
Schlichting streaming (Fig. 1.5). This boundary layer streaming will in turn generate
the large streaming vortices seen in the bulk of the fluid, the Rayleigh streaming. The
acoustic streaming is particularly pronounced when λ > > h > > δv, where λ is the
ultrasound wavelength and h is the microchannel height (perpendicular to the
direction of propagation of the standing wave) [19].
1. One dimensionally actuated Rayleigh streaming

In common acoustofluidic devices the motion in the acoustic force field of
particles (with properties similar to polystyrene) larger than the critical particles
diameter of 2 μm (BAW acoustophoresis actuated at around 2 MHz) is dominated
by the acoustic radiation force. The motion of smaller particles is, however, domi-
nated by the Stokes’ drag-induced acoustic streaming [25, 26]. At some positions in
the microchannel the resulting motion of the Stokes’ drag-induced acoustic stream-
ing counteracts the motion induced by the primary acoustic radiation force. For
larger particles, this presents no problem as the primary acoustic radiation force is
larger which keeps the focused particles in the nodal plane of the acoustic standing
wave. For smaller particles, however, this disables focusing of the particles when the
acoustic streaming drags the particles away from the nodal plane at the channel top
and bottom. The transition from acoustic radiation force-induced to acoustic
streaming-induced drag force motion is easily understood when considering that
the primary acoustic radiation force scales with the cube of the particle radius while
the acoustic streaming-induced drag force scales with the radius. Thus, as the particle
radius decreases the primary radiation force decreases faster than the streaming-
induced drag force.
2. Two dimensionally actuated Rayleigh streaming
When actuating the microchannel in two dimensions by two orthogonal reso-
nances the generated acoustic streaming velocity field is fundamentally different
compared to a single standing wave actuation [27]. The two-dimensionally actuated
streaming velocity field does not counteract the primary radiation force in the same
way as the classic Rayleigh streaming does. When actuated in only one dimension,
sub-micrometer particles are caught in the characteristic quadrupolar structure of the
streaming flow counteracting the focusing on these smaller particles (Fig. 1.5).
When simultaneously actuating in two dimensions the streaming pattern instead
takes the form of a single vortex (Fig. 1.6 (left)), no longer counteracting the
focusing of the smaller particles but instead rotating them and under the influence
of the primary radiation force they take on a spiraling trajectory towards the channel
center (Fig. 1.6 (right)). The occurrence of the circular streaming is dependent of the
oscillation phase shift between the side walls and top/bottom walls. Using this
finding Antfolk et al. reported that the critical particle diameter for acoustic particle
focusing could be reduced below 0.5 μm in diameter (for polystyrene-like particles)
when actuating the microchannel in two dimensions [27].
1.1.1.4 Size-Insensitive Separation
Most successful acoustophoresis separation experiments are dependent mostly on

the particle size differences, but the effect of the density and compressibility can also
be seen for example when Grenvall et al. [28] managed to separate monocytes from
granulocytes even though they are very similar in size.
Fig. 1.6 Superimposing horizontal and vertical λ/2 resonance modes and Rayleigh streaming (left)
in a square cross-section channel yields a particle trajectory that is driven by acoustic streaming and
the radiation force towards the channel center (right). (Adapted from Ref. [27])
In light of this, Augustsson et al. [12] recently reported on an innovative method,

iso-acoustic focusing, to perform size-insensitive acoustophoretic separation of cells
in inhomogeneous density liquid flow. The cells can be separated based on their
acoustic impedance that in turn depends on their density. Using this device, they
showed that the acoustic impedance of the breast cancer cell line MCF7 differs from
that of the different WBC populations. This indicates the possibility of a microfluidic
separation method less dependent on size that will not be biased towards only
recovering the larger cancer cells.
1.1.1.5 Medium Switching
Another acoustofluidic phenomenon that must be accounted for is the medium

switching effect. Similar to particles, whole fluids volumes can also be acoustically
manipulated. As most acoustofluidic cell separation experiments utilizes a particle-
free liquid in the microchannel centre into which the separated particles can be
focused from the side streams it is important to match to acoustic impedance of the
two separate carrier fluids accordingly. If two fluids of different acoustic impedance
stream alongside each other in an λ/2 acoustic standing wave field there will be an
acoustic force acting at the fluid interface such that the fluid of higher acoustic
impedance will relocate to the acoustic pressure node. This may have profound
impact on the outcome of acoustophoresis experiments. E.g. if the acoustic imped-
ance of the outer particle streams is higher than the central particle-free fluid the
entire particle fluid stream will relocate to the pressure node in the centre and drive
the particle-free liquid to the channel sides [29]. Thus, no separation has occurred
even though the impression might be that all target particles are now focused in the
centre. The medium switching effect might be seen for example when attempting to
separate cells from plasma if the acoustic impedance of the cell free centre liquid is
not properly matched. Medium switching occurs at very modest differences in
acoustic impedance (<1%) why the fluids used in acoustophoretic separations with
striated flows have to be properly tuned.
1.1.1.6 Biocompatibility
Acoustofluidics have been shown to be a gentle method to cells, not affecting the
viability or other cell functions [30, 31]. When separating platelets from peripheral
blood progenitor cells Dykes et al. reported that the platelets still retained their
capacity to become activated after the acoustic separation, and furthermore, that
there were significantly fewer activated platelets in the acoustophoretically separated
sample compared to control samples obtained from standard buffy coat platelet
concentrates. It was also shown that the blood progenitor cells retained their
colony-forming ability after the acoustic separation [32]. Similarly, Urbansky et al.
showed a preserved clonogenic capacity after separating hematopoietic progenitor
cells in a BAW device [33]. Further evidence that acoustic cell handling is gentle
was reported by Hultström et al. in studies of acoustic cell trapping where the
viability and proliferation was unaffected after acoustic trapping of COS-7 cells
for 30–75 min [34].
1.2 Blood Components
Applications of acoustophoresis has been dominated by the life science field and
much effort has been put into the separation of different cell types and/or blood
components from each other.
Fig. 1.7 Schematic of Platelet/ WBC separation

platelet separation from
WBC in an apheresis
product
Platelets
WBC
1.2.1 Platelets
As platelets display a very modest acoustophoretic mobility, several groups have

reported approaches to separate platelets from other blood cells by migrating these
other cells to the pressure node, leaving the platelets in the original streamline.
Dykes et al. removed platelets from peripheral blood progenitor cell products by
focusing the WBCs into a clean central buffer stream while the platelets followed the
plasma stream along the walls of a λ/2 resonator with a central and a single side
outlet configuration (Fig. 1.7). A depletion efficiency of 89% of the platelets and a
leukocyte recovery of 98% at a flow rate of 20 μL/min using a BAW device was
reported [32].
Using a SAW device, Nam et al. separated platelets from whole blood with a
recovery or 74.1% and a removal of 99.9% of the RBCs and WBCs. The device
could, however, only be operated at a flow rate of 0.25 μL/min [35]. Chen et al. later
proposed a parallel plate BAW device configured to perform an h-filter type sepa-
ration which could be operated in an impressive high-throughput mode of 10 mL/
min but with a modest separation performance, removing about 80% of the RBCs
and WBCs while recovering around 80% of the platelets [36].
1.2.2 WBC Sub-populations
A more challenging blood component separation is fractionation of different white

blood cell populations. Here, Grenvall et al. managed to separate lymphocytes,
granulocytes, and monocytes into three different populations. Using a BAW device,
they showed that 86.5% of the lymphocytes could be recovered with a purity of
95.2% and 68.4% of the granulocytes were recovered with a purity of 98.5%, and
83.1% of the monocytes could be recovered with a purity of 25.5% at a sample flow
rate of 8 μL/min and a cell throughput of 8000 cells/min [28].
When different cell types display overlapping acoustophoretic mobilities and are
not possible to separate based on their acoustophysical properties, affinity beads
directed towards a specific cell surface marker can be used. As the microbeads bind
to their target cell the bead cell complex display a different acoustic fingerprint and
will migrate at a different velocity as compared to the unbound cells, thereby
enabling acoustophoretic separation, analogous to magnetic bead based separation.
Using a BAW device Lenshof et al. utilized anti-CD4 affinity beads that gave the
target cell a higher acoustic mobility compared to the unlabeled lymphocytes of a
similar cell size. In this way 65 22% of the CD4+ T cells could be recovered with a
purity of 87 12% at a sample flow rate of 30 μL/min and a cell concentration of 107
cells/mL [37]. Similarly, Urbansky et al. also utilized affinity beads to separate CD8
+ T cells from peripheral blood progenitor cells in a BAW device. The mean purity
of the separated sample was 91.8 8% and a median separation efficiency of 63%
(range 15.1% to 90.5%) could be obtained at a sample flow rate of 60 μL/min and a
cell concentration of 107 cells/mL [33].
Utilizing a BAW device (Fig. 1.8) Urbansky et al. separated the mononuclear cell
(MNC) fraction of the WBCs, including monocytes and lymphocytes, from RBCs in
100x diluted blood samples. By optimizing the acoustic impedance of the medium,
thereby altering the relative acoustic mobility of the MNCs compared to the RBCs, a
high separation efficiency could be reported, where 99.4 0.4% of the collected
cells where MNCs with a contamination of 0.8 0.6% of RBCs. This corresponded
to at 390.6 169.8-fold enrichment of the MNC fraction and a recovery of
68% [38].
1.2.2.1 Integrated Acoustic Device for Monocyte Subpopulation

Isolation
Nivedita et al. proposed an integration of a spiral microfluidic device for Dean flow
and a BAW acoustic device utilizing microbubbles to size-selectively trap cells. The
device was used to separate a subpopulation of monocytes that were larger than
18 μm in diameter from 10x diluted whole blood. At a high flow rate of 1.1 mL/min
they were able to obtain a pure sample of only monocytes [39].
Fig. 1.8 Acoustic device for separating mononuclear cells from red blood cells. (Reprinted from
[38], Copyright (2017) The Author(s) licensed under CC BY 4.0)
1.2.3 Plasma and Plasma Proteins
Lenshof et al. demonstrated a way to obtain cell-free plasma from whole blood by
acoustically focus and sequentially removing the concentrated blood cells focused in
the center of a BAW device while retaining as much plasma as possible. At a sample
inlet flow rate of 80 μL/min plasma with only 3.65 * 109 cells/L could be produced,
which is below the recommended maximum threshold of 6 * 109 cells/L for plasma
transfusions set by the Council of Europe [40]. Later, the same platform was
integrated with an immunoaffinity-capture platform for the detection of PSA from
the plasma separated from whole blood. Spiked PSA could be detected in the
separated plasma at clinically significant levels of 1.7–100 ng/mL [41]. The
presented blood plasma isolation method fulfilled clinical standards for diagnostic
purposes, and if possible to integrate with an analytical step, the development may
become a valuable contribution in the field of clinical diagnostics.
In a similar way Tenje et al. used a BAW device to wash RBCs from plasma in an
attempt to produce blood plasma protein-free RBC concentrates important for blood
transfusion to patients suffering from IgA-sensitivity. While recovering 97% of the
RBCs they showed an almost complete removal of IgA with a concentration of less
than 0.25 μg/mL in the cell fraction, which is below the limit of detection and also in
accordance with the European guidelines. The RBC sample was diluted 20x before
processing and the sample inflow rate was 100 μL/min [42]. It should be noted that it
was critical for the separation to balance the acoustic impedance of the central buffer
versus the sample and ensure that medium switching did not occur. Although
showing a good performance in RBC isolation and background elimination, the
method still needs to show its value in terms of upscaling and throughput to meet
clinically relevant applications.
Fig. 1.9 Simple acoustic

hematocrit measurement
with optical readout.
(Reprinted from [44],
Copyright (2018), with
permission from Elsevier)
1.2.4 RBC Separation
Most acoustophoresis devices operate at flow rates around 1–100 μL/min. At the
lower end of this range the flow rate might not be clinically relevant for processing a
blood sample. Especially, if the target cells are less abundant in the blood sample. In
an attempt to show a high throughput device Adams et al. fabricated a parallel plate
BAW device that could be operated at an impressive flow rate of 16.7 mL/min. At
this flow rate, they showed that they could separate 2 μm and 10 μm diameter
particles with a recovery of around 80% of the 10 μm particles and a contamination
of 10% of the 2 μm particles [43]. When using this set-up to separate RBCs from
whole blood into PBS a recovery of 95% was reported at a flow rate of 16.8 mL/min.
It is however unclear if this separation actually occurred or if the recovery of cells at
the target outlet resulted as an effect of fluid relocation due to the differences in
acoustic impedance between PBS and whole blood as previously reported by Tenje
et al. [42]. No data was reported showing that the blood plasma background was
eliminated and since only the cell recovery and not the wash efficiency was
documented the actual separation was not concluded in the paper.
1.2.5 Hematocrit Measurements
Hematocrit measurements are commonly made by cell counting or centrifugation

followed by estimation of the packed cell volume. Using a simple BAW device,
Petersson et al. showed that acoustic hematocrit measurements were possible in just
2 s by acoustically focusing the blood cells in the channel center, separating it from
the plasma. The hematocrit could be detected by optically monitoring the cell-
focused area in the microchannel center with an error of less than 3%
[44]. (Fig. 1.9). The method was tested on a limited number of healthy volunteers
and the full clinical value of the method still needs to be clarified by benchmarking a
broader population of healthy and sick individuals compared to the golden standard.
1.3 Circulating Tumor Cells
Circulating tumor cells (CTCs) are among the most targeted rare cells found in
blood. Shed from a primary tumor they travel with the blood circulation to remote
tissues where they potentially can form metastases. Found in quantities as low as
1–10 CTC/mL they are challenging to detect but have a great diagnostic value and
interest to both clinicians and researchers. CTCs are sought after both for their
prognostic and diagnostic values, e.g. as a marker for the disease progression and
response to different treatments [45].
1.3.1 Cancer Cell Separation Based on a Positive Acoustic

Contrast Factor
In an early approach, Augustsson et al. [17] used a BAW device to separate spiked
tumor cells from WBCs based on the fact the that the tumor cells display a larger
average size. With the same configuration as in Fig. 1.8, the device could recover
85.4% of the cancer cells with a contamination of 0.7% of WBCs at a sample flow
rate of 70 μL/min. Later Antfolk et al. presented a BAW device with a simplified
fluidic setup based on the same separation principle. Simplification was achieved by
removing the center cell-free fluid inlet when performing the separation. In a first
channel section the cells were pre-aligned in two dimensions in two parallel streams
λ/4 from the channel side walls, before entering the separation zone that operated in a
λ /2 mode (and analogous to the separation in Fig. 1.8), where the tumor cells were
separated from the WBCs and collected in the central outlet. This device had three
obvious advantages.; (1) It facilitated the flow control of the device, which will
generate higher recoveries and purities of target cells; (2) It would also more easily
lend itself to integration with other unit operations, keeping the fluidic connections to
a minimum.; and (3) The need to match the acoustic impedances of fluids, to avoid
acoustic impedance mismatch when using multiple inlet streams, was eliminated.
Using this device, spiked cancer cells could be separated from WBCs with a
recovery of 86.5 6.7% of the cancer cells with 1.1 0.2% contamination of
WBCs at a sample flow rate of 100 μL/min. In addition to the mentioned advantages,
this device also made it possible to concentrate the recovered cells about 2.5-fold
instead of diluting them as devices relying on hydrodynamic pre-focusing often do, a
fact that is especially important for rare cell processing [16]. The original platform
presented by Augustsson et al. [17] was later improved to facilitate the processing of
clinical-scale samples of 10 mL of 2x dilute RBC-lysed blood spiked with 50 MCF-7
breast cancer cells in 2 h. In a high-recovery mode the cancer cell recovery was
Fig. 1.10 Separation and subsequent trapping of lung cancer cells A549 from RBCs. (Reprinted
from [47], Copyright (2015) The Author(s) licensed under CC BY 3.0)
86 2.3% with a WBC contamination of 0.57 0.14%, and in a high-purity mode

the cancer cell recovery was 62 8.7% with a WBC contamination of 0.1 0.07%.
This corresponded to cancer cell enrichment factors of 162 55 and 1445 1811,
respectively for the high-recovery and high-purity modes and demonstrated a clin-
ical potential for CTC enumeration based on acoustophoresis [46].
Antfolk et al. also presented an analogous integrated BAW device to simulta-
neously separate and concentrate spiked cancer cells from WBCs. It was reported
that MCF7 cells could be separated from WBCs with a recovery of 91.8 1.0% and
a contamination of 0.6 0.1% WBCs with a simultaneous 23.8 1.3-fold concen-
tration of the cancer cells. Correspondingly, DU145 cells could be recovered with an
efficiency of 84.1 2.1% with 0.2 0.04% contamination of WBCs while
concentrating the cancer cells 9.6 0.4-fold at a sample flow rate of 80 μL/min.
This might reduce the need for a separate concentration step, commonly performed
by centrifugation, before sample analysis and cancer cell enumeration which greatly
reduces the risk of losing rare target cells during this step.
In an attempt to take on-chip sample preparation further, Iranmanesh et al. [47]
presented a BAW device that simultaneously separated spiked cancer cells (lung
cancer cell line A549) from RBCs, trapped and concentrated the recovered cancer
cells and allowed for on-chip fluorescence image analysis of the trapped cells. 85.5%
of the cancer cells could be recovered with a purity of 100%, and the cells could be
concentrated 130 times in the trap (Fig. 1.10). The sample flow rate was, however,
only 4.5 μL/min making rare cell processing very time consuming in its current state.
It should also be noted that the experiments were made on an RBC suspension
without WBCs and the main challenge in CTC separation from blood is to discrim-
inate against the WBC background that has more similar acoustophysical properties
to CTCs than RBCs do.
Using a tilted-angle SSAW device, Li et al. [48] separated spiked cancer cells
from WBCs at a sample flow rate of 20 μL/min, i.e. >4 h for processing a 5 mL
clinical sample. While removing 99% of the WBCs 60–80% of the cancer cells could
be recovered. However, the system was tuned to operate at a high CTC recovery,
90%, (based on the cell line data) when running three metastatic breast cancer,
leading to a challenging background of 10% remaining WBCs. This should be
compared to the golden standard, CellSearch, that display a WBC depletion of
about log 3 to 3.5.
Fig. 1.11 Schematic of

acoustophoretic negative
selection using antibody
activated microbeads having
a negative acoustic contrast
factor (NCP). NCP
microbeads bind to WBCs,
forming a complex that
displays a net negative
acoustic contrast factor,
moving towards the pressure
antinode in the
microchannel, while cancer
cells (red), having a positive
acoustic contrast factor,
focus in the pressure node in
the center of the channel
1.3.2 Cancer Cell Separation Based on a Negative Acoustic

Contrast Factor
Faridi et al. used anti-EpCAM coated microbubbles to show that they could separate
microbubble-bound cells (HCT 116, a colon cancer cell line) from unbound cells.
The microbubbles display a negative acoustic contrast factor, i.e. they move towards
the pressure antinode in the acoustic standing wave field. At a sample flow rate of
3 μL/min they showed a 75% separation efficiency [49].
Using PDMS-based negative contrast factor particles (NCP), Cushing et al.
showed that they could separate cancer cells from WBCs bound to the anti-CD45
activated negative contrast particles. A separation efficiency of 99% and a cancer cell
recovery of 85–90% and a contamination of 1–2% WBCs, spiking 1000 MCF-7 or
DU145 cells per 100,000 WBCs, was demonstrated (Fig. 1.11) [50].
1.3.3 Cancer Cell Concentration
Rare cells, after isolation, are often dilute since they originate from large volumes of
sample. When handling dilute cell suspensions containing few target cells, or small
sample volumes produced by microfluidic processing, ordinary concentration tech-
niques such as centrifugation might not be practically usable due to inherent losses
Fig. 1.12 Acoustophoresis BAW concentration device operated in a recirculating mode.

(Reprinted with permission from [51]. Copyright 2015 American Chemical Society)
linked to centrifugation, i.e. no formation of a cell pellet or adsorption to centrifu-

gation tube walls. To this end, BAW acoustophoresis has been used to concentrate
cells and particles in order to facilitate subsequent analysis.
Nordin and Laurell showed that sequential concentration steps and
two-dimensional focusing was crucial to achieve a high concentration factor in a
continuous flow BAW device. The sequential concentration steps were used to
multiply a modest concentration factor achieved in each step to a high overall
concentration factor. The two-dimensional focusing allowed the particles to all be
positioned in the flow regime with the highest flow rate which reduced the time spent
in the acoustically chaotic trifurcation regions to a minimum, preventing them from
being deflected from their established flow trajectories and into the waste outlets.
Using these findings dilute cancer cell suspensions could be concentrated
195.7 36.2-fold with a recovery of 97.2 3.3% at a sample input flow rate of
200 μL/min [18]. Jakobsson et al. took the concentration possibilities even further
and using a recirculating BAW device they showed that RBCs could be concentrated
a staggering 1166 110 times with a 98.7% recovery, and DU145 cells and MCF7
cells could be concentrated 817 125 times with a recovery of 90.2%, and
519 115.7 times with a recovery of 81.7%, respectively (Fig. 1.12) [51].
1.3.4 Integrated Acoustic Devices for CTC Processing
Acoustophoresis systems have also been integrated with other microfluidic devices
to add even more to the usability of the devices. Kim et al. presented an integration of
a BAW concentration device and a dielectrophoresis (DEP) trapping device,
enabling simultaneous concentration, trapping and subsequent analysis of target
cells. As a consequence of the cell concentration step, the device also translated a
high flow rate into a lower flow rate adapted for the in-line integrated DEP trapping
chip that was dependent of a low flow rate to efficiently trap cells. Without the
concentration step, the low flow rate limit of the DEP trapping chip would be very
time consuming and in principle be non-applicable to clinical samples. When
integrating the two devices a higher sample flow rate could be used. The integrated
system could process samples with a tenfold higher flow rate as compared to the
DEP trapping chip alone, while still recovering over 90% of the cells at a sample
input flow rate of 40 μL/min [52].
An acoustic separation step was later added to the integrated rare cell concentra-
tion and DEP-trapping system. The totally integrated CTC system efficiently elim-
inated the risk of cell losses in each of the unit operations by enabling direct on-chip
tumor cell separation, concentration, trapping and labelling prior to identification
and enumeration. Prostate cancer cells (DU145) spiked into a sample of the periph-
eral blood mononuclear cell (PBMC) fraction, with a concentration corresponding to
their concentration in whole blood, were efficiently separated and trapped at a
recovery of 76.2 5.9% of the cancer cells and a minute contamination of
0.12 0.04% PBMCs while simultaneously enabling a 20x volumetric concentra-
tion at a sample flow rate of 80 μL/min [53].
1.4 Bacteria
1.4.1 Passive Bacteria Processing
The smaller size of bacteria compared to eukaryotic cells make them more challeng-
ing to process using microfluidics as the active forces often scales with the size of the
cell or particle. Many attempts of bacteria processing in microfluidic devices thus
aim to isolate them by actively removing the larger blood cells, leaving the bacteria
unaffected in the plasma fraction.
Ai, Sanders and Marrone [54] used SAW to separate E. coli spiked into
pre-isolated peripheral blood mononuclear cells (PBMCs). In the target outlet
95.65% bacteria and 3.92% WBCs could be found, while 7.24% of the cells
collected in the waste outlet were found to be bacteria and 91.48% were WBCs.
The sample was processed with a flow rate of 0.5 μL/min. Ohlsson et al. [55]
proposed a system combining a BAW device with an acoustic trapping device for
rapid bacteria separation from whole blood in sepsis samples. In the acoustophoresis
chip blood cells where focused and subsequently removed (plasmapheresis) leaving
the bacteria in the cell free plasma which were acoustically trapped and concen-
trated in the down stream trapping unit (Fig. 1.13). The trapped and enriched bacteria
were subsequently released directly into a PCR microchip for analysis. With a
sample flow rate of 80 μL/min they processed a blood sample of 1 mL (diluted to
70% whole blood) in less than 2 h while recovering ~11% of the bacteria and a
~0.1% RBC contamination, resulting in a detection limit of 1000 bacteria/mL blood.
Using a glass BAW device, Ngamsom et al. separated the bacteria
S. typhimurium, spiked at a rate of 107 CFU/mL, from 2x diluted horse blood. By
focusing the RBCs in the channel center at a sample flow rate of 10 μL/min, they
Fig. 1.13 Schematic of the components assembling the integrated sepsis diagnostics system
presented by Ohlsson et al. [55], where an initial acoustophoretic removal of blood cells produced
a clean plasma (plasmapheresis) containing bacteria that continued to an acoustic trapping unit
where bacteria were enriched and washed prior to release into a disposable PCR microchip followed
by DNA amplification and bacteria identification
could deplete around 99.8% of the RBCs while recovering around 10% of the
bacteria. (Fig. 1.14) [56] Presumably, the low recovery of the bacteria stems not
from an acoustic mobility velocity overlap but rather from a mismatch of the
acoustic impedances of the sample and the buffer solution where the phenomena
medium switching (mentioned above, see also [42]) occurs rather than a separation.
In the case of medium switching the sample fluid will, if it has a higher acoustic
impedance than the central buffer, entirely switch place with the central buffer rather
than to just allow the cells to acoustophoretically migrate across the stream lines into
the clean buffer. This phenomenon would explain for the fact that 90% of the
bacteria ends up together with the RBCs rather than staying at the sides even though
they are significantly smaller than the horse RBCs and thus have a much lower
acoustic mobility, ensuring that they would transition to the central stream much
later than the RBCs under conditions where no medium switching occurred.
Fig. 1.14 Acoustofluidic device for passive separation of bacteria from blood by depletion of the
RBCs. (Reprinted from [56], Copyright (2016), with permission from Elsevier)
1.4.2 Active Bacteria Processing
Although often more challenging, some attempts have been made to process bacte-
rial samples by actively moving them with external forces. Acoustophoresis devices
that focus particles in one dimension and are operated at a frequency of around
2 MHz handles larger cells efficiently. They can, however, not be efficiently used to
handle particles and rare cells smaller than about 2 μm in diameter, practically
meaning that most bacteria cannot be actively processed by acoustophoresis.
While the acoustophoresis-induced motion of larger particles is dominated by the
acoustic radiation force, the motion of smaller particles is instead dominated by the
acoustic streaming-induced drag force. Larger particles can, thus, be focused while
smaller particles instead are trapped in the streaming vortices inherent with the
acoustic field. When striving to solve this problem, Antfolk et al. found that while
simultaneously exciting two orthogonal resonances an acoustic streaming velocity
field was formed that no longer counteracted the primary radiation force, which
thereby allowed sub-micrometer particles to be focused (Fig. 1.6, section Two
dimensionally actuated Rayleigh streaming). The device was also used to focus
E. coli achieving a focusability of 0.95 0.35. Simulations and experiment showed

that the streaming velocity field was dominated by a large centered flow roll instead
of the four characteristic Rayleigh vortices seen when actuating the channel in one
dimension (Fig. 1.6) [27]. This achievement opens up the acoustophoresis field
against many microbiology applications not previously possible.
1.5 Conclusions
Acoustophoresis has been extensively used for processing biological samples with a
special emphasis on blood sample handling and processing. The method can be used
for separation, concentration/enrichment, buffer exchange, or alignment and has
been found useful for blood component separation, cancer cell separation, or hemat-
ocrit measurements amongst other processes. It has also been demonstrated that the
method can be used to process samples of a clinically relevant size in a relevant time.
Acoustophoresis is, however, limited to the size of particles that can be actively
processed, due to the acoustic streaming drag force. This limit has been lowered
through recent developments and is likely to be lowered more in the future. Caution
also has to be made to ensure that the different liquids used are clearly matched in
their acoustic impedances to avoid medium switching to occur, where two striated
fluids relocates in the acoustic field instead of just the intended cells or particles.
Even so, when considering these concerns during the experimental design,
acoustophoresis has proven to be a useful method with the ability to integrate with
other microfluidic or macroscale methods, why it can be anticipated to see
acoustophoresis solutions become an integral part of future cell processing systems
as well as bioanalytical and clinical diagnostic systems.
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Chapter 2
Microfluidic Technologies and Platforms
for Protein Crystallography
Masatoshi Maeki and Manabu Tokeshi
Abstract Protein crystallization and its three-dimensional structure analysis is

indispensable for understanding the protein function in the body and life phenom-
enon. Three dimensional structure of protein also plays important role for drug
discovery and it have been already used to design new drug. To determine the
three dimensional protein structure, protein crystallization conditions: concentration
of protein, kinds and concentration of precipitant, buffer, pH, temperature, and
additives must be optimized. In addition, high-diffraction quality protein crystals
are needed to determine the protein three-dimensional structure at high resolution.
However, optimization of the protein crystallization condition and preparation of
high quality protein crystals require the labor intensives and trial-and-error.
Microfluidics can provide the solution for the problems of traditional protein crys-
tallography. A lot of microfluidic based technologies and platforms have been
developed to utilize their unique characteristics. In this chapter, microfluidic tech-
nologies and platforms for protein crystallography is summarized. In particular, the
application of microfluidics for high-throughput protein crystallization condition
screening, controlling of protein crystal growth, and on-chip X-ray diffraction
experiment using microfluidic devices are overviewed.
Keywords Protein crystallography · Droplet microfluidics · Normally-closed

valve · Crystal growth · X-ray crystal structure analysis
2.1 Introduction
Biological sample is valuable and expensive due to the difficulty of sample prepa-
ration acquired from human patients, animals, cells, and bacteria. The acquired
sample contains many impurities, which have to remove by appropriate pretreatment
methods for each clinical diagnosis, assay, or experiment. In the pretreatment step,
M. Maeki (*) · M. Tokeshi

Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Sapporo, Japan

28 M. Maeki and M. Tokeshi
the target biological molecules isolate from the impurities using separation tech-
niques such as, centrifuge, electrophoresis, and chromatography. Generally, the
purification step requires multiple trials with different separation modes [1, 2]. In
the case of protein purification, affinity chromatography is the first choice for
tag-fusion proteins. After the affinity chromatography, ion exchange chromatogra-
phy and gel filtration chromatography are considered to obtain the high purity
protein sample. When we need a large amount or high concentration of target protein
sample, a scale scale-up of purification volume is required, because protein cannot
be amplified unlike the nucleic acids. In particular, preparation of membrane protein
is more complicated compared with soluble protein. From these reasons, preparation
of the high purity biological sample is labor-intensive and time-consuming process.
Microfluidic technologies and platforms can offer many advantages in biological
applications [3–6]. Reduction of sample consumption, high-throughput screening,
shortening detection time, distinguishing fluid characteristic are remarkable features
of microfluidic devices, and it facilitate the cell separation and single cell analysis
[7–9], biological assays [10–12], pharmaceuticals [13–15], drug screening [16–
18]. These are essential and powerful features for the biological applications,
especially for protein analysis, because the high purity protein sample is valuable
and expensive.
In the field of protein analysis, structure biology is a key research area to elucidate
the relationship between the protein structure and the protein function [19–21]. Pro-
tein can function by precisely folding into its native “three-dimensional” structure.
Conversely, misfolded proteins not only lose a function but also cause of the disease
like an amyloidosis [22]. Determination of the protein three-dimensional structure
provides us the essential information including protein-protein interaction, active
site of protein, and protein-ligand binding site. The protein structural information is
indispensable for the structure based drug design and development of molecularly-
targeted therapy [23–25]. To determine the protein three-dimensional structure,
X-ray crystallography [26–28], nuclear magnetic resonance spectroscopy (NMR)
[29], and cryo-electron microscopy (cryo-EM) [30] are gold standard analytical
methods. Although these are powerful analytical methods for protein structure and
dynamics measurement, 80 ~ 90% of protein structures have been determined by
X-ray crystallography.
In the protein X-ray crystallography, the preparation of high quality protein
crystals is remaining problem to determine the precise three-dimensional protein
structure. After the purification step, screening of the protein crystallization condi-
tions is carried out by the conventional protein crystallization method, such as the
hanging drop vapor diffusion method, sitting drop vapor diffusion method,
microbatch method, and liquid-liquid interface diffusion method, as shown in
Fig. 2.1 [31]. At the primary screening, the kinds and concentration of precipitant
solution, concentration of protein solution, kinds, pH, and concentration of buffer
solution, crystallization temperature, and additives are optimized to obtain protein
crystals. We can roughly predict the suitable crystallization condition from the
homology of proteins, which have been recorded in the Protein Data Bank (PDB).
However, protein crystals do not always appear the similar crystallization condition.
2 Microfluidic Technologies and Platforms for Protein Crystallography 29
Fig. 2.1 Schematic illustrations of the conventional protein crystallization methods. Vapor diffu-
sion method. A drop containing unsaturated precipitant and protein solution is placed in a crystal-
lization well containing a precipitant solution. The well is sealed with grease to prevent evaporation
and to allow vapor equilibration of the droplet and the reservoir. Equilibration of water vapor from
the protein-containing droplet to the reservoir solution causes the protein solution to reach a
supersaturation level, where nucleation and initial growth occur. (A) Hanging drop vapor diffusion
method. (B) Sitting drop vapor diffusion method. (C) Sandwich drop vapor diffusion method.
(Reprinted with permission from Ref. [31] with permission from MDPI)
There-fore, a comprehensive trial is unavoidable at the primary screening experi-

ment. Typically, 1 ~ 2 μL of protein solution is needed for one trial, and thus, a
several hundred microliter valuable protein solution is required for the comprehen-
sive crystallization condition screening.
Then the secondary screening is conducted based on the results of primary
screening to obtain high quality protein crystals. Although optimization of the
crystallization condition is significant, the control of protein crystal growth is
indispensable factor for obtaining the high quality of protein crystals. Growth
control of the crystal regardless of the proteins or inorganic materials, at the ground
environment is attracted in many researchers for improving the molecular packing.
Formation of the natural convection is the major reason to disrupt the ordered
molecular packing of crystal at the ground environment. For improving the quality
of crystals, the microgravity, electric field, and magnetic field are used to control the
crystal growth environment, even though specialized experimental apparatus are
required [32–37].
Microfluidic technologies and platforms can resolve the abovementioned prob-
lems using its unique characteristics [38–41]. Reduction of the protein consumption
is easily achievable by downsizing of the device dimension. Only nano to picoliter
volume of protein solution is needed for one trial, and the microfluidic device
enables the high-throughput screening of protein crystallization condition [42–
54]. In addition, the “micro-sized crystallization space” allow the control of protein
crystal growth different form the conventional crystallization environment [55–
61]. In this chapter, microfluidic techniques and platforms for protein crystallogra-
phy to accelerate the field of structural biology are summarized and the applications
of microfluidic devices combined with the recent synchrotron facilities are also
overviewed.
2.2 Principle of the Protein Crystallization and X-Ray

Diffraction Measurement
In brief, the protein crystallization experiment constructs: screening of protein

crystallization condition, preparation of high quality protein crystal, screening of
cryoprotection condition, and X-ray diffraction measurement. In the case of con-
ventional methods, a micropipette is used to prepare the protein crystallization
screening setups. Generally, 1 μL of protein solution and 1 μL of precipitant solution
is mixed to prepare the crystallization solution, and the droplet of crystallization
solution is placed into a well of microplate. The precipitant works to decrease the
solubility of proteins. The microplate setting the crystallization droplet is stored at
the appropriate temperature until forming the protein crystals. For example, the
hanging drop crystallization method is the most widely used for primary screening.
Characteristics of the method is that supersaturation of the crystallization solution is
gradually increased by the water-vapor diffusion from the crystallization droplet to
precipitant solution in the reservoir during incubation. Diffusion rate of the water-
vapor and the time necessary for reach the equilibrium can control by the mixing
ratio of protein and precipitant solutions. When the appropriate kinds and concen-
tration of precipitant solution is selected, protein crystals form in the droplet by
increasing supersaturation of crystallization solution. However, protein crystals do
not form at the suitable crystallization condition. Therefore, the optimization of
protein crystallization condition is the first barrier of the protein crystallization.
A nucleation of protein crystal occurs at the high supersaturation condition by
adding the precipitant solution. However, the moderate supersaturation condition is
preferable for the crystal growth, because the high supersaturation condition lead to
increase lattice defects. This is a major dilemma of protein crystallization process to
control the nucleation and crystal growth [62, 63].
X-ray diffraction experiment is also essential part of the protein crystallography.

Proteins are crystallized with hydrated water molecules, the percentage of water
molecules occupy 50% w/w in average. Thus, manipulation of the protein crystals
for the X-ray diffraction experiment difficult due to the fragility of protein crystals.
In addition, cryoprotection process to prevent the deterioration of protein crystals
during the X-ray diffraction experiment is also important for protein crystallography.
Current synchrotron facilities enabled X-ray diffraction data collection from the
micrometer-sized crystals by high energy and photon flux X-ray beam [64, 65]. How-
ever, the high energy X-ray beam caused the radiation damage during the measure-
ment process. To avoid the X-ray radiation damage, protein crystal undergoes the
cryoprotection and is measured at 100 K in cold nitrogen gas stream [66, 67]. Opti-
mization of cryoprotection condition and damage-less cryoprotection procedure are
indispensable for high quality X-ray diffraction data. Finally, the protein three
dimensional structure is determined from the X-ray diffraction data using a PC.
A variety of the microfluidic devices have been developed for protein crystallog-
raphy, which realize the effective crystallization condition screening, growth control
of the protein crystals, on-chip X-ray diffraction measurement, membrane protein
crystallography, time-resolved protein crystallography. Basically, microfluidic
devices for protein crystallography was miniaturization of the conventional tech-
nique. However, the applications of microfluidic devices for protein crystallography
have been expanding by the advances of the synchrotron facilities.
2.3 Microfluidic Devices for High-Throughput Screening

of Protein Crystallization Condition
Screening of protein crystallization condition is the one of the most significant

problems in protein crystallography. Screening for hundreds or thousands of protein
crystallization condition is necessary to prepare diffraction quality protein crystals.
In the case of the conventional protein crystallization screening, a robotic system
equipped with an automatic liquid dispenser is employed for high-throughput
screening. For example, mosquito® supplied from TTP LabTech Ltd. is a major
automated dispenser [56]. The pipetting range of sample is tens of nano litter to
micro litter, and the typical microplate is useable for the screening experiment.
However, these kinds of automated dispenser are astonishingly expensive, and
require a constant running cost.
In comparison with the conventional methods, microfluidic devices provide many
advantages for protein crystallization screening. Figure 2.2 shows the droplet-based
high-throughput screening system of protein crystallization condition
[43]. Ismagilov’s group first reported the droplet-based protein crystallization sys-
tem. Protein, precipitant, buffer, and additives (PEG) were separately introduced into
the microchannel, and droplets containing the all reagent at the defined concentration
were continuously generated by shearing the oil flow stream. The composition of
Fig. 2.2 Droplet-based high-throughput protein crystallization condition screening system. (a–c)
A schematic illustration of generation process of microdroplets including protein, NaCl, PEG, and
buffer. When the flow rate of NaCl is decreased and the flow rate of buffer solution is increased, the
supersaturation condition of the droplet is decreased. (d) Characterization of the idea shown in (a–
c). Flow rate of oil was 12 nL/s. Flow rates of aqueous solutions of PEG, salt, lysozyme, and acetate
buffer were varied between 0.8 and 5 nL/s. The total flow rate of the aqueous solutions was set at
15 nL/s. (e) Photographs of lysozyme crystals appeared in the droplets of variable composition.
(Reprinted with permission from Ref. [43] with permission from The American Chemical Society)
each reagent was defined by initial concentration of solutions and flow rates. When
the flow rate of NaCl was gradually decreased as shown in Fig. 2.2a–c, tens of
droplets containing different concentration of NaCl were automatically generated in
several seconds. The droplet volume was picoliter to nanoliter or-der and controlled
by the flow conditions. Figure 2.2d shows relative concentration of each reagent
within the droplets. Protein crystals formed at the optimal crystallization condition
ranges (Fig. 2.2e).
Fig. 2.3 (a) Schematic illustration of droplet formation process. (b) Continuous droplet generation
of 20 pL volume at a throughput of 4.5 s per droplet by sequentially aspirating a 20 pL fluorescence
solution and 80 pL of oil at flow rates of 2 and 8 nL/min. (c) Continuous generation of 1.6 nL
droplets containing five different dyes. (d) Droplets with different volumes of 20, 40, 160, and 1000
pL. (e) Droplets generation with different volumes (2.5, 4.5, 5.5, and 8.0 nL) and different kinds of
dyes. (f) Droplets of 2.5 nL volume with concentration gradient formed by diluting the green and
red dye solutions. (g) 2.5 nL droplets formed by sequentially introducing red dye, water, and blue
dye with different mixing ratios. The mixing ratios were 1.0:1.5:0, 0.5:1.5:0.5, and 0:1.5:1.0.
(Reprinted with permission from Ref. [68] with permission from The American Chemical Society)
An automated microfluidic droplets formation platform named DropLab has been

developed by Fang’s group. Schematic illustrations of droplets formation using
DropLab system is shown in Fig. 2.3 [68]. The minimum droplet volume using
the DropLab was ~ 20 pL and the operation speed was 4.5 s per droplet. The
concentration gradient between droplets was able to form by changing the dispens-
ing ratio of each reagent. Figure 2.4 shows the protein crystallization condition
screening and the optimization experiments using the DropLab system. In the case of
crystallization condition screening, 50 droplets with 12 nL containing different
precipitant and concentration solution in each were arrayed in a 10 cm long capillary
within 22.5 min. As shown in Fig. 2.4a–c, lysozyme crystals were appeared at the
suitable precipitant condition. Then, the optimization of the mixing ratio between the
100 mg/mL lysozyme solution and precipitant #1 (1.0 M NaCl and 25% PEG 6000
in 0.1 M sodium acetate buffer (pH 4.6)) were carried out to define the best
crystallization condition (Fig. 2.4d–e). Forty droplets array with different mixing
ratios of lysozyme to precipitants #1 were generated similarly with precipitant
screening.
The droplets which are 5 kinds of mixing ratios of lysozyme to precipitant with
4:1, 2:1, 1:1, 1:2, and 1:4, were prepared in a 10 droplet sequence. The best
lysozyme crystals were obtained at the mixing ratio of 1:2 (4 nL lysozyme and
8 nL precipitant #1).
A microwell-based protein crystallization platform is first reported by Quake’s
group in 2002 [47]. In the case of microwell-based crystallization platform, intro-
duction of sample solutions is carried out by pneumatic valve operation [69]. They
fabricated the protein crystallization device enabled 144 parallel condition screen-
ing. The microfluidic device was made from PDMS and sealed with glass substrate.
Fig. 2.4 Lysozyme crystallization screening using DropLab. (a) Schematic illustration of droplet
array for lysozyme crystallization condition screening using 50 different precipitants. (b) Photo-
graphs of 50 droplets containing lysozyme and 50 different precipitants in the droplet array of one
capillary after a 20 h incubation. (c) Enlarged views of droplets containing precipitant #1, #16, #17,
and #18 in (b). Lysozyme crystals were obtained in droplet #1. The second image of droplet #1 is a
polarized microphotograph. (d) Schematic illustration droplet array for optimization of mixing ratio
of protein to precipitant #1. (e) Photographs of 40 droplets (4 duplicates) in the droplet array with
different mixing ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 between lysozyme and precipitant #1. Droplet
volume was 12 nL. (Reprinted with permission from Ref. [68] with permission from The American
Chemical Society)
Different volume ratio of protein solutions and precipitant solutions were filled
separately to microwells via the normally closed valve. After introducing each
solution, the valves at the middle section (mixing valve) were opened to mix the
protein and precipitant solutions by interface diffusion. This crystallization method
is called as “free interface diffusion” in the field of protein crystallography. The
protein and precipitant solutions were gradually mixed by molecular diffusion, thus
the supersaturation of the solution can control by the opening time of mixing valve.
This type of microfluidic device enabled comprehensive protein crystallization
condition screening by only pipetting the protein and precipitant solutions at each
Fig. 2.5 (a) Photograph of SlipChip. (b) Schematic illustrations of reagent-loading procedure. (c)
Screening of the photosynthetic reaction center using SlipChip. 11 trials using the same precipitant
at different concentration ratios were demonstrated. (Reprinted with permission from Ref. [45] with
permission from The American Chemical Society)
inlet. The optimal protein and precipitant concentration are simultaneously explored
by the different volume ration of microwell sets and mixing time of the solutions. Six
different proteins such as, lysozyme, bacterial primase catalytic core domain, type-II
topoisomerase, thaumatin, xylanase, and glucose isomerase were crystalized using
the microfluidic device. In addition, a thaumatin crystal collected from the micro
wells (5 nL volume) after appearing the crystal were analyzed by X-ray diffraction
experiment and a high-resolution diffraction pattern is observed. This result suggests
that microwell platform was able to grow a diffraction quality protein crystal as well
as screening of the protein crystallization condition.
A unique protein crystallization condition screening device was reported from
Ismagilov’s group [45, 70]. The microfluidic device was named as “SlipChip”.
Figure 2.5 shows the schematic illustration of the SlipChip and its operating
photographs. Protein solution was loaded into the microchannel (bottom side) and
microwells (upper side) followed by slipped to divide the microchannel and
microwells (Fig. 2.5a–c). Then, precipitant solution was loaded into the other
microchannel and microwells, and the substrates are additionally slipped to mix
the solutions. There were also fabricated different sized microwells to realize the
high-throughput protein crystallization screening. Basically, the crystallization prin-
ciple of SlipChip was free-interface diffusion like microwell-based device men-
tioned above. They applied SlipChip to determine the crystal structure of glutaryl-
CoA dehydrogenase and dihydrofolate reductase/thymidylate synthase. These pro-
teins are diffracted at the resolution of 1.6 Å and 1.9 Å, for glutaryl-CoA dehydro-
genase and dihydro-folate reductase/thymidylate synthase, respectively. SlipChip
Fig. 2.6 Schematic illustrations of centrifugal microfluidic device with 24 crystallization array.
The device has 24 parallel microstructure units. Each unit comprises a protein metering structure, a
precipitant metering structure, and a crystallization chamber. All protein metering structures are
connected via a common channel to the sample inlet, and each precipitant metering structure is
connected via a feed channel to an individual inlet. Outlets of both the protein metering channel and
the precipitant channel are connected to the crystallization chamber. (Reprinted with permission
from Ref. [72] with permission from The American Chemical Society)
provide us the simple operation for protein crystallization screening, although the
mass productivity of device is lower than that of PDMS-based microfluidic devices.
Centrifugal microfluidic devices are also used for protein crystallography
[71, 72]. Wang et al. demonstrated the 24 parallel protein crystallization experiment
with one operation using a single microfluidic device (Fig. 2.6). The microfluidic
device was made from PDMS microchannel and glass substrate. They tested the
protein crystallization using lysozyme, xylanase, lipase B, glucose isomerase, and
thermolysin. A protein solution and precipitant solutions were introduced each
chamber by centrifugal and capillary force. Each chamber set of protein and precip-
itant solution were connected with microchannels. Therefore, the microfluidic device
was able to employ the vapor diffusion crystallization method. The supersaturation
of protein solution was gradually increased by water vapor diffusion similar with the
conventional hanging drop vapor diffusion method. For the lysozyme crystalliza-
tion, the microfluidic device got lysozyme crystals at 81 different conditions. This
result indicates the hits rate of the vapor diffusion chip is same as the conventional
hanging drop vapor diffusion method (76 hits). However, the vapor diffusion chip
allowed the rapid experimental setup (6–8 min for vapor diffusion chip, and
20–30 min for the conventional hanging drop vapor diffusion method) and reduction
of sample consumption (6 nL/trial for vapor diffusion chip, and 2 μL/trial for the
conventional vapor diffusion method).
2.4 Protein Crystal Growth in a Microspace Environment
Microfluidic device is useful for not only the protein crystallization condition
screening, but also the preparation of high quality protein crystal. When the opti-
mized protein crystallization is founded by the primary screening, improving the
quality of protein crystal is addressed by additional optimization of crystallization
condition screening. Typically, high diffraction quality protein crystals are not
necessarily obtained even with the crystallization condition is optimized. The high
diffraction quality crystal is defined as a good molecular packing single crystal (not
multiple or aggregated crystals), and it lead to obtain high quality X-ray diffraction
data. Crystallization environment is significant factor to prepare the ordered molec-
ular packing protein crystal. In the case of the ground-based protein crystallization
experiment, the natural con vection is the main factor of the formation of disordered
crystal. From these reasons, protein crystallization experiments at a special environ-
ment: a microgravity (at the International Space Station), a magnetic field, or an
electric field have been reported [33, 34]. These environments make it possible to
suppress the natural convection and the offer a diffusion-controlled protein crystal
growth environment. Microspace environments: the microdroplet and the microwell
are alternative ground-based crystallization platforms of the special crystallization
environment. In this section, microfluidic platforms for preparation of high diffrac-
tion crystal is introduced.
Advantages of the microspace environment on the protein crystallography are
producing one single crystal in a microdroplet or microwell (single crystallization)
and controlling protein crystal shape suitable for X-ray crystal structure analysis
[55–57, 73, 74]. Single crystallization is a technique for preventing an aggregation of
protein crystals to obtain high quality X-ray diffraction data. When a number of
nucleus or crystals formed in the crystallization droplet, each protein crystal cannot
grow enough to obtain good X-ray diffraction data due to the consumption of protein
molecules at the nucleation and early stage of crystal growth. In addition, one single
crystal must be exposed by X-ray beam to collect the “single crystal X-ray diffrac-
tion data”. From the viewpoint of X-ray data collection, stacked or aggregated
protein crystals: a needle-like crystal and a cluster-like crystal lowers the diffraction
quality and complicates the crystal structure analysis after the diffraction data
collection. In the case of these types of crystals, crystal growth control plays
important roles to prevent staking of each crystal.
Maeki et al. reported droplet-based protein single crystallization method. They
focused on the effect of droplet size on the protein crystallization and elucidated the
critical droplet size for generating one single crystal in the microdroplet both
theoretically and experimentally [56]. Figure 2.7 shows the concept of the single
protein crystallization method based on the microdroplet. After first nucleation of a
protein crystal in the droplet, the protein crystal grow in competition with further
nucleation until the supersaturation of the solution reaches the metastable region.
Thus, a one single crystal was generated in a droplet smaller than the critical droplet
size. Conversely, a large size droplet made it possible continuously nucleation rather
Fig. 2.7 Droplet-based high-throughput protein crystallization condition screening system. Con-
cept of the single crystallization method based on droplet microfluidics. (a) Droplet size < critical
size (Rc). (b) Droplet size > Rc. The critical size was estimated from the diffusion coefficient, the
initial concentration of protein, and the consumption rate of protein in solution. (Reprinted with
permission from Ref. [56] with permission from Wiley-VCH)
than the crystal growth after first nucleation. The microdroplet single nucleation
method is based on the mass balance of protein molecules and Fick’s laws of protein
diffusion. The critical droplet (Rc) size was able to calculate from the diffusion
coefficient of protein molecules (D), the initial concentration of protein in the droplet
(C0), and the consumption rate of protein by crystal growth (q), as shown in the
following equation:
Rc ¼ ð6DC 0 =qÞ1=2
To proof of concept, four model proteins: lysozyme (14 kDa), thaumatin (22 kDa),
glu cose isomerase (179 kDa, four subunits), and ferritin (440 kDa, 24 subunits)
were used for crystallization experiments. The Rc values were theoretically calcu-
lated as 600, 600, 450, and 200 μm for lysozyme, thaumatin, glucose isomerase, and
ferritin, respectively. In the case of lysozyme and thaumatin, single crystallization
enabled using 200 and 360 μm sized droplets, regardless of the supersaturation
condition. On the other hand, multiple crystals formed in the 500 μm sized droplet
after first nucleation at the high supersaturation condition. After the first nucleation,
protein concentration gradient formed in the microdroplet, regardless of the droplet
size, and it have been confirmed by in situ Raman spectroscopy. In other words, high
concentration of protein remains in the area far away from the crystal. The protein
concentration gradient gradually disappeared by molecular diffusion due to the
depletion of protein molecules around the formed crystal. In the case of small-
sized droplet, protein molecules could rapidly diffuse and incorporated to the formed
crystal compared with the large sized droplet. However, in the case of large sized
droplet, although protein molecules diffuse to the formed crystal, the time to
necessary to reach the protein molecules to the crystal became long compared with
Fig. 2.8 Photographs of protein crystals unsuitable for single crystal X-ray crystallography grown
without microseeds. (left) Photoactive yellow protein (PYP) and (right) cytochrome bo3 oxidase
(cyt bo3). (Reprinted with permission from Ref. [58] with permission from The Royal Society of
Chemistry)
the small sized droplet. For this reason, further nucleation enables in the region,
which protein molecules remains at high concentration. For the ferritin crystalliza-
tion, molecular diffusion of ferritin assumed slower than that of the small molecular
weight proteins: lysozyme and thaumatin. Therefore, multiple crystals form in the
droplet like the lysozyme and thaumatin crystallization using the large sized droplet.
These results suggest that a strategy for design of microfluidic devices to control the
protein crystallization behavior.
Stacking or aggregating of protein crystals are serious problems in the protein
crystallography (Fig. 2.8). Typically, the aggregations lower the diffraction quality
and complicates the structure determination process. To avoid the aggregation of
protein crystals, crystallization control based on the phase diagram is useful tech-
nique both the conventional methods and microfluidic methods. The aggregation
occurs by the nucleation onto the surface of protein crystals formed in the crystal-
lization droplet (or well). Thus, protein crystals incubated under the suitable crys-
tallization (supersaturation) condition can avoid the aggregations.
Kenis et al. reported a microseeding method using a microfluidic device [58]. Fig-
ure 2.9 shows a microfluidic approach for the protein crystallization experiment
using microseed. The microfluidic device composed four layers including PDMS
layers (50 μm for one layer) and cyclic olefin copolymer layers (50 μm for one layer).
The total thickness of the microfluidic device was 200 μm, which expected good
X-ray transmission efficiency. The microfluidic device enabled 24 crystallization
trials using 6 different concentration of diluted microseed solutions at once. The
solutions: the protein-precipitant solution and the microseed solutions were intro-
duced into the 24 microwells via the normally closed valves and mixed to transport
the microseeds to the crystallization solution under the metastable region (Fig. 2.9b).
Under the metastable region, protein crystal can grow without further nucleation.
Figure 2.9c–d shows the photographs of the crystals of (c) photoactive yellow
protein (PYP, soluble protein) and (d) cytochrome bo3 oxidase (cyt bo3, membrane
Fig. 2.9 (a) 3D perspective view of the microfluidic device with normally closed pneumatic
valves. The microfluidic device comprised of four layers: an impermeable cyclic olefin copolymer
top layer bonded to an PDMS control layer containing valves, a PDMS fluid layer to contain
protein, precipitant and seed solutions, and a COC bottom layer. (b) Schematic illustration of a
24-well array chip used for microseeding. Fluid layer is represented in blue, and the valve lines, V1,
V2 and V3, are colored based on their function. In the case of microseeding experiments, different
microseed dilutions and a pre-mixed protein–precipitant solution were introduced into the device
from inlets 2–7 and inlet 1, respectively, prior to on-chip mixing. (c and d) Results of the dilution
ratio screening of the seed solutions. At greater seed dilutions (lower seed concentration), PYP
crystals (c) grew into fewer, larger crystals and cyt bo3 crystals (d) grew into fewer, thicker crystals.
(Reprinted with permission from Ref. [58] with permission from The Royal Society of Chemistry)
protein). In comparison with the conventional crystallization method shown in

Fig. 2.8, the aggregations of protein crystals were able to prevent by the
microseeding method using microfluidic device. When the high concentration of
microseed solution was used, the small sized crystals formed in the crystallization
well. Conversely, 200 and 100 μm sized crystals for PYP and cyt bo3 were obtained
at the low concentration of microseed solution. At the microseeding process, the
competition of dissolution of microseeds and growth of microseeds occurs in the
crystallization solution and therefore, the appropriate dilution rate of microseed
solution allows the crystal growth of one or a few protein crystals in the solution.
Fig. 2.10 Photographs of protein crystals formed by the (a) microbatch method, (b, c) hanging
drop vapor diffusion method, and the (d–f) microfluidic chip method. (a, d) Lysozyme crystal, (b,
e) PsGK crystal, and (c, f) CPR–HO complex crystal. The scale bars represent 200 μm. (Reprinted
with permission from Ref. [55] with permission from The Royal Society of Chemistry)
They also optimized the microfluidic system with separated microchannels for
protein, precipitant, and microseed solutions to precisely control the mixing of
three solutions. By the improved microfluidic device, the microseeds solution was
transported to the crystallization solution (mixture of protein and precipitant solu-
tion) at preferable time. The resolution of bo3 was improved by the microfluidic
approach from 12 Å to 10 Å. Although, the diffraction resolution was not enough to
determine the three-dimensional structure, the microfluidic approach expects to
become a non-invasive and high throughput optimization method to obtain high
diffraction quality protein crystal after the first protein crystallization condition
screening.
Maeki et al. found the difference of protein crystal growth behavior depended on
the depth of crystallization chamber and demonstrated that the reforming of aggre-
gated protein crystal [55]. They also employed the microseeding method using the
microfluidic devices with normally closed valves. Lysozyme, glucokinase from
Pseudoalteromonas sp. AS-131 (PsGK), and NADPH- cytochrome P450
oxidoreductase-heme oxygenase complex (CPR-HO complex) as model proteins.
Figure 2.10 shows the photographs of protein crystals formed by the (a) microbatch
method, (b, c) hanging drop vapor diffusion method, and the (d–f) microfluidic
Fig. 2.11 (a, b) Photographs of the plate-shaped PsGK crystals formed in the microfluidic device
with 10 μm deep crystallization chambers. (c) Percentage of the plate-shaped PsGK crystals formed
under different mixing times. The scale bars represent 100 μm. (Reprinted with permission from
Ref. [55] with permission from The Royal Society of Chemistry)
devices. This result indicates that the microseeding method coupled with the
microfluidic device was a universal protein crystallization control technique. In
addition, they demonstrated the shape of PsGK crystal was dramatically changed
by the depth of the crystallization chamber. Although, the road-shaped PsGK crystal
formed in 50 μm depth microfluidic device, the plate-shaped PsGK crystal formed in
10 μm depth microfluidic device, as shown in Fig. 2.11. Mixing time (5–30 min)
between the crystallization solution and the microseed solution did not affect the
crystal shape. The three-dimensional structure of PsGK have been determined by the
road-shaped crystal formed by the conventional hanging drop vapor diffusion
method. Then the plate shaped PsGK crystal was also measured by the X-ray beam
at synchrotron facility. The plate shaped PsGK crystal diffracted at a resolution limit
of 2.8 Å and the crystallographic parameters: the space group and the lattice constant
were same as the rod-shaped PsGK crystal. For the X-ray diffraction measurement,
the plate-shaped crystal is easy to irradiate the X-ray beam on the targeted crystal,
because the irradiation area of crystal is larger than that of the rod-shaped crystal. The
thick cubic-shaped crystal provides the high diffraction intensity and ideally is the
best for the X-ray single crystallography. Theoretically, the plate-shaped crystal can
grow to the cubic-shaped crystal by the additional macroseediong method. Therefore,
the reforming method followed by the macroseeding may offer the solution for the
preparation of high diffraction quality crystal.
2.5 On-Chip Protein Crystal Structure Analysis and Other

Applications
X-ray diffraction measurement is the last experimental step on the protein crystal-
lography. When the X-ray diffraction data is obtained, the three-dimensional protein
structure is calculated using a personal computer or a super computer. The size of
protein crystals obtained by the microfluidic device is typically smaller than 200 μm
due to the limitation of crystallization space (microdroplet or microwell). Thus, a
synchrotron facility is widely employ for the X-ray diffraction measurement. Gen-
erally, X-ray diffraction measurement is carried out at the cryogenic condition to
reduce the radiation damage. The room-temperature measurement also enables, if
the diffraction data collected from the different protein crystals is able to merge each
other. Advances in X-ray free electron laser (XFEL), the serial femtosecond X-ray
crystallography at room temperature will be indispensable technique for elucidating
the protein structures and dynamics [75–78]. In any cases, optimization and selection
of the quality of protein crystals, characteristics of microfluidic device (material and
thickness), and the characteristic of X-ray beam (photon flux and beam spot size)
requires to collect high quality diffraction data.
Microfluidic devices for both of the on-chip X-ray measurements at cryogenic
and room temperature conditions have been reported in several papers. For the
on-chip X-ray measurement, the suitable substrate materials should be selected to
reduce the background signal. In addition, the thickness of the microfluidic device
also affects the X-ray diffraction intensity. Ismagilov et al. demonstrated that in situ
X-ray analysis using microdroplets collected into the glass capillary with 200 μm
inner, and 180 μm outer diameters [79]. Microdroplets containing thaumatin crystal
were directly exposed X-ray in 10 s beam at BioCARS station 14BM-C and 14ID-B
at the Advanced Photon Source at Argonne National Laboratory (USA). Thaumatin
crystals diffracted to 1.8 Å resolution and the crystallographic parameters are the
same as the reported thaumatin crystal prepared by the conventional crystallization
method. The background signal from the glass capillary is enough to low, because
they used 10 μm thickness-wall glass capillary. Teflon or PFA (perfluoroalkoxy
alkane) capillary (I. D. 200 μm, O. D. 360 μm, 80 μm wall thickness) was also
acceptable for the device material to determine the crystallographic parameters:
lattice constant and space group (BL 07 in the SAGA Light Source, Japan)
(Fig. 2.12) [80]. However, the background signal at the small angle was slightly
higher than that of thin-wall glass capillary. These results suggest that the thinner
microfluidic device is desirable for obtaining high signal to noise ration X-ray
diffraction data by on-chip measurement.
X-ray compatible COC-PDMS hybrid microfluidic device was developed by
Kenis’s group [52]. They evaluated the effect of substrate materials and thickness
on the X-ray transmission and confirmed that the COC-PDMS device showed the
transmission factor was more than 90%. Then, they carried out X-ray measurement
Fig. 2.12 (top) Lysozyme

crystal formed in the
microdroplet within
capillary. (middle and
bottom) X-ray diffraction
patters obtained by directly
irradiation of X-ray to the
lysozyme crystal within the
capillary. (Reprinted with
permission from Ref. [80]
with permission from The
Japan Society for Analytical
Chemistry)
using lysozyme crystal at room temperature in the oscillation range from 5 to +5
(10 per one lysozyme crystal, 1 oscillation step with 1 s exposure). Diffraction data
from multiple lysozyme crystals were merged to obtain a complete data set. The
diffraction data was compared with the lysozyme crystal prepared by the conven-
tional crystallization method over the resolution range from 50 Å to 1.55 Å. The
lysozyme crystal prepared by the conventional method underwent cryoprotection to
obtain a complete diffraction data set. The quality of the diffraction data using the
microfluidic device was compatible with the conventional crystallization and ana-
lytical method. Maeki et al. first demonstrated the integration of protein
Fig. 2.13 Comparison of the cooling situation of the microfluidic chip by cold nitrogen gas. (a)
Photograph of the in situ X-ray diffraction experiment using a standard microfluidic chip at 277 K.
(b) Photograph of a microfluidic chip that was cut to a size of 5 5 mm2. This microfluidic chip was
used for the X-ray diffraction experiment at 100 K. (Reprinted with permission from Ref. [81] with
permission from The American Chemical Society)
crystallization to on chip X-ray diffraction procedure including cryoprotection

[81]. Figure 2.13 shows the photographs of the X-ray diffraction experiment setup:
at (a) 277 K and (b) 100 K (cryogenic condition). They also used the COC-PDMS
microfluidic (240 μm thickness) device with normally closed pneumatic valves to
mix the cryoprotectant after appearing lysozyme crystals in microwells. The stan-
dard microfluidic device (3 4 cm2) was not completely placed in the cold nitrogen
stream and the crystals formed in the device could not apply the flash cooling. In
contrast, the microfluidic device optimized for the cryoprotection enabled the flash
cooling process and obtained complete diffraction data set from one protein single
crystal (oscillation range: 0 to 90 , 1 oscillation step with 30 s exposure at BL 07 in
the SAGA Light Source, Japan). They can avoid the X-ray radiation damage by the
cryoprotection and the lysozyme crystal diffracted to 1.5 Å resolution and the three-
dimensional structure was determined from one single crystal. The microfluidic
device with normally closed valves made it possible the step-wise cryoprotection
to reduce the osmotic shock at the cryoprotection. The stepwise cryoprotection
induced the improvement of diffraction data quality.
Perry et al. developed a graphene-based microfluidic device to reduce the back-
ground signal. Figure 2.14 shows schematic illustrations of fabrication and operation
procedures [82]. Unlike the polymer-based materials, the thickness of single layer
graphene is sub-nanometer. Therefore, the signal to noise ratio is expected
extremally increase, because of the decreasing of the background signal. They
confirmed that the 100 μm thick COC layer increased the attenuation and back-
ground scatter [83]. Typically, micro crystals shower the low diffraction intensity
compared with large-sized crystals, thus the graphene-based microfluidic device is
useful for the micro-crystallography. In addition, the graphene-based microfluidic
device can be applied not only for the X-ray crystallography and also the other
analytical methods including small angle X-ray diffraction (SAXS) and cryo-
transmission electron microscopy (cryo-TEM) to analyze the biological targets.
Fig. 2.14 Schematic illustrations of the device fabrication and operation procedures. A graphene
membrane on copper is coated with a layer of PMMA, and then released from the copper substrate
by etching. The graphene-PMMA film is floated on the surface of water and transferred to an
adhesive polyester support layer. This layer is adhered to a COC layer containing the cut-out pattern
for the microfluidic channels. Assembled device is held together by the adhesive layers defining the
window structures. (Reprinted with permission from Ref. [82] with permission from MDPI)
Fig. 2.15 Concept of

microdroplet-based protein
crystallization using
spontaneous emulsification.
(Reprinted with permission
from Ref. [84] with
permission from The Royal
Society of Chemistry)
Fukuyama et al. reported a droplet-based vapor diffusion protein crystallization

method [84]. They focused on the spontaneous emulsification to increase the
supersaturation in microdroplets. Figure 2.15 shows the concept of microdroplet-
based protein crystallization using spontaneous emulsification. They used lysozyme
as a model protein and microdroplets containing a crystallization solution: a mixture
of lysozyme and precipitant were dispersed into a continuous phase (Dodecane +
Span 80). The supersaturation in the microdroplets are gradually increased by the
spontaneous emulsification. When a 60 mM Span 80 solution was used as the
continuous phase, the microdroplet was shrunk 180 μm to 100 μm within 35 min.
The increasing rate of supersaturation was able to control by the concentration of
Span 80. Vapor diffusion method is most widely used protein crystallization method
in the conventional methods. The supersaturation is increased to reach the equilib-
rium to reservoir solution with increasing the incubation time. Therefore, the
crystallization condition is explored in wide supersaturation range. The

microdroplet-based vapor diffusion method provides many advantages in protein
crystallography by reducing the protein sample consumption and high-throughput
crystallization condition screening compared with the typical microdroplet-based
protein crystallization method.
2.6 Summary
This chapter summarized that the microfluidic techniques and platforms for protein
crystallography. Microfluidics has a great potential in the protein crystallographic
applications: high-throughput protein crystallization condition screening, low
amount of sample consumption, high quality single crystal preparation, and
on-chip X-ray diffraction measurement. A number of nano or picoliter-scale trials
are prepared by simple operation compared with the conventional crystallization
methods. In addition, the quality of protein crystals is able to improve using the
microfluidic device and environment without special apparatus. On-chip X-ray
crystallography using micro and nano protein crystals will be main research topic,
because of the recent advances of synchrotron facility. In particular, microfluidic
devices coupled with XFELs certainly accelerate the protein three dimensional
structure analysis. Novel microfluidic-based techniques and devices for protein
crystallography will be developed along with the progress of synchrotron facility
and diffraction data and statistical processing.
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Chapter 3
Application of SERS-Based Microfluidics
for In Vitro Diagnostics
Jinhyeok Jeon, Namhyun Choi, Joung-Il Moon, Hao Chen,

and Jaebum Choo
Abstract In this chapter, we describe SERS-based immunoassay techniques using

various types of microfluidic platforms. SERS is a highly sensitive detection modal-
ity, and microfluidic platforms provide many advantages such as automatic sampling
and reduced sample volume. Therefore, the integration of SERS with microfluidic
platforms offers wide applications in chemical or biological analysis. These novel
SERS-based microfluidic platforms provide a powerful clinical tool for highly
sensitive in vitro diagnostics.
Keywords Surface-enhanced Raman scattering (SERS) · Microfluidics · In vitro

diagnostics
3.1 Introduction
The development of fast and sensitive immunoassays for clinical samples has been a
critical issue in the area of early diagnosis of food-borne illness or infectious disease
[12, 22, 51]. Many efforts have been employed to develop sensitive detection
methods for specific biomarkers in human serum, including enzyme-linked immu-
nosorbent assay (ELISA) [42, 53], immunochemiluminescence assay (ICMA) [60],
and radioimmunoassay (RIA) [46], ELISA is the most widely used screening tool for
validating candidate protein markers, and various ELISA kits have been commer-
cialized for quantitative identification of specific biomarkers. In many cases, how-
ever, this approach does not meet the requirements for accurate assessment of a
specific biomarker at low concentrations. In ELISA assays, quantification of a
marker is achieved through observing enzyme-mediated color changes but a dis-
cernible color change of a target cannot be induced at low concentrations of a marker
J. Jeon · N. Choi
Department of Bionano Technology, Hanyang University, Ansan, South Korea
J.-I. Moon · H. Chen · J. Choo (*)
Department of Chemistry, Chung-Ang University, Seoul, South Korea

54 J. Jeon et al.
and consequently a microplate reader fails to distinguish the color change. Likewise,
RIA allows rapid and inexpensive screening of a clinical sample but the use of
radioisotopes and scintillation fluids restricts its application for practical detection of
a specific biomarker in human serum. ICMA is one of the most popular immunoas-
say techniques in clinical laboratories but it does not satisfy the low limit of detection
(LOD) for several hormone-related biomarkers such as sexual hormones (estradiol
and testosterone) or thyroid stimulating hormone (TSH). Recently, mass spectrom-
etry was adopted for more accurate and confident diagnostics in the clinical labora-
tory [47, 48]. However, despite a low detection capability of less than 10 pg/mL this
technique has several analytical disadvantages including long sample preparation
steps and the need for expensive instrumentation. For these reasons, mass spectrom-
etry is not considered suitable for routine clinical applications. Thus, there is an
urgent need for new technologies that can achieve efficient and sensitive detection of
target biomarkers in serum for use in routine clinical diagnostics.
Recently, surface-enhanced Raman scattering (SERS)-based assay platforms
have been increasingly considered as promising formats for the detection of various
biomarkers due to their high sensitivity and multiplex detection capability
[17, 21]. When SERS nano tags are used as detection probes, the Raman scattering
signals are greatly enhanced at active junctions known as “hot spots” as a result of
electromagnetic and chemical enhancement effects. Such enhancements have shown
great promise in overcoming the sensitivity problems inherent in the fluorescence or
luminescence detection of ELISA or ICMA assays. Moreover, SERS-based detec-
tion methods do not require the culturing or amplification steps inherent to bacteria
colony counting and PCR assays. To date, many different types of biomarkers
including proteins [8, 10, 11, 28, 29], viruses [2, 20, 40], and bacteria [13, 26, 44,
57, 63] have been studied using SERS-based assays.
The last decade has also seen great progress in the development of microfluidic
technology for use in chemical and biomedical sciences [43, 54, 61]. These devel-
opments have been driven by its advantages over conventional macroscale analytical
methods, including reduced consumption of reagents and mixing time, high analyt-
ical throughput, facile automation, and improved production conversion [34, 35,
37]. It has long been recognized that the system used for detecting the progress of a
reaction is a key factor in determining the applicability of a microfluidic system.
Because of the extremely small volume in a microfluidic channel, a highly sensitive
detection method is essential for monitoring the progress of a chemical or biological
reaction. Consequently, the development of SERS-based microfluidic platforms has
recently attracted significant attention in the biomedical sciences. The integration of
SERS, a highly sensitive detection modality, with microfluidic platforms, which
have many advantages over microscale methods [3, 23, 24, 32], offers significant
promise for chemical and biological experimentation. There are two primary types of
flow regimes in microfluidic platforms: continuous and segmented (or droplet)
flows. In this chapter we introduce various types of SERS-based microfluidic
platforms for the rapid and sensitive detection of biomarkers in serum.
3 Application of SERS-Based Microfluidics for In Vitro Diagnostics 55
3.2 Multiplex Immunoassay Using Array-Embedded

Microfluidic Channel
A gold-patterned microarray chip has been extensively used as a SERS-based

immunoassay platform but it has some technical problems [6, 16, 19, 28,
29]. First, it is not easy to optimize the homogeneous distribution of target bio-
markers on gold wells because the sequential processes including antibody/antigen
immobilization and washing steps are difficult to control manually using a micropi-
pette. Second, the repetition of washing steps required to remove non-specific
binding proteins makes this assay platform inconvenient. Finally, a tedious manual
dilution process is required for the immunoassay of various concentrations of target
biomarkers.
To resolve this problem, M. Lee and co-workers [30] developed a programmable
and fully automatic gold array-embedded gradient microfluidic chip that integrates a
gradient microfluidic device with gold-patterned microarray wells. Serial dilution of
the antigen marker can be achieved in a stepwise manner using microfluidic con-
centration gradient generators with N cascade-mixing stages [27–29, 39]. In this
channel, the desired concentrations can be achieved by controlling the volumetric
mixing ratios of two merging solutions in each stage. In addition, 30-μm gold-
patterned microarray wells (5 5) were embedded onto a glass substrate for
antibody immobilization on the surface. Use of this novel gold microarray-
embedded gradient microfluidic channel avoids the tedious manual dilution process,
and highly accurate immunoanalysis can be achieved with automatic flow controls.
The utility of this platform was demonstrated by quantitative immunoassay of the
alpha-fetoprotein protein marker. The total assay time, including incubation, wash-
ing steps and detection, was less than 60 min. Figure 3.1 shows the layout of a gold
array-embedded gradient chip for SERS-based immunoassay.
However, during the clinical sample analyses, the method described above is
affected by nonspecific binding of non-target species. To resolve this problem, K. K.
Reza and co-workers [41] developed a platform that utilizes a combination of
alternating current electrohydrodynamic (ac-EHD)-induced surface shear forces to
remove nonspecific molecules from the electrode surface. The use of ac-EHD
surface shear forces improves analyte transport across an antibody-functionalized
capture domain and simultaneously displaces nonspecific binding molecules from
the electrode surface [45, 52]. This capability enables rapid SERS immunoassays
that outperformed traditional pressure driven flow based assays. To demonstrate its
applicability, a microfluidic device containing five individual microchannels, each
with an array of asymmetric electrode pairs, was constructed. In a typical immuno-
assay, a complex biological sample containing multiple protein biomarkers and the
solution containing detection antibody conjugated to SERS nano tags are driven
through the devices sequentially under an ac-EHD field. In this study, simultaneous
capture and multiplexed detection was performed for human epidermal growth
factor receptor 2 (HER2), mucin 1, cell surface associated (MUC1), epidermal
growth factor receptor (EGFR), and mucin 16, cell surface associated (MUC16)
56 J. Jeon et al.
Fig. 3.1 Layout of a gold array-embedded gradient chip for the SERS-based immunoassay. The
illustrations in the enlarged circles represent the formation of sandwich immunocomplexes on the
surface of 5 5 round gold wells embedded in the gradient channel. (Reprinted with permission
from Lee et al. [30]. Copyright (2012) The Royal Society of Chemistry)
proteins that are overexpressed in breast, lung, and ovarian cancer, respectively.
Considering the extensive multiplexing ability of SERS immunoassays, the
presented approach with the ability to simultaneously detect four individual cancer
biomarkers within each channel can potentially detect a broad panel of cancer
biomarkers. Figure 3.2 shows a schematic illustration of the microfluidic channel
for the multiplex detection of protein biomarkers using ac-EHD induced SERS
detection.
For high throughput detection of multiple biomarkers, L. Wu and co-workers [59]
developed a SERS-assisted 3D barcode microfluidic system. Figure 3.3 shows a
schematic illustration of this immunoassay protocol. Multiple samples were dis-
pensed into parallel channels using a programmable syringe pump. Different anti-
gens in the sample were captured by the corresponding antibodies immobilized on
the surface of the channels. Next, a mixture of SERS nanoprobes was dispensed into
the channel and bound onto corresponding antigens to form immunocomplexes. For
quantitative evaluation of multiple biomarkers, a 3D barcode containing the spatial
and spectroscopic information was acquired from the variation of Raman peak
intensities (Fig. 3.3d, right). This array-embedded microfluidic technology could
be successfully used for the simultaneous detection of multiple biomarkers.
Wang et al. [56] developed a microfluidic device with multiple channels for the
duplex detection of pathogen antigens as illustrated in the photograph and scheme in
Fig. 3.4. Microfluidic channels were connected with three inlets and three outlets to
enable the simultaneous detection of multiple targets simultaneously. Each channel
contained gold patterns that could be used for the immobilization of specific
antibodies. Herein, NYscFv-350 antibody was immobilized on the surface in the
Fig. 3.2 Schematic illustration of multiplexed protein biomarker detection using ac-EHD induced
SERS-immunoassay. (a) Schematic illustration of typical immunoassay and false-color SERS
images, and (b) corresponding SERS spectra, for specific target capture and background response
from serum samples containing equal concentrations (10 pg mL1 for each antigen) of EGFR,
HER2, MUC1, and MUC16 antigens under ac-EHD field (f ¼ 1 kHz and Vpp ¼ 100 mV). Control
experiments were performed using devices functionalized without any capture antibody. Scale bar
is 10 μm. (Reprinted with permission from Reza et al. [41]. Copyright (2016) John Wiley & Sons,
Inc.)
first channel, and NYscFv-030 antibody was bound on the surface in the second
channel. In the third channel, NYscFv-350 and 030 antibodies at a 1:1 ratio were
immobilized to capture both antigens. Immunoreactions were performed, and detec-
tion antibody-conjugated silica-coated SERS nano tags were employed for readout.
The demonstrated duplex detection of 350 and 030 paves the way for a powerful
analytical platform with potential applications for high throughput multiplex detec-
tion of biomarkers.
3.3 Magnetic Bead-Based Immunoassay in Microfluidic

Channel
Magnetic beads have been extensively used for immunoassays because they can be
manipulated and separated using a magnetic field, strongly facilitating assay pro-
tocols [50]. When magnetic beads are used as a substrate, capture antibodies are
immobilized on the surface of the beads for use as mobile substrates. Their large
surface-to-volume ratio provides efficient capture of target antigens [36]. Antibody-
conjugated SERS nano tags are bound to target antigens captured by the magnetic
beads to form sandwich immunocomplexes [5, 58, 62]. Here, SERS nano tags are
used as detection probes for the highly sensitive quantification of target antigens.
The sandwich immunocomplexes can be immobilized on the wall of a microtube
58 J. Jeon et al.
Fig. 3.3 (a) Schematic illustration of the microfluidic system. The syringes are connected to
microfluidic channels through polyethylene (PE) tubes and controlled by programmable
microfluidic pumps. The upper PDMS mold was bonded with the antibody barcode substrate to
obtain the microfluidic platform. (b) Side view of the microfluidic system. The PE tube is preloaded
with samples and reagents, with each kind of fluid separated by air. The samples sequentially pass
the detection zones which are patterned with different antibodies in a process (known as “bubble-
based sample delivery.”) (c) The principle of sandwich immunoassays. The “antibody barcode” was
used to separate and capture different antigens. The SERS probes can be further captured by the
corresponding antigens, forming a sandwich structure. The different colours represent different
kinds of antibodies, antigens, and Raman reporters. (d) Multiplex immunoassay using the 3D
barcode chip (each channel was patterned with one antibody). The SERS spectrum in each unit of
the hybridization array is acquired and the measurement results are presented as a 3D barcode. The
different colors represent the Raman fingerprint of different reporters and the bar height reflects the
SERS intensity. (e) Multiplex immunoassay using the 3D barcode chip (each channel is patterned
with multiple antibodies). Different antigens in different samples can be identified according to the
2D spatial information and the characteristic Raman peaks, both of which are included in the 3D
barcode. (Reprinted with permission from Wu et al. [59]. Copyright (2015) John Wiley & Sons,
Inc.)
Fig. 3.4 (a) Photograph of the 3-channel microfluidic device containing gold patterns and (b)
schematic illustration of the SERS biosensor platform with NYscFv for duplex antigen detection.
(Reprinted with permission from Wang et al. [56]. Copyright (2014) American Chemical Society)
using a magnetic bar, and quantitative analysis can be performed by measuring the
characteristic Raman signals of a Raman reporter. This SERS-based immunoassay
overcomes the slow immunoreaction resulting from diffusion-limited kinetics on
two-dimensional substrates because of the large effective surface area of the mag-
netic beads. Nonetheless, this assay technique is inconvenient due to problems
associated with multiple manual washing steps, tedious manual handling of samples,
and difficulties in controlling assay conditions. To resolve these problems, SERS-
based microfluidic platforms were developed for more efficient analysis of bio-
markers. A highly accurate and reproducible immunoassay is possible by
maintaining a continuous flow and homogeneous mixing conditions in a
microfluidic channel.
Chon et al. [9] developed a gradient microfluidic channel to automatically achieve
serial dilutions of a target marker. This device has subsequently been used to
automatically serially dilute a target sample with buffer [4, 49]. For the formation
of sandwich immunocomplexes in a microfluidic channel, functionalized HGNs and
magnetic beads with antibodies were introduced into the channel. Next,
minisolenoids [19] were integrated into the microfluidic device for immobilization
of different concentrations of immunocomplexes. Here, minisolenoids were placed
close to each microfluidic channel to generate a magnetic field gradient to trap
magnetic beads in a flowing stream. The solenoids can be switched on or off, and
the field intensity can be tuned on demand. Finally, the SERS signal for sandwich
immunocomplexes immobilized on each microfluidic channel was measured using a
confocal Raman microscope. Consequently, a SERS-based immunoassay can be
automatically performed in a microfluidic channel. With this novel technique, the
tedious manual dilution process is eliminated and rapid and sensitive
immunoanalysis can be achieved. Figure 3.5 demonstrates the layout of the SERS-
based optofluidic sensor and its working scheme.
60 J. Jeon et al.
Fig. 3.5 (a) Layout of a SERS-based gradient optofluidic sensor integrated with solenoids. (b)
Formation of a sandwich immunocomplex between HGNs and magnetic beads. (c) Trapping of
sandwich immunocomplexes in a microfluidic channel. (Reprinted with permission from Wang
et al. [58]. Copyright (2010) American Chemical Society)
Li et al. [31] improved the detection sensitivity using magnetic focusing and
plasmonic coupling techniques. For this purpose, NiFe core-Au shell nanoparticles
(NPs) were synthesized by successive reduction of HAuCl4 using a seeded growth
approach. Using tunable magnetic core-shell NPs, effective magnetic focusing could
be achieved via an effective coupling between magnetic NPs and gold NPs in a
microfluidic platform. Figure 3.6 displays a schematic illustration of a SERS-based
microfluidic platform for magnetic focusing immunoassays.
During the process of magnetic focusing [33, 55], magnetic particles accumulate
on the bottom of the channel near the edge of the magnetic bar. This aggregation
induces a high density of electromagnetic “hot spots” from the nanogaps between
NiFe core-Au shell NPs and gold NPs. Figure 3.7 also shows the signal enhancement
effect according to the size of Au NPs. The peak intensity increases with the size of
Au NPs, and is maximized at a diameter of 60 nm. This result also shows good
agreement with theoretical simulation results for the electromagnetic field enhance-
ment in Fig. 3.7c.
Gao et al. [15] developed a novel SERS-based magnetic sensor for highly
sensitive detection of the anthrax biomarker poly-γ-D glutamic acid (PGA) [25] in
Fig. 3.6 Illustration of SERS detection of cancer biomarker CEA using functional nanoprobes
consisting of Au-coated NiFe magnetic nanoparticle (NiFe@Au), Ab1 (capture antibody), Ab2
(detection antibody) and RL (Raman label). (Reprinted with permission from Han et al. [19]. Copy-
right (2015) American Chemical Society)
human serum. For safe, sensitive, and rapid detection of a hazardous material in
human serum, they designed and fabricated a solenoid-embedded dual channel
microfluidic device to perform the PGA immunoassay in an automatic manner.
Figure 3.8 illustrates the schematic design of the microfluidic device used in this
study. The device consists of two parallel channel compartments: one for PGA
sensing and the other for control measurements. The sensing channel assays serum
for various concentrations of the PGA trace whereas the control channel only detects
PGA-free serum as an external standard. Both channels are also composed of three
compartments in the vertical direction. In the first compartment of the sensing
channel, serum including target trace PGA antigens and anti-PGA-conjugated mag-
netic beads were introduced into the channel via two inlets. In the second compart-
ment, PGA-conjugated AuNPs were introduced into the channel via the inlet located
in the middle part of the device. Here, PGA antigens and PGA-conjugated AuNPs
underwent competitive reaction with the antibodies on magnetic beads under flow
conditions. As shown in this figure, a staggered herringbone groove-shaped mixer
was incorporated into the channel to improve mixing efficiency. The third compart-
ment acted to trap and detect magnetic immunocomplexes. To achieve effective
trapping of the magnetic beads in the stream, two yoke-type mini-solenoids were
arranged at the end of each channel. After trapping the immunocomplexes, PGA-free
serum was introduced from the inlet located in the middle part of the device to wash
out unbound PGA antigens and AuNPs. Finally, the SERS signal was measured by
focusing the laser beam within each microfluidic channel. For the control measure-
ments, the assay and SERS detection were performed using the same procedure. In
62 J. Jeon et al.
Fig. 3.7 (a) SERS spectra of the sandwich complex of Ab1 conjugated NiFe@Au NPs-CEA
antigen-Ab2 conjugated Au NPs; (b) Plot of normalized peak intensity over total surface area at
1076 cm1 vs Au NP size. (c) Theoretical simulation of a dimer model for the sandwich complex in
terms of E-field enhancement around a small particle (area 1, squares), large particle (area 2, tri-
angles), and in the center (area 3, circles); insert: contour plots external to the dimers for 11–27 nm
(bottom), 30–27 nm (middle), and 60–27 nm (top) pairs. (Reprinted with permission from Han et al.
[19]. Copyright (2015) American Chemical Society)
this study, the external standard values for PGA-free serum were measured each time
using the control microfluidic channel, which greatly improved reliability by min-
imizing the influence of most experimental variables.
3.4 Segmented Flow-Based Immunoassay in Microfluidic

Channel
Gao et al. [14] developed a SERS-based microdroplet sensor for the rapid and
sensitive detection of hazardous materials. This sensor is composed of two droplet
compartments, one for the on-chip synthesis of fresh Ag NPs and the other for
droplet merging and SERS detection. Silver ions were nucleated and grown to larger
size silver nanoparticles in droplets, and then each droplet was synchronously
Fig. 3.8 (a) Schematic illustration of the solenoid-embedded dual channel microfluidic sensor for
SERS-based competitive immunoassay. The sensor is composed of two parallel channels: one for
PGA sensing (gray) and the other for control (red). (b) Optical images of the solenoid chip filled
with four different colours of inks. (c) Photograph of the capture area for magnetic
immunocomplexes. (Reprinted with permission from Wang et al. [55]. Copyright (2015) Elsevier
Inc.)
merged with a droplet containing target analytes for SERS detection. As shown in
Fig. 3.9, a high mixing efficiency among silver nitrate solution, distilled water, and
hydroxylamine hydrochloride/sodium hydroxide solutions was achieved in the first
compartment. Droplet formation resulted from induced shear forces at the interface
between the two different phases. After formation of droplets, aqueous samples in
each droplet were mixed by transport through the winding channels. Synthesizing
Ag NPs within droplets separated by an immiscible fluid environment allowed for
easy isolation of the reactants from the surrounding environment, thus avoiding
contamination. In the second compartment, each droplet was synchronously merged
with a droplet containing target analytes for SERS detection. The combination of a
droplet merging system with in situ Ag NP synthesis for on-line SERS detection is
expected to be a powerful analytical tool for the fast and reproducible bioanalysis.
Recently, Choi et al. [7] reported a fully integrated SERS-based microdroplet
platform for the automatic immunoassay of specific antigen. In this study, a novel
integrated microfluidic system that includes droplet generation, transport, mixing,
merging, and splitting modules was designed and fabricated. The device allows
efficient immunoreactions to be achieved through sequential droplet generation,
transport, and merging [1, 16, 18, 38], while wash-free immunoassays are realized
through the droplet splitting. This defines a conceptually new multifunctional
64 J. Jeon et al.
Fig. 3.9 (a) Schematic illustration of an integrated microdroplet channel for SERS detection of
DQ. The channel is composed of two compartments. The first is the synthetic section for Ag NP
synthesis (i) and the second is for droplet merging and SERS analysis (ii). (b) Expanded view of the
first compartment (i): nucleation of silver ions and large silver nanoparticles. (c) Expanded view of
the second compartment (ii): intersection for droplet merging and SERS detection. (d) Optical
images of the entire microdroplet channel filled with two different colors (red and blue) of ink.
(Reprinted with permission from Jang et al. [25]. Copyright (2014) The Royal Society of
Chemistry)
microfluidic platform capable of performing complex multistep immunoassays in

nanoliter volumes for the safe and sensitive detection of hazardous materials. To
validate this approach, the fraction 1 (F1) antigen for Yersinia pestis, which is of
significant interest to the defense and security communities because of its potential
use as a biological weapon, was chosen as a target protein marker. Figure 3.10
illustrates the schematic design of the integrated microfluidic channel used in the
study.
The device consists of six compartments. In the first compartment, aqueous
solutions of antigens and antibody-conjugated SERS nano tags are supplied from
two central inlets, and carrier oil is supplied orthogonally. Droplet formation results
from shear forces at the interface between the aqueous and oil phases (top image in
Fig. 3.10b). In the second compartment, antigens and detection antibody-conjugated
SERS nano tags in each droplet are efficiently mixed by transport through multiple
winding channels. Since the antibody–antigen reactions in each droplet are
Fig. 3.10 (a) Schematic design of the integrated SERS-based microfluidic channel composed of
six microdroplet compartments: (i) droplet generation from the shear force at the interface between
the aqueous and oil phases, (ii) droplet mixing for the first immunoreaction, (iii) droplet merging for
the formation of magnetic immunocomplexes, (iv) droplet mixing for the second immunoreaction,
(v) droplet splitting for the wash-free immunoassay, and (vi) Raman detection of unbound SERS
nano tags in supernatant solution droplets. (b) Extended images of (i) droplet generation, (iii)
droplet merging, and (vi) droplet splitting. (Reprinted with permission from Gao et al. [14]. Copy-
right (2017) American Chemical Society)
completely isolated it is possible to perform the immunoreaction of hazardous

materials safely while avoiding contamination from the surrounding environment.
In the third compartment, capture antibody-conjugated magnetic beads are intro-
duced into the inlet positioned at the middle of the chip, generating a second droplet.
Subsequently, the first droplet (containing the antigens and detection antibody-
conjugated SERS nano tag mixtures) and the second droplet (containing capture
antibody-conjugated magnetic beads) are sequentially merged through decompres-
sion in the wide merging channel (middle image in Fig. 3.10b). In this geometry, the
two droplets are pushed together before being pulled apart to initiate merging. To
ensure efficient and robust merging, the flow rates of the two droplets must be
precisely controlled. In the fourth compartment, immunoreactions between the
capture antibody-conjugated magnetic beads and the antigen-captured SERS nano
tags occur during passage through a second set of winding channels, with magnetic
immunocomplexes being formed in a droplet twice as large as the original droplets.
In the fifth compartment, a magnetic bar embedded in the channel initiates droplet
splitting. As shown in the bottom image of Fig. 3.10b, magnetic immunocomplexes
align on the bottom side of the droplet as each merged droplet passes close to the
magnetic bar, while unreacted SERS nanotags and antigens in the supernatant
solution remain on the other side of the droplet. Subsequently, each droplet is split
into a pair of daughter droplets, which respectively contain primarily magnetic
immunocomplexes and primarily unbound SERS nano tags. The critical parameter
66 J. Jeon et al.
that controls droplet splitting under such conditions is the capillary number, which
can be controlled by changing the droplet velocity since the surface tension and the
viscosities of both phases are constant. Specifically, the widths of the microchannels
containing the two daughter droplets were varied to control both the droplet velocity
and effective magnetic field strength. For the defined process, removal of unbound
SERS nano tags and antigens by washing was not necessary since they are automat-
ically separated from the magnetic immunocomplexes during droplet splitting. In the
final compartment, both daughter droplets pass through a set of winding channels for
particle dispersion. Finally, Raman signals of the unbound SERS nano tags in the
supernatant droplets are measured and analyzed for the quantitative analysis of
antigen. Such an integrated SERS-based microdroplet assay platform has significant
potential utility in the sensitive, rapid, and safe immunoanalysis of various hazard-
ous materials.
3.5 Summary
During the past decade, there has been growing interest in the biomedical application
of SERS-based microfluidics. In particular, SERS-based immunoassays are a poten-
tial clinical tool for highly sensitive and reproducible detection of multiple bio-
markers. In this chapter we describe three different types of SERS-based assay
platforms: (i) array-embedded, (ii) magnetic bead-based, and (iii) segment flow-
based microfluidic devices. These SERS-based microfluidic platforms have many
advantages, including low reagent consumption, short incubation time, wider con-
centration dynamic range, and cost savings compared with the conventional ELISA
technique. The SERS-based immunoassay technique can be used for highly sensitive
analysis of multiple biomarkers. With the use of appropriate antibody-functionalized
SERS-coding nanoprobes, the optical signals for specific immunocomplexes can be
amplified and analyzed using this novel technique. Consequently, this SERS-based
immunoassay platform is expected to be a powerful clinical tool for early disease
diagnosis.
Acknowledgements The National Research Foundation of Korea supported this work through
grant numbers 2017M3D1A1039287 and 2018M3A7B4071203. This work was also supported by
a grant of the Korea Health Technology R&D Project through the Korea Health Industry Devel-
opment Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant
number: HG18C0062) and by the Agency for Chemical & Biological Detection Research Center
(CBDRC).
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Chapter 4
Miniaturized Electrochemical Sensors
to Facilitate Liquid Biopsy for Detection
of Circulating Tumor Markers
Yi-Ge Zhou, Leyla Kermansha, Libing Zhang, and Reza M. Mohamadi
Abstract Miniaturized sensors to facilitate liquid biopsy for early cancer detection
and monitoring the disease progression have been unprecedentedly advanced in the
recent years, among which electrochemical sensors, offer advantages with their
features such as simplicity, fast response, low cost and capability for miniaturization.
In this chapter, we will provide an overview of recent advances in using miniaturized
electrochemical sensors for detection of circulating tumor markers, including circu-
lating tumor cells (CTCs), circulating nucleic acids (cNAs), and extracellular vesi-
cles (EVs). Representative examples will be given for each marker based on different
electrochemical approaches, and combination of electrochemistry with other tech-
nologies and strategies will be shown.
Keywords Electrochemical sensors · Liquid biopsy · Circulating tumor cell ·

Circulating tumor DNA · Exosomes
Y.-G. Zhou (*)

Institute of Chemical Biology and Nanomedicine, Hunan University, Changsha, People’s
Republic of China
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and
Chemical Engineering, Hunan University, Changsha, People’s Republic of China
L. Kermansha
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada
L. Zhang · R. M. Mohamadi (*)
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada

72 Y.-G. Zhou et al.
4.1 Introduction
4.1.1 Circulating Tumor Markers
Cancer is a group of diseases associated with the growth and spread of abnormal
cells. It is considered a localized disease in its early stage but becomes systemic once
the abnormal cells invade or spread to other parts of the body, causing metastasis,
which is responsible for 90% of cancer related deaths [7]. Few symptoms are evident
at the early stage of cancer; by the time the symptoms appear and progress to an
advanced stage, cancer is far more difficult to treat. Cancer survival rates, therefore,
tend to be very low due to the late-stage diagnosis as well as the limited access to
timely and effective treatment. Thus, there is an urgent need for early and accurate
detection of cancer. In addition, during the course of treatment, it is necessary to
monitor the progress of the disease. Ideally, this monitoring could be done using less
invasive blood tests. Once the blood sample is taken, it can be tested for several
biomarkers simultaneously.
A tumor biomarker refers to a biomarker found in serum, urine, or body tissues
that is altered in the presence of cancer. Specifically, circulating tumor markers
(CTMs) are known as biomarkers present in the blood circulation whose concentra-
tion is proposed as a diagnostic marker for quantitative real-time assessment of the
tumor burden. CTMs include cancer cells and a variety of biomolecules such as
proteins, enzymes, nucleic acids, and small vesicles [20, 76]. Detection of CTMs has
been acknowledged as “liquid biopsy” by its ability in dynamic and non-invasive
monitoring of cancer progression featuring convenience, high reproducibility and
low cost [28].
Great advancements have been made to the detection of CTMs, including fluo-
rescence methods [49], polymerase chain reaction (PCR) [34], enzyme-linked
immunosorbent assay (ELISA) [2], surface plasmon resonance [31], surface
enhanced Raman scattering (SERS) [39], electrochemical assay [14], colorimetric
assay [77], microcantilevers [61], and quartz crystal measurement (QCM) [53].
However, there remain inevitable challenges in the following aspects [20]. The
low concentration of biomarkers, the insufficient binding efficiency, and the low
signal transduction all make it difficult to perform early-stage cancer detection with
high sensitivity. Even when sensitivity is high, the detections may exhibit low
specificity due to a huge background of non-cancerous factors, causing false-positive
signals. Moreover, due to the cancer diversity and the biological variability,
confirming cancer by using only a single biomarker is not reliable.
4.1.2 Why Electrochemistry
Electrochemical approaches provide quantitative or semi-quantitative analytical

information by reporting the changes in the potential, the current or the impedance
4 Miniaturized Electrochemical Sensors to Facilitate Liquid Biopsy for. . . 73
that is generated from the interaction at the analyte-receptor interface. Compared to

other analytical methods, electrochemical sensors commonly show clinically rele-
vant sensitivity and specificity; in additions they feature simplicity, fast response,
low cost and the capability for miniaturization. Various types of voltammetry (such
as cyclic voltammetry, linear sweep voltammetry, stripping voltammetry, differen-
tial pulse voltammetry (DVP), etc.), amperometry, and electrochemical impedance
are the most widely used electrochemical detection methods. Electrochemical sen-
sors are usually married to new technologies to enhance their performances. To
further increase the sensitivity and specificity of the sensors, nanotechnology
and nanomaterials are commonly involved to construct nanostructured interfaces
[32, 85] or nanomaterial-based reporting system [42, 71]. Microfluidics that requires
lower volumes of samples and speeds up the interaction between the target analyte
and the recognition species, is often integrated with electrochemical sensors to
achieve multiplexing, automation and high-throughput detection [26, 67]. The
miniaturization and integration is promising for constructing portable devices and
realizing on-site analysis. In addition, technologies such as magnetic field [32],
ELISA [56] and surface plasmon resonance (SPR) [80] are also reported to be
combined to electrochemical sensors.
In this chapter, we focus on the advances of electrochemical strategies for the
detection of CTMs over the last 5 years. We will discuss examples of different
electrochemical approaches according to the category of circulating tumor markers
with special interests on CTCs, cNAs and EVs, aiming to demonstrate how the latest
developments meet the aforementioned challenges and highlight the remaining
problems that are urgent to be dealt with.
4.2 Circulating Tumor Cells
CTCs are shed from primary tumor and circulate in blood stream to seed metastasis
which is responsible for a majority of cancer-related deaths. Research on CTCs has
become a very active field in early cancer detection that is important for clinical
diagnosis, prognosis and cancer treatment.
Due to the fact that CTCs are very rare in blood, i.e. 1–10 CTCs per mL of whole
blood versus a few million white blood cells, isolation of CTCs from blood samples
with high yield, sensitivity and specificity remains a major technical challenge.
CellSearch is the only FDA-approved technology for CTC isolation taking advan-
tage of immunomagnetic separation method. In this method, CTCs bound to anti-
EpCAM-coated ferrofluid nanoparticles are captured by a magnetic field and later
detected by fluorescence imaging. Although CellSearch can reproducibly detect and
enumerate CTCs with an average recovery rate of no less than 80% [3], this
technology shows limitations in its EpCAM antibody enrichment strategy and
variable detection rates [23]. A great many techniques have been developed aiming
to achieve higher CTCs capture efficiency based on two main principles: physical
properties-based isolation [11, 13, 40] and immuno-based isolation [30, 51, 71]. The
use of microfluidic technologies is very common in both cases. Compared to

traditional methods, microfluidic technologies reduce the processing time to a
large extent and show advantages including low sample requirement, high through-
put, high sensitivity, large surface area-to-volume ratios, automatic operation and
multiplexed integration [54]. Magnetic separation has also been combined with
microfluidics to capture CTCs with higher purity, especially when a microfluidic
chip with specially designed geometry is used [46]. Most recently, an in vivo capture
of CTCs with a surface-modified vein indwelling needle [86] and a biomaterial
implant [4] were carried out in animal models. This technique can address the
problems such as small sampling volume and sampling error caused by the irregular
shedding of CTCs.
As previously mentioned, electrochemical methods provide attractive solutions to
CTC detection exhibiting simplicity, low cost, fast sensing, and clinically related
sensitivity and specificity (Table 4.1). Electrochemistry was recently reported to
release [82–84] and lyse [82] the captured CTCs for further analysis. A number of
advances have also been made in the electrochemical detection of CTCs using
various electrochemical techniques, including voltammetry techniques such as
cyclic voltammetry [59], linear sweep voltammetry [71], stripping voltammetry
[38, 42, 79], and DVP [88], electrochemical impedance [22, 44, 85], amperometry
[10, 29], and electrochemiluminescent (ECL) [74, 78, 87].
Voltammetry, for example, is one of the most powerful techniques among
electroanalytical methods in which information about an analyte can be obtained
by measuring the current with varying potential. Cyclic voltammetry (CV) is the
most common type of voltammetry measurement where the working electrode
potential changes with time according to a triangular waveform, and is widely
used to study redox processes reaction kinetics. A number of detections use CV as
readout method to report on the current difference from the electroactive labels
before and after CTCs are introduced to the electrode [59]. However, charging
current accompanying the redox current will compromise the sensitivity of the
sensors.
DPV is another kind of voltammetry that applies a series of regular voltage pulses
on the potential linear sweep. Since the effect of the charging current can be
minimized by sampling the current just before the potential is changed, DPV is
often applied to bio-detection and bio-analysis. By reducing background current
introduced by macromolecules or cells, DPV shows a much improved sensitivity
over normal voltammetry methods [60]. Kelley laboratory constructed a chip-based
assay with nanostructured microelectrodes (NMEs) to analyze and classify prostate
tumor cells in cultured cells and ultimately in a pilot study involving blood samples
from 16 prostate cancer patients. In this integrated chip, DPV was used to detect
RNA from lysed CTCs isolated by immune-magnetic beads [32]. The electrochem-
ical reporter system employed here was two different redox-active probes, Ru
(NH3)63+ and Fe(CN)64. The Ru(III) species accumulated at the sensor surface in
proportion with the hybridization of the negatively charged RNA targets to the
NME-tethered PNA probes and was electrochemically reduced to Ru(II) by applying
a DPV scan. The Fe(II) species was introduced to chemically regenerate Ru(III) from
Ru(II) for further electrochemical cycles, amplifying the signal. In another study,
Table 4.1 An overview of common electrochemical approaches for CTC detection

Electrochemical Electrochemical
approaches labels Biomarkers Detection limit References
Impedance Label-free Nucleolin 794 cells/mL Feng et al. [22]
Impedance Label-free N/A ~5 cells/mL Martinez-
Cisneros et al.
[44]
Impedance Label-free Glycan 12 cells/mL Zhang et al. [85]
580 cells/mL
ECL Tris No information 89 cells/mL Ding et al. [17]
(2,2-bipyridyl)
ruthenium (TBR)
ECL C-dot@Ag Folate receptor 10 cells/mL Wu et al. [78]
ECL NiS@CdS MUC1 12 cells/mL Wen and Yang
composite [74]
DPV Ru(NH3)63+/Fe EpCAM 10–100 cells/ Ivanov et al. [32]
(CN)64 (CTC capture) sensor
DPV Fe(CN)64/3 EpCAM 125 cells/sensor Moscovici et al.
[47]
DPV p-aminophenol CK18 2 cells/mL Safaei et al. [56]
DPV H2O2 N-glycan 10 cells/mL Chen et al. [12]
DPV Thionine EpCA ~34 cells/mL Zheng et al. [88]
~42 cells/mL
Apmerometry H2O2 EpCAM 10 cells/assay Hong et al. [29]
Apmerometry Proton EpCAM 8340 cells/assay Maltez-Da Costa
et al. [43]
Stripping CdTe and ZnSe EpCAM and 10 cells/mL Wu et al. [79]
QDs GPC3
Stripping CdTe QDs No information 50 cells/mL Liu et al. [42]
Stripping CdTe QDs MUC1 100 cells/mL Li et al. [38]
LSV AgNPs, CuNPs EpCAM, MUC1, 2 cells/sensor Wan et al. [71]
and PdNPs HER2, PSMA,
CK18 and
nucleolin
Bipolar N/A N/A 320 cells/mL Zhang et al. [87]
electrochemistry
Moscovici et al. developed a novel microfabricated glass chip that is able to count
prostate cancer cells by measuring the decreased DPV signals of the electroactive
molecule [Fe(CN)6]4/3 with the increasing number of cells specifically bound to
the electrode surface that hinders the electron transfer [47] (Fig. 4.1). Pulse
amperometry was also used for genetic profiling of single cancer cells by detecting
a HRP catalyzed electrochemical reaction where HRP was attached to a single
stranded DNA reporter probe [1].
Stripping voltammetry can sensitively analyze trace amount of electroactive
analytes in solution in two steps: a preconcentration/preaccumulation step to
Fig. 4.1 Cancer cell sensing approach. (a) Photograph of a microfabricated glass chip used for cell
detection. (b) Microscopic images of sensors with 50 μm, 150 μm, and 300 μm apertures. (c)
Schematic of apertures created on a glass chip that serves as cell sensors. An anti-EpCAM antibody
self-assembled monolayer is immobilized to gold surface as recognition layer. Upon binding of
DU145 prostate cancer cells to the anti-EpCAM antibody, the interfacial electron transfer reaction
of [Fe(CN)6]3/4 is hindered due to the blocking of the gold surface, resulting in a decrease in the
electrical signal. (d) DPV showing the decrease in electrical signal after target cancer cells are
bound to the sensor. (Figure taken from Moscovici et al. [47])
concentrate the target analytes to the electrode surface and a stripping step to strip the
preconcentrated analytes from the electrode and measure them in the form of current.
Stripping voltammetry may have lowest detection limit among the commonly used
electrochemical techniques due to the preconcetration/preaccumulation procedure
[37]. Anodic stripping voltammetry, cathodic stripping voltammetry and adsorptive
stripping voltammetry constitute the three commonly used stripping techniques.
Among those, anodic stripping voltammetry is most frequently applied to
biodetections, often with nanoparticles as labels, where nanoparticles are dissolved
into metal ions before being analyzed using anodic stripping measurement to
sensitively report the presence and level of target analytes [17]. The use of quantum
dots (QDs), such as CdTe [38, 42, 79], and ZnSe [79], has been extensively reported
as nanoparticle labels in surface marker based electrochemical sensing of CTCs.
QDs can be functionalized with small molecules and biomacromolecules to achieve
specific interactions with surface proteins of CTCs. After specifically bound to the
surface of CTCs, QDs are dissolved in HNO3 to form metal ions, such as Cd2+ and
Zn2+, which are determined using anodic stripping voltammetry to indicate the
presence and the number of CTCs they bind to. To more reliably confirm cancer
cells, in a recent study, Kang et al. used two kinds of QDs CdTe and ZnSe to
Fig. 4.2 Overview of the specific cancer cell detection using multi-nanoparticle approach. (a) Chip
layout and SEM image of an electrode after plating. (b) MNP-based electrochemical labels are
made specific to cell surface markers by conjugating MNPs to DNA aptamers or antibodies. (c)
Working principle of the electrochemical sensor, including cancer cells capturing, MNP labels
recognition, and electrochemical readout. (Figure taken from Wan et al. [71])
recognize two disease-specific biomarkers on the MCF-7 cell membrane [79]. By

combining a simple, portable and low- cost paper-based microfluidic immunodevice
with electrochemical-mediated signal amplification system, the present device
enabled ultrasensitive detection and capture of low-abundant MCF-7 cells with a
detection limit of 10 cells mL1.
Although anodic stripping technique had been successfully used in nanoparticle-
labeled CTC detection with high sensitivity, the direct determination of the nano-
particle labels without pre-dissolution and pre-deposition was never practiced in
previous studies. Wan et al. later developed a new, chip-based strategy that exploited
for the first time the direct electrochemical oxidation of metal nanoparticles (MNPs),
including Ag, Cu and Pd, to report on the presence of specific surface markers [41]
(Fig. 4.2). The electrochemical assay allowed simultaneous detection of multiple
different biomarkers on the surface of cancer cells while discriminating between
cancer cells and normal blood cells. Linear sweeping voltammetry (LSV) was used
as the electrochemical readout method in the detection, however, without a
pre-concentration step, this method may not be as sensitive as anodic stripping. By
applying NaBH4 as a signal amplification agent, the sensitivity of the electrochem-
ical assay was successfully maintained or even elevated by achieving a detection
limit of 2 cells per sensor.
Electrochemical impedance spectroscopy (EIS) is a highly sensitive electrochem-
ical approach that measures electrode surface changes by determining the resistive
and capacitive properties of materials bound to the electrode to probe complex
Fig. 4.3 Fabrication process for 3D tubular microsensors. (A) Sequence of the deposition layers:
(a) 20 nm Ge sacrificial layer; (b) 60 nm TiO2 layer; (c) 5 nm Cr layer and 10 nm Au electrodes on
top of the planar structure; and (d) rolling up the nanomembranes into rolled-up sensor. (B) A 3D
schematic representation of the experimental setup for in-flow sensing. (C) Scanning electron
microscopy (SEM) image of the 3D tubular microsensor. (D) Closed-up lateral view of the tubular
structure. Inset shows an FIB cut performed to the microtube to observe its cross section.
(Figure taken from Martinez-Cisneros et al. [44])
bio-recognition events with a simple and label-free strategy. However, non-specific

adsorptions may be very likely to limit the selectivity and sensitivity of this detection
method, thus the use of blocking agents to eliminate non-specific adsorptions is
important and common in electrochemical impedance detection [85]. EIS is widely
used in CTC detection by measuring the change of resistance after CTCs are
captured at the surface of an electrode [22]. Recently, Schmidt et al. reported an
ultracompact three-dimensional tubular structures integrating Au-based electrodes
as impedimetric microsensors for the direct in-flow determination of suspend HeLa
cells [44] (Fig. 4.3). Particularly, the microsensor was free from surface
functionalization, and showed a single cell resolution in a real-time manner. In
another report, a simple and label-free impedimetric cytosensor was fabricated to
detect MCF 7 cells, and a wide detection range from 30 to 1 106 cells per mL with
a detection limit of 10 cells per mL was reached. By cleaving the deoxyuridines
(dUs) of the capture aptamer, the cytosensor can be reused and the CTCs can be
collected and contributed for further analysis [62].
ECL is a combination of chemiluminescence and electrochemistry, where light
emission is initiated by a redox reaction occurring at an electrode surface. Due to its
simplicity, high sensitivity and label-free operation, ECL has been used as a pow-
erful technique for bioanalytical applications. Zhang el al. reported an
Fig. 4.4 Structure of D-BPE (dual-bipolar electrode) and working principle of BPE-ECL sensing
platform. (Figure taken from Zhang et al. [87])
electrochemiluminescent method with a bare gold electrode platform and magnetic

beads as isolation and purification tool that was able to detect Burkitt’s lymphoma
(Ramos) cells as low as 89 cells per mL [17]. Taking advantage of the nanostructured
materials, a highly sensitive electrochemiluminescent cytosensing was developed
using carbon nanodot@Ag hybrid material as probesand graphene nanosheets as
signal amplification agents for cancer cell detection with high sensitivity and selec-
tivity [78]. This ECL cytosensor showed superior cell-capture ability and exhibited a
wide linear range and a much lower detection limit of 10 cells/mL. Most recently,
Zhang et al. applied bipolar electrochemistry to ECL on a multichannel chip
platform to realize visual color-switch biosensing of HL-60 cancer cell by detecting
H2O2 produced by stimulating the cancer cells [87] (Fig. 4.4). The ECL intensity
was much higher because of electron transfer facilitation through one bipolar
electrode to the other, enabling quantitative detection of cancer cells and easy
Fig. 4.5 Capture and electrochemical detection of cancer cells. (a) Microfluidic chip design. (b)
COMSOL simulation of linear velocity gradient. (c) Immunofluorescent image of a captured cancer
cell on chip. (d) Image of a chip with 8 independent sensors and a zoomed in view of one sensing
chamber. (e) Cancer cell capture and ELISA construction. (f) Electrochemical readout.
(Figure taken from Safaei et al. [56])
discrimination from normal cells. Meanwhile, by using ratiometric method that

limited the interference factors, the possibility of false positive result was largely
decreased.
ELISA has been extensively employed in clinical diagnostics. However, this
technique usually requires relatively expensive test kits and bulky plate readers
that limit its applications. Electrochemical ELISA (EC-ELISA) that takes advantage
of the features of electrochemistry, such as simplicity, low cost and fast response, is
able to largely overcome the limitations of the conventional ELISA. Safaei et al.
recently reported a microfabricated system that integrated a sensitive EC-ELISA
method within a microfluidic cell capture system, enabling the reliable detection of
low levels of rare cancer cells in whole blood [56] (Fig. 4.5). Cancer cells were
captured by immune-magnetic method based on the previously reported velocity
valley (VV) chip with patterned microstructures [46]. Afterwards, the cancer cells
were functionalized on-chip with biotinylated anti-CK18 and streptavidin-alkaline
phosphatase (ALP) that converted p-aminophenyl phosphate to p-aminophenol,
which was electrochemically detected using DPV to indicate the presence and the
number of captured cells. In another report, Hong et al. presented an integrated
multifunctional system constructed by conductive disulfide-biotin-doped
polypyrrole nanowires (SS-biotin-Ppy NWs) for capture, release, and in situ quan-
tification of CTCs using EC-ELISA [29]. The well-ordered 3-D nanowires not only
significantly favored cell capture and release efficiency, but also showed high
sensitivity and specificity towards the amperometric detection.
Most recently, Cao et al. reported a new technique that combines electrochemis-
try with a nanochannel-ion channel hybrid array for efficient CTC capture and
sensitive detection [9]. In this work, CTCs were trapped in the ion cannel
immobilized with aptamer that coupled with the protein overexpressed on the
CTCs membrane, resulting in a dramatically blocked ionic flow through channels.
Therefore, instead of detecting the electroactive labels that are routinely used in
electrochemical detection of CTCs, this work monitored in real time the changed
ionic transfer behaviors caused by the varied mass-transfer property using LSV.
Micro- or nano-structured electrodes and the construction of nano-structured
electrode interface are very common in the electrochemical analysis of CTCs to
achieve better assay performance with the effects of their surface morphology, size,
optical and electronic properties, etc. Sage has applied the NMEs with the electrode
materials of gold or palladium to many cancer-related detections and clinical diag-
nostics [57]. This electrochemical platform, with large surface area and very fine
nanostructures, allows rapid and efficient analyte capture and more efficient mass
transfer, enabling to achieve excellent specificity and sensitivity, and fast sensing in
the detection of clinical samples at low concentrations. Taking advantages of their
large surface area, excellent conductivity, biocompatibility and flexibility, nano-
structured materials, such as carbon nanotubes [85], graphene [12], and soft
nanowires [29] were used to construct biocomposites modified at the surface of
the electrode with high stability and bioactivity for increasing analyte loading and
enhancing the electrochemical signal. Apart from nanostructured electrodes or
electrode surface modifications, nanoelectrocatalysts were designed and fabricated
to catalyze the reactions of the electrochemical labels with high activity and effi-
ciency for signal amplification of CTC sensors [43, 88]. Fe3O4@nanocage core
(Ag-Pd) nanohybrid NPs, for example, were fabricated that showed superior
electrocatalytic activities towards the nonenzymatic electrochemical reduction of
thionine, the electrochemical label [88] (Fig. 4.6). These nanoelectrocatalysts
showed signal-amplifying ability that could be used for ultrasensitive electrochem-
ical cytosensing, with the detection limits of ~34 and ~42 cells mL1 for MCF-7 and
T47D cells, respectively.
Electrochemical processes can be easily adapted and combined with other useful
technologies to achieve higher performance in biodetection by integrating the merits
and advantages of each technology. As is reviewed in the previous text, electro-
chemistry has been married to microfluidics [79], magnetic interaction [32], ELISA
[56], and nanotechnologies and nanomaterials [88]. Very recently, Wu et al.
presented a new strategy of the combination of SPR and electrochemical method
for real-time evaluation of live cancer cells [80] (Fig. 4.7). By monitoring the SPR
signal changes from the morphology and mass changes of adsorbed cancer cells and
the electrochemical measurement of the extracellular daunorubicin (DNR) residue
after being treated with cancer cells, the present strategy showed great potential in
evaluating therapeutic efficiency of bioactive agents to cells and monitoring relevant
treatment processes in clinical applications.
Fig. 4.6 (a) Scheme showing the fabrication of Fe3O4@AgPd hybrid NPs. (b) SEM image of
Fe3O4@AgPd hybrid NPs. The inset shows one Fe3O4@Ag Pd hybrid particle. (c) TEM image
of Fe3O4@AgPd hybrid NPs. The inset presents individual AgPd nanocages. (d) CVs of a bare
GCE, GCE/Fe3O4, and GCE/Fe3O4@AgPd. (Figure taken Zheng et al. [88])
4.3 Circulgating Nucleic Acids
So far, most of molecular-based analyses for cancer diagnosis are performed using
nucleic acids (NAs) extracted from nucleated cells by cell lysis. Unlike these nucleic
acids, cNAs, or cell-free nucleic acids (cfNAs), are found outside cells, primarily in
the serum and plasma with some in urine [24]. These nucleic acids such as DNA,
mRNA and microRNA that are released from tumors and circulate in the blood of
cancer patients have become a new generation of cancer biomarkers indicating the
malignant progression of the disease [58].
Fig. 4.7 Scheme of the combination of SPR and electrochemistry in real-time evaluation of live
cancer cells. (Figure taken from Wu et al. [80])
The detection of cNAs could serve as a liquid biopsy, potentially replacing tumor-
tissue biopsies in certain diagnostic applications, and has the potential to revolution-
ize the detection and monitoring of tumors in a non-invasive way [58]. However, the
concentrations of cNAs are typically low and very variable. Thus, early sequencing
technologies are often unable to detect cNAs and a number of efforts have been
made to increase the detection. The conventional and common methods of detecting
cNAs include PCR techniques, northern blotting, and fluorescent imaging [25, 64].
Unfortunately, these methods are normally expensive and time-consuming, and
require large amount of samples.
Electrochemistry-based NAs sensors, a key area in the scope of biomolecular
electrochemistry, are able to detect nucleic acid sequences or mutated genes associ-
ated with human disease by combining nucleic acid layers with electrochemical
transducers. Basically all common electrochemical methods have been applied to
NAs detection, in which amperometry [8], various voltammetry methods [19], and
electrochemical impedance spectroscopy (EIS) [63] are reported the most. Most of
electrochemical NAs sensors use label-based techniques that either covalently or
non-covalently (such as electrostatic adsorption) binds electroactive reagents to NAs
analytes. Label-free methods are also reported for this application, which are simpler
but are generally not able to reach comparable level of sensitivity as the label-based
methods. A number of literatures have reviewed the strategies of electrochemical
NAs sensors based on these two methods [19, 37, 52]. In this part we will focus on
new strategies employed in improving the performance of cNAs sensors.
Electrochemical NAs sensors are promising in constructing simple, accurate, and
inexpensive platforms for cancer diagnostics with high sensitivity [19]. However,
the crucial challenges still relate to further achieving high levels of sensitivity and
specificity on clinical samples, especially when dealt with cNAs. Although there are
currently not many studies concerning the electrochemical cNAs detections, smartly
designed strategies have been presented in existing papers to improve the perfor-
mance of the cNAs sensors, which often aim for signal amplification to enhance
sensitivity. For example, Labib et al. developed a protein-facilitated sensor for the
detection of miRNA signature of chronic lymphocytic leukemia (CLL) in human
serum by electrochemical detection of methylene blue (MB) molecules using CV
upon the hybridization of target miRNAs to two DNA adaptor strands bound to a
universal interfacial probe [36]. Glucose oxidase was introduced as electrocatalyst to
create multiple redox cycles for MB molecules in order to amplify the current from
the electrochemical reduction of MB. The developed sensor was capable of
distinguishing single base mismatches in the target miRNA and was employed for
profiling of three endogenous miRMAs characteristic to CLL. Higher sensitivity of
nucleic acid detection can also be achieved by the implementation of various
nanomaterials, which act either as nanoelectrodes (immobilization interface for
accumulation of increased amounts of nucleic acid probes), or as signal amplifiers
of the hybridization event [52]. By using NMEs as a platform and amino acid/nucleic
acid chimeras (ANAs) as probe molecules, Vasilyeva et al. reported a large-foot-
print electrochemical sensor that were able to capture large, slow-moving analytes
[70] (Fig. 4.8). Using DPV, the sensor proved to be ultrasensitive and specific in the
detection of mRNA derived from 10 cancer cells present in complex samples.
Besides, Wen et al. demonstrated a three-dimensional (3D) DNA tetrahedral
nanostructure-based interfacial engineering approach to enhance miRNAs binding
recognition at the gold electrode surface and drastically improve the detection
sensitivity by examining the electrochemical responses using CV and
chronoamperometry [75]. By employing this DNA nanostructure, the resulting
sensor can directly detect as few as attomolar miRNAs with high single-base
discrimination ability. Recently, Fang et al. developed an ultrasensitive and highly
specific electrochemical assay for miRNA-21 detection based on the selective
binding of Just Another Zinc finger proteins (JAZ) between a ssDNA capture
probe and a target miRNA [21]. In their work, the high signal amplification was
obtained through enzymatic amplification by alkaline phosphatase (ALP) conju-
gated to JAZ coupled with electrochemical-chemical-chemical (ECC) redox cycling
involving an ALP product (hydroquinone) mediated by Ru(NH3)63+/Ru(NH3)62+
redox system. The resulting detection limits for miRNA-21 in buffer and ten-fold
diluted serum can be achieved as low as 2 and 30 fM, respectively.
Other strategies to improve cNAs detection performance include the invention of
multi-mode detection [35] and the use of RNA binding viral protein as a selective
biorecognition element [8]. To be more specific, Labib et al. developed a three-mode
electrochemical sensor for miRNA analysis via direct hybridization with the target
miRNA, p19 protein binding, and protein displacement, using both square wave
voltammetry (SWV) and EIS measurement [35] (Fig. 4.9). The fabricated sensor
showed a detection limit as low as 90 molecules per sample of miRNA and a wide
dynamic range (from 10 aM to 1 mM). Very uniquely, the protein displacement
allows the detection of any type of miRNA. Using an RNA binding viral protein as a
Fig. 4.8 Chip-based sensors for detection of chronic myeloid leukemia cells. (a) Chip layout. (b)
SEM images of a 100 mm sensor formed by using gold electrodepositon on the surface of the chip.
(c) Sequence of steps for nucleic acids analysis. (d) RuIII/FeIII reporter group permits hybridized
nucleic acids to be detected. (e) Overall flow diagram of the analysis. (Figure taken from Vasilyeva
et al. [70])
selective biorecognition element, Campuzano et al. developed an amperometric

magnetobiosensor for miRNA quantification, which was able to detect 0.4 fmol of
a synthetic target and endogenous miR-21 in only 2 h in total RNA extracted from
cancer cells and human breast-tumor specimens [8].
The detection of cNAs for specific gene mutations, such as KRAS, is desirable
because these gene mutations happen very frequently in many tumor types and
contribute to tumor progression [58]. Recently, Das et al. reported an electrochemical
chip-based clamp assay that detected mutated sequences KRAS and BRAF directly in
patient serum, which was the first successful detection of cNAs without the need for
enzymatic amplification [15] (Fig. 4.10). Using a collection of oligonucleotides that
Fig. 4.9 Schematic representation of the 3-mode electrochemical sensor. (a) Modification of a
thiol-modified complementary probe onto a gold nanoparticles-modified screen-printed carbon
electrode for capturing miR-21. (b) A hybridization-based sensor showing an increase in the current
intensity by the binding of the target miR-21. (c) A protein-based sensor showing a large decrease in
current density by the binding of the p19 protein. (d) A displacement-based sensor showing a shift-
back in the signal due to the dissociation of p19 protein forced by the hybridization product of
miR-200 and its complementary probe. (Figure taken from Labib et al. [35])
sequester wild-type sequences in solution, the sensor allows only the mutated
sequence to bind to its surface. Benefiting from this principle, the electrochemical
clamp sensor exhibits excellent levels of sensitivity and specificity, and allows
accurate detection of mutated sequences in a collection of samples taken from lung
cancer and melanoma patients within 15 min readout time. On the basis of this work,
the same group later reported the electrochemical detection of mutated circulating
tumor DNA (ctDNA) in samples collected from cancer patients [16]. By employing
DNA clutch probes (DCP) to prevent reassociation of the denatured DNA strands and
ensure thereby that only mutated sequences associate with the detecting hybridization
events, it was possible to readout the presence of mutated ctDNA. The assay
exhibited excellent sensitivity and specificity of 1 fg/μL of a target mutation in the
presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a
level of 0.01% relative to wild-type.
Fig. 4.10 Scheme of the clamp chip for the electrochemical analysis of mutated cfNAs. (a)
Schematic representation of the clamp strategy. (b) Sequence of steps for sensor-based detection.
(c) Electrochemical readout using DPV method. (d) Microchip layout. (e) SEM image of a NME
sensor. (Figure taken from Das et al. [15])
4.4 Extracellular Vesicles
Many cell types, including tumor cells, release EVs. Exosomes (30–100 nm in
diameter) and microsomes (100–1000 nm in diameter) are small EVs secreted by
most mammalian cells which carry factors that facilitate intercellular communica-
tion. They are usually transported through biological fluids such as blood plasma
[55], body fluids [31], and urine [50]. Likewise, exosomes and microsomes that are
shed by tumor cells carry specific proteins, functional mRNAs, microRNAs and
DNAs. These biomolecules have been acknowledged as potential biomarkers
resource for non-invasive diagnosis of cancer without the need to access the tumor
directly. Given this fact about exosomes and microsomes, they have attracted
tremendous attention in the past decade. In the following section, we will focus on
reviewing exosomes in terms of their isolation, characterization and electrochemical
detection.
The isolation and characterization of exosomes and microsomes in clinical
samples with the presence of billions of blood cells is technically challenging.
Currently, the most commonly used isolation approaches for both exosomes and
microsomes are based on differential ultracentrifugation steps (often accompanied
by multi-step filtering). Since exosomes have much smaller sizes than microsomes,
the centrifugation condition is even more critical. A high-speed centrifugation (up to
200,000 g) for more than 10 h is required which makes this approach tedious and
time consuming [5]. Separation using affinity purification with specific antibodies is
another option, however, it depends on the presence of binding-target proteins [65].
Since CD63 is one of the intrinsic molecular subtypes of exosomes and EpCAM is a
ubiquitously expressed epithelial cancer marker, they are both commonly used as
markers for capturing exosomes. Some techniques have emerged based on this
separation principle, such as using affinity-binding beads and microfluidic
immunocapture platforms [27, 45]. He et al. developed a magnetic-bead based
microfluidic approach that allowed on-chip immunoisolation and in situ protein
analysis of exosomes directly from patient plasma [27]. This approach enabled
selective isolation of exosome subpopulations from ovarian and non-small-cell
lung cancer patients by targeting various surface markers. The intervention with a
dynamic force is also reported to combine with affinity purification method to
enhance the specificity of capture. As an example, a multiplexed microfluidic device
with high specificity for capture and detection of multiple exosome targets was
designed using a tunable alternating current electrohydrodynamic (ac-EHD) meth-
odology [67]. The generated nanoscaled fluid flow by electrical body forces within
nanometers of an electrode surface enhanced the specific capture efficiency and also
reduced nonspecific adsorption of weakly bound molecules from the electrode
surface.
The analysis and characterization of exosomes have been mainly focused on their
physical properties, protein expression and nucleic acids contents. Generally, phys-
ical properties such as size, concentration and morphology are obtained by optical
and non-optical methods such as scattering and electron microscopy [68]. However,
these methods, due to their intrinsic limitations in resolution, usually fail to quanti-
tate the level of exosomes in patient samples. Conventional optical methods for cell
analysis, such as fluorescence and dynamic light scattering (DLS), are not able to
provide reliable information due to their high detection limit [68]. Exosomal proteins
are typical markers for exosomes detection, and are mainly analyzed by western blot
analysis and ELISA [50]. Recently, Vaidyanathan et al. analyzed exosomes based on
their specific protein markers epidermal growth factor receptor 2 (HER2) and
prostate specific antigen (PSA) after successfully capturing exosomes using an
ac-EHD method on a microfluidic device [67]. By catalytically oxidizing peroxidase
substrate 3,30 ,5,50 -tetramethylbenzidine (TMB) with tagged horseradish peroxidase
(HRP) on the surface of captured exosomes, this detection method provides a simple
and rapid on-chip naked eye detection readouts and allows simultaneous detection of
multiple exosome targets. Unfortunately, surface protein based detections, in gen-
eral, have shortcomings such as poor sensitivity and highly manual workflows.
Another approach for exosome characterization is to detect exosomal nucleic
acids, such as miRNAs, mRNAs and DNAs by PCR and agarose gel electrophoresis
[66]. However, exosomes have to be lysed before detection, which adds to the
complexity of the testing. Very recently, Im et al. reported a label-free, high-
throughput assay based on transmission surface plasmon resonance through periodic
nanohole arrays functionalized with antibodies which enables profiling of exosome
surface proteins directly from ascites samples from ovarian cancer patients [31]
(Fig. 4.11). This approach offered improved sensitivity and allowed portable
Fig. 4.11 Label-free detection of exosomes with nano-plasmonic exosome (nPLEX) sensor. (a)
Exosomes secretion and high magnification transmission electron micrograph (TEM) (inset) show-
ing that exosomes from CaOV3 culture have a diameter ~100 nm. (b) Finite-difference time-
domain simulation showing the enhanced electromagnetic fields tightly confined near a periodic
nanohole surface which overlaps with the size of exosomes captured. (c) SEM image of the periodic
nanoholes in the nPLEX sensor. (d) A prototype miniaturized nPLEX imaging system for exosomes
analyses. (e) A representative schematic of changes in transmission spectra showing exosome
detection with nPLEX. (f) SEM image showing exosome capture by functionalized nPLEX.
(Figure taken from Im et al. [31])
operation while requiring much smaller sample amount, but the detection relied on
surface plasmon resonance instrument with a high level of cost and expensive
maintenance.
Clinical analysis based on electrochemical biosensors is promising to overcome
these limitations for their low cost, fast response and low material requirement. To
date, electrochemical approaches have not been widely used for the detection of
exosomes and other vesicles with clinically relevant levels of sensitivity, with only a
few examples including amperometry [33, 73, 90], DPV [81], LSV [91], electro-
chemical impedance [41, 48] in the existing studies. The strategy of reporting the
current decrease from the block of electron transfer by the introduction of target
exosomes has been commonly used. For example, Yadav et al. reported the con-
ventional immunoaffinity-based method to directly quantify the cancer-specific
exosomes from cell culture media [81]. Using the [Fe(CN)6]4/3 redox system, a
decrease in DPV current response was observed as the signal readout after
the addition of exosomes to the electrode and block the electron transfer of
[Fe(CN)6]4/3.
Aptamer-based biosensors (aptasensors) have drawn particular attention in the
applications in cancerous exosome analysis for their advantages in detection sensi-
tivity, speed, modest requirement of sample volumes and easy signal amplification
[72, 90]. Zhou et al. developed an electrochemical aptasensor to detect exosomes
from cell culture media [90]. In their study, aptamers specific to exosome transmem-
brane protein D63 were immobilized onto electrode surface, and MB labeled
probing strands hybridized with the aptamers to emit an electrochemical signal.
Upon the introduction of exosomes, the interaction of the aptamer-modified elec-
trode with exosomes carrying D63 resulted in the displacement of the probing
strands, causing the redox signal to decrease. Through a passivation procedure
using the blocking agents, non-specific adsorption was successfully prevented.
Furthermore, the assay was miniaturized by photolithography and integrated into
microfluidic devices, showing the low requirement of sample volume and simple
handling or processing steps. However, due to the flexibility of single-stranded
DNA, aptamers on electrodes tend to undergo self-assembled monolayer aggrega-
tion or entanglement, largely impeding the accessibility of the target exosomes. In
addition, the precise control of spatial orientation of single-stranded aptamers is also
difficulty to achieve. Recently, Wang et al. constructed a portable electrochemical
aptasensor that used DNA-based nanotetrahedron (NTH) for direct capture and
detection of hepatocellular exosomes [72]. They also used [Fe(CN)6]4/3 redox
system to signal the decrease of the electrochemical current upon the capturing of
cancerous exosomes using square wave voltammetry (SWV) method. The NTH
assisted structure allowed the oriented immobilization of aptamers that significantly
improved the accessibility of an artificial aptamer to suspended exosomes with
100-fold higher sensitivity when compared to the single-stranded aptamer-
functionalized aptasensor.
High-throughput measurement, such as flow cytometry, is of great importance
and has becomes a trend in a wide range of topics in biology and medicine. However,
flow cytometry tends to miss small vesicles (<200 nm) due to weak light scattering
[69]. In order to realize high-throughput detection of exosomes, Jeong et al.
constructed a portable sensor with multi-electrochemical channels for the detection
of exosomes from plasma samples of ovarian cancer patients [33] (Fig. 4.12). The
sensor was an EC-ELISA combining magnetic enrichment and enzymatic amplifi-
cation. As a proof-of-concept, the authors implemented an eight-channel device that
allowed for the simultaneous profiling of multiple protein markers within an hour,
showing high sensitivity and speed over conventional assays. EC-ELISA was also
Fig. 4.12 Integrated magneticelectrochemical exosome platform. (a) Sensor structure showing
the simultaneous measurement of signals from eight electrochemical channels. (b) Circuit diagram.
(c) Packaged device. (d) Schematic of assay. Exosomes are captured directly from plasma by
magnetic beads coated with CD63 and labeled with HRP. W, C and R represent working, counter
and reference electrode, respectively. HRP indicates horseradish peroxidase and TMB indicates
3,30 ,5,50 -tetramethylbenzidine. (Figure taken from Jeong et al. [33])
reported for the determination of exosomes elsewhere with a detection limit as low
as 200 exosomes per microliter [18].
Nanoparticle labeling was also used in exosome analysis. Zhou et al. extended the
MNPs labeling method to the non-enzyme detection of exosomes and microsomes,
which was based on direct electro-oxidation of MNPs to specifically recognize surface
markers of exosomes and microsomes [91] (Fig. 4.13). They also used a simple
centrifugation procedure to isolate exosomes and microsomes form cell culture super-
natant or directly from blood serum with a maximum centrifugation of 16,100 g for
30 min. Compared to the aforementioned detection methods, this strategy is fast in
response (within 10 s), simpler in instrumentation, and more cost effective. It also
provides the simultaneous detection of multi-markers. By testing the levels of
exosomal and microsomal EpCAM and PSMA derived from prostate cancer patients,
it was shown that exosomes and microsomes had potential for cancer diagnosis at
early stages. QDs were also reported as signal amplifiers in a stripping voltammetric
immunoassay for the detection of disease-specific exosomes [6].
Fig. 4.13 Schematic representation of the isolation and analysis of exosomes and microsomes: (a)
vesicles are captured to aptamer-modified sensors, (b) electrochemical detection of the captured
vesicles with Cu and Ag nanoparticles as markers. (Figure taken from Zhou et al. [91])
4.5 Conclusions and Perspectives
Non-invasive early detection of cancer markers has been the main focus of cancer-
control research over the past decades. In this chapter, we provide an overview of the
recent developments in the electrochemical detections of new circulating CTMs,
including CTCs, cNAs and EVs. We gave representative examples for each marker
and showed the combination of electrochemical approaches with other remarkably
progressed technologies and strategies, such as microfluidics, nanotechnology and
bioconjugation techniques. Although the performances of electrochemical sensors
have been much enhanced accordingly, there still exist obstacles that have to be
overcome before they can be applied to point-of-care diagnostics.
Firstly, electrochemical transducers, except EIS that measures the change in
electrochemical resistance, rely on the electron transfer from the electrochemical
labels of cancer biomarkers to electrode surface in order to report the signal. Since
most of the biomarkers, such as CTCs, proteins, and EVs are low in conductivity, the
development of new signal amplification strategies [15, 71, 90, 91] is highly desired
besides the common practice of constructing nanoscaled transducers with
nanomaterials. Secondly, nanostructured electrodes with high roughness factors
that aims for rapid and efficient analyte capture and more efficient mass transfer
may sometimes result in opposite effects. For example, at very rough electrode
surfaces the diffusion zones of the nanostructure may overlap or partially overlap,
leading to linear to partially linear diffusion over the entire surface and producing
less efficient mass transport [89]. In addition, the nanostructures with random sub-
structures may lead to uncontrollable analyte orientation and configuration on the
electrode surface, which may inhibit the transduction of the electrochemical signal.
Therefore, a rational design in nanostructures should be carefully considered before
the construction of the sensor. Thirdly, the efforts made to increase sensitivity also
bring about selectivity issues from interferences of non-specific adsorption that
usually occur when testing with complex biological matrices. Thus, the parameters
that determine the specificity of the electrochemical devices have to be further

optimized and refined to realize specific, reliable and simultaneous multi-marker
detection in complex biological samples.
Furthermore, most of the electrochemical sensors are merely proof-of-concept
demonstrations that are only practical to perform under optimized conditions in a
lab. There is still a long way to go to achieve portable and user-friendly point-of-care
diagnostics with complex real patient samples. High-throughput multiplexed sensing
with highly automated systems for sample handling is a big trend to achieve a
widespread use of point-of-care devices with fast response. Therefore, related
technologies have to be further advanced, such as the design of a miniaturized
multi-channel (hundreds and above) electrochemical circuit for high throughput
and the invention of a robot for automatic sample handling. There are also some
technologies that are well progressed but still exist constraints when integrated with
electrochemistry. Microfluidics, for example, allows considerable portability and
high capacity for device integration. However, it usually suffers from low reproduc-
ibility with electrochemical readout due to compact electrode configuration and
small amount of liquid volume.
In summary, there are considerable issues to be dealt with in constructing efficient
electrochemical platforms for portable, fast and cost-effective point-of-care diagnos-
tic devices. New advances and improvements are expected from the cooperation of
different communities to ultimately achieve this goal.
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Chapter 5
Spiral Inertial Microfluidics for Cell
Separation and Biomedical Applications
Ning Liu, Chayakorn Petchakup, Hui Min Tay, King Ho Holden Li,
and Han Wei Hou
Abstract The emergence of omics studies and single cell analysis in biomedicine
has advocated a critical need to develop novel cell sorting technologies to process
complex and heterogenous biological samples prior analysis. Spiral inertial
microfluidics is an enabling membrane-free cell separation technique developed
almost a decade ago for high throughput biophysical cell separation, and has since
been widely exploited for different biomedical applications. In this chapter, we will
provide a comprehensive review on spiral inertial microfluidics including (1) con-
ventional and microfluidic cell sorting techniques, (2) introduction to inertial
microfluidics and Dean-coupled inertial focusing, (3) classification of major spiral
devices, (4) summary of different biomedical applications, (5) recent advances in
next generation spiral cell sorters, and (6) highlight key challenges for future
research. With increasing advancement in microfabrication and computational sim-
ulation, we envision that spiral inertial microfluidics will play a leading role in
driving research and commercialization in clinical diagnostics, as well as other
research areas in chemistry and material sciences.
Keywords Inertial microfluidics · Cell separation · Biomedical diagnostics
N. Liu · H. M. Tay
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
C. Petchakup · K. H. H. Li
School of Mechanical and Aerospace Engineering, Nanyang Technological University,
Singapore, Singapore
H. W. Hou (*)
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
School of Mechanical and Aerospace Engineering, Nanyang Technological University,
Singapore, Singapore

100 N. Liu et al.
5.1 Introduction
Cell separation is an essential sample preparation step in biomedical research to

purify target cell population and minimize cell-cell interactions prior analysis. For
example, subpopulations of cells with unique biological signature and functions are
often found in tissue and clinical samples that are highly heterogeneous. It is
therefore necessary to isolate specific target cells to facilitate downstream biological
assays. Liquid biopsy or blood diagnostic is another important application as blood
is routinely obtained during medical checkup, and contains a myriad of information
on the health status of an individual. Efficient isolation of blood cell components
(white blood cells (WBCs), platelets) or rare diseased cells (cancer or bacteria
(~1–100 cells/mL)) from a large cellular background (~5 billion red blood cells
(RBCs)/mL) will hence greatly improve signal-to-noise ratio for unbiased and
accurate clinical assessment [1].
5.1.1 Conventional Separation Techniques
Conventional “label-free” separation approaches include membrane filtration and

centrifugation, which are widely available and simple to use. Membrane filtration
works by size-exclusion through a porous matrix which retains cells above a certain
size (larger than pore size) and allowing smaller cells to pass through. Target cells
can be collected either on the membrane or in the filtrate. Although useful as a
pre-enrichment step to remove cell aggregates, clogging issues persist in such
systems and it is non-trivial to retrieve the larger target cells from the membrane.
Variability in cell deformability can also affect size cut-off or optimal pressure
conditions [3, 4]. Differential centrifugation is yet another popular method which
separates cells based on size and density, but the isolation of rare cells (< 1000 cells)
is not practical and risk substantial cell loss. Furthermore, fluid shear stresses from
repeated centrifugation and resuspension can also damage or activate sensitive cells
(e.g. neutrophils) [5].
With the growing repertoire of monoclonal antibodies, affinity-based cell sepa-
rations methods such as fluorescence-activated cell sorting (FACS) and magnetic-
activated cell sorting (MACS) have become increasing popular among biologists.
Target cells are first immunolabelled with antibodies which bind to specific surface
markers prior separation. FACS relies on fluorophore-conjugated antibodies for
separation while MACS employs magnetic microbeads-conjugated antibodies. In
FACS, fluorescently-labelled cells are hydrodynamically focused to a narrow stream
and passed through a laser beam for signal interrogation. The resulting scatter and
fluorescence signals are used to discriminate different cells types based on their cell
size, granularity and surface markers. Target cells are subsequently identified based
on these biophysical signatures and electrostatically deflected into separate reser-
voirs for collection [2]. Being a well-established method with high sensitivity and
5 Spiral Inertial Microfluidics for Cell Separation and Biomedical Applications 101
Table 5.1 Comparison of conventional cell separation techniques

Separation
technique Principle Advantages Disadvantages
Label-free Filtration Size Simple to use Low purity
Centrifugation Density Relatively Low yield
cheap Non-specific
Requires manual post-
processing
Affinity- FACS Antibody High purity High cost
based MACS Antibody High yield Additional labeling steps
Highly May affect cell function
specific
throughput, it is often considered as the “gold standard” for cell sorting [3]. A
throughput of 2000–10,000 cells per second can be achieved with higher rates
sacrificing purity [4]. On the other hand, MACS achieve immuno-magnetic separa-
tion by applying an external magnetic field to extract the magnetic bead-bound cells
(positive selection) or eluting the non-labelled target cells (negative selection) [5]. In
contrast to FACS which enables multiplexed cell sorting, MACS is a bulk and binary
processing method, and does not provide individual cell analysis or multi-parametric
outputs. Moreover, both methods are usually labor intensive, time-consuming,
expensive, and the cell yield or recovery is highly dependent on user operations
(Table 5.1).
5.1.2 Microfluidics Cell Separation
With advancement in microfabrication, the birth of microfluidics or lab-on-a-chip

technologies since the 1990s has revolutionized chemical analysis and biological
assays through unique physical phenomenon and flow control in the microscale
[6]. The increasing demand for better and more sensitive assays have propelled the
development of many novel microfluidics separation strategies integrated with
single cell manipulation and analysis capabilities for point-of-care diagnostics
[3, 7, 8]. These miniaturized systems offer numerous advantages including reduced
sample and reagent consumption, faster processing time, high spatial resolution, low
device cost and high portability [9]. Consequently, microfluidics has become an
important toolbox for cell separation applications with the ability to achieve unprec-
edented size resolution and purity, and high throughput sample processing.
Generally, microfluidic cell separation techniques are classified into active and
passive methods based on the involvement of external fields (e.g. electric, optical,
acoustic, magnetic field) [8, 10, 11]. Active methods exploit external fields to impart
different forces on cells to achieve separation, and common examples include
magnetophoresis [12–14], dielectrophoresis [15], acoustophoresis [16, 17] and
102 N. Liu et al.
Table 5.2 Comparison of various passive separation techniques

Separation technique Mechanism/principle Separation criteria Throughput
Biomimetic Hydrodynamic force/ Size deformability 10 μL/h [19]
Fahraeus effect
Hydrodynamic Streamline manipulation Size shape 20 μL/min
[20]
>105/min [21]
Hydrophoretic filtration Pressure field gradient Size 4 103/s [22]
Inertial Lift force secondary flow Size shape ~106/min [23]
Microstructure (Pillars Laminar flow/perturbation of Size deformability 103 μm/s [24]
and weirs) flow 5 μL/min [25]
Surface affinity Specific binding to surface Size surface 1–2 mL/h [26]
markers biomarkers
Pinched flow fractionation Laminar flow (Hydrody- Size ~4 103/min
(PFF) namic force) [27]
20 μL/h [27]
optical sorting [18]. Passive separation methods rely on intrinsic hydrodynamic

forces during fluid flow which are modulated by microchannel design and flow
conditions (Table 5.2).
Numerous efforts have been focused on the development of passive separation
methods including deterministic lateral displacement (DLD) [24, 28], pinched flow
fractionation (PFF) [27, 29] and inertial focusing/microfluidics. Among these tech-
nologies, inertial microfluidics has emerged as a highly promising approach for size-
based cell separation due to its ease of operation and high separation resolution. The
first seminal work was reported by Di Carlo et al. in 2007 where they described
particle inertial focusing effects in microfluidics and its application for high through-
put size-based particles/cell separation [30]. This is shortly followed by the
Papautsky’s group who reported similar particle inertial focusing and Dean migra-
tion effects in spiral microchannels [23, 31]. With increasing understanding of the
particle inertial focusing behavior [32], many researchers have started working in
this exciting field to explore new frontiers and applications in fluid mechanics
research and biomedical applications. Unsurprisingly, several review papers on
inertial microfluidics have been recently published which give an excellent overview
of inertial microfluidics in different channel geometries [33–36]. Spiral
microchannel (hereafter termed as spiral inertial microfluidics) is one of the most
widely used designs for cell separation as it exploits both size-dependent particle
inertial focusing and secondary Dean-induced migration effects to achieve separa-
tion. In this chapter, we will first discuss the fundamental principles involved in
spiral inertial microfluidics. This is followed by a review of the major types of spiral
microfluidics devices and their separation principles. Next, we will provide an
overview of spiral cell sorting applications for various bio-entities (cells and mole-
cules) separation, and also highlight recent advances in spiral technologies with
novel designs or multiplexing capabilities. Finally, we will conclude with current
challenges and suggest future research directions in this area.
5.2 Theory
5.2.1 Stokes Flow
In fluid mechanics, the motion of viscous fluid can be described by the Navier-
Stokes equation as shown below:
ρð∂u=∂t þ u ∙ ∇uÞ ¼ ∇p þ μ∇2 uþf
where ρ represents the fluid density, μ is the fluid dynamic viscosity, u is the fluid
velocity field, p represents fluid pressure field, and f is the vector field of external
body forces imparting on fluid elements. ρ(∂u/∂t + u ∙ ∇u) corresponds to the
inertial forces, ∇p corresponds to pressures, and μ∇2u corresponds to viscous
forces.
To characterize fluid flow, the channel Reynolds number (Rc) is proposed as a
dimensionless quantity which describes the ratio of inertial to viscous forces:
ρU m Dh
Rc ¼
μ
where ρ represents the fluid density, μ is the dynamic viscosity of the fluid, Dh is
defined as the hydraulic diameter where Dh ¼ d in a circular channel (d represents
the diameter), or Dh ¼ 2wh/(w + h) in a rectangular channel (w and h denotes the
width and height of the rectangular cross section, respectively), and Um is the
maximum velocity of the fluid flow. When considering finite-size particles in the
channel flow, the particle Reynolds number (Rp) is helpful to describe the relation-
ship between the particles and the channel dimensions:
a2 ρU m a2
Rp ¼ Rc ¼
D2h μDh
where a is the particle size. Flow pattern in the channel will change when the size
ratio of particle to channel varies. For Rp 1, the viscous drag is dominant and the
particles are deemed as “point-particle”. As Rp increases, inertial effects become
more apparent in the channel flow. Laminar flow occurs when Rc is below a critical
value of approximately 2040 [37]. Due to the small channel dimensions (typically
less than 1 mm) in microfluidics, Rc is usually less than 100 and fluid flow is
completely laminar and the viscous forces of the fluid dominate the inertial forces
(Stokes flow). Hence, the inertial portion in Navier-Stokes equation is neglected for
most microfluidic systems by equating the left hand side of the equation to zero.
Stokes flow lies within laminar regime, but the inverse is not true [33, 34]. Recently,
the application potential of the long-ignored intermediate range flow (~1 < Rc < 100)
has received increasing attention and several inertial-based effects in microfluidics
devices include improved mixing and precise particle control [30, 33, 34, 38].
104 N. Liu et al.
Fig. 5.1 Particle

equilibrium positions in a
circular straight channel as
observed by Segre and
Silberberg in 1960s
5.2.2 Inertial Focusing
Segre and Silberberg reported the first observation of particle inertial focusing
effects and the earliest interpretation to explain this unintuitive phenomenon in
early 1960s [39, 40]. In their experiment, the randomly dispersed particles (~1 mm
diameter) were introduced into a cylindrical pipe (~1 cm diameter). After travelling a
distance of 114 cm, the particles were distributed in an annulus between the center
and the wall within the cross section of the pipe [40]. The mean radius of the annulus
was measured to be ~0.6 times the pipe radius, as indicated in Fig. 5.1.
This unique particle lateral migration effect was later found to be the result of the
interplay between two dominant inertial lift forces: the shear gradient induced lift
force pushing particles in the medium away from the channel center, and the wall
induced lift force repelling the particles away from the wall. Another important force
to consider is the Stoke’s drag force in secondary lateral flow. These forces will be
explained in more details below.
5.2.2.1 Wall Induced Lift Force (FWL)
For a particle flowing near the channel wall, the interaction between the particle and
the wall causes the particle to lag behind the fluid flow. In addition, the constricted
flow space between the particle and channel wall will cause the fluid flow at the top
side of the particle to be accelerated due to more streamlines diverted toward these
side. This creates a relative lower pressure than the “wall side” of the particle and a
lift force directed away from the wall is generated (Fig. 5.2a).
Fig. 5.2 Schematics illustration of the dominant forces experienced by particles in inertial
microfluidics (a) Wall induced lift force (FWL), (b) Shear gradient lift force (FSL), (c) Secondary-
flow drag force (FD)
106 N. Liu et al.
5.2.2.2 Shear Gradient Lift Force (FSL)
The shear gradient lift force arises from the curvature of the parabolic velocity
profile. As shown in Fig. 5.2b, the velocity magnitudes on each side of the particle
are different, leading to a pressure discrepancy between the top and bottom side of
the particle. Due to the existence of the pressure difference, a shear gradient lift force
is exerted on the particle which pushes it towards the channel wall (until it is
balanced by the wall induced lift force).
5.2.2.3 Net Lift Force (FL)
Following the discovery by Segre and Silberberg, matched asymptotic expansion

methods were proposed to determine the lateral lift forces acting on particles during
flow. Asmolov derived an analytical expression of the net lift force imparting on a
rigid particle (a/Dh 1) in a Poiseuille flow [41]:
ρU m 2 a4 μ2
FL ¼ f ðRc ; xÞ ¼ Rp 2 f ðRc ; xÞ
Dh 2 ρ
where f(Rc, x) is the lift coefficient which depends on the particle position within the
channel (x) and the channel Reynolds number (Rc).
Conventional theoretical predications are based on “point-particle” approximation
which neglects the size effect of particles. However, when the particle size approaches
the channel dimension, the disturbance to the flow will be affected by the particles. Di
Carlo et al. experimentally demonstrated that the net lift force varies with the position
in the microchannel [32]. For a particle of finite-size (0.05 a/Dh 0.2), the lift force
ρU m 2 a6
scaling relationship is modified as: F WL ¼ f WL ðRc ; xÞ near the channel wall,
Dh 4
ρU m a
2 3
and as F SL ¼ f SL ðRc ; xÞ near the channel center. The variation of lift forms is
Dh
attributed to the disparate fluid dynamics in different positions of the channel. Near the
center of the channel, the shear gradient lift force dominates, while the wall induced lift
force is more significant near the channel wall.
5.2.2.4 Secondary-Flow Drag Force
Besides the wall induced lift force (FWL) and the shear gradient induced lift force
(FSL), the secondary-flow drag force is the third major force responsible for particle
inertial migration and focusing effects. In 1928, William Dean reported the presence
of Dean vortices in curved channels due to the mismatch of fluid momentum within
the channel cross section as a result of the centrifugal acceleration acting on the fluid
flow [42]. Briefly, when fluid flows through a curved channel, the fluid velocity at
Fig. 5.3 Schematic of a pair of Dean vortices within a spiral channel with a rectangular cross
section
the center of the channel is higher than the sides due to the parabolic flow profile.
This introduces an additional momentum to the faster-moving fluid in the channel
center, and is pushed toward the outer wall (the concave wall) of the channel
curvature along the channel midline due to centrifugal acceleration. By conservation
of mass, secondary counter-rotating flows are induced to compensate the fluid
shifting. Hence, two symmetrical counter-rotating vortices are formed at the top
and the bottom of the cross-sectional plane (Fig. 5.3). Hereafter, the terms Dean flow
and secondary flow are used interchangeably [35].
The strength of the secondary flow can be characterized by a non-dimensional
Dean number (De):
rffiffiffiffiffiffi
Dh
De ¼ Rc
2R
where Rc is the channel Reynolds number defined as Rc ¼ ρUmDh/μ, here Um is the

maximum channel velocity, μ and ρ is the dynamic viscosity and density of the fluid,
respectively. The curvature ratio (δ ¼ Dh/2R, where R is the average radius of
curvature of the channel) implies a faster turn in the channel (i.e. smaller radius of
curvature R) and results in a stronger Dean flow.
For a given De, the expression of average transverse Dean velocity can be
determined by [43]:
U De ¼ 1:84 104 De1:63 ðm=sÞ
Accordingly, the Dean drag force experienced by a particle located in this flow can
be derived by assuming Stokes drag (Fig. 5.2c) and is expressed as:
F D ¼ 3πμU De a ¼ 5:4 104 πμDe1:63 a ðNÞ
In spiral or curvilinear channels, the interplay of the net inertial lift force (FL) and
Dean drag force (FD) gives rise to the Dean coupled inertial migration of particles.
To characterize particle inertial focusing in curved channel, Di Carlo et al. proposed
108 N. Liu et al.
a key parameter Rf, the ratio of shear gradient lift force to Dean drag force, to
describe the behavior [35, 44]:
F SL 1 a2
Rf ¼ /
FD δ D3h
It is well accepted that Rf > 0.04 (or a/ Dh > 0.07 [23]) to achieve particle inertial
focusing in microchannels. As Rf has a strong dependence on particle size (~a2), this
forms the basis for size-based particle separation in spiral devices since the particle
equilibrium separation can be modulated by tuning these forces. This is explained in
more details in the following section.
5.2.2.5 Dynamics of Particle Lateral Migration in Spiral Microchannel
Figure 5.4 depicts the schematic illustration of particle focusing dynamics in a

low-aspect ratio spiral microchannel. Due to the asymmetrical parabolic flow profile,
Fig. 5.4 Schematic illustration of particle migration dynamics in spiral microchannels

a steep shear gradient is generated along the vertical direction (height) of the
channel. The randomly distributed particles (1) are first pushed by dominant shear
gradient lift force (FSL) across streamlines towards the channel top and bottom
surfaces. Near the wall, the particles experience opposing wall-induced lift force
(FWL) and equilibrate into two broad bands at ~20% of channel height from surface
(2). Next, a rotational-induced lift force (FΩ), first proposed by Saffman [45], will act
on the particles and they begin to migrate laterally towards the centre face of the
channel wall to form two focused streaks (3). This lift force FΩ is usually considered
negligible compared with other lift forces (an order of magnitude lower than FWL and
FSL), but becomes important in radially-asymmetric (e.g. rectangular) channels as
particles exhibit spinning behavior in the presence of high (localized) shear rate close
to the channel wall [46]. Other groups have reported the particle size dependency of
FΩ (FΩ ~ a3) [47], and this effect is also recently used by Zhou et al. for particle
separation in straight channels [48, 49]. With increasing flow velocity, the strength
of the secondary Dean drag force becomes more significant and particles begin to
migrate towards the inner wall region in the direction of the Dean vortices (4).
Finally, a single focusing point is achieved near the inner wall region in each Dean
vortice as a result of the balance of net inertial lift force (FL) and Dean drag force
(FD) (5). As this equilibrium position is strongly dependent on particle size (Rf~ a2),
larger particles (a/h > 0.07, as Dh h in a low aspect ratio channel[50]) will focus
closer to the inner wall due to stronger inertial lift (FL ~ a3 vs. FD ~a) while smaller
particles are positioned further away from inner wall [23] (6). For all particle sizes, a
further increase in flow velocity will shift the focused particle streams back towards
the outer wall due to increasing FD [51]. Although an increase in flow velocity results
in a greater lift force (FL eU m 2 ) as compared to the Dean drag (FD eU m 1:63 ), the
particle movement away from the inner wall can be explained by a decrease in the lift
coefficient f(Rc, x) which is dependent on particle position within the channel [23].
5.3 Classification of Spiral Devices
Fluid mixing at the microscale poses a variety of challenges due to the dominant
viscous drag forces (low Re), and molecular diffusion remains the main transport
mechanism in this laminar flow regime. By taking advantage of the transverse Dean
vortices in curvilinear channels, spiral microdevices have been used as micromixer
to enhance fluid mixing [52, 53], and rotate cells in electroporation systems for
efficient gene delivery [54]. Since flow conditions and channel geometries can affect
the shape and magnitude of Dean vortices, these parameters have been extensively
investigated in spiral inertial microfluidics to enhance cell sorting capabilities. In this
section, we will describe four major types of spiral devices with different cells/
particles focusing mechanisms and features: (1) rectangular spiral microfluidics,
(2) trapezoidal spiral microfluidics, (3) double-inlet spiral termed as Dean Flow
Fractionation (DFF), and (4) High-resolution Dean Flow Fractionation (HiDFF).
110 N. Liu et al.
5.3.1 Rectangular Spiral Microfluidics
Spiral microchannels with rectangular cross-section are one of the most widely used
geometry, due to the well-established microfabrication techniques (photolithography
and deep reactive ion etching (DRIE)) which can generate uniform channel/feature
height and high aspect ratio vertical sidewall. The small feature size resolution
(~1–10 μm) in microfabrication also enables the design of complicated outlet
bifurcations (8–15 outlets) for multiplexed separation. In low-aspect ratio rectangu-
lar spiral channels, the regular cross-section geometry will lead to the formation of
symmetrical Dean vortices along the channel midline. Inertial forces are weaker
across the longer dimension (due to the blunting of the velocity profile) and particles
will first migrate along the shorter channel dimensions (channel height) to the top
and bottom surfaces due to higher shear rate [50]. Once particles have reached the
z-direction equilibrium positions, they will then migrate along the channel width to
the final equilibrium position near the inner wall region. Martel and Toner have
performed a systematic characterization of inertial focusing dynamics in spiral
microchannels of varying widths (Fig. 5.5a). Generally, as channel dimensions
(Dh) increase, particles experience less shear-induced inertial forces (blunting of
velocity profile) and more Dean drag force (~Dh1.5). They proposed slight modifi-
cations to Rf (ratio of FL/FD) by using UDean, ave in the equation to reduce variability
of Rf to ~1 for quality focusing in different spiral designs [55]. A straightened
composite image was also generated from empirically-determined particle focusing
images to more clearly visualize the particle focusing streak width and position along
the entire channel length (Fig. 5.5b).
As described previously, the interplay between inertial lift (FL) and Dean drag
forces (FD) is important to focus particles of different sizes at distinct equilibrium
positions. Early work by Kuntaegowdanahalli et al. [23] and Russom et al. [56]
clearly demonstrated the capability of spiral microchannels for continuous high
throughput size-based particle separation into different outlets. As shown on
Fig. 5.6a, large particles (a/h > 0.07) focused inertially close to the channel inner
wall due to dominant FL. At high flow conditions (De~10–15, ~3 mL/min), signif-
icant Dean drag force would move these focused streams farther away from the
channel inner wall based on particle size, with the largest particles being closest to
the inner channel wall. This phenomenon was exploited to separate closely-spaced
microparticles (10 μm, 15 μm, 20 μm) in a 500 μm wide Archimedean spiral device
(130 μm height) [23]. Xiang et al. also developed a smaller spiral device (160 μm
width, 50 μm height) for binary separation of 4.8 μm and 2.1 μm particles Fig. 5.6b
[57]. In addition, they also described the non-focusing behavior of smaller 2.1 μm
particles (a/Dh0.07) and the resultant particle-free regions at low flow conditions
(De~1–5). Besides particle sorting and filtration applications, inertially-focused
particle stream in rectangular spiral channels is used for cell self-ordering for
deterministic single-cell droplet encapsulation [58]. Noteworthy, the above men-
tioned Dean-coupled particle inertial focusing effects can also be applied in other
curvilinear channels with symmetrical cross section geometry including double
Fig. 5.5 Rectangular spiral microfluidics (a) Schematic illustrations of different flow profiles and
associated forces in curved channels of different widths, (b) Straightened image of the inertial
focusing behavior of 15 μm beads in a spiral channel. Dotted lines represent channel curvature
changes. (Reproduced with permission from Ref. [55])
spiral [59–61], serpentine [30], as well as soft microtubes (circular cross section)
coiled in planar or 3D spiral (helical) [62].
Recently, Nivedita et al. performed experimental and numerical simulation stud-
ies on fluid flow dynamics in spiral microchannels under high flow conditions. They
reported the presence of multiple pairs of secondary flow vortices at high Re (>100)
and De (~20–40), and defined a non-dimensional parameter termed as critical Dean
number (Dec) to describe this novel flow observations [63]. According to them, the
formation of additional Dean vortices was due to the large pressure gradient between
the high velocity area and the channel outer wall. Above Dec, the primary Dean
vortices were unable to maintain the pressure across the channel width. In order to
balance the pressure, the primary vortices would thus split to recirculate the fluid
near the outer wall region. This led to the formation of secondary Dean vortices
which can entrain particles or cells at higher flow rates (Fig. 5.6c).
112 N. Liu et al.
Fig. 5.6 Rectangular spiral microfluidics (a) Schematic illustration and fluorescence image indi-
cating multiplexed separation of 10 μm, 15 μm, 20 μm microparticles into different outlets in a
500 μm wide and 130 μm tall Archimedean spiral device. (Reproduced from Ref. [23] with
permission from The Royal Society of Chemistry) (b) CAD design and optical image of a 5-loop
spiral device (filled with red dye) fabricated in polydimethylsiloxane (PDMS). Fluorescent images
indicating tight focusing of 4.8 μm particles near the inner wall and the wider 2.1 μm particles band
at the channel centre. (Reproduced with permission from Ref. [57].) c Schematic and fluorescent
images of 10 μm particles (blue) and RBCs (red) entrapment in additional Dean vortices at high De
(~37). (Reproduced from Ref. [63] under Creative Commons)
5.3.2 Trapezoidal Spiral Microfluidics
Intuitively, one can modulate the Dean vortices to increase the separation distance
between particles of different sizes and enhance the separation resolution. Trapezoi-
dal cross section is an interesting design as the change in channel height from low
(inner wall) to high (outer wall) results in a Dean flow velocity gradient across the
channel width. Guan et al. first reported this behavior, and found that the skewed
Dean vortices were beneficial for particles separation as the gradient in FD across the
channel would led to a sharp transition of size-based focusing behavior beyond a
certain threshold flow rate [64]. Unlike in rectangular spiral channels where focused
particles streams gradually migrate towards the outer wall with increasing flow rates,
Fig. 5.7 Comparison of rectangular and trapezoidal spiral microchannels. CFD simulation and
experimental results (top and side view) of (a) 15.5 μm particles focusing behavior in rectangular
spiral microchannel (600 μm width, 80 μm height) and (b) 26.25 μm particles focusing behavior in
trapezoidal spiral microchannels (600 μm width, 80–140 μm height). (Reproduced with permission
from Ref. [64])
focused particles in trapezoidal spiral channels would “switch” to an equilibrium

position located at the outer half (deeper side) of the channel as flow rate increases
(Fig. 5.7). Side view analysis also revealed that these particles were trapped within
the Dean vortices at the outer wall region [64].
114 N. Liu et al.
Fig. 5.8 Binary particle separation in trapezoidal spiral microchannels (a) Schematic illustration of
particle focusing and trapping within the skewed Dean vortices in trapezoidal cross-section spiral
microchannel (b) High speed image at the outlet bifurcation showing separation of 18.68 μm and
26.9 μm particles (channel dimensions: 600 μm width, 80–140 μm height) (c) Flow rate character-
ization of size-based “threshold” flow rate to switch particles focusing to outer wall focusing.
(Reproduced with permission from Ref. [64])
As the threshold flow rate is a function of particle size, this novel focusing
behavior was used to achieve efficient binary separation (~92–96%) of particle
mixtures in a 2-outlet trapezoidal spiral channel (Fig. 5.8). This is ideal as one can
process samples at high throughput (~3–4 mL/min) and higher particle concentra-
tions (~107/mL) by minimizing the interactions between particles of different sizes.
This method was applied for separation of leukocytes (~10–15 μm) [65] and
circulating tumor cells (CTCs) (~15–20 μm) [66] from blood samples (RBCs
~6–8 μm), as well as macroscale filtration (~500 mL/min) of Chinese hamster
ovary (CHO) and yeast cells in bioprocessing [67]. However, a major limitation of
trapezoidal spiral microchannels is the use of micromilling [64] or 3D printing [68]
techniques to fabricate the channel molds with uneven channel heights. Due to the
relatively poor feature size resolution as compared to conventional microfabrication,
the devices are limited to a 2-outlet bifurcation and thus only used for binary
separation.
5.3.3 Double-Inlet Spiral/ Dean Flow Fractionation (DFF)
Rectangular and trapezoidal spiral microchannels are highly effective for size-based
particle and cell separation with working sample concentrations of ~105–7/mL, but
their use in blood-related applications is greatly limited by the large RBCs back-
ground (~45% v/v, ~109 RBCs/mL) as RBCs-RBCs interactions can severely affect
the cell focusing behavior and hence deteriorate separation efficiency [69]. Typically,
whole blood samples have to be diluted significantly (50–100 , ~0.1–2% hemat-
ocrit) which increases the processing time, making them unsuitable to process large
volumes of blood samples required in clinical settings.
In a landmark work by Bhagat et al.[31], they developed a 2-inlet spiral channel
to selectively introduce particle samples on the inner side of the channel with an
additional sheath flow. As the particles flow through the spiral device (100 50 μm
(w h)), the Dean vortices would transpose the smaller particles (a/Dh < 0.07)
towards the outer wall, and both inertial lift and Dean drag forces would equilibrate
the larger particles (a/Dh > 0.07) near the inner wall. This was used for complete
separation of 1.9 μm and 7.32 μm particles at De ¼ 0.47 (Fig. 5.9a). It should be
noted that unlike in 1-inlet rectangular spiral channels where all particles are
inertially focused near the inner wall and their final equilibrium positions differ
slightly based on size differences, the 1.9 μm particles did not undergo inertial
focusing effects in the dual-inlet spiral device and solely migrated to the outer wall
under the influence of Dean vortices. This was the primary reason for the signifi-
cantly wider 1.9 μm particles focusing band as compared to 7.32 μm particles
(Fig. 5.9a).
Inspired by this work, Hou et al. subsequently developed a 2-inlet 2-outlet spiral
device (500 μm (w) 160 μm (h)) for isolation of CTCs from whole blood, aptly
termed as Dean Flow Fractionation (DFF) [70] (Fig. 5.9b). Based on the size
difference between CTCs and blood cells [71, 72], the developed technique enables
inertial focusing of larger CTCs (15–20 μm) near the inner wall while smaller blood
components (RBC ~8 μm discoid; leukocytes ~7–12 μm) are solely affected by the
Dean drag and transposed towards the outer wall, thus achieving separation. The
authors further define this Dean-induced lateral migration in terms of ‘Dean cycle’
(DC) which can be modeled by COMSOL simulation (Fig. 5.9b). For instance, a
particle which is initially positioned near the microchannel outer wall and migrates
to the inner wall is said to have completed ½ Dean cycle (DC 0.5), and returning
back to the original position near the channel outer wall completes a full Dean cycle
(DC 1). The length for a complete Dean cycle migration (LDC) can be approximated
as LDC ~ 2w + h (where w is the microchannel width and h is the channel height). For
a given microchannel length, the particles can thus undergo multiple DC migration
with increasing flow rate conditions (Fig. 5.9b).
Notably, this separation principle is particularly useful for blood cell separation as
the additional sheath buffer in DFF device facilitates the Dean migration of large
volume of RBCs in a well-controlled manner. The authors reported that RBCs band
broadened with increasing hematocrit due to cell-cell interaction induced dispersion
116 N. Liu et al.
Fig. 5.9 Dean Flow Fractionation (DFF) using a 2-inlet spiral design (a) (left) Schematic illustra-
tion of the Dean-coupled inertial focusing separation principle in the 2-inlet spiral device. (right)
Composite fluorescent images and intensity linescans illustrating complete separation of 1.9 μm
(purple) and 7.32 μm (green) particles at De ¼ 0.47. (Reproduced from Ref. [31] with permission
from The Royal Society of Chemistry) (b) (left) Optical image of the DFF spiral microchannel
(filled with blue dye for visualization) used for CTCs isolation from whole blood. Fluid simulation
and particle tracking (blue streamlines) in the DFF spiral device at Re 50 (DC 1) indicating
complete recirculation of the fluid elements at the outer wall region (inlet) and back to the outer
wall (outlet) again. (right) Average composite fluorescence images and plot indicating equilibrium
position of 6 μm and 15 μm beads at different DC. (Reproduced with permission from Ref. [70])
and a final hematocrit of 20% was chosen as it resulted in negligible RBCs

contamination in the inner CTCs outlet (Fig. 5.10a). Compared to other inertial-
based microfluidic separation methods, 20% hematocrit implies ~2 dilution of
whole blood (original hematocrit ~40–45%). This translates to an unprecedented
processing time of ~20 minutes for 1 mL of whole blood (at 100 μL/min). Interest-
ingly, inertial focusing positions of cancer cells (MCF-7) remained similar in saline
solution and 20% hematocrit blood samples, indicating that the lateral migration of
RBCs did not affect their inertial focusing (Fig. 5.10b) [70]. The same group later
Fig. 5.10 High blood sample concentration processing in DFF (a) Averaged composite images and
intensity plot illustrate broadening of RBCs occupied regions (red dashed line) for increasing
hematocrit prior outlet bifurcation. Yellow dotted lines indicate position of channel walls, (b) Plot
and high speed image captured at the channel outlet (red dotted box) indicate similar focusing
positions of MCF-7 cancer cells suspended in PBS solution and 20% hematocrit blood samples at
DC 1. Shaded area (150 μm wide) corresponds to the dimension of CTCs outlet. (Reproduced with
permission from Ref. [70])
applied the DFF technology for label-free isolation of rare bacteria from whole blood
by collecting the Dean-induced migrated bacteria at the channel outer wall [73].
Compared to other spiral technologies, DFF is clearly more versatile as it can
achieve both size-dependent differential inertial focusing and multiplexed sorting of
larger particles, as well as isolation of smaller micro or nanometer-sized elements
through well-controlled Dean migration (a feat not possible with rectangular or
trapezoidal spiral microchannels). Figure 5.11 illustrates the Dean migration profiles
of 50 nm particles along the channel in DFF devices when introduced at either the
inner or outer inlet. In both cases, the particles are solely affected by the Dean drag
and migrated in the directions of the Dean vortices. By carefully tuning the sample to
sheath flow rate ratio, DFF could thus serve as an efficient buffer exchange system to
deplete smaller biological components including biomolecules (aptamers) [74] and
nanoparticles [75] from target cells with high efficacy.
5.3.4 High-Resolution Dean Flow Fractionation (HiDFF)
In biomedical research, there exists a critical need to develop novel separation tools
for sub-micron components in particle-based drug delivery system and purification
of circulating microvesicles. While DFF enables well-controlled, Dean-induced
migration of small microparticles (biomolecules, bacteria etc.) from inertially-
focused larger target cells, a major drawback remains in its inability to further
size-fractionate smaller microparticles (e.g. 1 μm vs. 2 μm) as they recirculate
118 N. Liu et al.
Fig. 5.11 Characterization of Dean-induced lateral migration of nanoparticles in DFF spiral

devices. 50 nm fluorescent bead sample is introduced at the a inner wall or b outer wall at the
inlet region. Average fluorescent stacked images indicate 50 nm bead positions along the channel.
Yellow lines indicate positions of channel wall. Corresponding schematic images of channel cross
section illustrate differences in bead migration pattern depending on initial position of beads.
(Reproduced from Ref. [76] under Creative Commons)
continuously due to Dean vortices. Inertial focusing of small particles is highly

challenging due to the requirement for smaller channel geometries (a/h > 0.07;
Dh ~10 μm for 1 μm bead focusing), and the large channel resistance would
cause flow-induced channel deformation [77]. A spiral sorter was developed to
inertially focus 2.1 and 3.2 μm microparticles, but the operating flow rate was low
(10 μL/min) due to large pressure drop [78].
To address these limitations, the Hou group recently developed a novel 2-inlet,
2-outlet spiral sorting technology termed as High-resolution DFF (HiDFF) [76]. Con-
trary to current inertial microfluidics technologies, this was based on a novel
phenomenon in spiral inertial microfluidics where particle transient innermost dis-
tance (Dinner) varied with size during Dean vortices-induced migration (Fig. 5.12a).
Briefly, small microparticles (a/ Dh < 0.07) introduced at channel outer wall expe-
rience Dean drag forces (FD) due to Dean vortices and migrate laterally towards
inner wall. As they migrate along the channel top or bottom, particles near the
surface experience size-dependent wall-induced inertial lift forces (FWL / a6) that
push particles away from the surface. Hence, particles flow at different fluid stream-
lines which leads to a differential transient innermost position (Dinner) at the inner
wall before they recirculate back towards the outer wall (Fig. 5.12b). Of note, since
Fig. 5.12 High-resolution Dean Flow Fractionation (HiDFF) (a) Schematic illustration of HiDFF
separation principle in a 2-inlet, 2-oulet spiral (300 μm (w) 60 μm (h)) device. Particles
introduced at the outer wall migrate laterally towards inner wall under the influence of Dean
vortices. As the particles migrate along the channel top and bottom, they experience size-dependent
wall-induced lift forces (FWL) that push larger particles away from the surfaces, (b) Subtle
differences in particle z-position (along height) lead to size-based transient innermost distance
(Dinner) at the inner wall which can be exploited for small particle separation. (Reproduced from
Ref. [76] under Creative Commons)
Fig. 5.13 Superior separation resolution of HiDFF (a) Fluorescence composite images and inten-
sity linescans indicating distinct innermost distance (Dinner) for particles of different sizes (b) High
speed, Z-imaging (40 magnification) of inner wall region at different planes indicates similar
Dinner along channel height. (Reproduced with permission from Ref. [76])
inertial focusing of particles is not necessary, channel dimensions are increased to

minimize clogging while improving throughput significantly (~100 μL/min).
To characterize the dependence of Dinner with particle size, the authors tested
beads of smaller diameters (50 nm, 1 μm, 2 μm and 3 μm) so that they would not
undergo inertial focusing in the device (a/ Dh <0.07). As shown on Fig. 5.13a, 50 nm
beads migrated completely towards the inner wall while larger beads exhibited
increasing Dinner at Re ~ 30–50. For all for bead sizes, there were negligible
differences in their Dean migration towards outer wall at Re ~ 60–70. High speed
imaging at the inner wall region was also performed to further understand the distinct
differences in Dinner by visualizing the particle flow position at different planes along
120 N. Liu et al.
the channel height. Composite brightfield images clearly indicated increasing Dinner
with particle size (~5.2 μm for 1 μm beads; ~17.4 μm for 2 μm beads; ~27.8 μm for
3 μm beads), a trend that was similar at different channel heights (Fig. 5.13b).
To determine if the subtle differences in Dinner during Dean migration can be
exploited for separating particles with closely-spaced sizes, the authors characterized
binary bead mixtures (2 μm and 3 μm beads; 1 μm and 2 μm beads) separation
performance using HiDFF at different sample to sheath flow ratios. Smaller particles
(with smaller Dinner) were positioned closer to inner wall and separated into the inner
outlet (outlet 1), while larger particles were sorted into outer outlet (outlet 2).
Separation efficiency improved significantly from 20% to 60% for 2 μm bead
isolation (2 μm and 3 μm bead mixture), and from 5% to 40% for 1 μm bead
isolation (1 μm and 2 μm bead mixture) at higher sheath flow, which translated to
an enrichment of the smaller particles by ~1000 and ~100-fold, respectively
(Fig. 5.14a, b). This was likely due to the smaller variation in particle initial
y-position (along channel width) which enabled them to migrate laterally as a tight
band towards the inner wall. As proof-of-concept for particle-based drug delivery
applications, polydisperse poly(lactic-co-glycolic acid) (PLGA) microparticles were
fabricated and fractionated into 3 different size groups (large, medium and small) in
a 2-step HiDFF separation. Particle size and morphology were characterized using
scanning electron microscope (SEM), which confirmed the distinct size differences
in each group (unsorted, 3.3 0.08 μm; large, 6.8 0.13 μm; medium,
1.7 0.03 μm; small, 0.89 0.03 μm) (Fig. 5.14c).
5.3.5 Summary
So far, we have described four major types of spiral devices used for high throughput
size-based particle separation. Each of these technologies has unique separation
features in terms of particle size range, throughput and sample working concentra-
tion. These factors should be carefully considered when designing spiral devices for
specific cells/particles sorting applications. Table 5.3 below provides a summary and
comparison of key features in different spiral types.
5.4 Cell Applications
5.4.1 Cancer Cells
Cancer is the leading cause of death globally, and cancer metastasis (spreading from
the primary tumor to secondary sites) is responsible for ~90% of cancer-related
Fig. 5.14 Tunable size fractionation of microparticles using HiDFF. Average fluorescent compos-
ite images and separation performance of smaller particles at different sample to sheath flow ratio
for (a) 2 and 3 μm, and (b) 1 and 2 μm binary bead mixture. Yellow dotted lines indicate positions of
channel outlet bifurcation, (c) Size distribution plot of drug-loaded poly(lactic-co-glycolic acid)
(PLGA) microparticles after a 2-step HiDFF separation into 3 sizes (large (>5 μm), medium
(2–5 μm) and small (< 2 μm)). Inset SEM images highlight distinct size differences between sample
(inlet) and different size groups. (Reproduced with permission from Ref. [76])
122
Table 5.3 Comparison of different spiral devices

Type
Rectangular spiral microfluidics Trapezoid spiral microfluidics Dean flow fractionation (DFF) High resolution DFF (HiDFF)
Principle
Size
~5–30 μm
~5–50 μm ~1–30 μm ~1–3 μm
Mode Multiplexed sorting Binary sorting Multiplexed sorting Binary sorting
Flow rate ~0.5–3 mL/min ~0.5–6 mL/min ~0.1–0.2 mL/min (sample flow) ~0.5–0.2 mL/min (sample flow)
[Cell] ~105–6/mL (~0.1–0.5% ~106–7/mL (~0.5–2% ~106–8/mL (~10–20% ~106–8/mL (~10–20%
hematocrit) hematocrit) hematocrit) hematocrit)
Applications Cell separation Rare cell isolation Rare cell isolation Microvesicle isolation
Particle concentrator Microfiltration Bacterial sorting Nanoparticles separation
Cell alignment and ordering Plasma separation Protein purification Bacterial sorting
Buffer exchange
N. Liu et al.
fatalities [79]. During metastasis, cancer cells are shed from primary tumors into the
peripheral blood and are known as circulating tumor cells (CTCs). Recent clinical
studies have shown that CTCs frequency and their genetic information can be used
as surrogate biomarkers to provide critical information for cancer diagnostic and
monitoring [80, 81]. However, the technical challenge for CTCs isolation lies in the
rarity of these cells (~1 to 10 CTCs/mL) in peripheral blood (~5 billion RBCs/mL)
[82–85]. CELLSEARCH® is the only FDA approved CTC separation technology
that uses antibodies to bind to epithelial cell adhesion molecule (EpCAM) on CTCs
surfaces, but EpCAM expression is highly heterogeneous which can lead to consid-
erable capture loss.
Microfluidic CTCs separation was first reported by Nagrath et al., where they
captured CTCs from patient blood using anti-EpCAM antibody functionalized
microposts (CTC-chip) [86]. Following this, the same group developed a herring-
bone device to increase the collision and capture frequency between CTCs and
antibody-coated surfaces [87]. However, the same issue related to cell surface
marker heterogeneity persists in microfluidic affinity-based cell sorting. Size-based
separation is hence preferred as it can significantly reduce cell loss and preserve cell
viability with its label-free sorting process. In most cancer types, the size of CTCs
(~10–20 μm) is larger than blood cells (WBCs ~ 8–12 μm; RBCs ~ 8 μm; platelets ~
2–3 μm), and this physical difference can be exploited in spiral inertial microfluidics
for high throughout cell separation.
To date, many spiral microfluidic devices have been developed for cancer cells
separation (Table 5.4) [23, 51, 60, 66, 70, 88–96]. Using Dean-coupled inertial
migration, Kuntaegowdanahalli et al. first reported the use of spiral microfluidics for
size-dependent cancer cell sorting (Fig. 5.15a) [23]. As proof-of-concept, they
separated a mixture of neuroblastoma (~15 μm) and glioma cells (~8 μm), and
achieved >80% separation efficiency at a high throughput of ~one million cells/
min. These results were comparable to the performances obtained using commercial
flow cytometry. To further improve the throughput, Warkiani et al. developed a
multiplexed spiral device (three devices stacked together) to isolate spiked cancer
cell lines from lysed blood samples. This high-throughput system can process
7.5 mL of lysed blood sample in 12.5 min and downstream fluorescence in situ
hybridization (FISH) analysis was successfully performed on the eluted CTCs
off-chip [94].
Recently, Guan et al. reported a slanted spiral device with trapezoid cross-section
(80 and 130 μm in the inner and outer channel height, respectively) for cancer cell
separation [64–66]. Due to the asymmetry of the channel cross-section, strong Dean
vortices were generated at the outer half (deeper side) of the channel, which shifted
smaller particles closer to the outer wall without affecting focusing of larger particles
at the inner wall (Fig. 5.15b). They successfully isolated three cancer cell lines,
MCF-7 (20–24 μm), T24 (16–17 μm) and MDA-MB-231 (10–15 μm) from whole
blood with high recovery rate (>80%) and purity (400–600 WBCs/mL; ~4 log
depletion of WBCs) [66]. In another study, Aya-Bonilla et al. designed a slanted
spiral microfluidic device to isolate melanoma CTCs from lysed blood sample and
obtained 80% recovery rate after one round of enrichment [98]. Kulasinghe et al.
124 N. Liu et al.
Table 5.4 Applications of spiral inertial microfluidics for cancer cell separation
No. Sample Spiral type Separation performance References
1 SH-SY5Y and neuroblastoma Rectangular Throughput: ~2 mL/ [97]
cells spiral, single min
inlet
2 Neuroblastoma and glioma Rectangular Throughput: ~106 cells/ [23]
cells spiral, single min 90% recovery
inlet
3 MCF-7, MDA-MB-231, HeLa DFF Throughput: 100 μL/ [70]
in 3 diluted whole blood min >85% recovery
(15% hct)
4 MCF-7, T24 and MDA-MB- Trapezoidal Throughput: 1.7 mL/ [66]
231 spiral min 80% recovery
5 1205Lu, A2058, SKMEL5, Trapezoidal Throughput: 1.7 mL/ [98]
UACC62; Melanoma clinical spiral min >55% recovery
samples
6 CAL27, RPMI2650, Trapezoidal Throughput: 1.7 mL/ [95]
UD-SCC9, MDA-MB-486; spiral min
HNC clinical samples 60–76% recovery
7 MCF-7 in 100 diluted blood Rectangular Throughput: 400 μL/ [93]
spiral, single min 75.40% recovery
inlet
8 MCF-7 and HeLa Rectangular Throughput: [60]
double spiral, 3.33 107cells/min,
single inlet 88.5% recovery
9 HeLa in 20 diluted blood Rectangular Throughput: [88]
double spiral, 2.5 108 cells/min
single inlet ~80% recovery
10 A549 (lung adenocarcinoma) Rectangular Throughput: 25 mL/h. [92]
double spiral, 74.4% recovery
single inlet
11 DU-145 (prostrate) Rectangular Throughput: ~1 mL/ [96]
spiral, single min 67% recovery
inlet
12 Breast and lung cancer clinical Multiplexed Throughput: >1.5 mL/ [90]
sample (lysed blood) DFF min
13 Breast and lung cancer clinical Multiplexed Throughput: >0.75 mL/ [91]
sample (lysed blood) DFF min 20–135 CTCs/mL
recovery
14 MCF-7 in leukocytes Rectangular Throughput: 550 μL/ [51]
suspension spiral, single min >86.8% recovery
inlet
15 MCF-7 (breast) Rectangular Throughput: >1 mL/ [89]
spiral, single min ~100% recovery
inlet
also performed CTCs isolation using trapezoidal spiral microfluidics in head and
neck cancer (HNC) patients, and reported the presence of CTC clusters (large group
with more than five CTCs) in a subset of these cancer patients [95].
Fig. 5.15 Isolation of circulating tumour cells (CTCs) from whole blood using spiral inertial
microfluidics (a) Distinct focusing and separation of microparticles (~15–20 μm, similar size
range to cancer cells) into different outlets due to size-dependent inertial lift (FL) and Dean drag
(FD) forces. Experimental results indicating efficient separation of SY5Y neuroblastoma cells from
smaller C6 glioma cells. (Reproduced from Ref. [23] – published by The Royal Society of
Chemistry) (b) Schematic illustration of CTCs isolation from blood using trapezoid spiral
microchannels FISH analysis of HER2 expression in recovered CTCs after microfluidics isolation.
(Reproduced from Ref. [66] – published by The Royal Society of Chemistry, under Creative
Commons) c Schematic illustration of Dean Flow Fractionation (DFF) for CTCs isolation. Clinical
validation of DFF in lung cancer patients. Fluorescence images and enumeration of isolated CTCs.
Isolation of CTCs clusters using DFF. (Reproduced with permission from Ref. [70]) (d) Schematic
illustration of a 1-inlet, 3-outlet spiral microfluidic device for cancer cell sorting. Due to the
dominant FD with smaller radius of curvature, cancer cells (MCF-7) focused inertially at the channel
centre, while smaller RBCs equilibrated near the channel inner wall. (Reproduced from Ref. [93] –
published by The Royal Society of Chemistry, under Creative Commons)
To increase blood processing throughput, Hou et al. developed a novel 2-inlet,

2-outlet spiral biochip for CTCs isolation based on DFF. Unlike other inertial
focusing devices, the DFF device inertially focused the larger CTCs while the
smaller hematologic cells (RBCs and leukocytes) were solely affected by Dean
drag forces (Fig. 5.15c) [70]. This enabled accommodation of high RBCs content
in the channel, which translated to a significant enhancement of RBCs processing
(~20% sample hematocrit) and efficient cancer cell recovery of >85%.
Huang et al. also reported a simple 1-inlet, 3-outlet, 5-loop spiral cell sorter for
CTCs isolation (Fig. 5.15d) [93]. Due to the smaller radius of curvature and
dominant FD, the larger cancer cells were recovered from the middle outlet, and
the smaller blood cells were sorted into the inner outlet. To test the efficacy of the
device, breast cancer cells (MCF-7) were spiked into diluted whole blood sample
(1:100) and the device was able to remove ~99% of the hematologic cells after
2 rounds of separation at a throughput of 400 μL/min. The authors also reported that
the processing capability can be further increased by using lysed blood samples.
126 N. Liu et al.
Table 5.5 Applications of spiral inertial microfluidics for stem cell separation
No. Samples Spiral type Separation performance References
1 Human mesenchymal Rectangular spi- Throughput: 3 mL/min [104]
stem cells (hMSCs) ral, single inlet (~15 106 cells/h)
2 Neural stem cells (NSCs) Rectangular spi- Throughput: 1 mL/min [106]
ral, single inlet >80% recovery
3 Neural stem cells (NSCs) DFF Throughput: 3 mL/min [107]
~93% recovery
5.4.2 Stem Cells
Stem cells are pluripotent cells that can be induced into other cell types using
physical and biochemical cues. The capabilities of self-renewal and differentiation
into other specialized cells have made stem cells highly important in regenerative
medicine. Unsurprisingly, an unmet need for stem cell sorting is to identify novel
biomarkers to isolate subpopulations that are more pluripotent (“more stemness”).
Most of the existing techniques are based on cell surface expression and labeling, but
this strategy is rather challenging due to the lack of well-established surface markers
and cell heterogeneity.
Recently, the biophysical properties of stem cells such as cell size, stiffness and
electrical properties have emerged as potential biomarkers for stem cell sorting [99–
101] (Table 5.5). One of the key physical parameters is cell size, whereby the
inherent size differences can be exploited for cell cycle synchronization
[102]. This physical-based method has direct advantages over common chemical-
based synchronization which can affect and possibly disrupt cell physiology and
metabolism [103]. Lee et al. first proposed a spiral cell sorter for high-throughput
cell cycle synchronization based on cell size differences (Fig. 5.16a) [104]. To
satisfy the inertial focusing criteria (a/h > 0.07), the channel height was set between
130 to 150 μm depending on cell types. As proof-of-concept, they demonstrated the
fractionation of human bone marrow-derived mesenchymal stem cells (hMSCs) into
enriched subpopulations of G0/G1, S and G2/M phases. From outlet 1, 70.4% of the
cell population collected were in S and G2/M phases (~24 μm) while 86.2% of the
cells in outlet 4 were from G0/G1 phase (~15 μm). The device throughput
(~15 106 cells/h) and cell viability (~95%) were much higher than those obtained
by conventional methods. In a follow-up study, the group successfully identified a
set of unique biophysical markers (small cell diameter, low cell stiffness and high
nuclear membrane fluctuations) for the isolation of multipotent stem cells
(Fig. 5.16a) [105].
Study of neural stem cells (NSCs) is pivotal for understanding disease progres-
sion and developing novel therapeutics for neurological diseases including
Alzheimer’s and Parkinson’s diseases [108]. Neurospheres, which are clusters of
hundreds to thousands of NSCs, are commonly used for in vitro study of neural
precursor cells [109, 110]. To induce stem cell differentiation or conduct clonal
analysis in this culture system, the original neurospheres are chemically and
Fig. 5.16 Stem cell fractionation using spiral inertial microfluidics (a) Design of the 9-loop spiral
microchannel for cell cycle synchronization (Adapted from Ref. [104] with permission from The
Royal Society of Chemistry). Size-based sorting of mesenchymal stromal cells (MSCs) into
different subpopulations for characterization of nuclear fluctuations (NF). (Reproduced with per-
mission from Ref. [105]) (b) Overview of neural stem cells (NSCs) separation process from mice
brain using spiral microfluidic device. Representative images of single NSCs and NSC clusters after
separation at 1 mL/min (scale bar: 50 μm). (Reproduced from Ref. [106] with permission from The
Royal Society of Chemistry) c Schematic illustration of iPSC-derived NSCs enrichment using a
2-loop spiral cell sorter. NSCs were collected in the middle outlets, while non-NSCs (wide size
range) were collected in all outlets. Enrichment factor and recovery of NSCs at different flow rates.
(Reproduced with permission from Ref. [107])
mechanically dissociated to produce single-cell suspension and plated under strin-

gent conditions for growth. However, the dissociated single cells are often contam-
inated by a small population of stem cell clusters, which can affect subsequent cell
identification and clonal analysis. An effective and rapid separation method is
therefore highly desirable to separate these single cells from cell clusters. As neural
stem cells (~8–14 μm) are smaller than cell clusters (~40–60 μm), Nathamgari et al.
developed a 1-inlet, 2-outlet spiral microdevice for size-based isolation of single
cells from chemically dissociated neurospheres (Fig. 5.16b) [106]. They showed that
at low flow rates (e.g. 1 mL/min), large particles (e.g. 38 μm beads or cell clusters)
were focused at the center of the channel while small particles (e.g. 7.7 μm beads or
single cells) were focused near the inner wall. When the flow rate was increased to
3 mL/min, the beads focusing behavior were reversed. They eventually used a flow
rate of 1 mL/min and reported that ~84% of single cells were isolated into outlet
1 and 2 (innermost outlets), while cell clusters equilibrated in the channel centre
(sorted into outlet 3–5). In addition, as neural stem cells are sensitive to shear stress,
a low working flow rate can help preserve the multipotency of the stem cells with
high cell viability rate (>90%).
128 N. Liu et al.
With the emergence of induced pluripotent stem cells (iPSC) technology, label-
free cell purification methods are highly important for iPSC-derived cells enrich-
ment. Song et al. designed a 2-inlet, 8-outlet DFF spiral device to enrich iPSC-
derived NSCs from a heterogeneous cell mixture based on cell size differences
[107]. They mixed NSCs (10–12 μm) and heterogeneously-sized non-neural cells
(6–19 μm) in a ratio of 1:1 (final concentration of 2 106 cells/mL), and processed
the sample at a flow rate of 3 mL/min. As expected, the NSCs focused into a tight
band and were sorted into the middle outlets (outlet 4 and 5), while the non-neural
cells were remained randomly distributed across the channel width and the larger
cells were sorted into the inner wall outlets (outlet 7 and 8). They reported ~2.1 fold
NSCs enrichment with a 93% recovery rate (Fig. 5.16c). A major limitation lies in
the low purity, as the device is unable to deplete non-neural cells of similar sizes
as NSCs.
5.4.3 Immune Cells
Neutrophils are the most abundant leukocytes in human blood and the key effector
cells of the innate immunity. They are also implicated in major diseases including
type 2 diabetes mellitus (T2DM) [111], cancer [112] and cardiovascular diseases
[113]. In TD2M, numerous neutrophil dysfunctions such as cell stiffening
[114, 115], impaired chemotaxis [116, 117] and phagocytosis [118] can lead to an
increased susceptibility to bacterial infections. Traditionally, neutrophils are isolated
using laborious methods such as density gradient centrifugation and RBC lysis,
which not only require a large blood sample volume (>10 mL), but are also prone to
induce neutrophil activation if not properly done. Commercial neutrophil isolation
kits that utilize immunomagnetic labeling have been developed to negatively select
untouched neutrophils (MACS xpress® (Miltenyi Biotec) and Easy SepTM
(STEMCELL Technologies)), but these kits are expensive and not practical for
large volume processing. Developing an efficient and cost-effective neutrophil
sorting strategy is therefore necessary for accurate phenotyping in neutrophil studies
and point-of-care testing. Table 5.6 shows a summary of spiral microdevices devel-
oped for this purpose.
Table 5.6 Applications of spiral inertial microfluidics for immune cell separation
No. Samples Spiral type Separation performance References
1 Monocytes in Rectangular spiral, Throughput: 1.1 mL/min [96]
10 diluted blood single inlet Recovery: not reported
2 Neutrophils in lysed DFF Throughput: 130 μL/min [119]
blood >90% purity
3 Leukocytes in Trapezoidal spiral Throughput: 0.8 mL/min [65]
200 diluted blood >80% recovery
4 Leukocytes in Rectangular spiral, Throughput: 1.8 mL/min [120]
500 diluted blood single inlet ~95% recovery
Capitalizing on the high separation resolution of DFF, Hou et al. developed a

4-outlet DFF device to purify neutrophils from whole blood without antibodies
labelling. The separation is based on subtle cell size differences among leukocyte
subtypes (neutrophils/monocytes (10–12 μm); lymphocytes (7–8 μm)) (Fig. 5.17a)
[119]. This device only required small amount of blood (finger prick; ~100 μL) for
neutrophil isolation, and sorted neutrophils also undergo simultaneous washing as
they were eluted in fresh saline solution due to the buffer exchange capabilities of
DFF. The group further characterized the rolling behavior of sorted neutrophils on
E-selectin using microfluidics. In their clinical validation using healthy subjects and
patients with T2DM, this developed microfluidic-based neutrophil sorting and
phenotyping strategy revealed a significant difference in neutrophil rolling pattern
between both groups, clearly suggesting neutrophil rolling speed as a potential
functional biomarker for inflammatory profiling in T2DM patients.
In general, conventional blood cell separation methods (centrifugation, FACS,
MACS) are laborious and highly dependent on user operation, and the phenotype of
isolated leukocytes could be altered if not done carefully. To address these issues,
Wu et al. developed a spiral microfluidic device with a trapezoid cross-section to
isolate the larger leukocytes (~8–12 μm) from diluted whole blood (RBCs ~6–8 μm)
[65]. The schematic in Fig. 5.17b illustrates the device design and working principle.
At optimized working conditions, the device can separate polymorphonuclear leu-
kocytes (PMNs) and mononuclear leukocytes (MNLs) from diluted human blood
(1–2% hematocrit) with high efficiency (>80%). In addition, the activation in the
device-sorted PMNs was negligible as compared to lysis method. Nivedita et al. also
developed an Archimedean spiral device with <8 cm focusing length for leukocytes
separation in diluted blood sample (1:500) at a flow rate of 1.8 mL/min, and achieved
a high separation efficiency (~95%) and throughput (up to 1 106 cells/min)
(Fig. 5.17c) [120].
5.4.4 Sperm Cells
Assisted reproductive techniques (ART) have benefited countless couples experienc-

ing infertility, and one major aspect of ART is to select healthy spermatozoa in vitro
fertilization (IVF). Traditionally, sperm cells are prepared using serial centrifugation
or the swim-up methods, but repeated handling and centrifugation can cause damage
to the sperm cells’ DNA, or lead to the production of reactive oxygen species (ROS)
[121, 122]. Conventional sperm cell sorting methods using microfluidics relies on
sperm motility, but the approach is unable to identify viable but non-motile sperms
for intracytoplasmic sperm injection, which is relevant for patients suffering from
severe or complete asthenozoospermia [122]. Taking advantage of the size differ-
ence between sperm and blood cell, Son et al. developed a spiral microfluidics sperm
cell sorter for non-motile sperms separation. (Fig. 5.18a) [123]. Sperm cells (1–2
million/mL) were mixed with RBCs (7–9 million/mL) and introduced into the spiral
channel. At a flow rate of 0.52 mL/min, 81.2% of the sperm cells were sorted into the
130 N. Liu et al.
Fig. 5.17 Immune cells isolation using spiral inertial microfluidics (a) Rapid size-based neutrophil
sorting and washing using DFF. High speed images indicating distinct focusing of larger neutro-
phils and smaller lymphocytes into different outlets. (Adapted from Ref. [119] under Creative
Commons), (b) Immune cell isolation using trapezoid spiral device. Intensity line scans indicating
distribution of polymorphonuclear leukocytes (PML), mononuclear leukocytes (MNL), and RBCs
across channel width (Reprinted (adapted) with permission from Ref. [65]. Copyright (2014)
American Chemical Society.) (c) Images of the 1-inlet, 4-outlet Archimedean spiral device
(<1 in.2). Focused streams of three particle populations (10 μm, 15 μm and 20 μm in diameter) at
a flow rate of 2.2 mL/min. Images of focused RBCs at the outmost loop of spiral channel with
10 and 200-fold diluted whole blood. (Reproduced with permission from Ref. [120])
outer two outlets, while 99% RBCs were separated into the inner two outlets. As
shown in Fig. 5.18b, the smaller sperm cells formed a broad focusing band near the
channel centre, which can be attributed to their asymmetrical and irregular shape
Fig. 5.18 Sperm cell isolation using spiral inertial microfluidics (a) Separation principle of sperm
cells from RBCs using spiral microchannels (b) Fluorescent images of the focusing positions of
sperm cells (blue) and RBCs (red) at a flow rate of 0.52 mL/min. (Adapted with permission from
Ref. [123])
Table 5.7 Applications of spiral inertial microfluidics for microbes and biomolecules separation
No. Samples/application Spiral type Separation performance References
1 Non-motile sperm cell Rectangular Throughput: 0.52 mL/min [123]
spiral, single 81% recovery
inlet
2 E.coli, K. pneumoniae, DFF Throughput: 1.7 mL/min [73]
P. aeruginosa, S. aureus, ~75% recovery
E. faecalis
3 Algal cells Rectangular Throughput: 3.2 mL/min 77% [125]
spiral, single recovery
inlet
4 Phytophthora ramorum Rectangular Throughput: 2 mL /min 95% [126]
sporangia spiral, single recovery
inlet
5 Antibodies in serum DFF Throughput: 130 μL /min [130]
>80% recovery
6 Aptamer DFF Throughput: 160 μL /min [74]
(~2 106 cells/min) 106
partitioning efficiency
(length of 4.79 0.26 μm and width of 2.82 0.23 μm). In contrast, larger RBCs
(~7.5–8.7 μm) inertially focused into a tight stream at the inner wall, thus achieving
separation (Table 5.6).
5.4.5 Microbes
Microorganism separation using spiral microfluidics (Table 5.7) is an area of

considerable interest for bacterial diagnostics and environmental monitoring
[124]. A major difference between microorganism and cell isolation is that microbes
are smaller (~1–3 μm) as compared to mammalian cells (~10–20 μm), and thus will
132 N. Liu et al.
Fig. 5.19 Microbe isolation using spiral inertial microfluidics (a) Bacterial isolation using DFF.
Plots indicating high bacterial recovery rate (>65%) at different bacterial loads and for low
abundance bacteria (~10–50 CFU/mL). (Reproduced from Ref. [73] – published by The Royal
Society of Chemistry.) (b) Experimental setup for bacteria separation using spiral microchannel.
Images of different algae species (Chlorella, Monoraphidium and Cyanothece) and their focusing
position across the channel width at 1.6 mL/min. (Reproduced from Ref. [125] under Creative
Commons) (c) Photograph of a multi-layered microdevice consisting of pneumatic microvalves,
spiral microchannel and fluid control channels. Significant enrichment of P. ramorum sporangia
from infested Rhododendron leaves using the developed spiral sorter (Adapted with permission
from Ref. [126]) (d) Spiral microfluidic channel for pathogens separation. Distribution of
C. parvum as a function of distance to the outer wall. (Reproduced from Ref. [129] under Creative
Commons)
experience less inertial (or drag) forces. Hence, channel dimensions have to be
scaled down significantly to achieve similar inertial focusing effects.
To overcome this issue, Hou et al. utilized the DFF technique to isolate low
abundance bacteria from whole blood based on cell size difference (Fig. 5.19a)
[73]. By using a sheath flow to “pinch” the bacteria-containing blood sample at the
inlet, they demonstrated well-controlled Dean migration of bacteria towards the
outer wall while larger blood cells remained inertially focused near the inner wall
to achieve separation. This approach enables continuous, species-independent
isolation of clinical bacteria isolates spiked at low concentrations (~10–50 CFU/mL)

from whole blood without affinity-based target labelling.
Besides bacteria, algae are also widely studied as they are often used as bio-
sensors for monitoring and detecting environmental changes. Traditional manual
algae identification method includes microscopy-based manual identification which
is time-consuming and limits the sampling resolution. To automate the identification
process, Schaap et al. developed a spiral inertial microfluidic device for algal cells
separation based on cell size and shape [125]. Three morphologically different
species of algae were used in the experiment: (i) Chlorella (spherical shaped,
diameter of 6.0 1.0 μm); (ii) Cyanothece (prolate spheroid shape,
15.6 2.3 μm in long axis and 11.1 1.0 μm in short axis); and (iii)
Monoraphildium (cylindrical shape, 54.6 14 μm in length, diameter of
3.14 0.6 μm). The authors found that the shape of the algae can affect inertial
focusing behavior in spiral channels. Even though Cyanothece and Monoraphidium
possess equivalent spherical diameter, they can be separated based on their geomet-
rical difference and a separation efficiency of 77% was achieved at a flow rate of
3.2 mL/min. The prolate spherical Cyanothece behaved similar to a 10 μm particle,
while Monoraphidium has an effective diameter of 3.14 μm in the plane perpendic-
ular to the flow, and thus experienced less lift forces as compared to Cyanothece.
Since Chlorella did not fulfil the inertial focusing criteria, they remained randomly
distributed spread across the channel. The experimental setup, algae images and
distribution across the channel cross section are shown in Fig. 5.19b.
Phytophthora ramorum is a fungal plant pathogen that infects a large number of
plant species and results in extensive damage to ecosystem. Hence, it is imperative to
detect and prevent the spread of this fungus. The P. ramorum generally presents an
ovoid shape with diameter ranging from 20 to 40 μm. To achieve efficient inertial
focusing effects, Clime et al. developed a spiral microfluidic platform with a channel
depth of 200 μm and width of 600 μm. The device was integrated with peristaltic
microvalves for fluid operation and process control. Using samples derived from
infested plant leaves, they were able to obtain 6.1-fold concentration of the fungi
(Fig. 5.19c) [126].
Another crucial environmental application is the detection of waterborne patho-
gens in drinking water. It is a challenging task as pathogens are usually present in
low numbers. For example, the presence of Cryptosporidium oocysts was reported
less than 10 per 10 liters in the recreational lakes in Amsterdam, The Netherlands
[127]. The standard waterborne pathogen monitoring process involves complex
procedures including filtration, immune-magnetic separation, fluorescence staining
and microscopy-based examination. These methods require long processing time
(several days), expensive equipment and highly trained expertise [128]. In a work by
Jimenez et al., they presented a 6-loop spiral focusing microchannel with depth of
30 μm and width of 170 μm to separate waterborne pathogens (Fig. 5.19d)
[129]. The device has two wide outlets to enhance particle positions discrimination
and separation resolution. To demonstrate the feasibility of this device, they sepa-
rated waterborne pathogen Cryptosporidium parvum, (~4–5 μm) at a high flow rate
of 500 μL/min with a separation efficiency of 100%.
134 N. Liu et al.
Fig. 5.20 Biomolecule separation using spiral inertial microfluidics (a) Workflow of multiplexed
proteins or cells sorting using DFF coupled with affinity-based bead binding. Results indicating
efficient separation of 3 major HIV antigen-specific antibodies, as well as the enrichment of bead-
bound CD3+ lymphocytes. (Reproduced from Ref. [130] under Creative Commons) (b) Schematic
illustration of the inertial microfluidic SELEX (iSELEX) for aptamer selection. Cells with bound
aptamers are focused along the inner wall while the unbound aptamers migrate completely to the
outer wall. (Adapted from Ref. [74] under Creative Commons)
5.4.6 Biomolecules
Purification of proteins or other biomolecules from complex background is essential

in many biomedical applications and molecular assays. Unlike mammalian cells or
bacteria, biomolecules are significantly smaller (nanometer scale) and hence chal-
lenging to establish inertial focusing effects. A possible strategy is to bind target
biomolecules to functionalized microparticles to “artificially enhance” their sizes
(Table 5.6). This unique method is presented in a work by Sarkar et al., where they
reported a multiplexed affinity-based protein separation platform using DFF
[130]. The working principle is shown in Fig. 5.20a. As proof-of-concept for HIV
diagnostics, HIV antigens p24, gp41 and gp120, were coated on beads of three
different sizes (10 μm, 4.5 μm and 1 μm) respectively to serve as capture agents. The
coated beads were then incubated with serum obtained from HIV-infected patients
and introduced into the DFF device. Based on inertial focusing effects, beads were
sorted into different outlets based on their size. The captured antibodies were then
eluted from the beads for analysis. Compared to traditional antibody purification
methods, this high throughput sample processing platform (104–107 beads/s,
milligram-of proteins) provided a ~ten-fold time reduction while enabling
multiplexed protein purification.
Aptamers are short nucleic acid or peptide molecules that can selectively recog-
nize distinct epitopes [131]. SELEX (systematic evolution of ligands by exponential
enrichment) is a combinatorial chemistry technique used for selecting binding
aptamers from a large random sequence pool in vitro, but this process is often
iterative and time-consuming [132, 133]. To increase the selection efficacy, Birch
et al. developed a novel microfluidic aptamer selection strategy based on the SELEX
principle and DFF, termed as I-SELEX (Fig. 5.20b) [74]. Briefly, the pre-incubated
cells and aptamer library mixtures were introduced into the spiral device. Due to
distinct size differences between RBCs and nucleic acid molecules, unbound
aptamers migrate along the Dean vortices towards the outer wall while the larger
cells focus inertially near the inner wall. By using a wider spiral channel, the large
separation distance between the unbound aptamers and aptamers-binding cells
resulted in a high partition efficiency of ~106, which is comparable to the traditional
“gold standard” capillary electrophoresis-based methods [134]. Besides high
throughput processing (~2 106 cells/min), the developed technology was also
used to identify novel high-affinity binding aptamer targets for malaria-
infected RBCs.
5.5 Recent Advances

5.5.1 Novel Spiral Designs and Microstructures
Inspired by trapezoidal spiral devices, one can modulate the Dean vortices to
manipulate particle focusing behavior by varying the channel geometries. For
example, Sonmez et al. utilized an asymmetric serpentine channel design in a spiral
device [135]. Due to the superposition of two different secondary flows induced by
serpentine and spiral geometries, they demonstrated an increase in focusing quality
of 9.9 μm beads as compared to conventional spiral and serpentine channels. The
device can achieve ~99.5% purity of sorted 9.9 μm beads at flow rate of 2.5 mL/min.
In recent studies, circular channels are used in spiral devices without relying on
conventional microfabrication techniques [62, 136]. This can be achieved by using
Tygon® tubing [136] or circular cross section PDMS tube [62] wrapped around
3D-printed barrel to form curvilinear or helical structures (Fig. 5.21a). A key
advantage of this strategy is that it is cheap and highly customizable as one can
easily change barrel size and tubing diameter to modify the radius of curvature and
channel dimension, respectively. Another attractive feature is that the tubings can be
connected in a “plug-and-play” mode to other PDMS based microfluidic devices,
136 N. Liu et al.
Fig. 5.21 Novel spiral designs and microstructures: (a) (left) Various designs of spiral inertial
microfluidics created using circular PDMS microtubes. (right) Characterization of particle focusing
efficiencies in different device designs. (Reproduced with permission from Ref. [62]) (b) (left)
Spiral channel with micropillar array for plasma extraction. (center) Schematic illustration of
separation principle. (right) Microscopic image showing efficient plasma extraction and RBCs
retention. (Reproduced with permission from Ref. [137])
thus enabling in-line integration with different modalities. Hahn et al. utilized
Tygon® tubing (inner diameter 190 μm, outer diameter 2 mm) in a helical spiral
device and demonstrated separation of 15 and 25 μm particles into different outlets
with recovery rates of 50.9 5.3% and 99.5 0.9%, respectively [136]. Xi et al also
fabricated various designs of serpentine and spiral devices using circular PDMS tube
(100 μm for inner diameter), and achieved high focusing efficiencies (>75%) for
particle size ranging from 10–25 μm (Fig. 5.21a) [62]. For novel designs such as
helical spiral and self-twisted spiral, an interesting feature is that Dean number (De)
and radius of curvature remain constant for each loop as opposed to the standard
planar Archimedean spiral devices [62]. The self-twisted spiral device also exhibits a
stronger Dean flow profile due to the smaller radius of curvature.
Several groups have also introduced patterned microstructures such as
micropillars or confined regions in their spiral channels [137, 138]. The presence
of these obstacles induces additional secondary flow which can be exploited to
manipulate particle position together with Dean flow to enhance separation effi-
ciency [34]. Geng et al. designed a micropillar array (1.7 μm gap) in spiral channel
for blood plasma extraction from diluted whole blood (Fig. 5.21b) [137]. Due to the
combinatorial effects of physical filtration in pillar array and lateral Dean flow
effects, “cell-free” plasma effectively filters through the micropillars towards the
channel outer wall while the blood cells are retained in inner wall. Another important
feature is the decreasing distance between pillars and the inner wall along the
Fig. 5.22 Novel spiral designs and microstructures 2: (a) (left) Schematic illustration of the spiral
device with stair-like cross-section. Cross-sectional view of the Dean vortices generated in the
device. (right) Particle size distribution from the inner outlet and outlets. (Reproduced with
permission from Ref. [138]) (b) (left) Spiral channel design with micro-obstacles. Velocity profiles
at the confined region in xy plane (right top) and zy plane (right bottom) using fluid simulation.
(Reproduced from Ref. [139] – published by The Royal Society of Chemistry)
microchannel to enhance the volume of extracted plasma. At an optimal flow rate of

10 μL/min, their device achieved 49.6% separation ratio (plasma volume to initial
sample volume) from 250 μL of 20 diluted blood. Ghadami et al. also developed a
novel spiral device with stair-like cross section in which the channel was divided into
3 sections namely inner, intermediate and outer wall [138] (Fig. 5.22a). Instead of
having two counter-rotating Dean vortices in conventional spiral devices, the Dean
vortices are positioned adjacent to each other with one located at the inner wall
section and another at the outer wall section. Similar to DFF, larger particles would
migrate towards the inner wall section while smaller particles remain in the outer
wall section. They reported that the device can achieve a large separation distance of
260 μm between 7 μm particles and 20 μm particles. Recently, Shen et al. investi-
gated various spiral designs with ordered narrow regions (Fig. 5.22b) [139]. As the
fluid flows into the narrow regions, the increasing fluid velocity provides additional
secondary flow acceleration which results in enhancement of particle focusing. To
demonstrate the separation performance of the developed devices, they successfully
sorted MCF-7 (97.5%) and HeLa (92.3%) cancer cells from diluted whole blood
138 N. Liu et al.
(~2.5% hct) at 6.5 mL/min. Furthermore, they also performed blood plasma extrac-
tion at a lower flow rate of 3 mL/min whereby the blood cells were focusing close to
the outer wall (removed through outer outlet) and plasma was extracted from other
outlets. They reported a high blood cells rejection efficiency (99.96%) and plasma
recovery (67.6%) from diluted blood (~2.5% hct) samples.
5.5.2 Integrating Multiplexing Spiral Devices
Unlike straight channel designs, a major limitation for spiral or curvilinear devices is
the difficulty for massive planar parallelization to achieve higher throughput. To
facilitate high volume processing, the spiral stacking strategy has been reported by
several groups [67, 91, 140–143]. Khoo et al. developed a multiplexed spiral
microfluidic device for label-free enrichment of CTCs at ultra-high throughput
[140]. The stacked device was fabricated by stacking three spiral channels vertically
with shared common inlets and outlets (Fig. 5.23a). To demonstrate the feasibility of
this device, the authors processed blood samples (7.5 mL) from 10 healthy donors
and 58 patients with metastatic breast or non-small cell lung cancer. The throughput
was significantly enhanced (20-fold higher) which translated to processing 7.5 mL in
less than 5 min with 100% detection sensitivity and high selectivity. Based on
similar concept, Warkiani et al. utilized a stack of 40 trapezoidal spiral devices for
CHO cell and yeast cells separation at 240 mL/min. [67]. Rafeie et al. later demon-
strated multiplexing of 16 trapezoidal spiral devices on one layer for blood plasma
separation from diluted blood [142]. They managed to achieve ~100% cell rejection
ratio for ~0.5%–1% hematocrit at an optimal flow rate of 1.5 mL/min for a single
device (24 mL/min for 16 devices). For stacking multiple devices, it should be noted
that the top surface of the PDMS devices needs to be flat to prevent leakages between
2 consecutive layers. Precise alignment of the inlet and outlet ports is necessary to
ensure that each device layer can receive equal flow distribution. In addition,
stacking of multiple devices can suffer from slight pressure variance and might
possibly affect individual device performance. To overcome these problems, several
groups have introduced custom design manifold to distribute fluid flow equally. For
example, Miller et al. developed a manifold to control pressure-driven flow in each
spiral device [141]. As shown in Fig. 5.23b, they successfully incorporated
20 devices which can provide a large separation size range (2–300 μm) at a
throughout of 1000 mL/min. Additionally, the system includes several unique
sample processing features such as cascaded channels to enhance separation effi-
ciency, and sample recirculation to increase purification and yield (Fig. 5.23c).
Fig. 5.23 Integrated multiplexing spiral devices: (a) Photo of a multiplexed spiral device by
stacking 3 spiral devices together for high throughput CTCs isolation. (Adapted from Ref. [140]
under Creative Commons) (b) Image of a stacked spiral system for large range particle separation
(c) Schematic illustration of cascaded sample processing using different spiral devices.
(Reproduced from Ref. [141] under Creative Commons)
5.5.3 Multiple Stage Spiral Device
Multiple stage spiral devices have been proposed to enhance sorting performance
[141, 144]. Robinson et al. developed a two-stage spiral device for RBC depletion
from diluted whole blood (2% hct). As a single spiral separation could not achieve
high WBCs purity, they added another stage of separation at the end of first spiral
device which has a bifurcation that equally splits into two smaller daughter spiral
devices; one for subsequent RBCs exclusion and another one for transporting sorted
RBCs to waste and balancing the fluidic resistances. They showed the device can
effectively eliminate RBCs from diluted whole blood with 30-fold increase in RBCs
depletion as compared to a single-stage spiral device [144].
140 N. Liu et al.
5.5.4 Closed-Loop Sample Processing
Besides multiple stage sorting, recirculation strategy is another attractive alternative

for processing of complex biofluid such as blood or high cell concentration samples
[141, 143, 145]. Briefly, sample recirculation is achieved by using peristaltic pump
to feed output eluent (containing target cells) back to the inlet sample reservoir so
that the suspension can pass through the device again. The recirculation strategy
provides several benefits. First, it continuously dilutes and removes the waste
materials that can otherwise affect the separation performance. Secondly, it will
enrich target cells into smaller volumes for subsequent processing without manual
handling. In most cell sorting applications, the target cells in the samples are usually
rare and do not contribute much to the sample concentration. Hence, the target cells
recirculation will not compromise the separation performance. Fig. 5.24a shows a
recirculation strategy developed by Ryu et al. [145]. In this work, they utilized spiral
inertial microfluidics with recirculation strategy to extract polymorphonuclear leu-
kocytes from patient-derived airway secretion or sputum. As sputum is highly
complex and heterogeneous among patient sample, a single step sorting is often
insufficient to achieve satisfactory separation performance. Recirculation can purify
target cell suspending medium while continuously eliminates mucin aggregates from
sample. The reported device retrieved ~95% PMNs from sputum from six patients
and provided superior performance over traditional Sputalysin (DTT) protocol
(Fig. 5.24a (right)). Kwon et al. recently described a closed-loop multiplexing spiral
system for perfusion culture of Immunoglobulin G1 (IgG1) CHO cells with high
IgG1 recovery rate (>99%), cell viability (>97%) and long term stability
(18–25 days) [143].
5.5.5 Integrating with Other Separation Modalities
Coupling spiral microfluidics with other cell sorting modalities was recently pro-
posed by Nivedita et al. [96]. In this work, they described a novel integrated platform
which comprised of a spiral separator and a second-stage cell sorter based on lateral
cavity acoustic transducer (LCAT). Briefly, angled lateral channel array was
designed to trap air as depicted in Fig. 5.24b. Acoustic field was applied to the
device which resulted in oscillation of the air/liquid interface and generation of
microstreaming vortices. When larger particles approached these microvortices, they
would be trapped into the circulating inner streamlines as smaller particles followed
along the outer streamlines with the bulk flow [96]. By adjusting excitation voltage
of acoustic actuator, the device can selectively capture different sized particles to
further enrich target cells. As proof-of-concept, they applied the device to trap larger
monocytes (>18 μm) from 10 diluted blood. Smaller RBCs and WBCs, and
intermediate sized cells (<18 μm) were eliminated to outlet 1 (spiral outlet) and
outlet 2 (LCAT), while enriched larger monocytes remained trapped inside LCAT
part. By flushing PBS through the channel, they were able to retrieve and concentrate
target cells with an enrichment of 987-fold. They also demonstrated separation of
DU-145 cells (>16 μm) from highly heterogeneous DU-145 population (7–28 μm)
and achieved 91.7% purity and 67.5% recovery rate of target cells. Noteworthy, this
work showed the unification of two separation techniques operating at different flow
rates (1500 μL/min and 25 μL/min). This was achieved by adjusting the pressure
drop/hydraulic resistance of the spiral outlet and transition region between 2 devices.
Another simple modification that can assist in enrichment of cells was proposed by
Wang et al. [92], in which they added a membrane filter at the outlet of the spiral
device to further enrich CTCs for immunostaining.
For blood cell sorting applications, it remains challenging to use whole blood as a
direct input for spiral devices due to the high RBCs concentration (~5 billion/mL)
and secondary cell-cell interactions are known to have adverse effects on sorting
performance [69, 146]. This makes off-chip sample pre-processing (dilution or RBC
lysis) a prerequisite prior spiral separation. Recently, Ramachandraiah et al. pro-
posed an integrated spiral device for leukocyte fractionation with on-chip RBC lysis
(Fig. 5.24c) [146]. Whole blood and hypotonic solution (deionized water) were
introduced into the device to selectively lyse the RBCs. The integrated lysis chamber
with expansion chambers helped increase residence time for proper RBCs lysis. The
lysed blood was then washed with sheath buffer stream (PBS) to prevent negative
effects such as leukocytes swelling. Finally, the focused sample stream proceeded
through the double spiral channel to fractionate out granulocytes, monocytes, and
lymphocytes with a purity of 86%, 41% and 91% respectively. Such integrated
device is favorable for point of care systems as it facilitates user operation and is less
time-consuming process compared to sequential sample processing. Furthermore, it
reduces cell loss that can occur during sample handling between each process. It
should be noted that the abovementioned device required three syringe pumps to
operate which can be a major drawback for point of care applications.
5.6 Conclusions and Future Outlook
Spiral inertial microfluidics has emerged as a superior separation technique for high
throughput particle sorting in biomedical applications and clinical diagnostics. Since
the introduction of inertial microfluidics by Di Carlo et al. [30] and the first
demonstration of spiral microchannels for particle filtration [147] a decade ago,
enormous efforts by us and other groups have focused on exploiting this technology
for different cell sorting applications, achieving higher throughput by multiplexing,
and enabling small micro and nanoparticles separation. Compared to other
microfluidics systems, the key advantages of spiral devices include simplicity in
microfabrication (3D printing is possible [68]), low clogging issues due to large
channel dimensions, high separation resolution, and scalability for macro-scale
volume processing.
142 N. Liu et al.
Fig. 5.24 (a) (left) Recirculation spiral microfluidics strategy (right) Images and plots showing
sputum and purified samples using their system (C-sep) and DTT protocol. (Reprinted (adapted)
In this chapter, we have provided a comprehensive review on spiral inertial

microfluidics and covered related topics including (1) conventional and microfluidic
cell sorting techniques, (2) introductory theory in inertial microfluidics and Dean-
coupled inertial focusing, (3) classification of major spiral devices, (4) sorting of
cells and biomolecules using spiral technologies, and (5) recent advances in next
generation spiral cell sorters. Noteworthy, we have also provided tables (in different
sections) to highlight key principles and features in different spiral designs, as well
as comparing separation performances in various cell-based applications. We
believe this broad overview will be invaluable to readers who are new to the topic
and keen to use spiral cell sorting technology.
As the spiral inertial microfluidics community continues to grow, there are
several important areas that researchers can contribute to enable novel applications
and improve understanding on the underlying physics. Being a size-based separation
technology, its remains challenging to purify cells of closely-spaced sizes or with
other biophysical differences (cell deformability, electrical properties etc.). To
enhance sorting specificity, it is important to couple spiral inertial microfluidics
with other active or passive sorting techniques which exploit other biophysical
parameters such as electrical properties (dielectrophoresis), magnetic properties
(magnetophoresis) and mechanical properties (acoustophoresis). The biggest chal-
lenge is the flowrate mismatch between spiral device and other techniques as high
flow conditions commonly used in spiral channels may not be suitable for other
sorting techniques. As cell heterogeneity is becoming increasingly appreciated,
another major technological improvement is to integrate biosensing capabilities in
spiral sorters to facilitate high throughput real-time single cell analysis. If successful,
we are confident that these next-generation integrated spiral platforms will be of
great significance for biomedical applications and commercialization opportunities.
Finally, improvement in imaging modalities to characterize particle equilibrium
positions in 3D at high flowrates can help to elucidate the underlying physical
mechanisms and inertial focusing dynamics [148]. A deeper quantitative analysis
of particles inertial focusing in spiral channels can also validate simulation studies
and machine learning models to optimize spiral designs and enhance separation
capabilities. We envision that with increasing advancement in microfabrication and
computational techniques, spiral inertial microfluidics will continue to play a leading
role in driving biomedical research by enabling novel and tunable separation tech-
nologies with more precision and user-defined separation features.
⁄
Fig. 5.24 (continued) with permission from Ref. [145]. Copyright (2017) American Chemical
Society.) (b) (left) Schematic of integrated spiral device with sequential lateral cavity acoustic
transducer. (right) Microstreaming vortices in LCAT channel for particle trapping. (Adapted with
permission from Ref. [96]) (c) (top) Workflow and image of the integrated spiral device with
on-chip RBC lysis. (Reproduced from Ref. [146] – published by The Royal Society of Chemistry,
under Creative Commons)
144 N. Liu et al.
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Chapter 6
Worms on a Chip
Han-Sheng Chuang, Wen-Hui Wang, and Chang-Shi Chen
Abstract Advancement in miniaturization in recent years has enabled high-

throughput, in-parallel, rapid, and precise operations in modern medical and biolog-
ical research. Although numerous biomimetic devices have been inspired by nature
cues, the artificial gadgets still cannot be on a par with their natural counterparts.
Caenorhabditis elegans (C. elegans), the smallest multi-cellular model animal, has
become a popular platform for drug screening, biosensing, genetic engineering,
neuroscience, developmental biology, and so forth since its first debut made by
Sydney Brenner nearly five decades ago. The nematode C. elegans features small
size, transparency body, fully sequenced genomes, high genetic similarity with
humans, short life cycle, and simple neural network. The combination of
C. elegans and microchip can prompt promising uses in some aspects. To cope
with the new demands, the scientific community has endeavored great efforts to
meet all sorts of worm maneuvers, such as sorting, immobilization, long-term
imaging, confined culture, and biomechanics. The proposed manipulation repertoire
then leads to realizations of a wide applications. Examples may include drug
screening for pharmaceutics, point-of-care testing (POCT) for diseases, and funda-
mental research. Although worms-on-a-chip (WoC) appears to remain in its infancy
stage of development, intensive research has gradually unveiled novel possibilities
in many potential fields. This chapter aims to introduce the current development of
WoCs and provides examples according to their categories. Pros and cons will be
addressed in the end. Some practical uses will also be suggested for the future
prospects.
H.-S. Chuang (*)

Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan
Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan
W.-H. Wang
State Key Laboratory of Precision Measurement Technology and Instrument, Department of
Precision Instrument, Tsinghua University, Beijing, China
C.-S. Chen
Department of Biochemical and Molecular Biology, National Cheng Kung University, Tainan,
Taiwan

152 H.-S. Chuang et al.
Keywords C. elegans · Microchip · Screening · Model animal · Microfluidics
6.1 Introduction
The nematode Caenorhabditis elegans is a well-known model animal in research

fields, such as neuroscience, genetic engineering, and drug discovery. C. elegans
was firstly studied and introduced to the world by Sydney Brenner in 1963 [1]. The
tiny multi-cellular organism measures around 1 mm in length and about 4 pg in wet
weight [2]. In addition, the worm possesses only 302 neurons, 959 somatic cells, a
short life cycle (~3 days), and more than 60% genetic similarity with human being.
In 1998, C. elegans was even the first animal to have its genome fully sequenced in a
pilot project led by John Sulston and Bob Waterston. All of the above features make
C. elegans a unique model animal in preliminary research. With such advantages,
Sydney Brenner once praised C. elegans a nature’s gift to science in his Nobel
lecture in 2002. Until now, several Nobel prizes have been awarded to those
scientists who devoted to the C. elegans research, including Sydney Brenner, Robert
Horvitz, and John Sulston (the 2002 Nobel Prize in Physiology or Medicine),
Andrew Fire and Craig Mello (the 2006 Nobel Prize in Physiology or Medicine),
and Martin Chalfie shared the 2008 Nobel Prize in Chemistry. These facts make the
tiny animal a real giant in science.
After more than five decades of intense development and support from the
science community, C. elegans has become a great platform used in a broad
spectrum of fields. To facilitate the research based on the worm, a worm bank,
named Caenorhabditis Genetics Center (CGC), has been established at University of
Minnesota to provide various mutants and genetically-engineered worms to all
worm breeders around the world. Instead of conventionally tedious breeding, now-
adays researchers only need to place their orders for specific worms and will receive
them in a few days, thus saving time and effort in their work. Moreover, some online
encyclopedias, such as WormBook, WormBase, and WormAtlas, can also guide
beginners to start their work in a timely fashion. A clear trend shows that rapidly
growing researchers have been brought into this area recently not only because of the
significance of the worm but also the friendly environment. Of the researchers, the
engagement from engineers especially throws unique impact to this community by
introducing new tools and mindsets.
The prior studies [3–8] have revealed that C. elegans can respond to many stimuli
with chemotaxis, thermotaxis, electrotaxis, magnetotaxis, phototaxis, and more. The
capability of multiple responses implies that various sensory neurons are involved
inside the worm’s body. The basic communications between sensory neurons and
motor neurons can form different pathways to activate relevant motions mentioned
above. The locomotory gait of C. elegans is a sophisticated behavior in response to
environmental changes [4–10]. Through image recognition of the gait changes, one
can further understand the interactions between worms and their environmental
stimuli. In anatomy, most worms are hermaphrodite, leaving only 5% of the total
6 Worms on a Chip 153
population to be male. Therefore, the self-reproduction results in slow process in

genetic diversity. The highly genetic similarity between parents and offspring avoid
inconsistency in experiments involving with several generations. Nevertheless,
individual worms still can behave differently from each other even receiving same
stimulus. The difference makes C. elegans an ideal model for investigating the
correlation between decision making and the formation of neuronal network.
Although C. elegans is preferably cultivated on agar, they perform amphibious
adaption in aqueous environments [11]. According to prior studies [12, 13], worms
show no significant differences in lifespan, physiological conditions, and biochem-
ical reactions between both situations. However, worms growing in an aqueous
environment are more likely to result in vigorous undulating motion instead of
crawling. Although the mechanism remains not very well understood yet, some
studies suggest that viscosity change may form a mechanical stimulus to the worm’s
sensory neurons and then trigger the subsequent gait switch. Of the studies, some
evidences show the gait change is rather a binary switch between swimming and
crawling than a progressive shift [14]. In spite of the fact, aqueous media remain a
popular culture environment for the study of C. elegans.
As C. elegans becomes the spotlight of model animals, many skills have been
exploited to enhance the repertoire of the tiny little animal. In addition, their fast
reproduction and easy maintenance provide a great statistical basis over other model
animals. As compared with artificial intelligence, the natural-born primitive organ-
ism remains to outperform human technology in all aspects. For example, the
neuronal network constructed by the 302 neuron cells imparts C. elegans sufficient
ability to deal with various daily activities, such as foraging, mating, avoidance from
adverse situations, sensing stimuli, decision making, and interactions with other
worms or environments. As a result, the worm draws not only attention from
biologists but also engineering scientists in recent years. By properly incorporating
C. elegans into a microchip, the worm has potential to work as a multi-functional
sensor, a drug carrier, a sophisticated animal platform, an actuator and more
(Fig. 6.1). This chapter will begin with worm biology to introduce the uniqueness
of the worm and then extend to some state-of-the-art WoCs. Next, we will look into
some applications that require such kind of special worm chips and their niches. At
last, we will finalize this chapter and envision some unmet needs and possible
developments in the near future.
6.2 Worm Biology
6.2.1 Fundamental Features
The nematode Caenorhabditis elegans is a free-living and tiny multi-cellular organ-

ism that inhabits on rotted vegetables or fruits in nature. It was firstly studied by
Sydney Brenner and introduced to the world as a great genetic model animal
[15, 16]. Unlike its other parasitic cousins, such as threadworms, flukes, hookworms,
Fig. 6.1 Conceptual illustration of the universal WoC platform
Fig. 6.2 The life cycle of

C. elegans at 25 C
and so forth, C. elegans has been proven to be non-hazardous, non-infectious,

non-pathogenic, and thus safe in laboratory use. For research purposes, C. elegans
is usually cultivated on agar and fed with non-pathogenic bacterium, Escherichia
coli strain OP50. In general, the features that bring C. elegans on the spotlight are
focused on their small size (~1 mm for adult worm and 959 somatic cells), trans-
parent body, short life cycle (~3 days), a small number of neurons (302), and fully
sequenced genome.
The distinct developmental stages of C. elegans include egg, larva, and adult
(Fig. 6.2). In the larval stages, worms can be even divided into L1, L2, L3, and L4
according to their physiological phenotypes. At 25 C, the growing periods for L1,
L2, L3, and L4 stages are estimated to be 12, 7, 7, and 9 h, respectively. These
predictable growth periods are therefore references used to synchronize worms. To
this end, adult worms need to be bleached by strong basic solutions, such as alkaline
hypochlorite, to remove their cuticles and leave eggs. Subsequently, all eggs will be
synchronously hatched from the same time point and grow till the desired develop-
mental stage. Notably, C. elegans may detour to a special larval stage, named dauer,
when the worm experiences starvation before L2. The dauer worms are more durable
to environmental stress, so they can survive for at least a month without food. This
capability imparts them a high survival rate during critical conditions. As for the
worm’s appearance, the body lengths of L1, L2, L3, and L4 are estimated to be
around <250, 360–380, 490–510, and 620–650 μm, respectively. In addition, the
body length for a young adult worm is 900–940 μm and will become 1110 μm–
1150 μm for a fully-grown adult worm. Especially, L4 stage worms can be explicitly
recognized by a crescent mark loomed in the mid-body resulting from the intrusion
of vulva.
6.2.2 Anatomy and Physiological Phenotypes
An adult C. elegans nematode comprises an alimentary system (mouth, pharynx,

intestine, rectum, anus), a reproductive system (gonad, uterus, spermatheca, vulva in
the hermaphrodite; gonad, seminal vesicle, vas deferens, cloaca in the male), a
nervous system (302 neurons in the hermaphrodite, a cluster of synapses, and a
nerve ring in the head), and an excretory system (a group of four cells believed to
control osmolarity and the elimination of waste) (Fig. 6.3a and b). The cellular
mitosis of somatic cells process only proceeds during the developmental stages. A
total of 1030 somatic cells are generated in a hermaphrodite worm’s time course of
development. However, the number reduces to 959 after 131 cells are programmed
to death in the later worm stage. Anatomically, the worm is simple in body
constituent but sophisticated in functions. The animal is often described as a series
of concentric tubes comprising cuticle and internal organs separated with a
pseudocoelomic cavity (Fig. 6.3c). As a result, mass transfer in worm’s body
including metabolites and nutrients is primarily dependent on diffusion due to lack
of a circulatory system. Three sets of muscles are responsible for controlling the
foraging, locomotion, and egg laying. All the worm body movements and sensory
activities are coordinated by the neuronal network out of the 302 neurons.
Apart from most organisms, wild-type C. elegans has two unique sexual forms,
male and hermaphrodite. They can reproduce not only by mating but also self-
fertilization. In nature, male worms only occur 0.1% of the overall population. As
compared with the hermaphrodite in appearance, a male worm is usually distin-
guished by its fan-shaped tail. Since the male is not considered the majority of the
Fig. 6.3 Anatomy of C. elegans. (a) Anatomical features of hermaphrodite and (b) male worms. (c)
Cross-sectional view of hermaphrodite in the anterior region. The schematic illustrates the four
muscle quadrants surrounded by the epidermis and cuticle with the intestine and gonad housed
within the pseudocoelomic cavity. (Reprint permission for all sub-figures from http://www.
wormbook.org) [19]
worm population, unless mentioned otherwise, we will focus on hermaphrodite in

the following content. Each hermaphrodite worm can produce around 300 eggs in its
lifespan. The gonad of hermaphrodites forms an ovotestis that firstly produces
haploid amoeboid sperm stored in the spermatheca in the L4 stage, and then the
germline switches fate to produce much larger oocytes near adulthood. Young adult
worms are mature in their germlines and start to lay eggs from adult day 1 to around
adult day 6. Thereafter, the worm can keep living for another week or two. The short
lifespan makes the animal easy for any life-long study.
Wild-type C. elegans performs undulating crawling to migrate on solid surfaces.
Forward, backward and U-turn are three common locomotory gaits that worms
generally maneuver. In a liquid phase, however, worms switch to thrashing-like
swimming instead. The rate of body bends for N2 worms is around 1.5–2 Hz in
average. In addition, a special behavior, termed quiescence/lethargy, can also be
observed on C. elegans while they are in transition from one developmental stage to
another. Although the mechanism remains not fully understood, lethargy is believed
to be a sleep-like process during larval stages in C. elegans [12, 17]. Worms
deprived of sleep can lead to growth defects or death.
Food uptake of C. elegans relies on constant pharyngeal pumping in the head.
The pharyngeal pumping is usually associated with the healthy state of C. elegans.
The pumping rate progressively declines after worms enter the late adulthood.
Egg-laying requires 16 muscle cells, including 4 vm1 and 4 vm2 vulval muscle cells
and 8 uterine muscle cells [18]. Both vm1 and vm2 muscle cells are arranged in
X-shape patterns around the vulva. The uterine muscle cells are responsible for
pushing eggs out of the vulva by constricting the uterus. Of these cells, the 4 vm2
muscle cells are of particular importance to the vulval opening since only ablation of
them causes an egg-laying defect. Nevertheless, loss of functions in these muscles
increases the risk of eggs hatched internally in their parental worms, a.k.a. bag of
worms (BOW). The occurrence of BOW is not rarely seen in nature. Age-related
degeneration is a major cause of this phenotype. However, other factors from
environmental toxins or genetic abnormality may trigger BOW as well.
6.2.3 Nervous System
Neurons in C. elegans differentiate in three developmental stages. The first stage is

during the proliferation phase of embryogenesis, the second is at the late L1 stage,
and the third is at the late L2 stage. According to the cells’ loci in the hermaphrodite
C. elegans, the nervous system is usually distributed in the anterior, the ventral cord,
and the pharynx. Despite the simplicity of the worm’s nervous system, its high
degree of cellular diversity still astonishes researchers in many ways. In a general
sense, all neurons in adult C. elegans can be classified into four functional catego-
ries: sensory neurons and motor neurons, interneurons, and polymodal neurons.
Sensory neurons are specialized to sense a wide variety of environmental stimuli,
including but not limited to mechanical, electrical, chemical, optical, and thermal
variations. The perception of environmental cues is accomplished through
24 sensillar organs and various isolated sensory neurons (see Table 6.1). Each
sensillum contains ciliated endings of one or more neurons. Most sensilla are located
in the head, except for posterior deirids and phasmids. Motor neurons are responsible
for locomotion, alimentary, and reproductive systems by making synaptic contacts
onto muscle cells. The common behavioral phenotypes are forward, backward, and
U-turn movements on agar or swimming/thrashing in liquid media. The motor
neurons can also work together with sensory neurons in response to external inputs
from gentle body touches or environmental vibration/tapping. Interneurons are
bridges that connect different types of neurons. They function as information pro-
cessors by receiving signals from one or more neurons and conveying decisions to
other target neurons. They work as a primitive brain in modulating neuronal
functions and process information. In addition to the three abovementioned neuron
categories, polymodal neurons are some special neurons that can perform multiple
circuit functions, such as ASH, M3, NSM, DVB, etc. An idealized network depicted
in Fig. 6.4 is used to interpret the basic principle of a neuronal circuit for chemotaxis.
Upon interactions and coordination between specialized neurons, C. elegans can
maintain its life for all sorts of activities. To aim at the worm-based biosensors, the
following paragraphs will focus on some common locomotory responses prompted
by different sensations.
Table 6.1 Functions and input signals of some common sensory neurons
Neuron Signal Sensed Functions
ASE Water-soluble chemicals Chemotaxis (major)
AWC Volatile odorants Attractive chemotaxis, Lifespan, Navigation
AWA Volatile odorants Attractive chemotaxis, Lifespan (minor)
AWB Volatile odorants Avoidant chemotaxis
ASH Touch, Chemicals, UV light, Nociception: Osmotic avoidance, Nose touch
Osmolarity avoidance, Chemical avoidance, Light
avoidance
ASI Water-soluble chemicals, Dauer Dauer formation, Chemotaxis (minor),
pheromone Navigation
ADF Water-soluble chemicals, Dauer Dauer formation, Chemotaxis (minor)
pheromone
AFD Temperature Thermotaxis
ASG Water-soluble attrantants (Na+, Cl, Dauer formation (minor), Lifespan, Chemo-
cAMP, biotin), Hormonal signaling taxis (minor)
ASJ UV light, Electric field, Dauer pher- Dauer formation and recovery, Chemotaxis
omone), Hormonal signaling (minor), Lifespan, Electrotaxis, Phototaxis
ASK UV light), Hormonal signaling Avoidance (minor), Chemotaxis (minor),
Lifespan, Navigation, Phototaxis
ADL Food Avoidance (minor), Social feeding
URX, Oxygen, Food Aerotaxis, Social feeding
AQR,
PQR
PHA, Water-soluble chemicals Avoidance (Antagonistic)
PHB
Modified from the WormBook [20]
Fig. 6.4 An example of idealized neuronal network for chemotaxis in C. elegans

6.2.3.1 Phototaxis
Light sensation is one of the vital sensory organs in most animals. Phototaxis refers
to a locomotion in response to optical stimuli. Unfortunately, C. elegans is a soil
animal which lacks visionary capability to aid them navigating in the dark environ-
ment. However, this understanding was changed by Ward et al.’s study [10]. Ward
and his colleagues investigated the negative phototaxis of C. elegans in 2008.
Surprisely, they discovered that C. elegans can sense light via ciliated amphid
neurons and maneuver repulsive reactions to it in a dose-dependent manner. Seven
neurons, ASJ, AWB, ASK, ASH, ASI, AWC, and ADL, were verified to be involved
with the avoidance response. Among them, ASJ, AWB, ASK, and ASH neurons
seemed to show the strongest association with the head avoidance. In conclusion,
Ward et al. indicated that C. elegans photoreceptor cells are primarily mediated by
the CNG channels and the second messenger cGMP for the phototransduction.
Notably, this phototaxis of C. elegans is associated with light wavelength and optical
intensity. In another study, Edwards et al. [4] stated that C. elegans showed strong
avoidance in relation to low wavelengths, for instance ultraviolet light. This partic-
ular relationship was inferred to the worm’s physiological need of survival. Since
sunlight exposure appears to be fatal to worms, sensing the right direction to migrate
then becomes an essential ability to keep them safe from harm in the dark soil. By
associating the noxious low wavelengths with the sunlight, the phototactic strategy
can form immediately in worms to steer them away from the danger.
6.2.3.2 Chemotaxis
Chemosensation is vital to worms’ survival since it can help worms find food, avoid
noxious conditions, develop appropriately, and mate with male worms. The sensory
neurons are mostly distributed in the cilia. There are roughly 16 pairs of amphid and
phasmid neurons involved in chemosensation. The chemosensory neurons are
interconnected with motor neurons to form so called “chemotaxis”. To activate the
reactions in C. elegans, target olfactory molecules must be firstly sensed by the
neurons to trigger the subsequent signaling pathways. Therefore, C. elegans can be
trained as a miniaturized robot to seek specific target molecules in an unknown
environment. Neto et al. [21] attempted to use C. elegans as a biosensor to detect
pulmonary tuberculosis (TB) through the exhaled breath from patients. Their study
showed that TB-specific volatile organic compounds (VOCs), including methyl p-
anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, could be
sensed by C. elegans and induced corresponding chemotactic responses. The
approach improved the conventional diagnosis of TB which relies on a combined
approach of clinical symptoms, a chest X-ray and sputum based laboratory testing
including smear microscopy, bacterial culture and molecular methods. However, the
observed olfactory response to the purified TB-specific VOCs demonstrated only
preliminary feasibility. More work still needs to be done in order to verify the
repeatability when the four VOCs are isolated from wild type M. tuberculosis and
when spiked into human breath specimens.
6.2.3.3 Electrotaxis
In addition to odorants and light, C. elegans can sense electric fields and orient
themselves to the negative poles as well. This tendency of movement toward the
cathode in an electric field is then termed electrotaxis. Gabel and colleagues [6]
firstly revealed the worm’s electrosensory behavior and attributed the response to the
amphid sensor neurons. Their in-depth investigation showed that ASJ and ASH were
strongly stimulated when the worms’ heads pointed to the anode in an electric field.
ASJ and ASH apparently dominated the navigation and avoidance, respectively. As
mentioned previously, these two neurons also play roles in mediating phototaxis and
chemotaxis of C. elegans because ASJ typically responds to environmental cues
during development and ASH is responsible for several nociceptive reflexes.
According to their finding, a possible scenario as to how worms form their
electrotactic orientation is by moving their heads randomly in an attempt to look
for a direction that can lower the activity of these electrosensory neurons. Once the
direction is found, they will orient their bodies to align with the electric field and
move to the cathode. Although the physiological needs for such electrotaxis remains
enigmatic, it may possibly be inferred to the inherent nature from foraging or mating
lures the worms to move. Later, the electrotaxis of C. elegans was further applied in
a microfluidic chip [22]. Evidences suggested that the effective electric field inten-
sity changed with the worm’s developmental stages. For worms younger than L3,
they showed low response to the electric stimuli possibly because of immature
sensory neurons. As worms aged (young adult), the required minimum intensity
declined. The different responses to electric fields thus provide a way for size sorting
[23–25].
6.2.3.4 Thermotaxis
Of all sorts of sensations, temperature is a unique factor that can regulate C. elegans
growth rate and healthy states for the whole lifespan. Prior research [7, 26] has
observed that Caenorhabditis elegans tends to migrate toward its cultivation tem-
perature or isothermally tracks along that specific temperature in a spatial thermal
gradient. The tendency of migrating up the thermal gradient is termed positive
thermotaxis; otherwise, it is termed negative thermotaxis. It is a quite straightforward
strategy to study the navigational skills in C. elegans to understand the plasticity and
programming of sensor-motor circuits in a small nervous system [27]. In the
thermotactic model, thermophilic neurons (AFD and AIY) and cryophilic neuron
(AIZ) are involved in the behavior. Notably, worms were only tested between 15 C
and 25 C and the gradient was as shallow as 0.2 C/cm. It is obvious that
temperature higher or lower than the thresholds that worms can bear will certainly
harm the animals.
6.2.4 Worm Cultures
6.2.4.1 Growth on Agar
In general sense, C. elegans needs to be cultivated at a specific temperature and fed

with sufficient food, E. coli OP50, on the nematode growth medium (NGM) agar
dish. To this end, a drop of 0.1 mL of E. coli is firstly seeded on agar in a large petri
dish with a pipette. Noted that the lawn of E. coli should be spread in the center of the
dish to prevent worms from crawling to the edge. After seeding, the lawn of E. coli
will grow overnight at room temperature or at 37 C for 8 h [28]. Since the worm’s
growth is strongly regulated by its culture temperature, worms are grown in an
incubator to ensure temperature is well controlled. Worm culture usually starts from
eggs after bleach to achieve synchronization. The growth period in each stage can
then be estimated according to the set temperature.
When worms reach adulthood, they will be able to lay eggs for the first 5~6 days.
To avoid food shortage and interference from self-offspring, worms need to be
transferred to a new agar dish daily to maintain the consistency. Several methods
can be chosen for such purpose. A quick and convenient method is “chunking”,
which a chunk of agar from an old dish is cut and removed to a fresh dish. Another
method is to pick single worms with a picker connected with a platinum wire
(or eyelash) on one end. Between transferring, the platinum wire needs to be
sterilized with fire to avoid contaminating the worm stocks. This method is
performed when specific worms are selected. With sufficient food and space in a
well-controlled environment, most worms can live up to 3 weeks.
6.2.4.2 Growth in Liquid Media
Since C. elegans is an amphibious animal, it can also be cultivated in liquid media.

At macroscale, liquid cultures of C. elegans are usually grown on S medium using
E. coli OP50 as a food source [29]. It is best to grow only one generation of worms in
liquid before the worms are harvested. Whereas, growing worms for more than one
generation, overcrowding can lead to dauer formation despite the presence of food.
As a result, keeping food supply, E. coli OP50, sufficient in the liquid media is vital
for a long-term worm culture. Worms usually consume food more and faster because
of vigorous locomotion. Therefore, the amount of food needed should take into
consideration the duration that worms are grown. As a reference, a 62.5 mL of liquid
culture stock can support worms from a 100-mm petri dish to grow for
4~5 days [28].
At microscale, the maintenance is diverse due to various microchips and appli-

cations. Nevertheless, the amount of liquid determines the length of time that worms
can survive. Without supplying fresh liquid with nutrient, single worms can live up
to 4 days in a confined aqueous droplet as small as 125 nL. Otherwise, worms can
live as long as those which are grown in an open or circulating environment.
Notably, food appears to be associated with the worms’ survival. Under proper
condition, oxygen level and consumption can reach a safe equilibrium through gas
exchange at the interface [12].
6.3 Basic Maneuvers for Worm Maintenance

6.3.1 Sorting
C. elegans worms at different developmental stages have distinct phenotypic behav-

iors and nerve responses [25]. Sorting C. elegans is of great significance in studying
these stage-specific behaviors. Therefore, it is important to construct a rapid and
accurate C. elegans sorting method. Manual picking is a preferred maneuver when
the number of selected worms is small. However, the maneuver becomes time-
consuming and labor-intensive as dealing with a large population. The commercially
available automated sorter such as the BIOSORT/COPAS is massive and expensive,
reducing its prevalence in the routine laboratory use [30]. Recently, microchip
technology, relying on specially-designed micro-structure to sort C. elegans at
different developmental stages, has emerged as an attractive and enabling platform
to overcome these problems [31].
To date, sorting C. elegans on a microchip usually relies on the following three
features [32]. (1) Fluorescence signal expression. For example, green fluorescent
protein (GFP) expression in the URX neurons is consistent, but expression in AQR
and PQR neurons is stage-specific, which can be used to classify C. elegans
[33]. (2) Size difference. The life cycle of C. elegans consists of four developmental
larvae stages (L1–L4) and an adult stage. In each stage, the nematode possesses
different yet characteristic body sizes, morphological and anatomical features, which
can be used to classify C. elegans [34]. (3) Behavioral phenotypes. It is known that
C. elegans has distinct electrotactic responses to electrical fields. In the presence of
an electric field, worms at different stages tend to crawl to the negative pole with
different angles due to their deflecting electrotaxis [24].
Numerous sorter microchips using the electrotactic features have been developed.
Chung et al. [35] fabricated a multivalve-integrated microfluidic chip that could
identify the phenotypic features of the C. elegans under investigation and sort them
one by one based on automatically classifying the fluorescent markers with custom-
ized software (Fig. 6.5). Worm screening throughput was achieved as high as several
hundred worms per hour and the sorting accuracy reached over 95%. The microchip
relies on a set of on-chip valves to control the suspension of C. elegans and differs
from previous work in the integration of image-based feedback that enables robust
Fig. 6.5 Multivalve-integrated microfluidic microchip for C. elegans sorting. (a) Micrograph of
the microchip full with different dyes showing active regions. (b–d) Video frames showing the
chamber awaiting a worm to enter (b), with the worm loaded to prevent another from entering (c),
and the worm exit from the chamber while a second worm automatically coming in (d). (Reprint
with permission from [35]. Copyright 2008 Springer Nature)
Fig. 6.6 Size- and electrotaxis-dependent sorting microchips. (a) Size-dependent sorting micro-
chip. (Reprint with permission from [36]. Copyright 2015 Royal Society of Chemistry.) (b)
Electrotaxis-dependent sorting microchip, with its schematic structure and working principle.
(Reprint with permission from [24]. Copyright 2015 Royal Society of Chemistry)
operation, which is critical for high-throughput sorting. Particularly, the system is

empowered with a customized control program to automate the entire processes of
image acquisition and identification without human intervention. However, the
microchip requires sucking C. elegans for still and high-magnification imaging, in
which mechanical immobilization of the worm may cause traumatic damage and the
sequential working mode limits the throughput.
Dong et al. [36] proposed a microfluidic chip for size-dependent sorting of
C. elegans (Fig. 6.6a), making use of the external pressure-deformable cross-sec-
tional shape of PDMS channels that run through two neighboring on-chip worm
chambers. By setting the control pressure properly, the system separated C. elegans
of specific developmental stages from a mixed worm population. The efficiency
reached nearly 100% and throughput was up to 3.5 worms per second. The chip
comprises two layers of PDMS structure, where one is for fluidic control and the
other is for valve pressure control (green in Fig. 6.6a). Sandwiched between the
fluidic and control layers is a thin layer of deformable PDMS membrane. In the
fluidic layer, two isolated worm chambers C1 and C2 are connected by four parallel
transfer channels. The effective cross-section and shape of the channels are regulated
by a pneumatic control chamber orthogonally placed above. In this way, the transfer
channels function as adjustable valves for size-dependent filtering. The microchip
appears to be simple, highly efficient, and flexible.
Wang et al. [24] proposed a microchip (Fig. 6.6b) to realize the sorting of a
population of C. elegans of different stages based on the stage-specific deflecting
electrotaxis of C. elegans. Through investigation of the electrotactic angles of
C. elegans at different electric field strength, the PDMS-agarose hybrid microfluidic
chip was configured to have a number of circle-evenly-spaced sorting channels with
a 5-degree interval in the range of 50 to 50 . The chip was assembled simply by
covering the surface of an agarose gel with the PDMS chip treated with oxygen
plasma to produce a hydrophilic surface, providing a highly efficient, cost-effective,
and harmless method.
We envision that the microchips for sorting would follow the path towards
integration, high-throughput, automation, and standardization [37]. More functional
modules are expected to be integrated in one microchip including sorting as the very
first sample preparation procedure. To meet the needs of biologists, who are poten-
tial end-users of the microchip technology, sorting would be made high-throughput,
automated and standardized to use. In this way, microchips will find more
applications.
6.3.2 Immobilization
Worm immobilization is an essential step in many applications, such as high-

resolution imaging [38], sorting [39], monitoring neuronal responses after various
stimuli [40], laser ablation of neurons [41], and exogenous gene microinjection
[42]. Considering that the vulnerability of C. elegans, gluing and anesthesia [43]
are often performed in conventional worm immobilization. However, labor-
intensive, low-throughput, and irreversible nature limit their practical uses. Recently,
quite a few high-efficiency and smart immobilization techniques have been pro-
posed, including mechanical constriction [44, 45], valve-based aspiration [46],
anesthetic gas (e.g., carbon dioxide) [47]. These methods generally work well for
particular application cases. However, there still exist unwanted limitations. Typi-
cally, mechanical constriction methods could immobilize C. elegans at certain places
but without on-demand function. Valve-based aspiration methods need complex
fabrication procedure and peripheral control. Moreover, these two methods easily
injure C. elegans. Anesthetic gas methods could long-time completely immobilize
worms, but they will influence synaptic transmission and have detrimental effects on
sub-cellular neuronal transport. By contrast, thermosensitive hydrogel methods and
dielectrophoresis (DEP) methods could eliminate issues of gluing and implement
low-injury, rapid, reversible immobilizing. Here we introduce such two methods for
immobilizing C. elegans.
Fig. 6.7 Thermosensitive hydrogel methods for immobilizing C. elegans. (a) Schematic diagram
of a protocol for immobilization of worms in a PDMS-glass microfluidic device. (b) Time-lapsed
snapshots showing a worm trapped and immobilized in the microfluidic channel. Quantitative
analysis of locomotion of the worm by tracking head position according to the time. (Reprint
with permission from [49]. Copyright 2014 Royal Society of Chemistry) (c) Close-up view of the
optoelectric device. (Reprint with permission from [50]. Copyright 2017 Elsevier)
6.3.2.1 Thermosensitive Hydrogels
Thermosensitive hydrogels can be used to immobilize worms by changing temper-

ature to modulate the phase of the hydrogels. Pluronic F-127 is a thermo-reversible
and biocompatible copolymer. By adjusting the ratio of PF-127 in a dissolving
medium, its gelation temperature can be tuned to the worm’s culture temperature
(15–25 C) [48]. Therefore, a slight temperature change can rapidly immobilize
worms by constraining them in a gel. C. elegans appears to be unaffected and stay
healthy. Hwang et al. applied a photo-absorbing layer to the substrate to raise
temperature for gelation of the PF gel. This immobilization strategy requires only
a normal white light source [49] (Fig. 6.7a). It can be configured to selectively
immobilize a specific worm on demand by integrating a spatial light modulator.
Fig. 6.7b shows an immobilized worm after 60 s of illumination.
Despite the abovementioned benefits, this method requires a long gelation time
(45~60 s) and cannot immobilize/recover worms rapidly. Later, Chuang et al. [50]
achieved faster worm immobilization based on bulk heating rather than surface
heating by the optoelectric effect. The optoelectric device is composed of two
indium-tin-oxide (ITO) glass plates separated by a spacer (Fig. 6.7c). One of the
glass plates is coated with a photoconductive layer, which serves as an optical
switch. Selected worms were fully or partially immobilized in 4 s and recovered in
1 s. This rapid and reversible immobilization technique provides insight to worm-
based applications that require long-term and constant monitoring, such as drug
assays.
The microchips with thermosensitive hydrogels feature simple, versatile, and
biocompatible immobilization of C. elegans. Localized illumination allows selective
immobilization of specific worms of all developmental stages at normal physiolog-
ical conditions.
Fig. 6.8 Dielectrophoresis methods for immobilizing C.elegans. (a) Photograph of planar elec-
trodes microfluidic device. (b) An adult worm trapped in the electric field. (Reprint with permission
from [53]. Copyright 2011 Royal Society of Chemistry.) (c) Photograph of C-PDMS microfluidic
device. (d) Immobilization results for C. elegans of different stages under different conditions.
Multiple time-lapse images are superimposed in one picture for three states before/on/after immo-
bilization. (e) Bright-field and corresponding fluorescent images of immobilized C. elegans, whole-
body imaging at 10X and segmented high-resolution imaging at 60X. (Reprint with permission
from [54]. Copyright 2018 Elsevier)
6.3.2.2 Dielectrophoresis
Numerous studies have demonstrated C. elegans immobilization with

thermosensitive hydrogels. However, exposing to the hydrogel over a long period
of time retards the growth of worms. To avoid the drawbacks, rapid, low-injury,
reversible, and easy-to-implement immobilization techniques remain intensively
pursued. Due to some notable merits such as little harm to organisms and fast
response in action, DEP has been widely used to manipulate DNA, proteins and
cells [51, 52]. C. elegans in a non-uniform alternating electric field can be polarized.
The polarization DEP forces induce a torque to align the body with the electric field
lines and thus immobilizes the worm. Chuang et al. [53] micropatterned a pair of
planar gold electrodes on a glass slide (Fig. 6.8a). The worms were trapped at the
location of the maximum electric field intensity. The magnitude of DEP forces can
be readily controlled by adjusting the electric signals and worms can be trapped and
released on demand (Fig. 6.8b).
Planar electrodes have showed great success to immobilizing worms. However,
the electric field distribution is distance-dependent for planar electrodes. Accord-
ingly, the field intensity significantly attenuates as worms stay away from the
electrodes. This physical limitation impedes the immobilization of C. elegans. In
order to alleviate the electric non-uniformity, Huang et al. [54] proposed an
immobilization microchip with 200-μm-thick electrodes, which were made of car-

bon black PDMS (C-PDMS, carbon black nanoparticles mixed with PDMS)
(Fig. 6.8c). The two parallel thick C-PDMS electrodes multiplex as the microfluidic
channel sidewalls. When C. elegans swam inside the channel, switching on/off the
electrodes could easily immobilize/release them. Through experiment, C. elegans of
different stages were immobilized on demand in the microchannel, proving the
efficiency and applicability of the method (Fig. 6.8d). High-resolution imaging of
C. elegans was also demonstrated as a supportive evidence (Fig. 6.8e).
Both thermosensitive hydrogels and DEP technology provide simple, low-injury,
versatile, and biocompatible capabilities to immobilize C. elegans. They can be
integrated on microchips and have been demonstrated for immobilization of
C. elegans of all developmental stages without physical damages. How to develop
these immobilization platforms for more worm assays needs inputs from the
C. elegans biologists.
6.3.3 Confined Cultures of C. elegans
Long-term cultures are crucial for investigations of C. elegans. The basic require-
ment for long-term survival of C. elegans is adequate nutrition supply, including
oxygen and food. At present, C. elegans research is mainly carried out in liquid or on
agar plates. Worms are bred in a large number of groups based on the traditional
research method. As a result, plenty of reagents is needed and experiments are time-
consuming, labor-intensive, and low throughput. Moreover, accurate transferring,
controlling, and individual worm tracking are difficult. To tackle the problems, a
wide variety of microchips for cultivating C. elegans were developed
[31]. Polydimethylsiloxiane (PDMS) is commonly used in the microchip fabrication
because of good permeability for gas exchange and therefore suitable for long-term
cultures of worms [32]. There are two main ways to create a microenvironment for
confined culture of C. elegans. One is to use channel-formed micro-chambers to trap,
house and grow C. elegans. The other is to use micro-droplets to encapsulate
C. elegans and transfer the droplets to a micro-chamber for confined cultures [31].
6.3.3.1 Microchannels
Channel microfluidics offers a continuous flow system, enabling long-term confined

cultures for C. elegans because of the ability to exchange nutrients and excretions
[55]. Krajniak et al. [56] proposed a channel-microchip that allowed long-term
cultures of C. elegans starting at L1 stage (Fig. 6.9). The microchip enables to
breed, individually track and repeatedly image C. elegans at high resolution while
supporting normal development for C. elegans of any stage. The microchip is able to
provide both nutrients and proper gas exchange to multiple worms bred in individual
chambers, which are adopted to separate worms at all times, avoid cross-
Fig. 6.9 Two-layer channel-microfluidic microchip for confined cultures of C. elegans. (a) The
heating layer. (b) The fluidic layer. (c–d) Culture chambers. (e) The cross section of a valve.
contamination and achieve single-worm analysis. Made of PDMS, the microfluidic

chip has two layers including fluidic layer and heating layer. The fluidic layer has
flow inlets, outlets and the individual culture chambers. The heating layer contains
flow-control valves and temperature-control heating conduits. During loading pro-
cess, C. elegans and culture medium are delivered to the chamber from the loading
inlet, such that C. elegans enter and float in the individual culture chambers full with
food and supplemental cholesterol. During analysis process, PF127 (thermo-
reversible and biocompatible copolymer, which can realize solution-to-gel transition
when temperature is tuned) solution is flowed into the device from the PF127 inlet.
The phase of PF127 can thus be tuned by controlling the flow rate and temperature of
the fluids in heating-layer channels. At high temperature, PF127 is converted into gel
which is capable of immobilizing C. elegans, so high-resolution image for analysis is
accessible. At low temperature, PF127 is converted into solution, thus C. elegans can
still be bred in the individual chambers.
The primary function of the device was designed to monitor specific develop-
mental processes. However, the device seemed to be more excellent in maintaining
culture conditions. In this channel-microfluidics device, early L1 stage C. elegans
worms were loaded and maintained for up to 36 h until they became young adults.
Moreover, the microchip can be scaled-up in terms of the array number to realize
higher throughput.
6.3.3.2 Microdroplets
Operation processes in channel microfluidics require a longer time and thus consume
large amounts of reagents. Moreover, channel microfluidics cannot control flow
across channels independently [57]. As a result, droplet microfluidics becomes an
Fig. 6.10 Droplet-microfluidics microchip for C. elegans cultures. (a) Schematic diagram of the
droplet chip. (b) Individual C. elegans loading, encapsulation and substance exchanges by using
droplet operations. (Reprint with permission from [58]. Copyright 2015 Royal Society of
Chemistry)
alternative method for confined cultures of C. elegans because of its discrete nature.
Unlike the continuous channel microfluidics, droplet microfluidics creates discrete
microdrops using immiscible phases. Moreover, droplet microfluidics can control
each droplet independently, thus guaranteeing that each microchamber can be
manipulated individually. As a result, droplet microfluidics can reduce the amount
of reagents and processing time while offering greater potential for increased
throughput and scalability than channel microfluidics [55]. Taking these advantages,
Wen et al. [58] proposed a droplet-microfluidic microchip, which can not only
handle individual worms flexibly through a controllable microenvironment, but
also conduct high-throughput analysis for setting up bio-chemical conditions
(Fig. 6.10). A series of droplets can be generated precisely with adjustable size.
Then, they are used to encapsulate individual C. elegans and provide a collection of
external cues to study the worm’s behavioral response Integrating four different
functional modules, including a sample-loading unit, a microvalve control unit, a
droplet trapping array, and a worm collection unit, the droplet chip allows on-chip
manipulations of worm sampling and long-term confined cultures, enabled by
efficient nutrient exchange within the droplets. Parallel microfluidic channels are
deployed and switched on/off to analyze multiple strains of individual C. elegans
simultaneously. Sufficient substance exchange within the droplet is achieved by
applying continuous coalescence and splitting steps between the interface of worm-
in-droplet and multiple medium plugs, meeting the needs of long-term culture of
C. elegans. This droplet-based microfluidic system is powerful for long-term study
of developmental process of worms, in applications such as animal test in multiple
drugs or environmental toxins.
To date, great progress has been made in the confined cultures of C. elegans on
microchips even though it remains in the infancy stage. We envision integrating
more analysis modules alongside cultures will be a challenge to face with. However,
this step will boost the significance of confined culture modules in the real world.
6.3.4 Long-term Imaging
Long-term imaging is commonly applied in the environmental impact on worm

development, nerve damage and regeneration, and the molecular pathways related to
complex human dysfunctions [59, 60]. To achieve long-term imaging, specific worm
individuals must be identified due to the need of repeated observation, thus it is
important to culture and track animals individually [61]. Moreover, the microchip
should have the capability of maintaining the normal development of C. elegans
[56]. When immobilization is needed, for example, to observe specific small features
such as cells/organs/nerves inside C. elegans, the immobilization module should be
harmless to the animal.
Various microchips that can achieve long-term imaging have been proposed.
They can be classified into two kinds based on whether an immobilization module is
required. Normally, if imaging-based parameters may be obtained when the worm
moves, like C. elegans locomotion metrics, forces, and body size, immobilization is
unnecessary or prohibited. In this case, the key point is to guarantee that C. elegans
can be cultured and tracked individually. If imaging-based parameters cannot be
obtained unless C. elegans stays still, like C. elegans internal cells/organs/nerves,
immobilization is required and the key thing is to ensure C. elegans maintains
normal physiology that would not hamper imaging function under immobilization.
6.3.4.1 Microchips Without Immobilization
Churgin et al. [62] made a microchip called the WorMotel (WM). As shown in
Fig. 6.11a, the WM is configured with hundreds of individual wells to house single
worms. Each well is filled with necessary living materials including standard agar
liquid media and bacterial food to support long-term cultivation and imaging of
uniquely identified animals. Depending on the application, the WM can be imaged
continuously or intermittently. Activity analysis could characterize aging and devel-
opment of C. elegans. The worms could live more than 10 days in the WM. However,
due to the liquid culture environment used in the WM, the operation of the microchip
is challenging.
To avoid liquid handling, Pittman et al. [61] made a microchip called Worm
Corrals that could culture 100~200 C. elegans individually, from hatching to death,
on a solid gel medium supported by a microscope slide. The structure of the system
is shown in Fig. 6.11b. Each animal occupies a single pad of food bacteria adsorbed
to a PEG hydrogel, which is supported by a polyethylene frame attached to the glass
slide. PDMS silicone cured atop the device binds covalently to the PEG gel wherever
those materials are in direct contact (i.e. all locations except the positions of the food
pads); thus each animal is confined while still movable on the two-dimensional
surface of its food pad by covalent bonds in all directions. To implement hatching to
death culture, one single egg is loaded first into each individual pad. This is done by
dipping drops of bacteria medium onto the pad and then loading one egg into the
medium by pipette.
Fig. 6.11 Microchips with no immobilization module needed. (a) Design of the WM chip and C.
elegans’ activity data. (Reprint with permission from [62]. Copyright 2017 eLIFE) (b) Worm
corrals, with schematic of the chip, components, fabrication process, and cultured C. elegans.
6.3.4.2 Microchips with Immobilization
Reliable worm immobilization is highly demanded when specific parts or small

features need to be monitored over a period of time. Hulme et al. [63] made a
microchip that could not only culture worms individually in the chamber, but also
immobilize worms when imaging. Using microfluidic “clamps” to immobilize
worms periodically and temporarily, the microchip is useful to image individual
worms from L4 stage until death in separate channels. The structure is shown in
Fig. 6.12a. Only a worm in the early stage of L4 could get into the circular chamber
due to the width of the right microchannel, then after 6 h at 24 C, the worm becomes
too large to exit the chamber from the right microchannel when the nutrient liquid is
provided from the left. By reversing the direction of flow from right to left, the worm
could be directed into the clamp for periodic, temporary immobilization. After
imaging, the worm could be squeezed back to the chamber by directing flow from
left to right.
Imaging C. elegans nervous system is of particular importance and interest,
especially in investigations of the response of C. elegans to external stimuli, such
as environment constraint and odor/chemical. Here we introduce two such classic
microchips [38]. The first one is the behavior chip (Fig. 6.12b), which permits the
correlation analysis of the worm locomotion in association with its neuronal activity.
The behavior chip consists of a worm trap, which is a straight channel and differs
from the above-mentioned tapered clamp. The trap dimensions were optimized to
accommodate young adult worms. One design trick was that the width of the trap
gradually decreases to nearly half at one end to prevent worm escape. Using the
behavior chip, the group successfully imaged calcium transients in the AVA inter-
neuron and used the data to explain how the neuron activity correlates with the
direction of the traveling body wave. The second microfluidic device is called the
olfactory chip (Fig. 6.12c), which integrates a worm trap channel with a microfluidic
Fig. 6.12 Microchips with immobilization module for C. elegans imaging. (a) Clamp structure for
C. elegans immobilization with culture chambers. (Reprint permission from [63]. Copyright 2009
Royal Society of Chemistry) (b) The behavior chip and its working for C. elegans neuron imaging
associated with body wave propagation. (c) The olfactory chip and its working for C. elegans
neuron imaging associated with stimulus solution. (Reprint permission from [38]. Copyright 2007
Springer Nature)
chemical delivery module. The trap channel for the young adult worm is the same as
the behavior chip, but the constriction at one end is smaller. Therefore, when the
worm is loaded, the very end of the nose protrudes out of the trap channel into the
chemical delivery channel with flowing stream of the control or stimulus solution.
The group applied high osmotic strength stimuli to the worm and simultaneously
recorded calcium changes in ASH sensory neurons.
Microchips for long-term imaging often require chambers/wells/channels to
culture worms, which may or may not be immobilized by compression or
temperature-driven sol-gel transitions. It is crucial for the microchips to be able to
maintain the normal physiological conditions for C. elegans during long-term
imaging. With the aid of existing culture and immobilization techniques, long-
term imaging of low-intensity fluorescent signals from neurons or rather strong

bright-field signals for the whole animal has been made possible. We envision
these microchips would find wide applications in drug testing and animal behavior
screening.
6.3.5 Biomechanics
Like all animals, C. elegans is subject to continuous and a wide range of mechanical
loadings during its daily life. The importance of C. elegans mechanics is highlighted
by previous studies suggesting that the worm’s locomotion patterns and foraging
behaviors are strongly dependent on mechanical and osmotic stresses [64]. To date,
studies of C. elegans mechanics focus on two aspects: one is to investigate its
anatomy structure of cuticle, measuring its internal pressure or body stiffness; the
other is to investigate C. elegans mechanical response to external conditions,
measuring mainly its forces and locomotion parameters in the interplay with external
environment. This chapter concentrates on the latter in which the worm is treated as
an animal as a whole.
There are different microchips to measure forces, but all based on the displace-
ment or bending of sensing elements, which get touch with the worm body [65–
68]. Park et al. [65] fabricated a silicon piezoresistive cantilever which was utilized
as a force-displacement measurement system (Fig. 6.13a). The worm is immobilized
on the substrate and its body is touched by the cantilever tip, whose displacement is
measured and converted to corresponding force. The system can measure forces
between 100 nN and 1 mN for tip-worm distance of up to 100 μm with a resolution
of 12 nN when the measuring frequency is between 0.1 Hz and 100 kHz. More
recently, a microchip was developed with SU-8-based pillars. The pillars work as
cantilever, and their tip gets touch with the worm body, but their bottom base is
deployed with four 90 -spaced gold resistors as strain gauges (Figs. 6.13b and c)
[66]. Measured by proper electronic instrumentation, the resistance change of the
strain gauges gives the force applied at the tip of the pillar. Featuring sub-μN force
resolution from 1 Hz to 1 kHz, >25μN range, kHz acquisition rate and biocompat-
ibility, the device is smart in terms of using the electrical signal to map the force.
However, the structure complexity, heat dissipation and optical occlusion from gold
resistors cause unwanted interference to the locomotion pattern and instrumentation.
To overcome the drawbacks, a simpler force sensor was proposed by Ghanbari
and colleagues [69]. The system still consists of an array of micropillars, but the
pillars are made of transparent and elastic PDMS only (Fig. 6.14a). Correspondingly,
the deflection of the cantilever-like pillar is measured from a vision-based algorithm,
rather than from strain gauges. Subsequently, the force can be resolved from the
deflection using the deflection-force model (Fig. 6.14b). The microchip provides a
powerful platform for measuring continuous, multi-point forces of C. elegans, which
can maintain its normal locomotion patterns. Experimental results showed that a
resolution of 2.07 μN was achieved, enabled by the sub-pixel resolution for visual
Fig. 6.13 Three microchips for measuring C. elegans forces. (a) Schematic of nematode measure-
ment system with a piezoresistive cantilever. (Reprint with permission from [65]. Copyright 2007
PNAS) (b) SU-8-based pillar microchip viewed from the top. (c) The force sensing pillars, indicated
by the arrows, are surrounded by passive spacer pillars and posts. (Reprint permission from
[66]. Copyright 2009 Royal Society of Chemistry)
Fig. 6.14 PDMS pillar arrays for measuring multi-point forces. (a) The schematic of a worm
deflecting PDMS micropillars while moving between them, and real pictures of the channel with
pillars. (b) Schematic of the bending pillar for force analysis. (Reprint with permission from
[69]. Copyright 2012 IOPScience)
tracking of the pillar deflection. 13 worm samples were experimented a maximum

force of 61.94 μN was observed from 1571 data points.
Later, Wang and colleagues [70] extended the work to explore how the force
pattern is correlated with locomotion patterns. To enable the research, they borrowed
the ‘artificial dirt’ concept by using the pillars to mimic the worm’s soil-habitat
environment. To incur different locomotion patterns, the microstructured pillar
arrangement and spacing were adjusted. Two different pillar configurations were
used in the microchip (Fig. 6.15a), including the ‘honeycomb’ (HC) design and the
‘lattice’ (LC) design. The spacing values were chosen to allow free movement of the
Fig. 6.15 PDMS pillar arrays configured for extended locomotion analysis and optigenetic manip-
ulation. (a) PDMS device with two sets of micropillar configurations and two different spaces
between the pillars. (Reprint with permission from [70]. Copyright 2013 Royal Society of Chem-
istry.) (b) Schematic view of the optogenetic platform. The microscope condenser provides bright-
field light, the projector provides optogenetic light, the motorized stage actuates tracking of
C. elegans, and the computer controls the hardware elements (e.g., camera, stage, and projector)
and relevant image processing software. (Reprint with permission from [71], Copyright 2015 AIP
Publishing LLC)
worm inside the matrix of pillars. Interesting experimental results were obtained, and
one is that the worm had smaller undulation frequency and locomotion speed for HC
design than the LC counterpart, which was consistent with literature. They also
provided evidence that the natural sinusoidal movement of C. elegans remains
similar in the device, despite the presence of the PDMS micropillars.
Recently, Wang et al. [71] reported on the development of an optogenetic system
(Fig. 6.15b) integrating optical illumination for neuron manipulation and a
microfluidic chip with soil-environment-mimicking and force-measuring micro-
pillar arrays, taking advantage of both optogenetics and microfluidics. The micro-
chip is exactly the same structure as previously reported [70], but integrated with
four control and instrumentation modules including illumination pattern generation,
pattern projection, automatic visual tracking of the worm, and force measurement.
They proposed a customized image-processing algorithm to isolate the worm’s
optical contour from the neighboring pillars, and track and extract its skeleton
automatically. In return, the skeleton information is used to provide instantaneous
position of the worm body such that specific illumination pattern can be projected
onto the device through the lighting source. The system was demonstrated to work
well and be useful for revealing subtle force and locomotion difference between
C. elegans under normal or intensified lighting environments.
Overall, the biomechanics studies of nematodes may be promising though, more
improvements still can be expected. One urgent point, for example, is to standardize
the definition of behavioral parameters for nematode locomotion and forces. There-
fore, data from different laboratories can be comparable. This will rely on more close
collaborations between engineers and C. elegans biologists in the future.
6.4 WoC Applications in Environmental and Biomedical

Fields
From the past to the present, humans have long been inspired by nature in all aspects.
Despite the rapid development in technology, the diversity and complexity of natural
species remain second to none. As compared with numerous biomimetic artifacts to
date, the nature-born life may not be outstanding in a single domain. However, they
are more robust and flexible to deal with multiplexed information in the real world.
As a result, when lab-grown tissues still suffer from expressing full functions, the
tiny nematode can certainly provide more comprehensive functionality than its
counterparts. Unlike other higher animals, C. elegans is easy in maintenance,
small in size, low in cost, but sophisticated in functions. They have been widely
used in medical and biological applications. To take advantage of this multi-cellular
organism, more researchers have attempted to incorporate C. elegans into different
microchips to carry out a worm-based analysis system. Indeed, such kind of WoC
can hence show us more possibilities in brand-new perspectives.
6.4.1 Drug Screening
Drug screening is usually a heavy-duty and lengthy process in the conventional

pharmaceutics. In order to identify and optimize potential drugs for clinical trials,
large libraries of chemicals must be screened beforehand. To this end, high through-
put assays are always needed to improve the effects. Cells have long been used as
targets to respond to the stimulations from different drugs [72–74]. However, there
remains a gap between single cell analysis and animal tests. Although higher animals
can provide more comprehensive information than single cells, high cost, long
turnaround time, ethical issues, and sophisticated mechanisms hamper their preva-
lent use in reality. Instead, C. elegans is well known as a simplest multi-cellular
organism in between single cells and higher animals. Therefore, the nematode can
serve as an ideal candidate to provide biological cues in the selection of potential
drugs. Putting the concerns into consideration, various worm-based drug screening
studies have been emerging in recent years [75–77]. Chemotaxis is a common
physical expression to show the worm’s perceptions in the tested media. The visual
responses may involve with developmental, physiological and behavioral changes.
The olfactory system of the worm works as a major receptor to steer worms from an
adverse environment to a favorable one. In general, numerous chemosensory neu-
rons are interconnected by synapses in a compact neural circuit, allowing the worm
to respond to sophisticated chemical stimuli. Of the 11 pairs of amphid
chemosensory neurons (Fig. 6.16), the AWA, AWB, and AWC neurons are spe-
cialized for volatile odorants [78, 79], while ASE neurons are responsible for
detecting salts and small soluble molecules [80]. Chemical and osmotic avoidance
may be triggered by ASH, ASK, ADL, PHA, and PHB neurons. In particular, ASH
Fig. 6.16 (a) Head neurons in C. elegans and their roles for ascaroside-mediated phenotypes,
downstream signaling pathways, and identified receptors (Reprint with permission from http://
www.wormbook.org) [89]. (b) Cilia morphology of the sensory neurons in the head. (Reprint with
permission from http://www.wormatlas.org) [90]
is a polymodal nociceptor analogous to pain-sensors in vertebrates [81–84]. It was

found that high osmolarity, chemical repellants, or nose touch can all induce a sharp
increase of the calcium level in ASH, activating a rapid response of avoidance.
Repulsion from detergent and heavy metals, besides ASH, may also involve with
additional ASK and AWE neurons. Parasitic environment and soil have vigorous
metabolic activities, the ability of sensing oxygen and carbon dioxide becomes vital
to the worm’s survivorship in the nature. Prior studies [85–87] have shown that
aerotaxis due to oxygen content can be associated with the URX, AQR, and PQR
neurons. The neurons have a strong tendency to keep worms staying in an area of
oxygen levels between 4% and 12% [87, 88]. According to Gray et al.’s investiga-
tion [87], shifting of the oxygen content can regulate the worm’s social behaviors,
such as clumping.
Luo et al. [91] once conducted a motility assay of C. elegans in an array of open
wells to show their olfactory responses to odorants. Cycles of alternating streams of
airborne isoamyl alcohol (IA)/benzaldehyde (BZ) and clean air were supplied to the
wells. They found that a low dose (dilution ratio D ¼ 104) of IA and BZ can elicit
attractive movement in wild-type worms. On the contrary, however, a relatively high
dose (dilution ratio D ¼ 102) of IA and BZ elicits repulsive movement in wild-type
worms. AWC and AWB neurons were identified capable of mediating the repulsion
and attraction, respectively. In addition, the chemosensory neurons showed a clear
dose-dependent trend as well.
Shi et al. [92] reported a microfluidic system composed of a droplet array
(Fig. 6.17a). By trapping single worms in a droplet in the upstream and transport
them to the droplet array in the downstream, they can achieve single-worm resolu-
tion while maintaining a high throughput. The innovation of this device is that no
interference is incurred between worms, allowing single-worm tracking in a long
timeframe. However, the encapsulation rate remains limited by the Poisson distri-
bution. The microchip was used to investigate the neurotoxin, 1-methyl-4-
phenylpyridinium (MPP+). The toxin causes the symptoms of Parkinson’s disease
in vertebrates by impairing their dopaminergic neurons. Since prior study has
indicated that IC50 of MPP+ on worms was 11 mM, they attempted 3 mM and
5 mM of MPP+ on their microchip. The result revealed that 5 mM and 3 mM still
exhibited significantly differences in stroke frequency and omega turn on worms.
The slight changes in behaviors can be distinguished by the microchip. Later, similar
design of static droplet arrays was adopted and modified by Aubry et al. [93]
(Fig. 6.17b). With the flow chamber, they investigated worm’s behavior while
having them exposed to different concentrations of an anthelmintic drug,
tetramisole. A dose-dependent and temporal drop in thrashing was also observed
at concentration of the drug higher than 10 mM. The concentration showed even the
same effect on the drug-resistant strain, CB211. In addition, the same group used the
system to test a pheromone-enriched culture media. 77% of male worms were
successfully evoked acute mating phenotypes (looped tail and curved tail) in such
kind of man-made environment. After experiments, all worms could be safely
unloaded from the droplet traps.
Nanoparticles have been extensively used in our consumer products and medical
applications for a long while. However, their toxicity to animals remains controver-
sial and debatable. C. elegans provides a good animal model to implement simple
toxicity assays. Among so many nanomaterials, silver nanoparticles (AgNPs) have
been widely used in our daily life, such as cosmetics, clothing, household items,
medical devices, and food packaging [94, 95]. The broad prevalence of AgNPs thus
draws the public attention to their environment impact to human health. Kim et al.
[96] proposed a simple C. elegans-on-a-chip to rapidly monitor the toxicity
(Fig. 6.17c). A mutant strain CL2122 which carries the mtl-2::gfp gene was used
to report the existence of AgNPs. The mtl-1 and mtl-2 genes are two verified
isoforms of metallothionein to detoxify heavy metals, such as Cd, Cu, Cr, Pb, Co,
Ni, As, and so forth in C. elegans [97–100]. The worm’s body size and green
fluorescent intensity were measured and compared with the control. The
Fig. 6.17 (a) Top: Schematic of a droplet-based microfluidic system integrating a droplet generator
and a droplet trap array. Bottom: Photograph of the microfluidic trap array immobilized with
180 droplets. Serial images showing an individual worm encapsulated in a droplet. (Reprint with
permission from [92]. Copyright 2008 Royal Society of Chemistry.) (b) display of worms’ body
size, migrating distance, and specific gene expressions for Ag nanoparticle uptake and biotoxicity
on the C. elegans-on-a-chip. (Reprint with permission from [93]. Copyright 2017 Royal Society of
Chemistry.) (c) Droplet array platform for screening acute behavioral responses of C. elegans to
chemical stimulation. (Reprint with permission from [96]. Copyright 2017 Springer Nature)
concentration of AgNPs as low as 5 μg/mL can be detected. The uptake of AgNPs in

C. elegans led to reduction in body size and strong transcription of the mtl-2 gene.
However, higher concentration due to aggregation of nanoparticles impeded the
worm’s uptake, resulting in low adverse effects apart from the expectation. Never-
theless, the paper suggests high throughput and dose-dependent measurement can be
done on such kind of droplet-based microfluidic system.
6.4.2 Biosensors for Diseases/Environmental Changes
C. elegans possesses 302 neurons, it can therefore sense a wide variety of stimuli,
including electrical, chemical, optical, mechanical, magnetic, gravity, and so forth.
Since the worm is small in size, it is particularly feasible for micro, precision, and
versatile sensing applications. As compared with the cutting-edge microsensors
[101–107] and microactuators [108–111] developed so far in the research frontiers,

C. elegans is obviously a perfect candidate. The use of animals to monitor environ-
ment or diseases is not whimsical. As a matter of fact, birds were commonly used by
miners in mining sites to provide early warnings of noxious gases in the past; dogs
are often trained to detect drug smuggling and explosives in airports nowadays;
some dogs and cats can tell whether some people are ill by smelling special odors
from humans [112, 113]. Therefore, it is convenient to take advantage of these super
powers owned by animals to serve as sensors in fields of interest. The benefits of
such worm biosensors may include, but not limited to, (1) consistent expressions due
to rare genetic mutation via self-reproduction, (2) reliably statistical analysis due to
ease of cultivating a large worm population, ensuring statistical correctness,
(4) robustness, and (5) low failure rates. However, uncertainty and accuracy may
still be a concern in such kind of animal-based detections.
Cancer detection by C. elegans, termed Nematode Scent Detection Test (NSDT),
was firstly investigated by a Japanese group, Hirotsu and his colleagues [114]
(Fig. 6.18a). To achieve non-invasive, economical, painless, and rapid cancer
screening, urinary samples were collected from patients with various cancers (breast
ca., gastric ca., colorectal ca., prostate ca., etc). High sensitivity (95.8%) and high
specificity (95%) were obtained from all stages in the pilot runs. The measurement
sensitivity could even reach 100% for those early stage samples. They observed that
wild-type C. elegans was still attracted to 107 dilutions of the cancer-cell medium.
Although the exact molecules that triggered the worm’s chemotaxis remains unclear,
the worm’s corresponding neurons were found likely to rely on AWC and AWA
neurons. The promising animal tests therefore lit up a hope for more disease pre-
screens based on C. elegans. Later, Tee et al. [115] developed another worm assay,
termed C. elegans Sepsis Detection Assay (CESDA), to detect whether a person is
under infection or sepsis though patient’s urinary sample. To avoid interferences
from cancer factors, all tested samples were diagnosed cancer-free at the measure-
ment moment. In their study, sepsis could be detected in as early as 20 min while
infection could be identified within 40 min among 45 subjects. However, as cancer
and infection share many T-cell mediated processes, it is too early to conclude
whether the both cancer and infection detections may lead to the same olfactory
molecules. Further investigation will be necessary.
Metallic ions, pH level, temperature, and gases are part of the environmental
constituents. Their fluctuations may alter the homeostasis of all animals. Therefore,
C. elegans is equipped with all kinds of sensory neurons to deal with changes of
those factors. Evidences have revealed that worms are attracted to oxygen (O2) and
repelled from carbon dioxide (CO2). For instance, Bretscher et al. [116] reported that
well-fed C. elegans avoids elevated CO2 levels above 0.5%, which is ten-fold higher
than the atmospheric CO2 level. The sensation of CO2 in the worm is promoted by
the cGMP gated ion channel subunits TAX-2 and TAX-4. Notably, the CO2
avoidance was found to be distinct from the avoidance of low pH. Wakabayashi
et al. [117] stated that C. elegans employs two behavioral maneuvers, reversal and
Fig. 6.18 (a) Box plots and dot plots of chemotactic responses of wild-type C. elegans to urine
samples from control subjects (n ¼ 218) or cancer patients (n ¼ 24). (Reprint with permission from
[114]. Copyright 2015 PLOS.) (b) Assay plate and acidic pH avoidance behavior. Schematic
drawings of (i) long reversal avoidance, (ii) short reversal avoidance, (iii) gradual curve avoidance,
and (iv) deep curve avoidance behavior. (Reprint with permission from [117]. Copyright 2015
BioMed Central.) (c) Tracks of wild-type and laser-operated worms on a thermal gradient ranging
from 17 to 25 C. Sub-figures (a–c) are responses of worms cultivated at 15 C, 20 C, and 25 C,
respectively. Other sub-figures (d–j) display tracks of different thermotactic phenotypes. (Reprint
with permission from [120]. Copyright 1995 Springer Nature)
curve, to avoid acidic pH. The sensation of pH may be, in part, regulated by ASH
and ASK neurons (Fig. 6.18b). The temperature responses of C. elegans was firstly
discussed by Hedgecock et al. in 1975 [118]. Later, Jurado et al. [119] further
delineated the thermal migration behavior of C. elegans in response to different
thermal gradients. The work was based on their earlier finding [120] associated with
the worm’s thermosensation and neural plasticity (Fig. 6.18c). In Jurado et al.’s case,
N2 worms were preconditioned at 17 C and 23 C before evaluations. When the
worms were placed in the middle of a plate with a linear thermal gradient from 17 C
to 23 C, they tend to move toward their preconditioned temperatures. However,
they discovered that these responses were strongly affected by thermal gradients. In
a shallow thermal gradient (0.5 C/cm), thermophilic and cryophilic worms all
migrated to their reconditioned temperatures. Whereas, only cryophilic worms
returned to their preconditioned temperature in a steep thermal gradient (1.2 C/
cm). The difference was attributed to the natural protection mechanism encoded in
the blind soil animals. In soil, steeper and warmer direction usually imply near the
ground surface, which is very likely to endanger their lives by being heated by sun;
conversely, cooler direction may imply deeper into the ground. Nevertheless, the
appropriate temperature distribution may only be between 16 C and 25 C [118].
6.4.3 Neurosciences and Disease Modeling
Neurons are basic units to constitute a complex neural system in animals. The
understanding of the roles of neurons can help scientists unveil the secrets of mind
mapping, neuronal network, and neurodegenerative diseases. In this respect,
C. elegans is apparently a good choice due to its manageable number of neurons.
Laser ablation has been employed to study specific neurons in C. elegans. Consid-
erable papers [6, 7, 10, 121–123] have revealed their findings on the roles of
neurons. It is clear that neurons are not functioning alone in maintaining the
worm’s daily activities. By forming multiple neuronal networks, they are able to
perform more complicated actions. This feature imparts C. elegans the ability to
make more sophisticated decisions as encountering tasks. In order to understand
such kind of intelligence, Qin and Wheeler [124] explored the learning capacity of
C. elegans with three microfluidic mazes, including six T-maze, complex U-maze,
and continuous T-maze. Two behavioral assays, group exploration and reward-
related associative learning, were investigated. In their findings, worms’ exploratory
behavior was strongly affected by food (E. coli). Without food, however, the chance
of exploration in each outlet was nearly equal. Surprisingly, this behavior could be
acquired by learning. In the associative learning assay, worms’ memory was pro-
gressively enhanced by first few trials with reward, leading to faster decision-
making. Followed by similar trials without rewards, a high rate of the same worms
could still make the right choice. Especially, the neurotransmitter dopamine was
found to boost the learning. However, the memory seemed to lose rapidly as the
no-reward trial continues. Nevertheless, this pilot study showed an insight to use the
simple nematode for the future learning research.
Unlike single cell analysis, C. elegans is a multi-cellular model organism. As an
alternative solution, the worm is suitable for studying neurodegenerative diseases.
Transgenic worm models have been established for various neurodegenerative
diseases, such as Alzheimer’s disease (AD) [125], Parkingson’s disease
(PD) [126, 127], Huntington’s disease (HD) [128], and Amyotropic Lateral Sclerosis
(ALS) [129]. For such kind of research, long-term monitoring (>24 h) is usually
needed to collect enough information for accurate time-resolved analyses. To this
end, Cornaglia et al. [130] came up with a multi-functional microdevice to realize
automated longitudinal monitoring of in-vivo neurodegenerative disease on
C. elegans (Fig. 6.19a). The microdevice was composed of a heat sink, a ring-
shaped thermoelectric module, a chip thermalization frame, a PDMS microfluidic
chip, a glass coverslip, a thermally insulating plate, and a resistive temperature
sensor. Their successfully-bred worms could grow from a desired developmental
stage within the device up to 4 days. During the breeding, fresh food, E coli
suspension, could be continuously supplied into the confinement chamber to main-
tain the worm’s growth. Eggs were washed away to keep the observation free from
unnecessary interferences. Pluronic F127 hydrogel was injected into the chamber to
tentatively immobilize worms for acquiring high-resolution fluorescent images of
protein aggregation inside the worms’ bodies. The reversible gelation of PF127 was
controlled by changing the chip temperature with the ring-shaped thermoelectric
Fig. 6.19 (a) Schematic representation of the main constitutive components of the thermoelectric
microfluidic chip for reversible worm immobilization. (Reprint with permission from [130]. Copy-
right 2016 BioMed Central.) (b) Channelrhodopsin-2 was specifically expressed in the osmo-
sensitive neuron, ASH. Worms were tracked via a small cluster of neurons expressing a red
fluorescent protein located in the head. ASH was photoactivated whenever the worm’s head entered
the annular region. (Reprint with permission from [140]. Copyright 2011 PLOS)
module. In the study, ALS transgenic C. elegans strain (AM725) was employed to
monitor the dynamics of protein aggregation, SOD1-YFP, in the body wall muscle
cells. Constant growing aggregates were found in all the disease models. However, it
was verified that a treatment with an antibiotic, doxycycline, by activating UPR on
the ALS transgenic worms effectively slowed down the progressive loss of their
motility.
In addition to the neuron-based disease models, it is also well known that genes
store biological traits and can be passed from generations to generations. Since the
genome of C. elegans has been fully sequenced, specific genes in worms can be
intentionally modified by genetic engineering to obtain anticipated strains. Cur-
rently, numerous transgenic strains, such as AD, PD, HD, ALS worms, have been
established and stored in the CGC for order. Apart from that, another genetic
engineering measure, optogenetics, has been emerging in recent years. Optogenetics
is an innovative technology combining optics and genetics to manipulate the activity
of individual neurons. Channelrhodopsin and Halorhodopsin are the two primary
light-sensitive opsins used in the majority of optogenetic research. Nagel et al. [131]
was the first group to demonstrate the activation of selected channels with blue light
by expressing cRNA encoding Channelrhodopsin in oocytes of Xenopus laevis, in
the presence of all-trans retinal. They found Channelrhodopsin can be a promising
tool to mediate a large light-switched H+ conductance to manipulate electrical and
proton gradients across cell membranes simply by illumination. The idea was then
rapidly prevailing in many research fields, such as PD [132, 133], AD [134], HD
[135], and epilepsy [136–138]. As for C. elegans, the use of optogenetics can allow
in-vivo observation of specific neurons without even removing them by microsur-
gery. Narayan et al. [139] once showed graded transmission between AFD and AIY
neurons by expressing Channelrhodopsin-2 (ChR2) in AFD using gcy-8 promoter.
They found that the amplified signal in AFD was scaled in a quantitative manner.
Faumont and colleagues [140] also studied locomotion of freely-moving C. elegans
generated by distinct neuronal circuits using ChR2 (Fig. 6.19b). ChR2 was
expressed specifically here in the osmo-sensitive neuron, ASH, to mimic the
avoidant response from the worms. Their system can be used to create virtual
environments by optogenetic activation of sensory neurons or to image activity in
identified neurons at high magnification.
6.4.4 Parasitiology
Parasites, such as hookworms or whipworms, are rampant in undeveloped areas due

to poor hygiene. Failing to control parasites may eventually cause life-threatening
diseases in animals as well as humans. Unfortunately, like super bugs, some
parasites also have gradually developed resistivity to drugs through natural evolu-
tion, weakening effectiveness of some anthelmintics. New anthelminitic treatments
are urgently needed, but the current drug development pipeline remains very inad-
equate [141]. Although lab-bred C. elegans is recognized as hazard-free animals and
can be safely used in non-bio-environments, it shares many biological similarities
with its parasite relatives. The similarities make the worm a good candidate in the
studies relevant to parasitiology. Among them, how anthelmintics can effectively
affect parasites are extensively investigated in pharmacies. Chen et al. [142] char-
acterized movement phenotypes of an animal parasite, Oesophagostomum dentatum,
with a microfluidic device (Fig. 6.20a). They successfully found drug sensitivity
Fig. 6.20 (a) Microfluidic chamber and larvae. The four-channel microfluidic device was fabri-
cated to study nematode locomotion. There are vertical markers to help calibrate the swimming
velocity. (Reprint with permission from [142]. Copyright 2011 Cambridge University Press.) (b)
Top: Illustration of the fabricated T-shaped microfluidic device showing the three electrode ports
and sample electric fields at the respective ports. Bottom: Worms are inserted in the left port and
their preference to move towards either cathode is tracked with the imaging unit. (Reprint with
permission from [143]. Copyright 2011 Royal Society of Chemistry.) (c) Top: Microfluidic EPG
recording device. Channels were filled with a dye to aid visualization in this image. Bottom:
Enlarged view of a single recording module, with an A. ceylanicum L4 positioned tail-first in the
worm trap. (Reprint with permission from [144]. Copyright 2016 Elsevier.) (d) Different trapping
positions of J2 G. pallida generate electrical signals with different amplitude in the presence of
2 mM 5-HT. (Reprint with permission from [145]. Copyright 2014 Royal Society of Chemistry)
doses of levamisole for two drug-resistive C. elegans mutants, SENS and LEVR,
within 45–60 min. They believed their microfluidic assay offered an improved and
higher level of resolution. Carr et al. [143] also once reported a microfluidic platform
for high-sensitivity and real-time drug screening on C. elegans (Fig. 6.20b). On this
chip, a multi-parameter microfluidic bioassay has been developed to observe the
innate locomotion of worms along with their transient and time-resolved responses
to an anthelmintics, levamisole. Percentage of worms responsive, percentage of
worms leaving the well, mean post-exposure velocity, and time until
unresponsiveness were measured. To ensure worms can move in and out of the
drug well as expected, electrotaxis was used as a guiding force in the research. With
this new platform, the measurement duration of dose response was considerably
improved from several hours in conventional worm motility assays to less than an
hour. As compared to drug resistant strains, levamisole-sensitive N2 worms were
paralyzed in a median duration of 0.4 min after exposing to 100 μM levamisole.
Many anthelmintic drugs target at proteins associated with neurotransmitter
receptors and ion channels, so characterizing the electrophysiological functions
can help the development of new drugs in parasitology. To understand electrophys-
iological signals and phenotypes of parasites, several microfluidic platforms were
proposed. Weeks et al. [144] designed a microchannel to trap a single worm in a
desired position and then inserted electro probes to measure their
electropharyngeograms (EPGs) (Fig. 6.20c). EPGs reveal electrical activity of mus-
cles and neurons of pharynx, which is important for feeding in worms. With the
microchip, the worm’s EPGs against environmental stimuli can be continuously
recorded. Two parasite species, A. ceylanicum and A. canium, relevant to human
health were removed from their hosts and measured in vivo. The study showed that
such a new technology is capable of providing massive and real-time EPG informa-
tion to assist evaluations of anthelmintic drugs on worms and understanding of the
worm’s electrophysiological statuses depending on their EPG readouts.
In addition to parasites in host animals, plants may be infested by invader worms
as well. Plant parasitic nematodes (PPNs) are worms that infest the roots of their host
plants impairing plant viability and reducing the yield of crops. For feeding, a stylet
is protruded from a worm to stab into the plant root. However, the study of stylet
activity remains technically challenging. To understand the underlying biology,
electrophysiological recordings can commit some valuable information. A
microfluidic device, named StyletChip, built up in such a purpose was developed
by Hu et al. [145] (Fig. 6.20d). A PPN, G. pallida, was used to demonstrate the
feasibility of the device. Recordings were made on the nematode within 2 days from
hatching. Each worm was trapped at the test region with a positive pressure by
arranged valve controls. 5-hydroxytryptamine (5-HT) is a drug used to stimulate the
worm’s stylet thrusting action. By 2 mM 5-HT was applied to the trapped worm,
rhythmic electrical signals with a frequency of 1 Hz was recorded. The study showed
that the proposed StyletChip was suitable for defining the actions of chemicals on
stylet and median bulb activity with a view to developing new anthelmintics for crop
protection.
6.5 Summary and Outlook
Since C. elegns was firstly introduced to the scientific community, it has been proven
itself a quite successful and valuable model animal in all aspects over the past
decades. Several scientific breakthroughs originated from C. elegans research have
brought significant impact in our world. For instances, biocompatible green fluores-
cent proteins (GFP), insulin-like signaling pathway (ILS) for the secret of longevity,
knocking down the genetic expressions with the biological tool, RNAi, and more.
Nevertheless, challenges also arise when complex data, more information, and new
tasks await processing in a shorter timeframe. Demands for new technologies to
meet the new challenges are elevated. Fortunately, the advent of microfluidics has
been progressively revolutionizing the worm research community. These novel
microfluidic chips offer several advantages over conventional approaches:
(1) maintaining well controllable micro-environments, (2) creating reproducible
experimental conditions, (3) automating tedious experimental protocols, and
(4) enabling high-throughput studies. The addition of the engineering elements
extends advanced possibilities of the worm development. By taking advantaging
of the worm’s responses to different stimuli, strategies for size sorting, immobiliza-
tion, long-term imaging, microsurgery, confined breeding have been well developed.
The concept of WoC is even prevailing in broad applications, not only limited to the
group of worm breeders nowadays. Drug screening, biosensors, disease modeling,
parasitiology, neuroscience, and genetic engineering have long been benefited from
C. elegans for decades. With WoC, however, all the applications appear to boost in a
new era. Eventually, it can be expected to see the growing in WoCs as a promising
branch of the C. elegans research in the near future.
6.5.1 Unmet Challenges
Despite the promising possibilities of the integration of microfluidics and C. elegans,

there are still many hurdles laying ahead before the concept can really come to
practice in the worm community. First, a general-purpose microfluidic platform
remains undeveloped yet. Actually, most worm chips proposed so far in the papers
are designed for specific goals, resulting in the use of microfluidics in C. elegans
labor-intensive, time-consuming, costly, and high expertise needed. The conse-
quence makes the technology very diverse and dependent on each lab, impeding
its prevalence in the market. Second, worms are thought to be terrestrial animals
although they can be amphibious. However, most microfluidic platforms require to
culture worms in an aqueous environment, which may come to suggestions different
from terrestrial cases. Third, how well can worm research be relevant to human
health remains under debate. Although worms are well known to have more than
60% gene similarity with humans, it’s not completely sure of whether the underlying
genetic pathways preserved across species as diverse as humans and worms. Some
of them have been proven to exist in both species [146, 147], but more are still
waiting for confirmation. For a scientific standpoint, however, the uncertainty can be
clarified faster when the microfluidics are incorporated.
6.5.2 Future Prospects
As many possibilities have demonstrated for WoCs to date, we envision more new
gadgets to enrich the worm arsenal. Several routes are expected to receive focuses of
the future prospects in WoCs. They are further addressed below:
(a) Automation of worm processing
Ease-to-use is always a concern in the worm research. Because of the nature of
biological complexity, worm assays usually require plenty of expertise and training
to complete worm processing. Automation accompanying with graphical user inter-
faces (GUIs) can be a great solution to ease researchers’ life. Automation can also
facilitate laymen to deal with the worm research. Despite the ideology, the major
hurdle is attributed to the worm’s diversity and uncertainty. Accordingly, a large
number of data becomes key to obtain reasonable statistical analysis. The recent
hotline concerning artificial intelligence (AI) perhaps provides an insight to the
future development. With deep learning of the worm behavior, optimized automa-
tion can be tuned to increase the efficiency of the worm processing.
(b) High throughput, robustness, and high reliability for industrial applications
Unlike normal lab research, being eligible for industrial applications usually
implies that a technique is sufficiently mature to proceed from theory to practice.
In this phase, high throughput, robustness, and high reliability are particularly
emphasized. Some potential fields, such as pharmaceutical and agriculture, have
studied C. elegans as a model animal in drug tests for neurodegenerative diseases
[148–150] and anthlmintic screening for parasites [151, 152]. However, the overall
investigations still heavily relied on manual operations, limiting the yield and
reliability. The introduction of powerful microdevices can certainly promote the
throughput by several orders of number. Moreover, the reduction of labor can
improve the reliability as well.
(c) An integrated platform to enable versatile goals
To this end, considerable methods have been conducted and discussed. Among
them, the mostly novel proposal goes to a technique termed “LEGO microfluidics”
[153]. Apart from the conventional way, module bricks of various microfluidic
components were built in advance. When a specific assay is designated, the selected
modules were assembled to form a complete platform for that task. Although such a
highly integrated chips have not emerged yet, its appearance is for sure just a matter
of time. Nevertheless, the conceptual work has been realized in many existing
MEMS-based devices [154–158]. The pioneering idea of programmable lab on a
chip was firstly attempted by Urbansiki in 2006 [159] and then extend to more areas
[160, 161]. With all necessary components built on the same chip, general-purpose
tasks can be done. The concept is to minimize the barriers from hardware, so that the
core work can be executed with only software programming. Obviously, the use of
such an integrated platform not only imparts WoCs versatility but also eliminates the
gaps between labs, reducing the turnaround time in most worm studies.
Acknowledgements This work was supported by the Ministry of Science and Technology in
Taiwan under the grants 107-2221-E-006-054-MY3 and 107-2622-E-006-022-CC2. W.-H.Wang is
grateful to the financial support from the One-Thousand Young Talent Program of China.
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Microfluid Nanofluid 15:647–659
Chapter 7
Microfluidic Devices for Gamete Processing
and Analysis, Fertilization and Embryo
Culture and Characterization
Séverine Le Gac, Verena Nordhoff, and Bastien Venzac
Abstract Assisted reproductive technologies (ART) include all techniques used to

achieve pregnancies not only in case of human infertility, but also for the in vitro
production of embryos in the livestock industry, and for the conservation of endan-
gered species. Focusing on human ART only, the total number of babies born
worldwide has been estimated to more than 7 million, and ART are more and
more utilized as a consequence of an increase in human infertility. However, at the
same time, even if ART are now used as routine clinical treatments, the success rate
remains low, with less than 30% clinical pregnancies. Furthermore, the ART preg-
nancy rates are now stagnating, showing a need for new improvements. Finally,
current protocols are lacking standardization and automation, and they are still
dependent on the skills of highly trained personnel. In that context, microfluidics
can offer a new paradigm in the ART field, by providing integrated and automated
platforms. Furthermore, the use of microfluidics can introduce new approaches by
performing some steps of the entire protocol. In this chapter, we summarize and
discuss microfluidic developments in the field of ART, and specifically devices for
gamete analysis, selection and processing, fertilization and embryo culture.
Keywords Assisted reproductive technology · Gamete processing and analysis ·

In vitro embryo manipulation · Integrated platforms · Microfluidics
S. Le Gac (*) · B. Venzac

Applied Microfluidics for BioEngineering Research (AMBER), MESA+ Institute for
Nanotechnology, TechMed Center, University of Twente, Enschede, The Netherlands
V. Nordhoff
Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Münster,
Germany

198 S. Le Gac et al.
7.1 Introduction
Assisted reproductive techniques (ART), which comprise all techniques utilized to

conceive offspring using (semi-)artificial ways have been utilized for 40 years to
overcome human infertility. Next to this, ART are also widely used for the in vitro
production of bovine and porcine embryos in the livestock industry as well as for the
conservation of endangered species. Infertility affects 15% of the couples in the
developed countries, and this figure continues to increase as a consequence of our
modern lifestyle and repeated exposure to chemicals causing infertility such as
endocrine disruptors. Currently, ART are well-established practices, and have
become clinical routines in nearly all parts of the world. As a great honor for the
ART field, the Nobel Prize in Medicine or Physiology was attributed to Sir Robert
Edwards, one of the “medical fathers” of the first born IVF baby worldwide, Louise
Brown. Since then, according to the European Society for Human Reproduction and
Embryology (ESHRE, www.eshre.eu), more than 7 million babies have been con-
ceived using ART worldwide. Furthermore, it has been estimated that during the
years 2008–2010 over 1.1 million children have been born as the result of the various
ART treatments [1], and, following the increasing infertility rate the number of
treatment cycles has been continuously rising [2]. Equally important is the use of
ART in the multi-billion dollar livestock industry with about half a million bovine
embryos produced in vitro in 2013 [3]. The most commonly used techniques
worldwide are intrauterine insemination (IUI), in vitro fertilization (IVF) and
intracytoplasmic sperm injection (ICSI), as discussed later in this chapter.
In spite of this continuously growing number of ART cycles, the so-called “take-
home baby rate” remains still low, with less than 30%. Furthermore, the year-to-year
enhancement in the pregnancy rates reported in the last decades was not observed for
the first time in 2008 [4], which may indicate that the techniques used in ART and for
the selection of gametes have reached a saturation level regarding success rates.
Altogether, these facts call for new technological developments and alternative
approaches for the ART routines. Another main issue encountered with ART next
to the low success rates is the higher chance for multiple pregnancies and multiple
birth rates (www.eshre.eu; ca. 17.5% in 2014 in the EU), which can be accounted by
the lack of reliable approaches to identify embryos with the highest developmental
competence at the end of the pre-implantation development period. The implemen-
tation of the policy for elective single embryo transfer, or eSET, has helped decreas-
ing the multiple pregnancy incidence, but it requires, more than ever, the availability
of reliable means to grade and identify top-quality embryos before transfer. Next to
this, almost all procedures in an ART protocol are entirely manual and differ from
lab to lab. This lack of standardization together with the manual and subjective
character of the processes explain the sometimes significant discrepancy in success
rates between IVF centers.
In this chapter, we discuss how microfluidic technology could revolutionize the
field of ART by providing not only new approaches to conduct the different steps of
the process, but also integrated and automated platforms. We will first present in
7 Microfluidic Devices for Gamete Processing and Analysis. . . 199
detail an entire ART protocol, using the most widely used insemination techniques,
IUI, IVF and ICSI. Next, after a very brief introduction to microfluidic technology,
we will highlight some of the key-features this technology presents, and which are of
particular interest for the field of assisted reproduction. Following this, we will
review selected platforms developed to conduct different individual steps of an
ART routine; namely, the analysis, characterization and/or selection of the female
and male gametes; the fertilization and/or cloning; the in vitro culture of the resulting
embryos and, finally, their characterization in the view of identifying embryos with
the highest developmental competence. In every section we will discuss the prom-
ises and identified shortcomings of microfluidic technology and the applicability of
the so far reported platforms for true clinical routines. Finally, in the conclusion, we
will introduce additional examples of microfluidic platforms either capable of
performing an entire IVF process or developed for other promising applications
related to ART.
7.2 A Standard ART Routine, as Performed in an ART

Center
Although ART techniques have developed over the last decades and are differently
managed and used in every ART clinic and lab, some basic methods are equal
everywhere and form the basis for assisted reproduction the control of the ovarian
cycle. In a regular ovulatory cycle of a woman (between 26 and 35 days) the growth
of one follicle is stimulated by endogenous hormones (mainly Follicle-stimulating
hormone, FSH) and, respectively, a single oocyte is ovulated 40–44 h after a
Luteinizing hormone (LH) peak in the middle of the cycle. For assisted reproduction
the female cycle is often supported or stimulated in a controlled manner by admin-
istration of exogenous FSH. In case of simple treatment options like timed inter-
course or insemination (intrauterine insemination, IUI) only low doses of FSH are
applied. Successful follicular growth is followed by ultrasound of the ovaries and if
one or two follicles are confirmed ovulation is induced by a single high dose of
human Choriogonadotropin (hCG), which mimics the natural LH peak. The couple
can then either go for timed intercourse 24–36 h after ovulation induction or for
insemination, with the placement of a prepared semen sample into the uterine cavity
36 h after application of the hCG dose.
If insemination is not the treatment of choice or has not resulted in a pregnancy,
in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI), the
two major in vitro treatment options have to be used (Fig. 7.1). IVF is the treatment
of choice if a female factor is the cause for the infertility, e.g., obstruction of
the oviducts or uncontrolled growth of endometrial cells in other body areas outside
the uterus (endometriosis). ICSI is indicated if a male factor has been
diagnosed. However, ICSI is also the treatment of choice if IVF has failed. For
Sperm selection
Sperm
analysis
Intra-cyto-
plasmic
sperm
Follicle injection
puncture Cumulus cell
removal
In-vitro fertilization Embryo culture
Fig. 7.1 Schematic representation of a conventional ART medical routine, using either IVF
(in vitro fertilization) or ICSI (intra-cytoplasmic sperm injection) for the fertilization. Oocytes are
retrieved from a woman’s ovary, and incubated with processed sperm in a Petri dish for fertilization
if the sperm count and motility are in the upper normal range. Otherwise, cumulus cells are removed
around the oocyte, a single sperm is selected based on motility and ICSI is performed. The resulting
embryos are kept a few days in culture until their intrauterine transfer for further development.
Illustration by Dr. Venzac
IVF and ICSI women are treated with higher doses of hormones to achieve multi-
follicular growth in both ovaries. This multi-follicular growth is necessary as not all
oocytes may be of equal quality or possibly not mature enough to be used for IVF or
ICSI. Additionally not all oocytes are fertilized or result in normally developing
embryos or may although of good quality lead to a pregnancy.
Oocytes with their surrounding cumulus cells (cumulus-oocyte-complex, COC)
are retrieved from the follicles 35–36 h after hCG application by an ultrasound-
guided transvaginal puncture of both ovaries. In case of an IVF cycle the COCs are
incubated with 25,000–100,000 motile spermatozoa from a prepared semen sample
in droplets of 30–100 μL, often covered by light mineral oil to omit evaporation of
fluid. For ICSI, the oocytes have to be separated from the cumulus cells by gentle
denudation using an enzyme (hyaluronidase) and small pipettes. Mature oocytes
(in Metaphase II stage) are then injected each with a single immobilized spermato-
zoon using a small injection needle. Fertilization success, meaning the appearance of
a fertilized oocyte (pronucleus stage) is checked 15–18 h later. Further cultivation in
10–50 μL droplets [5–7] of special culture media for 2–5 days leads to the produc-
tion of pre-implantation embryos, of which normally only one, sometimes also two
or three (depending on quality and transfer policy of the respective country) is
(or are) transferred back to the uterus of the patient. A biochemical pregnancy is
confirmed by measuring β-hCG in blood, while a clinical pregnancy is checked via
detection of the heartbeat of the fetus by ultrasound 1–2 weeks later.
For all ART treatments, meaning IUI, IVF and ICSI, the ejaculate has to be
prepared. The ejaculate consists of two major fractions, a sperm-rich first fraction
from the testes followed by a mostly seminal fluid fraction from the accessory glands
[8]. Sperm have to be separated within 1 h from the seminal fluid as the latter
contains components (e.g., prostaglandins, zinc) and non-sperm cells that may
damage intact spermatozoa. In the case of natural conception, these components
and non-sperm cells cannot pass the cervical mucus at time of ovulation. However,
in ART natural barriers are bypassed either by direct insertion of the sperm into the
uterus of the women (IUI) or direct exposure of the oocytes to the sperm sample, so
that these components become obstacles for the achievement of a pregnancy by
possibly damaging sperm cells [9].
Sperm samples are typically prepared using different techniques, depending on
the sperm concentration and motility in the native ejaculate. The choice of the
method depends on which ART treatment is used [9]. For normozoospermic
samples (meaning normal concentration (WHO reference value is 39 million/
mL), normal motility (32% progressive motility) and normal morphology (4%))
the WHO recommends in their latest handbook [9] to use a simple washing step:
the ejaculate is mixed with medium, centrifuged, the supernatant discarded and the
pellet is simply resuspended with medium. In the swim-up procedure, which is
ideal for samples in the normal range, the ejaculate is centrifuged, next overlaid
with medium, and the motile sperm swim during an incubation step into the
medium. If the semen sample presents a lower concentration or reduced sperm
motility (oligoasthenozoospermia; oligo: reduction of concentration, astheno:
reduction of motility) the WHO recommends the use of density gradients. Specif-
ically, different layers of medium with various densities are prepared in a tube and
the sperm sample is centrifuged through these different layers. Typically, sperma-
tozoa accumulate in the pellet at the bottom of the tube, this pellet is re-suspended
in medium for further use. Using the gradient approach high numbers of motile
sperm are obtained, while the swim-up methodology provides lower number of yet
highly motile sperm cells [10]. In very severe cases of
oligoasthenoteratozoospermia (terato: reduction in morphology) with extremely
low sperm numbers (<100,000 sperm in the complete sample) or in case of
azoospermia (absence of spermatozoa in the ejaculate) it might even be necessary
to retrieve sperm directly from the testes. During an operation the testis capsule is
opened and the tubuli seminiferi, where spermatogenesis takes place, are screened
using a microscope to detect areas with high probability of intact spermatogenesis.
Small testicular biopsies are taken for testicular sperm extraction (TESE). Biopsies
can be used either fresh or frozen to retrieve intact spermatozoa for ICSI. Nor-
mally, motility is the clear evidence of vitality and only vital spermatozoa will give
reasonable fertilization rates. However in low quality TESE samples, it may
happen that only immotile spermatozoa are retrieved. In this case additional
methods for detection of vitality have to be used [11].
7.3 Why Microfluidics for ART?
Microfluidics, as pointed out by Georges Whitesides, can be defined as “the manip-

ulation of fluids in channels with dimensions of tens of micrometers” [12]. For now
more than two decades, this technology has been gaining more and more popularity,
especially in the fields of biomolecule analysis and cell biology, and microfluidic
devices are currently routinely used in healthcare and diagnosis, particularly for
point-of-care applications. The success of microfluidics can be explained by the
enhanced capabilities this technology brings about. Volumes in microfluidic devices
are dramatically reduced down to the low nanoliter range. Therefore, analysis can be
conducted on smaller-sized samples, and cell experimentation can easily be down-
scaled to the single cell level [13, 14]. Processes, including analyses, are much faster
at the micrometer scale, and they are better controlled as a result of both the large
surface-to-volume ratio and the unique properties of the flows [12]. For cellular
studies, these characteristics associated with the high confinement level found in
these devices enable to closely emulate conditions found in vivo [15]. At that scale, it
is also straightforward to create dynamic conditions, diffusion-based delivery of
substances [16, 17] or gradients [18]. Furthermore, microfluidic devices present a
high level of integration. One device can for instance comprise a series of identical
systems (horizontal integration), which is highly attractive for assay parallelization.
Alternatively, a series of operations can be implemented in one single device
(vertical integration), to perform a complete (analytical) process. Furthermore,
microfluidic structures can be combined with add-on capabilities (smart integration),
e.g., for sensing of biochemical or physical parameters, to control the temperature, or
for fluid actuation. Finally, these devices, which are realized using either conven-
tional microfabrication techniques derived from the field of microelectronics or
through replication approaches originating from the polymer industry, can be pro-
duced at a large scale, which drastically reduces their price and promotes their use as
disposables.
Microfluidic technology is highly attractive for the field of ART, specifically to
perform the different in vitro steps of the ART routine, namely from semen
analysis and selection until embryo scoring and selection, including the fertiliza-
tion step and the embryo pre-implantation culture. Some of the most promising
features of microfluidic technology for ART are its high level of integration and its
amenability to process automation, which would eventually allow implementing
the whole ART routine in a fully automated single platform and additionally lead
to standardization of the treatment across laboratories [19, 20]. Furthermore, using
fully integrated platforms, the manipulation of the gametes and embryos would be
minimized, together with human intervention, associated errors and risks of con-
tamination, and, subsequently, the success rate of the treatment would be
enhanced. Another main expected outcome of using microfluidic technology is a
decrease in the treatment costs: not specifically due to the smaller volumes of
medium required and the price of the devices, but also through an increase in the
treatment success.
In the following sections, we will successively review microfluidic advances for

the preparation of the gamete samples (oocytes and sperm), for the fertilization, and
for the embryo culture and characterization. Finally, in the conclusion, new trends of
fully integrated devices able to perform multiple steps of the ART protocol, or to
support spermatogenesis in vitro will be briefly presented.
7.4 Gamete Preparation and Fertilization
7.4.1 Oocytes
In the frame of conventional in vitro fertilization routines, there is little or no

preparation and selection of oocytes, as happens for the semen, before fertilization.
As mentioned in Sect. 7.2, a commonly performed step consists of removing the
cumulus cells surrounding the oocytes in the cumulus-oocyte-complex. However,
there is evidence in the literature that pre-selecting the oocytes before fertilization
does enhance the quality of the resulting embryos, and could be an alternative
strategy to the currently used approach to select embryos after their
pre-implantation culture and before their intra-uterine transfer. Furthermore, for
ICSI-based fertilization, it may be advantageous to characterize the oocytes to ensure
they are at the right maturation stage for fertilization (also before removal of the
cumulus cells).
Various microfluidic platforms have been developed for the different purposes of
oocyte denudation, oocyte characterization and/or selection, as well as oocyte
maturation, as discussed in the following. Next to this, dedicated capabilities have
been proposed for the controlled transportation and manipulation of oocytes in a
microfluidic device, and for the fertilization step as well as cloning (discussed in
sect. 7.4.3).
The first examples of microfluidic platforms for the manipulation of oocytes
aimed at removing the cumulus cells around the oocytes, which is typically done
in vitro, either before fertilization (in case of ICSI) or 15–18 h after IVF, when the
oocyte is still at the zygote stage. Conventional approaches in vitro rely on an
enzymatic treatment using hyaluronidase or a mechanical treatment, through
pipetting or vortexing. The mechanical treatments in particular have been proven
to be stressful for the zygotes. As an alternative approach, Zeringue et al. proposed a
microfluidic protocol for the mechanical denudation of bovine oocytes, in a better
controlled and gentler way, as illustrated in Fig. 7.2a. Their device comprised a long
channel equipped with 2 constricted areas, followed by a 90 turn with smaller side
channels [21]. While traveling through the constriction, the cumulus cells got
loosened around the zygotes, and they could be entirely removed by applying
suction from the side channels placed in the right angle turn. Rotation of the
cumulus-oocyte-complex in the turn of the channel was found to be essential for
efficient removal of all the cumulus cells. Comparison of this novel microfluidic
approach with a vortex-based strategy revealed that embryos undergoing the
B- Oocyte Force sensor

A- Oocyte
mechanical
denudation
characterization
Actuator
D- Oocyte
C- Oocyte optical
sorting characterization
Viable
oocyte
AC
CURRENT
Non-viable Glass µ-
oocyte pipette
Optical fiber
Fig. 7.2 Example of microfluidic platforms developed for the manipulation, characterization and
sorting of oocytes. (a) Oocyte denudation in a microfluidic channel equipped by a constricted area
and suction side-channels (illustration inspired by [21]); (b) Evaluation of the mechanical properties
of oocytes using on-chip actuated beams (illustration inspired by [24]); (c) Dielectrophoretic-based
sorting of oocytes (illustration inspired by [26]). (d) Optical characterization of oocytes using
on-chip integrated optical fibers (illustration inspired by [27]). Illustrations by Dr. Venzac
microfluidic treatment were developing better, with higher cell counts than their
counterparts having experienced vortexing [22]. Furthermore, the vortex group of
embryos exhibited a peak in their transcriptomic activity just after removal of the
cumulus cell, which may be indicative of their stressed state. Using a microfluidic
format allows precisely controlling the shear exerted on the oocytes/zygotes by fine-
tuning the dimensions of the microfluidic structures as well as the flow and negative
pressure applied in the different channels to efficiently remove the cumulus cells,
without compromising the viability of the oocytes/zygotes.
Developing novel integrated schemes for oocyte characterization has widely been
explored, with three main applications in mind: (i) to determine the maturation stage
of the oocyte before fertilization, (ii) to confirm fertilization after incubation with the
semen sample, and (iii) to evaluate the oocyte viability. All microdevices reported
for oocyte characterization rely on two approaches, using either mechanical sensors
or optical means, which are successively discussed below.
The rationale behind the mechanical approach is that the properties of the
zona pellucida, which is like a shell surrounding the oocyte, change during the
different maturation stages through thickening [23], as well as after fertilization by
“hardening”. Furthermore, zona pellucida hardening has also been observed on
“aged oocytes”, which are of lower quality. In short, the mechanical properties of the
zona pellucida directly correlate with the oocyte maturation stage and quality. A
variety of mechanical sensors have been proposed and integrated in microfluidic
devices to measure the mechanical properties of oocytes. In a first example,
Wacogne et al. proposed a SU-8-based microtool consisting of a free-standing
cantilever [24]. The oocyte was forced against a beam, and its mechanical properties
derived by jointly measuring its deformation as well as the beam deflection
(Fig. 7.2b). The system, which was tested on human oocytes, could be automated
for successive characterization of a group of embryos. In an alternative and more
straightforward approach, Luo et al. implemented a constriction in a microfluidic
channel, through which oocytes could be forced and would deform [25]. In this
device, mouse oocytes were exposed to well-defined flow and shear, by taking
advantage of the microfluidic format. Here again, from its deformation, the mechan-
ical properties of the oocyte could be determined.
Similarly, optical sensors have been developed to assess the oocyte maturation
stage and the “fertility index” in a minimally invasive way. Optical fibers were
integrated in a microfluidic device (Fig. 7.2d) to measure the transmission spectra of
individual oocytes [27, 28]; the different maturation phases of the oocyte could
successfully be distinguished by determining the minimum transmission intensity
and the associated wavelength. Altogether, this approach allowed monitoring the
oocyte maturation and deciding on the best moment for fertilization.
In the view of the selection of oocytes, dielectrophoresis (DEP) has been applied
in a microfluidic format for the automated and non-biased identification and isolation
of viable oocytes (Fig. 7.2c) [26, 29]. Viable and fertile oocytes were found to move
faster. Oocytes with no or little dielectrophoretic motion gave rise, after fertilization,
to embryos with a poor developmental competence, while in case of fast oocytes,
reduced polyspermy was observed and higher blastocyst rates and cell numbers were
found.
As recently reviewed by Yanez and Camarillo, key-advantages offered by
microfluidic technology for the oocyte characterization and selection compared to
better established approaches for the mechanical characterization of cells (e.g., AFM
and optical tweezers) [23] are its amenability for automated and high-throughput
measurements, and the possibility to design integrated platforms for on-line fertili-
zation of the oocytes. Next to this, in a microfluidic format, not only active elements
like beams, for instance for mechanical measurements can be used, but also passive
elements can be explored such as well-defined integrated constrictions and even
more simply, a well-controlled flow. Furthermore, the fact that most of the materials
used to fabricate microfluidic devices are transparent, they allow direct and contin-
uous optical examination of the specimen during the measurements.
An essential aspect for this on-chip oocyte characterization is the capability of
displacing the oocytes in the device in a gentle yet accurate manner. The Gharbi lab
reported two different silicon-based strategies for, respectively, long-distance trans-
portation and accurate capture of human oocytes at a characterization site
[28, 30]. For long-range displacement, the oocyte was trapped in a translator
consisting of a wet-etched cavity in a silicon free-standing beam, and motion of
this translator was controlled using a dedicated stage. Accurate positioning of the
oocytes was achieved by applying a negative pressure through micronozzles in the
bottom substrate of the device. Nakahara et al. proposed another gentle protocol for
controlled transportation of mouse oocytes in an open microfluidic device using a
vibration-induced flow, towards an integrated on-chip robot for their mechanical
characterization [31].
A last step of oocyte treatment, which has more recently been explored in a
microfluidic format is the in vitro maturation of the oocytes. Normally, this step is
performed in vivo through extensive hormonal treatment of the patient, as discussed
in Sect. 7.2, with however a risk for significant complications such as the ovarian
hyperstimulation syndrome. Alternatively, the oocytes can be exposed to a similar
hormonal treatment in vitro, before fertilization, through exposure to gonadotropin
[32]. Berenguel-Alonso et al. reported a COC-based platform (COC – cycloolefin
copolymer), into which oocytes could be trapped with the help of a micromachined
filter and exposed to maturation medium in the device [33]. Oocytes could easily be
retrieved from the device, thanks to careful design of the microfluidic circuitry, for
off-chip fertilization. A typical advantage of microfluidics for the oocyte maturation
is the spatial and temporal control this technology provides for the perfusion of
solutions. Furthermore, the mature oocytes could subsequently be fertilized in situ
by replacing the maturation solution by prepared semen.
7.4.2 Semen Preparation, Analysis and Selection
In the frame of an ART routine, different operations are performed on the male
gametes, including the semen preparation and selection for subsequent ART steps,
as described in Sect. 7.2. Next to this, microfluidic systems have also been developed
for fertility diagnosis, in the form of either lab-based or home-testing devices.
A male factor is involved in 40–50% of all infertility cases. This male factor can be
caused by a variety of pathologies, and associated with different semen sample
compositions, ranging from a total absence of sperm to a lower concentration of
motile sperm in the ejaculate. Sperm analysis is therefore one of the first actions
taken when a couple encounters fertility issues. The main assays focus on sperm
characteristics, such as the concentration in the ejaculate, the motility of the sperm,
their vitality and morphology, and their DNA integrity as detailed below. The thresh-
old for a normal semen sample is considered to be 39 million cells per millilitre, as
evaluated using a counting chamber or by automated treatment of microscopic images
using a system called CASA (computer-assisted semen analysis). The motility is the
second main parameter, which is also measured with a CASA system. Motility is a
strong indicator for a successful fertilization. A semen with a good quality has to
contain more than 32% of motile sperm [9]. Sperm cells can also be classified
according to their motility, the latter being characterized as progressive (straight and
fast movement), non-progressive and immotile. The vitality of the sperm is typically
detected using classical dye exclusion or hypotonic swelling assays [9].
acrosomal region
Coiled tail
Bent midpiece
Abnormal
Absent tail
Thin head
Microcephalous
Cytoplasmic
Abnormal post-
acrosome
Normal
sperm
Morphological
defect
droplet
of sperm 50% 33% 21% 6.6% 6% 4.6% 6.6% 1.6% 1%
Fig. 7.3 Main morphological defects observed in sperm, with the average percentage of cells
presenting this defect in samples from 1001 male partners of pregnant women. Illustration inspired
by [34], and made by Dr. Venzac
Morphological defects (size of the head, double-headed, double-tailed, etc.) are

easy to observe qualitatively (Fig. 7.3), but their quantification is more delicate,
especially because a semen sample is still considered normal if only 4% of the sperm
cells have a normal morphology. Moreover, the relationship between morphological
defects and pregnancy rate is still not clear [35]. Finally, DNA integrity, which can
be probed using a comet assay or a sperm chromatin structure assay, has been
identified as a good predictor of embryo quality after IVF [36].
None of these analyses is currently automated; they all suffer from a lack of
standardization, they use expensive equipment and they are time-consuming.
Microfluidics is a mature technology, which has been proven to be attractive for
diagnostic purposes, for other medical applications, as well as for the development
of a variety of point-of-care devices [37]. In that frame, microfluidics not only can
help to simplify, automate and even improve the diagnostic of male infertility, but
also provides new opportunities to perform semen analysis at home. The so-far
developed microfluidic systems for this purpose of semen analysis can be divided
into lab-based systems and point-of-care devices.
First, microfluidic devices have been mainly developed to quantitatively evaluate
the sperm concentration and/or motility in semen, in an automated way, in a
specialized laboratory. A number of reviews has extensively discussed
microfluidics-based analysis of sperm for interested readers [38, 39], and here, we
will mainly focus on two different systems. Using impedance measurements of
micrometre-sized objects flushed in a microchannel, Segerink et al. successfully
distinguished sperm from other cell debris and white blood cells [40]. Furthermore,
to derive the concentration of sperm cells in samples, a known amount of polysty-

rene beads was added to the raw semen and both the beads and sperm were
counted. This approach eliminates the need to know the flow rate in the channel.
The same system was next used to distinguish normal from morphologically
impaired sperm that present a cytoplasmic droplet on the flagella [41]. Chu et al.
proposed a simple passive system, in which a semen sample and medium are
injected next to each other in a microfluidic chamber. After about 10 min, half of
the motile sperm is expected to have migrated into the medium compartment
[42]. The device was next subjected to a centrifugation step: each semen fraction
formed a pellet in the device and the pellet areas were calculated to derive the
concentration and ratio of motile sperm. The results with this device strongly
correlated with those obtained with a CASA system. As for now, none of the
microfluidic systems reported for semen analysis have been developed beyond the
proof-of-concept stage. However, the performance and ease-of-use of these
devices are promising for their future clinical translation as diagnostic tools.
Still, one of the main advantages of microfluidic devices remains their portability
and their suitability for home-testing and as point-of-care devices. As a consequence,
no medical appointment is needed for a first qualitative testing, which decreases the
stress and anxiety related to testing in a specialized clinic as well as the price of the
male fertility test. Point-of-care testing could also be of great interest for remote
locations or developing countries with limited access to medical centres. Different
technologies have been proposed for low-cost, easy-to-use and easy-to-readout
assays for home-based semen analysis, as recently reviewed [43]. Lateral flow
assays, which use the same principle as the pregnancy test, are already commercial-
ized to determine at home the sperm concentration (e.g., SpermCheck; https://www.
spermcheck.com/). They only give a qualitative estimation of the sperm concentra-
tion, and must be therefore followed by a quantitative and complete analysis in a
clinical laboratory in case of low sperm count. Furthermore, other main parameters
(morphology, motility and viability) are not examined in this assay. Other systems
based on the staining of sperm, which are to swim outside a sample semen drop (e.g.,
SwimCount Sperm Quality Test, https://www.swimcount.com/), provide a qualita-
tive concentration of motile sperm, with an accuracy of 95% as compared to the Gold
standard (CASA).
Two other alternative microfluidic strategies offer multi-parametric, cheap and
easy-to-use diagnostic. For instance, an automated CASA was implemented in a
microfluidic chamber: sperm count and motility are being recorded and determined
using the camera of a smartphone. Sample loading in this device is power-free and
entirely manual. This system allows characterising the concentration, motility and
velocity of the sperm with results similar to the traditional CASA [44]. Paper
microfluidics, with its promises of ultra-low cost and ease-of-use, has also been
applied for semen analysis. One device, reported by Nosrati et al. successfully
determined the concentration of live and motile sperm in a sample [45]. Their
assay relies on a colorimetric principle: an enzyme produced by live sperm converts
a yellow tetrazolium dye into a purple compound. Next to this, the device includes a
membrane with small pores, through which only motile sperm can pass. Therefore,
only the motile sperm crossing the membrane could reach the dye and induce a
colour change, reaching an accuracy similar to CASA. The sperm DNA integrity,
which is acknowledged as the best predictor for a successful fertilization, was also
measured in a paper microfluidic system using an electrokinetic separation of intact
and damaged genomic DNA after on-chip sperm lysis [46]. In contrast to the
aforementioned commercialized systems, and despite their promising performance,
all these paper microfluidic systems are still at the proof-of-concept stage. Commer-
cialization is also not foreseen, which is a difficulty shared with most of the paper
microfluidic devices.
Even for perfectly fertile men, the ejaculated semen always contains a large
amount of sperm with impaired characteristics. In a normal sample (according to
the WHO definition), up to 68% of sperm can be immotile, and up to 96% can
present morphological defects (See Fig. 7.3). As mentioned in sect. 7.2, during
natural fertilization, sperm are subjected to a strong selection pressure in the female
track (cervix/uterus/oviduct) [47, 48]. Specifically, sperm must be able (i) to swim
independently, against a flow of oviductal fluid and in a complex environment in
terms of topology and fluid composition, (ii) to escape the female immune system;
(iii) to correctly reach the oocyte (long-range thermotaxis and short-range chemo-
taxis); (iv) to undergo hyperactivation and modification of their head composition
(process also known as capacitation) after interactions with the oviduct epithelium.
From an initial quantity of several million cells, only a few hundred of them
actually reach the oocyte, and all the immotile sperm presenting strong morpholog-
ical defects are eliminated stepwise in the journey through the female reproductive
track [48]. Arguably, similar selection has to be implemented in vitro to isolate the
best sperm for subsequent IVF, and the unique optimal sperm for ICSI. Current
selection techniques (e.g., swim-up, density gradient), as discussed in sect. 7.2,
merely sort sperm according to their motility and morphology, since morphologi-
cally abnormal sperm might not reach the swim-up medium. Furthermore, the
centrifugation steps involved in the process are stressful to the sperm and may result
in the production of reactive oxygen species (ROS) and, in turn, to DNA fragmen-
tation [49]. Using microfluidics, the best sperm can be isolated using milder condi-
tions and avoiding the stressful centrifugation steps, as illustrated by a number of
devices proposed by several research labs.
A large number of microfluidic systems focus on passively selecting motile sperm
in microchannels [50], by recovering the fastest swimmers and highly motile ones at
the end of a microchannel. For instance, Nosrati et al. reported a circular high-
throughput system with 500 microchannels, able to process 1 mL of raw semen in
less than 20 min, and to isolate only motile sperm with a significant increase in DNA
integrity (80%) compared to the raw sample, which is to be compared to the
traditional 50% achieved using conventional methods involving centrifugation
[51]. A similar device exploiting the swimming ability of motile sperm through a
membrane having 8-μm pores provided an improvement in the DNA integrity,
motility speed and morphology of the sperm compared to the standard swim-up
technique [52]. Actually, sperm selected according to their motility always show
better DNA integrity compared to the raw semen specimen, as most of the damaged
sperm in the raw semen are not able to move efficiently anymore. Compared to more
centrifugation-based traditional methods, the production of ROS and DNA damages
are significantly decreased when using microfluidic devices, thanks to the use of
gentler handling conditions. However, a large amount of the selected sperm is still
morphologically deficient or damaged.
Using microfluidic devices, other selection criteria have been explored, which
cannot easily be implemented using conventional dishware, to further improve the
quality of the selected sample. In vivo, spermatozoa are able to direct themselves by
following gradients of either factors secreted by the oocyte (short-range chemotaxis)
[53], or temperature between the uterus and the fertilization location (long-range
thermotaxis) [54], or even against weak flows of oviduct fluid (rheotaxis) [55].
Microfluidics, which provides a fine control over chemical and physical gradients
(e.g., temperature gradients) [18], as well as on flows, is the perfect format to design
novel types of sperm selection devices, making use of different sorting strategies.
For instance, chemotaxis has been combined with a motility screening assay, where
swimming sperm in branching channels could choose to follow a gradient of factors
either created by addition of chemical such as acetylcholine [56], or generated by
cultured cumulus cells [57] or even directly by an oocyte [58]. The general finding of
these systems is that the chemo-attracted sperm represents only a small fraction of
the total initial cell numbers, i.e., around 10%. Thermotaxis has also been explored
as a means to quantify viable sperm, but not for selection [59]. Finally, Wu et al.
proposed a high-throughput sorter (up to 200,000 sperm per minute) making use of
the sperm cell ability to swim against a flow [60]. In a tapered channel, in which a
gradient of fluid velocity is created, sperm that swims against the flow accumulate at
the position corresponding to their maximum swimming speed, while non-motile
cells and debris were flushed away by the flow (Fig. 7.4).
However, for all these examples, only proof-of-concept experiments have been
reported. Furthermore, the DNA integrity, morphology and fertilization rate of the
isolated sperm using these various taxis processes were not examined. Altogether,
while showing great promises, these techniques still need further and more
extensive clinical validation before they can effectively be utilized in the frame
of ART routines.
In the case of ICSI-based fertilization, a single spermatozoon is required, and
has to be chosen. For now, the choice is based on the cell motility and morphology,
as evaluated by the embryologist under a microscope. However, this step is labor-
intensive, time-consuming and dependent on the skills of highly trained personnel.
Therefore, the availability of a fully automated system able to characterize and
select individual sperm would be of great interest, especially if multiple parameters
could be considered for this step (e.g., cell vitality, DNA damage). To that end, de
Wagenaar et al. [61] trapped individual sperm cells in geometrical constrictions in
a microfluidic system, so as to evaluate the cell viability and chromosomal content
using fluorescence microscopy. However, the sperm of interest could not be
Cumulus-oocyte
complex
Fig. 7.4 Sperm selection in microfluidic systems based on different physical principles. (a)
Motility (motile sperm in black, non-motile sperm in red); (b) Chemotaxis, where chemotactic-
sensitive sperm (in blue) is attracted by a gradient of factors generated by a cumulus-oocyte-
complex (illustration inspired by [57]); (c) Rheotaxis, where motile sperm swims against the flow
while others cells are flushed away (illustration inspired by [60]). The top drawings correspond to
the initial situation after loading of the semen and the bottom drawings to the final situation after the
selection process. Illustrations by Dr. Venzac
retrieved from the device for subsequent ICSI. Alternatively, using opto-electronic
tweezers immotile sperm were distinguished from dead cells in a microfluidic
chamber for extreme cases of oligoasthenoteratozoospermia. Live sperm were
successfully attracted to the opto-electronic tweezers, while dead cells were
repelled [62]. When applied to ICSI, single sperm analysis can be a powerful
strategy to identify the best gamete for subsequent fertilization. However, for
extreme cases where a TESE (testicular sperm extraction) is needed, the resulting
semen preparation is often composed of a small amount of often sperm in a large
number of other germ cells, red and white blood cells, somatic cells and debris.
Therefore, the initial sample should first be purified to yield a new sample
containing only sperm, which could subsequently be analyzed at the single-cell
level [63]. Microfluidics, which is an established technology for the isolation for
rare cells [64] (as demonstrated for instance for circulating tumor cells [65] or
bacteria), as well as for single cell analysis [10, 14] is without a doubt very
attractive to provide integrated platforms for the isolation of the sperm from
TESE samples, and their on-line characterization.
7.4.3 Fertilization and Cloning
After processing and analysis of the gametes, fertilization takes place using either
IVF or ICSI. A few examples of microfluidic devices are found for this fertilization
step. A first device was developed for porcine IVF; incubation of oocytes with a
large number of sperm led to the penetration of multiple male gametes into the egg
cell (polyspermic fertilization). Following this, two systems described by Clark et al.
and Suh et al. consisted of a simple straight channel equipped with a constriction to
trap the oocytes in the center of the channel. A gentle flow was applied to bring the
sperm into the vicinity of the oocyte, decreasing thereby the contact time between
the gametes and in turn polyspermy [66, 67]. Sperm sorting has directly been
combined with fertilization in a handful of systems. Sano et al. used for fertilization
sperm sorted according to their motility [68]. The developmental competence of the
fertilized oocytes produced in their system was higher than using conventional IVF
in a Petri dish and the monospermy rate improved.
Next to this, dedicated microfluidic approaches have been proposed for cloning
purposes or genetic manipulation. For instance, Zeringue et al. exposed oocytes in
a microfluidic device to a well-defined flow of lyzing acid solution to soften and
remove the zona pellucida around the oocyte [69]. The Arai lab integrated
magnetically actuated microrobots in a microfluidic platform for high-throughput
oocyte enucleation and cloning [70]. The device specifically incorporated a
microgripper for the capture of the oocyte and a microknife to rupture the zona
pellucida. Mechanical squeezing of the oocyte thereafter allowed ejecting the
nucleus out of the oocytes. Using this technique, enucleated oocytes were found to
remain viable and gave rise to viable embryos after (off-chip) cloning.
7.5 Embryo Culture and Characterization
Next to semen analysis, embryo manipulation and in vitro culture is the second main
aspect of an ART routine, which has been implemented in a miniaturized and/or
microfluidic format. Interested readers are referred to recent reviews by us and
others, which are dedicated to the steps of in vitro embryo culture and pre-transfer
characterization [71–73], for more detailed information.
7.5.1 Embryo Culture
For the specific in vitro steps involving the embryos, namely their pre-implantation
culture and characterization microfluidics exhibits a number of specific advantages.
First, the single cell study capability offered by microfluidics is essential in the frame
of the eSET policy since the use of microfluidics can allow culturing individual
embryos in separate nanoliter structures and simultaneously monitoring in situ their
development over time throughout the entire pre-implantation period. Embryo
characterization would not be restricted to basic morphological criteria through
simple imaging, but could include advanced morphokinetic studies and time-lapse
imaging combined with metabolic measurements to yield a comprehensive picture of
the embryo’s developmental potential. Furthermore, from a culture perspective,
using this single embryo approach issues associated with degenerating embryos
that can have a bad influence on their healthy counterparts as found in group culture,
would be entirely alleviated. Finally, the high control on the flow and on any
physical and chemical parameter made possible at the micrometer-scale is ideally
suited to finely tune the in vitro embryo microenvironment and to possibly refresh
growth medium towards the possible design of an in vivo-like situation.
A variety of miniaturized platforms have been reported in the literature for the
in vitro culture of pre-implantation mammalian embryos, although most of these
platforms were tested on animal models. These platforms can be classified in
different groups (Fig. 7.5): (i) microwell arrays, (ii) microfluidic devices, and (iii)
semi-microfluidic platforms allying microfluidic structures with microliter volumes
as conventionally used in an ART routine.
In microwell arrays, whose concept resembles that of the conventional well-of-
the-well devices [74] embryos are individually captured in micrometer-sized wells,
which are machined in the bottom of a plastic culture dish. While each embryo is
isolated in one microwell, all embryos in the dish are in contact with each other
through the culture medium. Furthermore, culture typically takes place in microliter
volumes. The second category of platforms relies on the use of confined nanoliter
volumes found in microfluidic structures, such as microchannels [77–79] and
microchambers [75, 80, 81]. Finally, in the last category, embryo culture takes
places in a conventional microliter droplet, while liquids are actuated and/or
refreshed using microfluidic means in the form of an integrated peristaltic pump
[82] or electrowetting-on-a-dielectric (EWOD) principle [76]. Most of these plat-
forms are fabricated from PDMS, an elastomer material widely used in the field of
Fig. 7.5 Microfluidic and microfabricated platforms for the culture of mammalian embryos can be
classified in 3 distinct categories: (a) microwell arrays (illustration inspired by [74]); (b)
microfluidic structures (illustration inspired by [75]); and (c) semi-microfluidic platform, using
here microliter-volumes and electrowetting-on-dielectric actuation for the dynamic culture of
individual embryos (illustration inspired by [76]). Illustrations by Dr. Venzac
microfluidic technology, which has proven not to be toxic to the developing

embryos. Alternatively, glass and silicon have been employed to produce embryo
culture devices.
Typically, embryos are kept in culture for 3 days until reaching the 8-cell stage or
up to 4–5 days, a time at which they are expected to reach the blastocyst stage.
Thereafter, they are scored upon developmental rate and stage and, sometimes, cell
counts. In two reports using microfluidic technology [75, 82] the full-term develop-
ment of the in vitro cultured embryos was included in the embryo scoring to fully
validate the potential of the proposed platforms and protocols. Overall, when
compared to conventional droplet culture, microfluidics better supports embryo
development: embryos are developing faster, they reach earlier the blastocyst stage
and higher blastocyst rates are found. These higher developmental rates are generally
attributed to the confinement found in these microfabricated structures that better
emulate the in vivo conditions. Furthermore, the concentration in growth factors,
which are secreted by the embryos and which are essential to their development is
expected to be much higher in these small volumes. This last aspect has notably
proven to be interesting to achieve single mouse embryo culture in a nanoliter
chamber [75]. A conventional microliter-sized droplet format does not support
single embryo development, in part because of the limited availability of these
growth factors [83]. In contrast, in a nanoliter and highly confined environment
growth factors produced by the embryo accumulate in its close vicinity, promoting
thereby its development.
While one of the key-advantages of microfluidic technology would be to be able

to downscale the pre-implantation culture to the single embryo level in a nanoscale
environment, embryo culture has mostly been reported on groups of at least
10 mouse embryos [58, 77, 79, 82, 84] or, in one example, a pair of mouse embryos
[81]. Only a few examples of microfluidic single embryo culture are found. For
instance, Esteves et al. demonstrated viable single mouse embryo culture in nanoliter
chambers in a first attempt of screening of in vitro culture parameters in a
microfluidic format [75], and a similar device was next applied on donated human
embryos [80]. In more recent work and using a completely different approach
individual mouse embryos were successfully cultured in 1-μL droplet periodically
actuated by EWOD [76].
Another promise of microfluidic culture compared to conventional droplet
culture, is the possibility to implement dynamic culture conditions with tailored
perfusion of fresh medium and/or growth factors. However, so far mostly static
conditions have been tested, and embryos were cultured in microwells,
microchambers or microchannels. Only a few reports actually describe dynamic
culture, using either continuous flow, or (periodic) medium refreshment, or even
physical shaking of the embryos. For instance, Hickman et al. implemented
continuous pumping of liquid with a constant flow-rate [77]. Esteves et al. pro-
posed one-time medium refreshment in the device using passive pumping [85] at a
well-defined point [75]. Alternatively, Heo et al. developed a device with pulsatile
delivery of fresh medium in a 10-μL volume, where embryos were cultured [82]. In
another example, embryos were displaced in a channel presenting constrictions to
emulate more closely the structure of the oviduct with the help of a tilting stage
[78]. Finally, EWOD was used to regularly mechanically stimulate individual
embryos in a 1-μL droplet of culture medium [76]. Comparison of static and
dynamic culture shows contrasting results depending on the exact culture protocol.
Continuous medium perfusion and the use of constrictions were associated with
impaired development of the embryos [77, 78]. In contrast, periodic or one-time
medium refreshment as well as regular embryo agitation resulted in enhanced
blastocyst rates and cell numbers, and, in some cases better fetal rates
[75, 82]. In those cases, the beneficial effect of the flow may be accounted for by
the mild mechanical stimulation exerted on the embryos during medium refresh-
ment, since this is expected to positively influence their development [86] while a
too strong flow would result in embryo apoptosis [87].
7.5.2 Embryo Characterization
The last step of the ART routine which has been explored in miniaturized devices is
the scoring of the pre-implantation embryos, either based on metabolic parameters or
through continuous imaging and the use of morphokinetic parameters. Metabolic
parameters provide information on the physiological status of the embryos, and they
can be assessed in a non-invasive way using integrated sensors [88] and analytical
modules, or on-line analysis by coupling the microdevice to spectroscopic tech-

niques [89–91]. While individual embryos have a low metabolic activity, the use of
small volumes as found in microfluidic devices will give rise to relatively large
variations and detectable changes. Practically so far, metabolic measurements have
focused on two metabolic parameters: the embryo oxygen consumption and their
utilization of basic substrates (glucose, pyruvate and lactate), as discussed in the
following.
The first ever introduced metabolic marker to evaluate embryo developmental
competence is its oxygen consumption [92, 93]. Oxygen is an overall marker for the
embryo metabolism, and based on previous studies a low oxygen consumption is
linked to developmental arrest or embryo degeneration, while a too high consump-
tion is an indicator for damages at the molecular level. Furthermore, an embryo
oxygen utilization directly correlates with its developmental stage and presents a
significant increase upon reaching the compaction stage. The embryo respiratory
activity is typically characterized by measuring changes in the dissolved oxygen
concentration in the culture medium, either electrochemically through oxygen
reduction or optically using oxygen-sensitive probes (Fig. 7.6).
In the first case, the dissolved oxygen concentration is measured amperometrically
in the vicinity of the embryo(s) with the help of (micro)electrodes, which are integrated
in the embryo culture device. Two electrode configurations were tested: a linear series
of 4 independent microelectrodes [94], or a ring-shaped sensor in the center on which a
single embryo was placed [95]. In both cases, the oxygen concentration was mapped at
different distances from the embryo and the embryo respiratory activity was derived
from this measured oxygen depletion pattern using the spherical diffusion theory [96].
In the alternative strategy using an oxygen-sensitive light emitting probe, the
dissolved oxygen concentration can be determined from variations in either the
High [O2]
Substrate probe
Soluble probe
fluorescence
fluorescence
OXYGEN
SENSOR Low [O2]
Fig. 7.6 Schematic representation of various embryo characterization schemes, which have been
introduced in a microfluidic format: integrated electrodes to evaluate the embryo oxygen consump-
tion, oxygen-sensitive probes, either added to the culture medium or embedded in the bottom of the
device, and using time-lapse imaging in situ in the culture system. Illustration by Dr. Venzac
emitted light intensity or the fluorescence lifetime [97]. In a first report, O’Donovan
et al. added a water-soluble oxygen-sensitive complex directly to the embryo culture
medium and measured differences in oxygen consumption of embryos at the 2-cell
and blastocyst stages in group culture [98]. Most of the time however, these
complexes are not soluble in water but in organic solutions. Therefore, Komori
et al. supplemented polystyrene material used as the bottom layer of their microwell
array device with similar Pt-Porphyrin-based oxygen-sensitive dye [99] to follow the
respiratory activity of individual mouse embryos.
Other metabolic parameters, which have been analyzed to evaluate the embryo
quality also in a microfluidic format, are these three substrates: glucose, pyruvate and
lactate. Embryos consume pyruvate and glucose, depending on their developmental
stage and produce lactate, so that mapping the variations in the concentrations in the
three substances provides information on the embryo metabolism and development.
These three substrates are typically quantified through enzymatic assays by
analyzing products of an enzymatic reaction, either electrochemically or optically,
depending on the set of enzymes used and the products formed. Using oxidase-type
enzymes (e.g., glucose oxidase for glucose analysis) hydrogen peroxide is produced,
which can be quantified electrochemically. If dehydrogenases are used, in contrast,
typically fluorescent or colored substances are generated. Using three different
substrate-specific dehydrogenases, the substrate utilization of 10 individual mouse
embryos was analyzed off-line from spent culture medium in an automated manner
using parallelized nanoliter loop reactors in a microfluidic device [100]. In a second
platform culture medium was automatically sampled from a funnel structure, where
embryos were kept in culture [101]. Here again, spent culture medium was mixed
with an enzymatic cocktail on-chip and the glucose consumption of groups of mouse
embryos quantified through optical measurements.
Finally, embryo development can be monitored in microfluidic and miniaturized
devices using continuous imaging or so-called time-lapse microscopy, by taking
advantage of the transparent properties of the materials of which the devices are
made. In particular, time-lapse imaging has been reported for microfabricated well
array platforms [102–104].
7.6 Conclusion and Perspectives
Microfluidics undoubtedly presents high potentials for the field of ART. Namely, for
individual steps of the ART procedure microfluidics allows automating processes
while offering new opportunities. As reviewed in this chapter, these opportunities
are in the form of new analysis and selection schemes for semen samples, and more
in vivo-like conditions for the culture of pre-implantation embryos in well-defined and
dynamic environments for instance. However, one key feature of microfluidics has not
fully explored to date, which is the possibility to implement complex processes
comprising multiple steps as found in a complete ART routine. Only few examples
of integrated platforms combining different steps of the entire ART treatment are
found [58, 84]. For instance, Ma et al. developed a device in which up to 16 individual
Fig. 7.7 Example of an integrated platform, in which the entire IVF process can be performed.
Sperm interact with a mature epithelium grown on chip on a porous membrane (intermediate blue
layer), to be capacitated, before they fertilize an oocyte. The resulting pre-implantation embryo is
cultured on-chip in close vicinity of the oviduct epithelium. Illustration by Dr. Venzac
oocytes were individually trapped in octacolumn structures and fertilized in situ by

sperm selected on-chip for their motility and ability to cross obstacles. The resulting
embryos were cultured in the same device, after medium refreshment in the entire
platform, during the pre-implantation period yet under static conditions, and could
easily be retrieved one by one from the device for further development and/or analysis.
In a more recent example, and with the motivation to create a more biomimetic
system to enhance the fertilization rates and the quality of the in vitro produced
embryos, an oviduct was re-created in a compartmentalized microfluidic device and
it was next successfully applied to perform an entire IVF process [105, 106]. First, a
functional bovine oviduct epithelium was grown on a porous membrane, while being
exposed to a continuous flow of growth medium, in both the apical and basolateral
compartments (Fig. 7.7). Oocytes and sperm were separately injected on the apical
side of the device for the process of fertilization, just after on-chip capacitation of the
sperm by the oviduct epithelial cells. Embryos were kept in culture during the
pre-implantation period in the close vicinity of the epithelium, and subjected to
continuous perfusion of medium. Importantly, these embryos were found to present
less molecular aberrations than their in vitro counterparts grown in a conventional
Petri dish, which opens new avenues for the field of in vitro embryo production.
Along the same idea of exploiting the biomimetic environment provided by a
microfluidic format, the process of spermatogenesis has recently successfully been
recapitulated in a microfluidic device [107, 108]. In the frame of an ART procedure,
the possibility to produce sperm in vitro from testis tissues is of particular interest for
male patients that suffered from cancer at an early age of their life, and whose
testicular stem cells were cryopreserved. Samples of mouse seminiferous tubules
were successfully cultured for up to 6 months in a shallow chamber, while being
exposed to a continuous flow of fresh nutrients. A porous membrane was placed
between the tissues and the channel in which the flow was applied, to protect the
tissues from any shear that would damage it. The on-chip generated sperm was
functional and successfully used for fertilization through micro-insemination of

mouse oocytes. Following this success one can expect similar development for the
in vitro production of the female gametes and their maturation in a microfluidic
device, starting from cryopreserved ovary tissues from female patients that were
diagnosed with cancer at an early age.
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Chapter 8
Microfluidic Organs-on-Chips
to Reconstitute Cellular Microenvironments
Yu-suke Torisawa
Abstract Recent advances in microsystems technology and tissue engineering have

led to the development of biomimetic microdevices to model key functional units of
human organs, known as organs-on-chips. By mimicking natural tissue architecture
and microenvironmental chemical and physical cues within microfluidic devices,
this technology realizes organ-level function in vitro that cannot be recapitulated
with conventional culture methods. Since the physiological microenvironments in
living systems are mostly microfluidic in nature, microfluidic systems facilitate
engineering of cellular microenvironments. Microfluidic systems allow for control
of local chemical gradients and dynamic mechanical forces, which play important
roles in organ development and function. This organ-on-a-chip technology has great
potential to facilitate drug discovery and development, to model human physiology
and disease, and to replace animal models for efficacy and toxicity testing. This
chapter shows an overview of the organ-on-a-chip technology to recapitulate cellular
microenvironments and especially focuses on bone marrow-on-a-chip that enables
culture of living bone marrow with a functional hematopoietic niche as a novel type
of approach to develop organs-on-chips.
Keywords Organs-on-chips · Microfluidics · Tissue engineering · Cellular

microenvironment
8.1 Introduction
8.1.1 Cellular Microenvironment
Cellular functions are precisely controlled by their specific microenvironment where

they normally reside. The microenvironment contains a complex set of structural,
Y.-s. Torisawa (*)

Hakubi Center for Advanced Research and Department of Micro Engineering, Kyoto
University, Kyoto, Japan

228 Y.-s. Torisawa
chemical, and mechanical signals which are necessary to maintain cellular viability
and function [1–3]. Since stem cells cannot maintain their stemness without specific
stem cell niches [3–5], cellular microenvironments are crucial for maintaining
cellular function, whereas current conventional culture methods do not contain
these microenvironmental cues correctly. Because of this microenvironmental gap,
cellular functions and responses in vitro are very different from those in vivo, and
thus conventional 2D cultures cannot accurately predict cellular functions and
responses inside the body [6–8]. It is necessary to reconstitute cellular microenvi-
ronments for developing reliable in vitro methods. Development of biomimetic
microdevices that can recapitulate tissue structure and microenvironmental cues
could be a useful platform to facilitate drug discovery and development and to
develop predictive models of human physiology and diseases.
8.1.2 Cellular Microenvironments Within Microfluidic

Systems
The physiological microenvironments inside the body contain largely microfluidic

structures such as vascular networks and sinusoids. Thus, the use of microfluidic
systems facilitates engineering of cellular microenvironments. Cell culture condi-
tions in the microfluidic systems are very different from those in conventional
culture dishes (Fig. 8.1); the volume of culture medium in the dish culture is much
greater compared to cellular volume, whereas a majority of the volume in a tissue is
taken up with cells inside the body [9]. This difference in fluid volume to cell volume
ratio causes differences in the chemical microenvironment. The volume of culture
medium in the microfluidic systems is nanoliter-scale which is similar to in vivo.
Because of physiological ratios of cells to liquid volumes, microfluidic systems
enable to maintain cellular interactions based on autocrine and paracrine signals,
whereas dish cultures do not maintain these signals [9, 10]. This feature also
facilitates the formation of local chemical gradients that may otherwise not be
possible to be studied. Furthermore, microfluidic devices can apply fluidic shear
stress, enabling reconstitution of the physiological mechanical microenvironment
inside the body [11].
8.1.3 Control of Chemical Microenvironments Using

Microfluidic Systems
Microfluidic cell culture systems are typically made from poly(dimethylsiloxane)

(PDMS) using the soft lithography technique [12]. Microfluidic systems have been
used to engineer chemical microenvironments such as concentration gradients of
biochemicals. Gradients of biochemical signals (e.g. growth factors, hormones,
8 Microfluidic Organs-on-Chips to Reconstitute Cellular Microenvironments 229
Fig. 8.1 Cellular microenvironment in a microfluidic culture and a conventional dish culture. (a)
Cells cultured in a dish are maintained in a large volume of culture medium under static condition.
(b) Cells cultured in a microfluidic device are maintained in a small volume of culture medium
under static or dynamic conditions. Concentration gradients can be generated by flowing chemicals
or cellular secretion and consumption and also fluid flow can generate shear stress within a
microfluidic device
morphogens, and chemokines) play an important role in a wide range of biological

processes including development, immune response, wound healing, and cancer
metastasis [13, 14]. Although these biochemical gradients are very difficult to
produce and maintain in conventional culture systems, microfluidic systems can
generate arbitrary shapes of biochemical gradients [15, 16]. Thus, these microfluidic
systems to generate concentration gradients have been used to study of immune
responses, cancer metastasis, and stem cell biology [16–19]. Recently, development-
on-chip has been developed by mimicking spatial and temporal chemical environ-
ments found in vivo during neural tube development using a microfluidic device
[20]. This system was able to maintain simultaneous opposing and/or orthogonal
gradients of developmental morphogens, resulting in neural tube patterning analo-
gous to that observed in vivo.
Chemical gradients can be generated using cellular secreted factors by patterning
different types of cells within microfluidic devices (Fig. 8.2). Cancer metastasis was
modeled in a compartmentalized microfluidic device in which cancer cells were
hydrodynamically patterned in a microchannel at spatially defined positions relative
to source cells that secrete chemokines and sink cells that scavenge the chemokines
(Fig. 8.2a) [21]. This system enabled to recreate a physiological cancer microenvi-
ronment, resulting in efficient chemotaxis under much shallower chemoattractant
gradients than previously possible in other in vitro systems. These concentration
230 Y.-s. Torisawa
Fig. 8.2 Microfluidic co-culture systems to recreate chemical microenvironment. (a) A cancer
metastasis model was engineered in a microfluidic device consisting of two PDMS microchannels
separated by a porous membrane. Cancer cells (blue) were hydrodynamically patterned in the top
channel at spatially defined positions relative to source (red) and sink cells (green), which generated
chemoattractant gradients that induced cancer cell migration. (Reproduced from Ref. [21] with
permission from the Royal Society of Chemistry). (b) A microfluidic system to form a 3D
perfusable vascular network. The device consists of a central vessel channel, two adjoining media
channels, and the outermost fibroblast channel. The vascular network (red) covered by pericytes
(green) was formed in the central channel with assistance from the lateral fibroblasts. (Reproduced
from Ref. [26]. Copyright 2015, Public Library of Science)
gradients of cellular secreted factors are very difficult to be maintained in conven-

tional dish cultures. Using 3D cell culture techniques with microfluidic devices, 3D
functional microvascular networks can be engineered [22–27]. Jeon’s group has
developed a microfluidic device which enables spatially patterned 3D co-culture of
endothelial cells with stromal fibroblast cells [23] known to induce angiogenesis and
support vascular formation (Fig. 8.2b) [25]. This system was able to model natural
cellular programs found during development and angiogenesis to form perfusable
networks of intact 3D microvessels. This system enabled the formation of 3D blood
vessels with perfusable lumina that are similar in 3D architecture, intact barrier
function, long term stability and salient biochemical markers to their in vivo
counterparts. This model can be used to study interactions between pericytes and
endothelial cells [26] and the role of interstitial flow during the formation of
neovessels [27]. Since vascular networks are crucial to the maintenance of cellular
viability and function in tissues and organs [28], engineering perfusable 3D vascular
networks that can deliver nutrients and oxygen as well as cells could be a powerful
platform to develop in vitro systems. This method has recently been applied to novel
methods to vascularize 3D cell spheroids where a perfusable vascular network
connected to microchannels is formed [29, 30]. These microfluidic devices may
provide a novel approach to culture 3D tissues as well as organoids [31].
8.2 Organs-on-Chips to Mimic Cellular

Microenvironmental Signals
Organs-on-chips are microfluidic cell culture devices made from elastomer, typically
PDMS. By recapitulating tissue architectures and chemical and mechanical micro-
environments, these microfluidic devices reconstitute organ-level cellular function-
ality not possible with conventional culture methods [32–35]. Especially, these
devices can mimic biomechanical signals which cells normally experience inside
the body.
8.2.1 Lung-on-a-Chip
A representative example of organs-on-chips is the lung-on-a-chip which reconsti-

tutes alveolar function in the human lung by mimicking tissue-tissue interface and
breathing mechanical environment (Fig. 8.3a) [36]. This system consists of a
compartmentalized microfluidic device in which human alveolar epithelial cells
are cultured in apposition with human pulmonary microvascular endothelial cells
on a thin porous ECM-coated PDMS membrane that resembles the in vivo alveolar-
capillary interface. This system is integrated with a mechanical actuation system to
mimic breathing motions by applying cyclic suction to distort the PDMS
microdevice which cyclically stretches the thin PDMS membrane. This biomimetic
microdevice enables the reconstitution of organ-level cell responses not normally
observed in conventional culture systems, such as recruitment of immune cells in
responses to bacteria. Moreover, this system has revealed unexplained adverse
effects of breathing-induced mechanical forces; the cyclic breathing motions
increased cellular uptake of silica nanoparticles and translocation of nanoparticles
from the alveolar airspace to the vascular compartment. This effect was confirmed in
a mouse ex vivo model. This system also enables to model pulmonary edema
induced by toxicity of the cancer drug interleukin-2 (IL-2) [37]. Clinical relevant
concentration of IL-2 administration into the vascular channel caused continuous
232 Y.-s. Torisawa
Fig. 8.3 Microfluidic organs-on-chips to recapitulate tissue architecture and mechanical microen-
vironment. (a) Lung-on-a-chip recapitulates the alveolar-capillary interface by culturing human
alveolar epithelial cells on top of a thin porous PDMS membrane and human capillary cells on
bottom. Breathing motions are recapitulated by applying cyclic suction to the side chambers, which
deform the PDMS membrane to which the cell layers are attached. Administration of interleukin-2
(IL-2) into the vascular channel resulted in fluid leakage into the alveolar channel. (Reproduced
from Ref. [44]. Copyright 2018, Nature Publishing Group). (b) Gut-on-a-chip. Photograph (A) and
a schematic illustration (B) of the device in which human Caco-2 intestinal epithelial cells are
cultured on a thin PDMS membrane to form 3D villi-like structures by applying peristalsis-like
cyclic mechanical strain as well as fluid flow. (C) Micrograph of Caco-2 cells cultured for 6 days in
the Gut-on-a-chip. Scale bar, 100 μm. (Reproduced from Ref. [43]. Copyright 2015, Public Library
of Science). (c) Primary human small intestine-on-a-chip. A confocal image showing a cross-
section of primary intestinal epithelium immunostrained (magenta, F-actin; yellow, Ki67; green,
Muc5AC). Optical images of primary intestinal epithelium cultured on-chip for 12 days under
continuous flow compared with a static culture condition. Formation of intestinal villi-like struc-
tures occurred only in the presence of flow. (Reproduced from Ref. [44]. Copyright 2018, Nature
Publishing Group)
leakage of vascular fluid into the alveolar channel and complete flooding of the
airspace (Fig. 8.3a). Importantly, this system revealed that the mechanical forces
produced by breathing motions contribute to the development of increased vascular
leakage and pulmonary edema. Thus, this system enabled the identification of novel
therapeutics that inhibited mechanotransduction pathways. The lung-on-a-chip
microdevice faithfully recapitulates organ-level cell function and predict physiolog-
ical and pathological responses in vivo.
This system has been applied to a human lung small airway-on-a-chip which
contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an
underlying microvascular endothelium that experiences fluid flow [38]. This system
enabled active synchronized cilia beating and mucociliary transport whose velocity
was nearly identical to that in healthy human lung airway. Exposure of the epithe-
lium to IL-13 in this device reconstituted the goblet cell hyperplasia, cytokine
hypersecretion, and decreased ciliary function of asthmatics. The small airway-on-
a-chip generated with epithelial cells from individuals with chronic obstructive
pulmonary disease reconstituted features of the disease such as selective cytokine
hypersecretion, increased neutrophil recruitment, and clinical exacerbation by expo-
sure to viral and bacterial infections. Thus, this device allows for analysis of organ-
level lung pathophysiology in vitro. Furthermore, this device enabled modeling of
smoke-induced lung injury by connecting to a smoking instrument that inhales and
exhales whole smoke from burning cigarettes in and out of the epithelium-lined
microchannel of the device [39]. This system was used to compare the effects of
inhaled smoke on chips containing bronchiolar epithelium isolated from normal
lungs or from lungs of chronic obstructive pulmonary disease (COPD) patients. This
model led to identification of ciliary micropathologies, COPD-specific molecular
signatures, and epithelial responses to smoke generated by electric cigarettes. This
smoking airway-on-a-chip provides a tool to study normal and disease-specific
responses of human lung to inhaled smoke across molecular, cellular, and tissue-
level responses.
8.2.2 Gut-on-a-Chip
A similar compartmentalized microfluidic system was used to create a human

gut-on-a-chip system that mimics physiological peristaltic-like motion and fluid
flow of the intestine (Fig. 8.3b) [40–43]. The device consists of two layer of cell
culture channels separated by a thin porous PDMS membrane in which human
intestinal epithelial Caco-2 cells (colorectal carcinoma line) are cultured. Caco-2
cells cultured with fluid flow and cyclic mechanical distortion in the microfluidic
device spontaneously formed 3D structures that resemble the architecture of the
intestinal villus. This system enabled to reproduce the intestinal barrier function,
high levels of mucus production, and intestinal absorptive characteristics, which will
be useful for drug testing as well as development of intestinal disease models. This
system also enabled long-term co-culture of various gut microbes with the epithelial
cells by recapitulating the dynamic mechanical microenvironment, whereas conven-
tional culture methods cannot maintain co-culture of microbes. Lack of mechanical
signals was shown to trigger bacterial overgrowth similar to that observed in patients
with ileus and inflammatory bowel disease. Thus, the human gut-on-a-chip can be
used to analyze contributions of microbiome to intestinal pathophysiology and
disease mechanisms in a controlled manner.
Caco-2 cells are widely used to estimate intestinal barrier function; however,
these cells are cancer-derived cells and have some issues such as enzymatic activity.
Recently, primary human small intestine-on-a-chip has been developed using
234 Y.-s. Torisawa
intestinal biopsies [44]. Primary intestinal epithelial cells harvested from 3D

organoids are cultured in a microfluidic device with human intestinal microvascular
endothelium in a parallel microchannel under fluid flow. The intestinal epithelium
formed villi-like structures lined by polarized epithelial cells that undergo multi-
lineage differentiation (Fig. 8.3c). Transcriptomic analysis demonstrated that the
intestine chip more closely mimicked whole human duodenum than duodenal
organoids. This study demonstrated that recapitulation of the dynamic mechanical
environment and tissue-tissue interface enabled the formation of 3D villi-like struc-
tures as well as the maintenance of intestinal functions. Since this system can
maintain normal human intestinal function, it will be a powerful platform to study
drug pharmacokinetics and to model human intestinal diseases.
8.2.3 Kidney-on-a-Chip
In vitro systems to model kidney functions are crucially needed to predict drug-
induced kidney injury which is often observed in pharmacotherapy; however current
available models do not recapitulate the biological functions of the kidney and poor
predictions of drug-induced kidney injury [45]. A kidney proximal tubule-on-a-chip
microdevice was developed that is lined by human proximal tubule epithelial cells
exposed to physiologically relevant fluidic flow [46]. Mechanical forces caused by
fluid flow have been recognized as a key determinant of cellular functions in the
kidney. This microfluidic device consists of a luminal flow channel and an interstitial
compartment separated by a thin porous membrane on which the kidney cells are
cultured. This device demonstrated that low levels of fluid shear stress (0.2 dyn cm-2)
similar to that observed in the collecting ducts and proximal tubules of the kidney
enhanced differentiation, increased molecular and drug transport functions, and
produced more in vivo-like toxicity responses. Thus, this kidney proximal tubular
model can mimic the structural, mechanical, transport, absorptive, and physiological
properties of the human kidney. A 3D flow-directed kidney proximal tubule system
has also been developed using a microfluidic device [47]. Human proximal tubular
epithelial cells are seeded onto collagen extracellular matrix within a microchannel
so that the cells self-assemble to form a 3D tubular structure. This system recapit-
ulates the perfusion delivery and transport pathway of a solute which is perfused into
a surrogate vascular channel, diffuses through the pseudo-interstitial space, and
undergoes uptake and efflux across the epithelial barrier into the flowing perfusate
in the tubular luminal channel. This model replicates the polarity of the proximal
tubule, retains polarized expression and function of protein essential for reabsorptive
and secretory transport, responds to physiological stimuli, and performs critical
biochemical synthetic activities. This system provides a platform for modeling of
renal drug clearance and drug-induced nephrotoxicity.
A kidney glomerulus model has recently been developed using a compartmen-
talized microfluidic device with human iPS (induced pluripotent stem) cell-derived
podocytes which can produce glomerular basement-membrane collagen [48]. This
device enabled to recapitulate the natural tissue-tissue interface of the glomerulus

and the differential clearance of albumin and inulin by co-culturing podocytes and
human glomerular endothelial cells under physiological fluid flow and cyclic
mechanical strain. This device demonstrated that application of physiological rele-
vant mechanical cues further augmented podocyte differentiation and maturation.
Since existing immortalized podocyte cells and cultures poorly mimic glomerular
function, this device could provide a better in vitro system for predicting nephro-
toxicity and therapeutic development.
8.3 Bone Marrow-on-a-Chip to Reconstitute Hematopoietic

Niche Physiology
Bone marrow is the only permanent hematopoietic organ in humans. Given the
importance of bone marrow as the source of all blood cells, an in vitro culture system
that can reconstitute function of living bone marrow will be a useful platform. The
bone marrow microenvironment contains a complex set of cellular, chemical, struc-
tural, and physical cues necessary to maintain the viability and function of the
hematopoietic system [4, 49]. This bone marrow microenvironment regulates the
function of hematopoietic stem cells (HSCs), facilitating a balance between self-
renewal and differentiation into progenitors that generate all mature blood cells.
Current in vitro culture methods do not accurately model bone marrow physiology;
hence, studies relevant to the hematologic system are usually conducted in animals
[50–52]. Engineering an artificial bone marrow capable of reproducing the bone
marrow microenvironment could be potentially used to study blood development
and physiology, model diseases, and serve as a platform for drug development and
toxicity studies.
8.3.1 Engineering of Bone Marrow In Vivo
Because of the complexity of the bone marrow microenvironment necessary to

support HSC function as well as blood production, it has not been possible to
recapitulate the complex bone marrow microenvironment as well as blood forming
functions in vitro [52–55]. To overcome this challenge, we used a tissue engineering
approach to first induce formation of new bone containing marrow in vivo, and then
we surgically removed it whole and maintained it in a microfluidic device in vitro
(Fig. 8.4a) [56]. New bone formation was induced in the subcutaneous tissue of a
mouse by implanting a PDMS device containing cylindrical hole filled with a
collagen gel and bone-inducing materials (demineralized bone powder and BMPs).
This method produced a cylindrical shaped bone containing a bony cortex and an
internal trabecular bone network that was filled with a normal appearing marrow
236 Y.-s. Torisawa
Fig. 8.4 Bone marrow-on-a-chip. (a) PDMS devices containing a cylindrical chamber filled with
bone-inducing materials were implanted subcutaneously on the back of a mouse for 8 weeks and
then surgically removed. The engineered bone marrow that formed within the PDMS device was
placed into a similar shaped central chamber in a microfluidic system and then maintained in culture
in vitro. (b) Low- (left) and high-magnification views (right) of histological hematoxylin-and-eosin-
stained sections of the engineered bone marrow formed in the PDMS device at 8 weeks following
implantation (top) compared with a cross section of bone marrow in the normal mouse femur
(bottom). (Reproduced from Ref. [56] with permission from the Nature Publishing Group)
with a blood cell composition virtually identical to that of natural bone marrow
(Fig. 8.4b). Previous reports showed that tissue engineering approaches with the
bone-inducing materials resulted in the formation of marrow largely inhabited by
adipocytes [57–59] which are known to inhibit hematopoiesis [60]. To reduce
adipocyte content in the marrow, we used a PDMS device to restrict access of
cells or soluble factors from the overlying adipocyte-rich hypodermis to the bone-
inducing materials while maintaining accessibility to the underlying muscle tissue
through the lower opening. Thus, the use of PDMS devices enabled to engineer bone
marrow that resembles to natural bone marrow (Fig. 8.4b). We demonstrated that our
method produced bone containing marrow with a hematopoietic cell composition
nearly identical to that of natural bone marrow. Immunohistochemical analysis also
confirmed that endothelial and perivascular cells as well as hematopoietic stem cells
were located in their normal spatial positions in the engineered bone marrow. The
presence of key cellular and molecular components of the hematopoietic niche
indicated that the cellular content of the engineered bone marrow closely resembles
the natural bone marrow microenvironment.
Several approaches have been reported that bone marrow can be engineered
ectopically in mice. Human mesenchymal stem cells (MSCs) were seeded into
collagen-based scaffolds and cultured in vitro to form a hypertrophic cartilage tissue

[61]. When the engineered cartilage tissues were implanted into mice for 12 weeks,
these tissues formed fully-fledged bone containing marrow with hematopoietic stem
and progenitor cells which can reconstitute multilineage long-term hematopoiesis in
lethally irradiated mice. Matrigel has also been used to engineer bone marrow
ectopically [62]. When human MSCs were suspended in Matrigel and subcutane-
ously injected into immunodeficient (NSG) mice, MSCs spontaneously formed a
bone marrow cavity through a vascularized cartilage that was progressively replaced
by hematopoietic tissue and bone. This humanized MSC-derived microenvironment
permitted homing and maintenance of long-term mouse hematopoietic stem cells
(HSCs) as well as human HSCs after cord blood transplantation. This xenotrans-
plantation model also enables robust engraftment of primary acute myeloid leukemia
samples at levels much greater than those in unmanipulated mice [63]. Direct
intraossicle transplantation accelerated engraftment and resulted in the detection of
higher leukemia-initiating cell frequencies. These humanized ossicle xenotransplan-
tation systems allow to model wide variety of human hematologic diseases. There-
fore, human MSCs can induce the formation of bone marrow ectopically in vivo
when they are implanted with bone-inducing materials. The in vivo engineering of
bone marrow offers a new approach for analysis and study of hematopoiesis and
hematologic disease.
8.3.2 In Vitro Culture of Bone Marrow with a Functional

Hematopoietic Niche
Various in vitro culture systems have been developed to maintain and expand HSCs
and hematopoietic progenitors because the expansion of HSCs would greatly
improve bone marrow transplantation and facilitate the development of advanced
cell therapies for many blood disorders and malignant diseases [64–68]. Bone
marrow stromal cells are mostly used to mimic the bone marrow microenvironment
in vitro [68]. Recently, 3D culture systems have been develop using scaffolds or
hydrogels in which bone marrow stromal cells are cultured with hematopoietic cells
[53, 69–72]. Bone-derived materials have also been used as a scaffold to recreate the
bone marrow microenvironment [73]. Frozen human bones were decellularized,
demineralized, and cut into pieces to make bone-derived scaffolds so that cells can
be cultured with those scaffolds in conventional culture plates. Bone marrow stromal
cells such as mesenchymal stromal cells (MSCs) and osteoblasts were cultured
within the bone-derived scaffolds with hematopoietic stem and progenitor cells
(HSPCs), resulting in enhancing expansion of HSPCs compared with conventional
2D culture systems. Microfluidic culture systems with scaffolds also have been
developed to maintain HSPCs for a long time. A 3D co-culture model based on a
hydroxyapatite coated zirconium oxide scaffold consisting of human MSCs and
HSPCs enabled the culture of HSPCs for 4 weeks within a microfluidic device
238 Y.-s. Torisawa
[74]. Although HSCs rapidly expand after transplantation in vivo, in vitro studies
indicate that control of HSPC self-renewal and differentiation in culture remains
difficult. There is currently no method to recreate the entire bone marrow microen-
vironment and to expand hematopoietic stem cells in vitro because of the complexity
of the bone marrow microenvironment.
As a proof of concept, we tested whether the engineered bone marrow could
maintain a functional hematopoietic system in vitro [56]. The engineered bone
marrow formed 8 weeks after implantation was surgically removed from the
mouse, perforated with a surgical needle to permit fluid access, placed in a similar
shaped chamber in a microfluidic device, and perfused continuously with culture
medium for in vitro culture (Fig. 8.4). Flow cytometric analysis of cellular compo-
nents of the cultured ‘bone marrow-on-a-chip’ revealed that the presence of hema-
topoietic stem and progenitor cells in similar proportions to those of freshly
harvested bone marrow for up to 7 days on-chip. In contrast, bone marrow cultured
on a stromal feeder cell layer in a culture dish, which is the current benchmark,
exhibited a significant decrease of long-term HSCs and a concomitant increase in
hematopoietic progenitor cells relative to freshly isolated from natural mouse bone
marrow. Thus, the long-term HSCs, which are the only cells capable of self-renewal
and multilineage potential, appeared to be differentiating into more specialized
progenitor cells in the static stroma-supported culture system as previously reports
noted [68–72]. Thus, the bone marrow-on-a-chip enabled maintenance of a signif-
icantly higher proportion of long-term HSCs while more effectively maintaining
distribution of hematopoietic progenitors as well as mature blood cells. Moreover,
because the engineered bone marrow autonomously produces factors necessary to
maintain hematopoiesis, this system was able to maintain HSCs and hematopoietic
progenitors in normal proportions in the cultured chip without the addition of
exogenous cytokines. Importantly, the full functionality and robustness of this
engineered bone marrow tissue was confirmed by demonstrating that bone marrow
cultured on-chip could be used to fully reconstitute the entire blood system when
transplanted into lethally irradiated mice. Cells harvested from the engineered bone
marrow after in vitro culture successfully engrafted the mice at a similar rate to that
of freshly isolated bone marrow and repopulated all differentiated blood cell line-
ages. Thus, the bone marrow-on-a-chip retains fully functional, self-renewing,
multipotent HSCs as well as a functional hematopoietic niche in vitro. Furthermore,
because of microfluidic perfusion culture, this bone marrow-on-a-chip can produce
blood cells continuously and release them into the microfluidic circulation, while
maintaining HSCs and hematopoietic progenitors in normal in vivo-like proportions
inside the microfluidic device for at least 2 weeks in culture [75]. This system was
able to induce red blood cell production by adding erythropoietin (EPO) which is
known to stimulate erythrocyte formation. When EPO was administrated in the bone
marrow chip, we detected a continuous increase in the number of erythrocytes
released in the outflow. Thus, this bone marrow-on-a-chip can be used to study
and test continuous blood cell production in vitro. This method enabled to maintain
an intact 3D bone marrow microenvironment with functional hematopoietic cells
in vitro.
8.3.3 Testing of Drugs Using Bone Marrow-on-a-Chip
To evaluate whether the maintenance of an intact bone marrow microenvironment

could be effective for efficacy and toxicity testing, we tested whether the bone
marrow-on-a-chip can be used to test radiation toxicity and pharmacological coun-
termeasures that might help radiation injured bone marrow repair itself
[56, 75]. Despite an active search for medical countermeasures to radiation toxicity,
no effective drug has been approved by FDA that could help protect or reconstitute
the hematopoietic system [76]. Current drug development studies rely on animal
studies; however, these animal models rarely reflect human response [77]. Thus,
there is a critical need for predictive in vitro models to test effects of radiation
countermeasures. When the bone marrow-on-a-chip was exposed to varying doses
of γ-radiation, it showed a significant radiation dose-dependent decrease in the
proportion of HSCs and hematopoietic progenitors which closely mimics what is
observed in the bone marrow of live irradiated mice. In contrast, the stroma-
supported static culture exhibited suppressed responses and was significantly more
resistant to the effects of radiation toxicity. We then evaluated radiation-protecting
effects of two potential therapeutic proteins, granulocyte-colony stimulating factor
(G-CSF) [78] and bactericidal/permeability-increasing protein (BPI) [79], which
have been reported to accelerate recovery of hematopoiesis after radiation-induced
bone marrow failure in vivo. When G-CSF was administrated to the bone marrow
chip 1 day after exposure to γ-radiation, we detected a significant increase in the
number of HSCs and progenitors within the bone marrow chip compared to
untreated irradiated bone marrow chips, and furthermore we detected significant
increases in the number of HSCs, progenitors, and myeloid cells in the outflow of the
chips. When BPI was administrated to the bone marrow chip 1 day after exposure to
γ-radiation, it also significantly increased the number of HSCs and myeloid cells in
the outflow of the chips. Thus, these radiation countermeasure drugs were able to
accelerate recovery of blood cell production after radiation injury in the bone
marrow-on-a-chip in vitro as they have previously been reported in vivo. In contrast,
when a conventional static culture was exposed to γ-radiation, it was not able to
detect any effect of BPI. Therefore, the bone marrow-on-a-chip effectively mimics
the in vivo response of living bone marrow to radiation countermeasure drugs,
whereas the conventional static bone marrow cultures do not. These results suggest
that the presence of a hematopoietic microenvironment is crucial for modeling
radiation toxicity and testing effects of potential countermeasure drugs in vitro.
Thus, it is because conventional culture methods do not accurately recapitulate the
bone marrow microenvironment that drug development studies currently rely on
animal models for efficacy and safety testing. Since the bone marrow-on-a-chip
system contains a functional bone marrow microenvironment, this system can mimic
physiological responses to radiation countermeasure drugs normally only observed
in vivo and thus this system could offer a novel approach to replace animal testing.
Therefore, recapitulation of cellular microenvironment is crucial for efficacy and
240 Y.-s. Torisawa
toxicity testing and thus the organ-on-a-chip technology has great potential to
generate reliable predictions of drug efficacy and toxicity in humans.
8.4 Culture of iPS Cells in Organs-on-Chips
The organ-on-a-chip technology has been used to culture human pluripotent stem
cells including induced pluripotent stem cells (iPSCs) [80]. A microfluidic method
has been reported to induce functional differentiation of human pluripotent stem
cells on-chip [81]. This method demonstrated that extrinsic signal modulation
though optimal frequency of medium delivery can be used as a parameter for
improved germ layer specification and cell differentiation. This method enabled
accurate spatiotemporal control of the soluble microenvironment around cells
through regulation of periodic perfusion frequencies and achieved effective induc-
tion of hepatocytes on-chip with higher activity of hepatocyte specific functions
under optimized perfusion conditions.
A disease model has been reported to reproduce disease pathophysiology in vitro
using the organ-on-a-chip technology. A heart-on-a-chip was used to replicate
contractile pathophysiology of Barth syndrome (BTHS) using engineered myocar-
dial tissue assembled from BTHS or control iPSC-derived cardiomyocytes (iPSC-
CMs) [82]. When iPSC-CMs were cultured onto thin elastomers micropatterned
with fibronectin lines for 5 days, the iPSC-CMs self-organized into laminar aniso-
tropic myocardium tissues. During electrical field stimulation, control iPSC-CM
tissues contracted rhythmically, whereas BTHS iPSC-CM tissues developed signif-
icantly lower twitch and peak systolic stress compared to controls, demonstrating
that BTHS engineered myocardial tissues replicate the BTHS myopathic phenotype.
Importantly, this method enabled to model disease correction; treatment of BHTS
iPSC-CMs with TAZ modRNA for 5 days restored contractile function of the
cardiomyocyte tissues to levels comparable to those of controls. Patient-derived
iPSCs have considerable potential to facilitate study of human disease and enable
therapeutic screening [83]; however, currently there is the lack of in vitro models that
reproduce disease pathophysiology. Thus, the use of organ-on-a-chip systems with
the iPS cell technology could be a powerful platform to develop in vitro disease
models [84].
Microfluidic systems have recently been utilized to engineer blood cells from
pluripotent stem cells. To produce platelets, microfluidic bioreactors have been
developed by mimicking the bone marrow and blood vessel microenvironments
[85, 86] because platelets are generated from megakaryocytes in the bone marrow
vasculature in vivo [87]. These bioreactors recapitulate extracellular matrix compo-
nents, stiffness, endothelial cell contacts, and vascular shear forces. These systems
demonstrated that physiological shear stress triggers proplatelet production and
platelet release. By recapitulating physiological microenvironments, these bioreac-
tors enabled the production of functional human platelets from megakaryocytes
derived from adult hematopoietic progenitor cells as well as human iPSCs. The
iPSC-based technology has recently enabled to produce stable immortalized mega-

karyocyte progenitor cell lines [88]. These iPSC-derived megakaryocyte progenitor
cells can be expanded, cryopreserved, and differentiated into functional platelets.
Producing platelets from iPSCs in vitro could offer donor-free blood products as
well as genetically modified products and provide a way of autologous
transfusion [89].
Microfluidic systems that can recapitulate biomechanical forces have also been
used to engineer HSCs. It has been demonstrated that biomechanical forces, such as
blood flow-induced shear stress, play an important role in hematopoietic develop-
ment and the emergence of HSCs [90, 91]. The use of microfluidic systems enabled
to apply physiological fluid shear stress (5 dyn cm 2), demonstrating that fluid shear
stress endows long-term multilineage engraftment potential on early hematopoietic
tissues not previously shown to harbor HSCs [92]. This study revealed that effects on
hematopoiesis are mediated in part by a cascade downstream of shear stress that
involves stimulation of prostaglandin E2 (PGE2)-cyclic adenosine monophosphate
(cAMP)-protein kinase A (PKA) signaling axis. Using mouse embryonic stem cells
differentiated in vitro, it has been demonstrated that fluid shear stress increases
hematopoietic colony-forming potential and expression of hematopoietic markers
[93]. Thus, the use of the organ-on-a-chip technology that can recapitulate cellular
microenvironment and biomechanical signals could facilitate engineering of HSCs
[94]. Recently, production of HSCs from iPSCs has been achieved using transcrip-
tion factors as well as in vivo transplantation [95]. Human iPSCs were differentiated
into hemogenic endothelial cells in vitro and then treated with cocktails of transcrip-
tion factors to endow the potential to engraft multi-lineage hematopoiesis in vivo.
This method requires in vivo microenvironment to mature HSCs, suggesting signals
from the microenvironment is necessary for HSC maturation. Therefore, the organ-
on-a-chip technology may provide a way to produce HSCs in vitro by mimicking the
microenvironment inside the body.
8.5 Future Prospects
Organ-on-a-chip microdevices produce organ-level function in vitro by recapitulat-

ing natural tissue arrangements and microenvironmental signals. Since these
microdevices are created using human cells including primary human cells and
cell lines as well as pluripotent stem cell-derived cells, these devices can predict
human responses to medications, which provides reliable prediction of drug efficacy
and safety in humans. These systems have also potential to serve as a platform to
differentiate pluripotent stem cells into specific cell types. The use of iPS cells with
organ-on-a-chip microdevices is especially attractive because iPS cells provide
sources of patient-specific cells and enable genome editing. These systems enable
to model human physiology and disease and further will provide opportunities to
model organs that have not yet been possible to be studied in vitro.
242 Y.-s. Torisawa
As a novel approach to develop organs-on-chips, we have developed the bone

marrow-on-a-chip in which an organ, bone marrow, is engineered inside a device
in vivo and then maintained in culture. This system can sustain an intact bone
marrow with a functional hematopoietic microenvironment in vitro, enabling to
mimic complex tissue-level responses to radiation toxicity and to therapeutic agents.
This system provides an interesting alternative to animal models because this offers
the ability to manipulate individual hematopoietic cell populations or to insert other
cell types such as cancer cells in vitro as well as in vivo before analyzing the response
of the intact bone marrow to relevant clinical challenges. It is also possible to
generate human bone marrow models by engineering bone marrow in immunocom-
promised mice and inserting human hematopoietic cells or human leukemia cells
[63]. Therefore, this method offers a new approach for analysis of drug responses
and toxicities and for study of hematopoiesis and hematologic diseases.
These organ-on-a-chip microdevices can be integrated via microfluidic linkage to
model physiological interplay between different organs. Body-on-a-chip systems
have been developed by integrating several organ models (typically 2~4 types) into a
microfluidic device [96–100]. These systems realize to estimate the adsorption,
distribution, metabolism, and elimination (ADME) of drugs in vitro. For example,
when a drug was introduced into a microfluidic device, the drug converted in a liver
compartment into its reactive metabolites and then circulated to other organ com-
partments such as a lung compartment or a cancer compartment to detect drug
toxicity or effect. It might be possible to develop ‘human body-on-a-chip’ consisting
of fluidically linked chambers representing different organs that could reliably
predict drug toxicity and efficacy inside the human body in the future. Although
the organs-on-chips have great potential, this technology still requires further vali-
dation and improvement. For integration of different organ chips, it requires a novel
common culture medium, such as blood substitute, that can be perfused through the
entire system to maintain viability and functions of all the cells on a chip. Further-
more, although PDMS devices are widely used, the use of PDMS could raise a
problem for drug discovery applications because it absorbs small hydrophobic
molecules. By improving materials, cell culture techniques, and tissue engineering
techniques, organs-on-chips could provide novel approaches for drug discovery and
development, which could reduce or replace animal testing in the future.
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Chapter 9
In Vitro Tissue Construction
for Organ-on-a-Chip Applications
Yuya Morimoto, Nobuhito Mori, and Shoji Takeuchi
Abstract Functional living tissues, reconstructed in vitro, have been demanded as

grafts for regenerative medicine and drug test models for pharmacokinetic study. For
the reconstruction of the functional living tissues, cellular structures with the shapes
of point, line and plane are attractive building blocks to form macroscopic cellular
tissues. Microfluidics are suitable techniques in the preparation of the cellular
structures with design flexibility and high productivity. Owing to their shape con-
trollability, the cellular structures are able to be assembled into macroscopic tissues
in various dimensions using microfluidic devices. Furthermore, the integration of
in vitro constructed cellular tissues and microfluidic devices provides organ-on-a-
chips to evaluate the change of cellular functions in response to applied drugs. This
chapter introduces microfluidic fabrication methods for cellular structures with the
shapes of point, line and plane, and methods to manipulate and assemble the cellular
structures for formation of macroscopic cellular tissues. Furthermore, we show that
organ-on-a-chip, combination of the cellular tissues and microfluidic devices, can
find applications in biological studies and the analysis of pharmacokinetics and
toxicology.
Keywords Microfluidics · Biofabrication · BioMEMS · Tissue manipulation ·

Functional analysis
9.1 Introduction
There is a need for in vitro reconstruction of functional living tissues as transplant-

able grafts for regenerative medicine and as an alternative to animal pharmacokinetic
models [1, 2]. To reconstruct functional living tissues, tissue engineering methods
are necessary because cells cannot form three-dimensional (3D) structures sponta-
neously when cultured on a culture dish. To assemble cells into 3D structures,
Y. Morimoto · N. Mori · S. Takeuchi (*)

Institute of Industrial Science, the University of Tokyo, Tokyo, Japan

248 Y. Morimoto et al.
Fig. 9.1 Illustration of point-, line-, and plane-shaped cellular structures fabricated by microfluidic
techniques. Microfluidic methods facilitate mass and reproducible construction of cellular struc-
tures, which are then available as building modules to form macroscopic cellular structures.
(Figures are reprinted with permission from [7], ©2015 Elsevier)
cultures of cells in 3D biocompatible scaffolds are required [3, 4]. Hydrogels,

especially the extracellular matrix (ECM), are commonly used as a material for 3D
biocompatible scaffolds [5, 6]. The hydrogel scaffolds enable cells to adhere onto
their surfaces and/or be embedded as cellular structures.
The cellular structures are categorized into three types according to their shapes:
point-, line-, and plane-shaped cellular structures (Fig. 9.1) [7]. For the preparation
of the various-shaped cellular structures, microfluidic devices are well suited
because they can manipulate hydrogel solutions in a controlled manner by providing
design flexibility and high productivity [8–10]. Moreover, microfluidic devices
allow for the cell-laden hydrogel structures to be assembled into macroscopic
cellular structures. The fabricated cellular structures are available as transplantable
grafts for treatment of endocrine disease and repair of defective sites.
Furthermore, by integration of the cellular structures with microfluidic and
microfabricated devices, we can fabricate organ-on-a-chips with in vitro constructed
cellular structures [11, 12]. The organ-on-a-chips allow us to measure mechanical
functions of the cellular structures such as contractile force and permeability; the
chips facilitate the evaluation of cellular functions that are difficult to measure using
common biological methods. Therefore, the organ-on-a-chip achieves in vitro drug
testing by the evaluation of cellular functions depending on the types and concen-
trations of applied drugs.
In this chapter, we present an overview of (i) microfluidic fabrication methods for
point-, line-, and plane-shaped cellular structures; (ii) methods for manipulating
cellular structures for assembly into macroscopic cellular tissues; and (iii) organ-
on-a-chip applications of the cellular structures.
9 In Vitro Tissue Construction for Organ-on-a-Chip Applications 249
9.2 Microfluidic Fabrication of Cellular Structures
For the fabrication of various-shaped cellular structures, hydrogel scaffolds with

shapes corresponding to shapes of the cellular structures are necessary. Microfluidic
devices allow for the flow control of hydrogel solutions, resulting in the production
of hydrogel scaffolds with various configurations such as point, line, and plane
shapes. In this section, we introduce microfluidic devices for the formation of
hydrogel scaffolds according to the classification of each shape, and also describe
the resulting cellular structures by culturing cells on and/or in the hydrogel scaffolds.
9.2.1 Point-Shaped Cellular Structures
Point-shaped cellular structures are defined as cellular beads in spherical shape and
cellular blocks in a polyhedron shape. A simple fabrication method of cellular beads
is the formation of cellular spheroids by aggregating cells. Because most kinds of
cells tend to aggregate without their adhesion to the culture substrate [13], the size of
the cellular spheroids is controllable depending on the number of aggregated cells.
Therefore, the production of uniform cellular spheroids is achieved by enclosing the
same number of cells in hanging-droplets [14, 15] and microwells [16, 17]. By
combination with microfluidic devices, the hanging-droplets and the microwells
allow the formation of cellular spheroids in a high-throughput manner, and addition
of drugs to cellular spheroids and collection of secretions from cellular spheroids
[18, 19]. In addition, microfluidic devices achieve aggregation of cells without
enclosing cells by cell manipulations with external force [20, 21]. Usage of micro-
rotational flows and magneto-Archimedes effect in a paramagnetic medium
succeeded in collecting cells for the production of cellular spheroids of a desired
size (Fig. 9.2). Thus, microfluidic devices are a useful tool for the mass formation of
size controlled spheroids and biological evaluation by addition of reagents.
Cellular beads are also constructed by cell culture with hydrogel beads. In cell-
laden hydrogel beads, their sizes are determined according to the size of the hydrogel
beads. In addition, ECM can be used for the hydrogel, allowing the cells to be
cultured with an appropriate culture condition. For the formation of size-controlled
hydrogel beads, quasi-two-dimensional (2D) microchannels, such as T-shaped and
flow-focusing channels, have been widely used [22–24]. Both microchannels allow
for the formation of hydrogel droplets with high uniformity and production rates by
infusing oil and a hydrogel solution. After gelation of the hydrogel droplets,
hydrogel beads are formed (Fig. 9.3a, b). In this process, microfluidic devices
allow for the size control of the hydrogel beads depending on the configurations of
the flow channels and the flow velocities. However, the wettability of the channel
surface can influence the possibility of droplet formation in the quasi-2D
microchannels, and the formation of hydrogel droplets becomes difficult when the
channel surface is hydrophilic. In addition, even when the channel surface is
Fig. 9.2 (a) Conceptual illustration of cellular spheroid formation using micro-rotational flows.
When cells suspended in culture medium are introduced into the device, the cells aggregate into a
cellular spheroid. (b) Sequential images of the cellular spheroid formation using micro-rotational
flows. After cell rotation occurs throughout the entire area with high-speed flow, cells start
aggregating in the center of the device (~120 s) when the flow speed is reduced. Scale bars,
200 μm. (c) Conceptual illustration of cellular spheroid formation using the magneto-Archimedes
effect. A magnetic force acts on cells within the paramagnetic medium, and then the cells aggregate
to areas with a lower magnetic flux density. (d) Sequential images of cellular spheroid formation
using the magneto-Archimedes effect. Cells gradually aggregate in the gap of the magnetic array.
(Images are reprinted with (a, b) permission from [20], ©2010 Elsevier, and (d) permission from
[21], ©2011 AIP Publishing LLC)
hydrophobic, the formation of droplets is sometimes difficult due to the adsorption

of proteins and saccharides onto the channel surfaces in cases of extended usage.
Some 3D microchannels, such as the axisymmetric flow-focusing devices
(AFFDs) and capillary microchannels, have been proposed as solutions for the
wetting problem on the channel surface. In the AFFD, hydrogel droplets are pre-
pared without contacts onto channel surfaces because a hydrogel solution is covered
with oil (Fig. 9.3c) [25–28]. The sizes of the hydrogel beads due to the gelation of
hydrogel droplets are controlled by the flow velocities and dimensions of the
AFFDs, similar to the quasi-2D microchannels. Additionally, in the formation of
hydrogel beads using a capillary microchannel, hydrogel droplets formed at the
microchannel tip are gelated by direct injection into a gelling agent [29–31]. This
method does not require pumps for liquid delivery, in contrast to AFFDs and quasi-
2D microchannels. The infusion of hydrogel solution by centrifugation allows for
Fig. 9.3 (a, b) Schematic illustration of quasi-2D microchannels for the production of hydrogel
beads. (a) T-shaped microchannels and (b) flow-focusing microchannels. (c, d) Schematic illustra-
tion of 3D microchannels for the production of hydrogel beads. (c) Axisymmetric flow-focusing
device (AFFD) and (d) capillary microchannels
the formation of hydrogel beads with a uniform diameter (Fig. 9.3d). By using
capillary microchannels, the compartmentalization of hydrogel beads is also
achieved by the placement of multiple microchannels (i.e.: θ tube geometry) at the
tip as well as through the infusion of different solutions [32]. In this method, since
hydrogel beads can be formed easily without oil, it is possible to use them as culture
scaffolds without any damage to the cells due to residual oil.
Depending on the microfluidic methods used for the formation of the hydrogel
beads and gelation mechanisms of the hydrogel, the types of constructible hydrogel
beads are determined. Favorable gelation mechanism used in microfluidic methods
are ionic cross-linking used in alginate gel formation, inherent phase transition by
heat used in collagen and gelatin gel formation, and reactions with photoreactive
crosslinkers used in synthetic polymers such as polyethylene glycol (PEG). The
AFFDs and the quasi-2D microchannels exhibit good affinity with all types of
gelation mechanisms, since gelation stably occurs in oil. As a result, these
microfluidic devices allow for the formation of alginate [22, 26, 27], PEG
[23, 24], and collagen gel beads [33]. On the other hand, capillary microchannels
are also available for the production of hydrogel beads by ionic cross-linking, and
are not available for the formation of hydrogel under inherent phase transition due to
the slow speed of the gelation. Therefore, the capillary microchannels have been
widely used for the production of alginate gel beads.
Fig. 9.4 Cellular beads fabricated from hydrogel beads. (a) Cell-encapsulated alginate gel beads.
(b) Cellular beads fabricated by culturing cells on collagen beads. (c) The 3D hierarchic co-culture
beads, where the inner cells (green) are dermal fibroblasts and outer cells (red) are epidermal
keratinocytes, are fabricated by culturing the different cells with collagen beads. (Images are
reprinted with (a) permission from [22], ©2007 John Wiley and Sons, (b) permission from [33],
©2011 John Wiley and Sons, and (c) permission from [34], ©2012 John Wiley and Sons)
Cell-laden hydrogel beads can be constructed with cells cultured on the hydrogel
beads or in cell-encapsulated hydrogel beads formed by the addition of cells into the
hydrogel solution before gelation. Owing to the size uniformity of the hydrogel
beads, uniform cell-encapsulated alginate gel beads and cell-adhered collagen gel
beads were constructed (Fig. 9.4a, b) [22, 33]. Moreover, seeding another type of
cell on cell-encapsulated hydrogel beads can be performed to construct 3D hierar-
chical co-culture in the beads. This method has been used for the construction of
fibroblast-hepatocyte co-culture beads and dermal fibroblast-epidermal keratinocyte
co-culture beads (Fig. 9.4c) [34]. In both co-culture beads, cellular functions were
improved due to cell-cell interactions, indicating that the 3D hierarchical co-cultured
beads can be used as high-throughput experimental tools for pathological and
physiological studies under 3D co-culture conditions.
Cellular blocks in polyhedron shape, another type of point-shaped cellular struc-
ture, have also been fabricated in microfluidic devices for high-throughput prepara-
tion (Fig. 9.5) [35, 36]. Light exposure to hydrogel solution containing a
photoreactive crosslinking agent in the microfluidic devices, so-called flow-lithog-
raphy, allows for the continuous formation of cellular blocks, the shape of which is
determined according to exposure patterns controlled with photomasks or digital
mirror devices. By using flow-lithography, cell-encapsulated PEG blocks can be
produced in a continuous flow by controlling their shape via columns, triangle poles,
and square poles.
9.2.2 Line-Shaped Cellular Structures
Cellular fibers and tubes, which are line-shaped cellular structures, are constructed
by cells cultured with hydrogel fibers or tubes. Hydrogel fibers and tubes easily
formed from cylindrical flows of hydrogel solution in microfluidic devices. For the
Fig. 9.5 (a) Illustration of the fabrication process for polyhedron-shaped cellular structures. (b)
Images of fabricated cellular blocks with various shapes. The shapes of the cellular blocks were
controlled by the shapes of the exposure patterns. Scale bars, 10 μm. (Images are reprinted with (b)
permission from [35], ©2006 Nature Publishing Group)
Fig. 9.6 (a, b) Fabrication process for hydrogel fibers using (a) capillary microchannels and (b)
coaxial fluidic devices. (c) Cell-laden alginate gel fiber fabricated by the extrusion method. Green
shows living cells and red shows dead cells. (d) Fabrication process for hydrogel tubes using
coaxial fluidic devices. (e) Scanning electron microscopy image of an alginate gel tube. (Images are
reprinted with (c) permission from [38], ©2011 Royal Society of Chemistry, and (e) permission
from [43], ©2009 John Wiley and Sons)
formation of hydrogel fibers, the extrusion of hydrogel solution flows from capillary
microchannels have been widely used [37–39]. By introducing the flows into a
gelling agent, hydrogel fibers are constructed (Fig. 9.6a). In other fabrication
methods for hydrogel fibers, microfluidic devices allow for the formation of coaxial
flows with the hydrogel solution as the inner flow and gelling agent as the outer flow,
whereby the hydrogel solution is gelated in the coaxial flows, and a hydrogel fiber is
formed (Fig. 9.6b) [40–42]. Since the diameters of the hydrogel fibers are control-
lable without length limitations depending on the configuration of the microfluidic
channels and the flow rate ratio, cellular fibers can be prepared with various
Fig. 9.7 (a) Fabrication process for core-shell hydrogel fibers with cell-laden collagen. (b–e)
Images of core-shell hydrogel fibers using (b) mouse fibroblasts, (c) rat islet cells, (d) human
adipocytes, and (e) human induced pluripotent stem cells (iPSCs). Scale bars, 200 μm. (Images are
reprinted with (b, c) permission from [44], ©2013 Nature Publishing Group, (d) permission from
[45], ©2015 John Wiley and Sons, and (e) permission from [47], 2017 licensed under Creative
Commons Attribution 4.0 International License)
dimensions by culturing cells with the hydrogel fiber (Fig. 9.6c) [38]. On the other
hand, hydrogel tubes are formed by using microfluidic coaxial flows with gelling
agent or culture solution as the inner flow, hydrogel solution as the intermediate
flow, and gelling agent as the outer flow (Fig. 9.6d). In the hydrogel tube formation,
when cells are embedded in the hydrogel solution, cell-laden hydrogel tubes are
constructed as a cellular tube (Fig. 9.6e) [43]. For example, the use of vascular
endothelial cells enables the formation of cellular tissues. Dimensions of the cellular
tubes are also variable according to the configuration of the microfluidic channels
and the flow rate ratio.
The construction of fiber-shaped alginate gel structures that included cell-laden
collagen solution at the core was achieved by microfluidic formation of coaxial flows
with cell-laden collagen solution as the inner flow, alginate solution as the interme-
diate flow, and a calcium chloride solution, which is a gelling agent for alginate
solution, as the outer flow (Fig. 9.7a) [44]. Here, owing to cover by the alginate gel
structure, the gelation of collagen is possible regardless of the slow rate of gelation,
allowing for a core-shell hydrogel fiber with cell-laden collagen as the core and
alginate gel as the shell to be constructed. After the cells were cultured with the fiber,
cellular adhesion to each other in the collagen to form a cellular tissue resulted in the
formation of a core-shell cellular fiber. Because cells were cultured in collagen
having high cell compatibility, line-shaped cellular tissues were successfully
constructed using various kinds of cells such as fibroblasts [44], islet cells [44],
adipocytes [45], smooth muscle cells [46] and human induced pluripotent stem cells
(iPSCs) [47] (Fig. 9.7b–e).
Fig. 9.8 (a) Fabrication process for hydrogel fibers containing cells by using a mold with fiber-
shaped grooves. (b, c) Cellular fibers constructed by culturing cells in the hydrogel fibers. (b)
Mouse skeletal muscle fiber, and (c) Human iPSC-derived cardiomyocyte fiber. Scale bars, 1 mm.
(Images are reprinted with (b) permission from [48], ©2013 Elsevier, (c) Reprinted from [49],
©2016 Royal Society of Chemistry)
Cellular fibers can also be formed by patterning cell-laden hydrogel structures

using molds with fiber-shaped grooves (Fig. 9.8a) [48, 49]. In this method, the
dimensions of the cellular fibers are controlled by the geometries of the grooves in
the molds. In addition, this method allows for the integration of cellular fibers with
artificial components when cell-laden hydrogel structures are patterned in state of
placement of the components in the hydrogel solution. In the case of embedding
anchors at the ends of the hydrogel structures, the cellular fiber can be cultured
without shrinkage because the anchors maintain its shape. Therefore, the cellular
fiber with anchors are well suited for the formation of muscle tissues, which have
strong traction force. As a result, by patterning skeletal myoblast-laden matrigel
structures or human iPSC-derived cardiomyocyte-laden collagen structures with the
molds and placing anchors at the ends of the structures, skeletal muscle fibers and
human iPSC-derived cardiomyocyte fibers can be constructed without deformation
caused by their traction force (Fig. 9.8b, c).
9.2.3 Plane-Shaped Cellular Structures
As a method for constructing plane-shaped cellular structures, cells cultured on

temperature-responsive culture dishes have been widely used [50, 51]. In this case,
cells adhered onto the culture dish are released when the dish is cooled. Due to its
characteristics, by cooling the dish with the cells in a confluent state, a cellular sheet
is obtained. In addition, by patterning the cells on the culture dish, a cellular sheet
with a heterogeneous arrangement of cells is constructed. Although temperature-
responsive culture dishes are a useful tool to form cellular sheets, their productivity
and design flexibility are limited to the number and size of the dish.
To increase the productivity and design flexibility of cellular sheets, the use of
microfluidic devices is an effective means. Microfluidic devices with a flat channel
Fig. 9.9 (a) Conceptual illustration of the fabrication process for cell-laden hydrogel sheets using
microfluidic devices. (b) Fluorescent image of patterned cardiomyocytes in an alginate gel-collagen
sheet. Scale bar, 2 mm. (Images are reprinted with (a, b) permission from [52], ©2012 John Wiley
and Sons)
allow for a planar flow of cell-laden hydrogel solution and contact to a gelling agent
(Fig. 9.9). Thus, after gelation of the hydrogel solution flow, cell-laden hydrogel
sheets are constructed continuously without length limitations [52]. Moreover, the
microfluidic devices enable control for the placement of different cells in the planar
flow, resulting in the formation of cell-laden hydrogel sheets with arrangement of
different cells [53].
Furthermore, plane-shaped cellular structures are also constructed by using a
special culture frame with anchors. When culturing a cell-laden hydrogel structures
in the frame to fix the periphery of the structure, shrinkage of the hydrogel structures
due to cell traction force could be prevented, and plane-shaped cellular structures
could be constructed (Fig. 9.10) [54]. Although it is difficult to release the cellular
structures from the frame, this method allows for the integration of the cellular
structures with external machines such as a pump via the frame. Therefore, by
making tube connectors as anchors on the frame and connecting pumps with
vessel-like structures, perfusion cultures of the plane-shaped cellular structures are
now available. This method is appropriate for the construction of skin tissues
requiring it to be cultured at an air-liquid interface because the nutrients are supplied
by the perfusion culture.
Fig. 9.10 (a) Conceptual illustration of the fabrication process for plane-shaped structures fixed
with anchors. (b) Skin tissue with vessel-like structure. After perfusion culture, the skin tissue
repelled water. Scale bar, 5 mm. (Images are reprinted with (a, b) permission from [54], ©2017
Elsevier)
9.3 Macroscopic Tissue Engineering by Assembling

Cellular Structures
The above-mentioned various-shaped cellular structures are attractive modules for

reconstructing organomimetic and homogeneous dense macroscopic cellular tissues.
When assembling the cellular structures, microfluidic techniques facilitate the pre-
cise control of cell arrangements by using appropriate methods depending on the
shapes of the cellular structures. In this section, we introduce the microfluidic
techniques used to assemble cellular structures according to the classification of
each shape.
9.3.1 Assembling Point-Shaped Cellular Structures
In the assembly of point-shaped cellular structures, especially cellular beads, the

molding method has been widely used [33, 55–57]. By culturing cellular beads
packed in a mold, the cellular beads are integrated by cell adhesion, resulting in the
construction of macroscopic cellular tissues. In this state, since nutrients and oxygen
can be supplied from the gap between the cellular beads, and central necrosis due to
the depletion of nutrients and oxygen is prevented in the cellular tissues. In addition,
shapes of the fabricated cellular tissues are transcribed from the mold shapes.
Therefore, using various shaped molds, the molding method can be used for the
construction of a millimeter to centimeter-sized cellular tissues with a human-doll
shape, mouse-brain shape, or lotus root shape (Fig. 9.11a–c).
To arrange the cellular beads in the cellular tissues, bioprinting is a promising
method for the construction of macroscopic cellular tissues by assembling cellular
beads. In bioprinting, printers or liquid dispensers eject and stack the cellular beads
[33, 58, 59]. In this process, cellular beads can be assembled by selecting a type and
Fig. 9.11 (a–c) Cellular tissues fabricated by the molding method. (a) Fluorescent image of a
human doll-shaped cellular tissue made of fibroblast-laden collagen beads. Green shows living cells
and red shows dead cells. (b) Mouse brain-shaped cellular tissue made of neurospheres. (c) Lotus
root-shaped cellular tissue made from fibroblast-laden cellulose beads. (d–f) Cellular tissue fabri-
cated by bioprinting. (d) Tube-shaped tissue made of fibroblast-laden collagen beads. (e) Branched
cellular tissue made from human skin cellular spheroids. (f) Ring-shaped cellular tissue made of
collagen beads containing various typed cells. (g) Scanning electron microscopy image of the
convex block. (h) Guided motion of the convex block along the concave rail. (Images are reprinted
with (a, d, f) permission from [33], ©2013 John Wiley and Sons, (c) permission from [57], ©2012
John Wiley and Sons, (e) permission from [58], ©2009 Elsevier, and (g, h) permission from [62],
©2008 Nature Publishing Group)
placing them at arbitrary positions (Fig. 9.11d, e). Using these properties, ring-
shaped tissues compartmentalized by cell types and tube-shaped tissues have been
achieved with bioprinting (Fig. 9.11f).
To precisely control the arrangement of the cellular beads, microfluidic tech-
niques capable of manipulating the cellular beads have been used. Since microfluidic
devices are effective for manipulating cellular beads one by one, fluid force in
microchannels can move cellular beads [60] or cell-laden PEGDA rings [61] to
construct ordered cellular tissues. Alternatively, microchannels with concave rails
have been proposed for assembling cell-laden convex PEGDA blocks since the
concave rails can guide the motions of the convex blocks (Fig. 9.11g, h) [62]. By
combining the rail networks and variable shapes of the PEGDA blocks, this
approach can be used for the construction of complex-shaped and heterogeneous
cellular structures.
9.3.2 Assembling Line-Shaped Cellular Structures
Reeling and weaving are suitable for the assembly of line-shaped cellular structures.
A millimeter-sized cellular tissue can be easily constructed by reeling cellular fibers
around a support such as a rod or plate (Fig. 9.12a–c) [39, 42–44]. In this state,
reeling cellular fibers with different typed cells can provide 3D co-culture conditions
in the cellular tissues (Fig. 9.12d). For assembling cellular fibers without the support,
the cellular fibers can be weaved [44]. Since microfluidic operations allow for the
manipulation of cellular fibers accurately, weaving the cellular fibers can be
Fig. 9.12 (a, b) Reeled chitosan fibers with hepatocytes around plates. (c) Helical tube released
from a rod after reeling it around the rod. (d) Co-cultured helical tube fabricated by reeling cellular
fibers containing HepG2 cells and NIH3T3 cells. (e) T-shirt-shaped cellular tissues fabricated by
weaving cellular fibers with fibroblasts, hepatocytes, and small lung carcinoma cells. (Images are
reprinted with (a, b) permission from [39], ©2010 Royal Society of Chemistry, and (c–e) permis-
sion from [44], ©2013 Nature Publishing Group)
Fig. 9.13 (a) Formation of a channel (white arrow) fabricated by dissolving alginate gel after
embedding a poly-L-lysine-coated alginate fiber in collagen gel containing human neonatal dermal
fibroblasts. (b) Blood vessel-like structure fabricated by dissolving an alginate gel fiber with smooth
muscle cells and endothelial cells in a collagen structure. (Images are reprinted with (a) permission
from [63], ©2016 Elsevier, and (b) permission from [64], ©2008 American Chemical Society)
achieved by combining microfluidic channels and a loom for microsized fibers

(Fig. 9.12e). Moreover, using cellular fibers with different typed cells, the method
has been successfully used for the construction of centimeter-sized 3D cellular
tissues with compartmentalization according to cell types.
Furthermore, the cellular fibers and cellular tubes can be used to form blood
vessel-like structures for the supply of nutrients in macroscopic cellular tissues. It is
well known that lumens can be formed in the cellular tissues by dissolving the
hydrogel with enzymes after embedding hydrogel fibers or tubes in the tissues
(Fig. 9.13a) [63]. When cellular fibers or tubes containing vascular endothelial
cells are embedded and the hydrogel is dissolved, cell-covered lumens can be formed
as blood vessel-like structures by cell adhesion to the luminal wall surface
(Fig. 9.13b) [64]. Construction of blood vessel-like structures in macroscopic cellu-
lar tissues is important for maintaining the cell viability of the tissue for extended
culture times.
9.3.3 Assembling Plane-Shaped Cellular Structures
For the assembly of plane-shaped cellular structures, stacking and reeling are
suitable methods. Stacking cellular sheets have been widely used to produce mac-
roscopic cellular tissues such as myocardial and liver [50, 51, 65]. In addition, by
reeling overlapped cellular sheets with different types of cells, millimeter-sized
vascular tubes hierarchized with vascular endothelial cells, smooth muscle cells,
and fibroblasts have been successfully formed (Fig. 9.14) [53]. Thus, these methods
are attractive for the construction of macroscopic cellular tissues with simple shapes.
Fig. 9.14 3D macroscopic tubular tissues fabricated by reeling a cell-laden sheet with location
control of various cell types. Red shows endothelial cells, green shows smooth muscle cells, and
blue shows fibroblasts. (Images are reprinted with permission from [53], ©2012 John Wiley and
Sons)
9.4 Organ-on-a-Chip Application
By integrating cellular tissues with microfluidic systems, devices referred to as

“organ-on-a-chip” have been developed. Organ-on-a-chip systems mimic physio-
logical conditions such as spatiotemporal chemical gradients, fluid shear stress,
cyclic stretch, and interactions between various types of tissues and organs.
Organ-on-a-chip systems are used not only as models of organs and pathology,
but also as platforms for drug discovery since organ-on-a-chip systems can better
emulate pharmacokinetics and toxicology rather than conventional 2D cultures. In
this section, we introduce various organ-on-a-chip systems according to the
microfluidic features.
9.4.1 Organ-on-a-Chip Based on Tissue-Trapping Structures
Many studies have integrated microfluidic devices with tissue-trapping structures

such as microwells and dynamic microarrays to fabricate organ-on-a-chip systems.
In these systems, cellular beads are able to be arrayed owing to the tissue-trapping
structures, and are also suitable for high-throughput drug screening. As an example
of a system utilizing microwells, a microfluidic device with a distributive
microfluidic channel network and microwells designed to capture cancer cells and
form cellular spheroids has been proposed (Fig. 9.15a) [66]. Using this device,
Fig. 9.15 Organ-on-a-chip systems based on a tissue-trapping structure. (a) System composed of a
distributive microfluidic channel network and microwells designed to capture cancer spheroids. (b)
Dynamic microarray device to trap cancer spheroids. (c) Liver-on-a-chip fabricated by trapping
hepatocytes into two lines. (Images are reprinted with (a) permission from [66], ©2012 Royal
Society of Chemistry, (b) permission from [68], 2014 licensed under Creative Commons Attribu-
tion 3.0 Unported Licence, and (c) permission from [69], ©2011 AIP Publishing LLC)
human breast cancer cells were distributed into 80 microwells via the microfluidic
channel network and the spheroids trapped in the microwells were obtained. After
formation of the spheroids, a cytotoxicity test of the anticancer drugs was performed
by treating the spheroids with the drugs and performing a MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay via the
microfluidic channel network.
Dynamic microarray is also available to trap cellular spheroids (Fig. 9.15b)
[67, 68]. The dynamic microarray is composed of a trapping channel and a bypass
channel that enables trapping cellular spheroids one-by-one. Using dynamic micro-
array device, spheroids consisting of cancer cells were trapped and arrayed. The
trapped spheroids were treated with anticancer drugs via the microchannel, and the
viability of the spheroids was measured by confocal-based live/dead cell imaging
[67], or by apoptosis assay based on caspase-3 activity with a normal plate reader
[68]. These researches indicated that the methods to evaluate chemosensitivity can
be chosen in the organ-on-a-chip systems based on dynamic microarray according to

the situation; confocal imaging when spatiotemporal information is important, and
apoptosis assay with plate reader when the simplicity of the procedure has high
priority. The dynamic microarray was used not only for cellular spheroids but also
for cell-laden hydrogel beads. For example, the dynamic microarray can trap skin
cellular beads, which was composed of type-I collagen gel beads, embedded human
fibroblasts, and coated by human epidermal keratinocytes [34]. Owing to the ability
of continuous observation that is one of advantages of the dynamic microarray, the
secretion of type-VII collagen from the skin cellular beads was analyzed with
individual beads trapped. By analyzing a large amount of the beads at once using
the dynamic microarray devices, this system might be a screening tool of skin drugs
and cosmetics.
The trapping structure has also been used to align hepatocytes to form line-shaped
liver tissues (Fig. 9.15c) [69]. The cell culture area was designed to trap and align
hepatocytes into two lines, and was separated from the main perfusion channel by a
wall containing a series of long narrow slits that mimics the endothelial barrier. The
aligned hepatocytes formed bile canaliculi along the cell culture area, and the
excretion function was evaluated by the CDFDA (5-(and-6)-carboxy-20 ,7-
0
-dichloro-fluorescein diacetate) assay. Thus, by trapping tissues using the device
with endothelial-like channels, microfluidic devices can be used for evaluating drug
metabolism and excretion as an alternative to animal testing.
9.4.2 Organ-on-a-Chip Based on Membrane-Integrated

Channel
In order to reconstruct epithelial structures and functions in vitro, arranging plane-

shaped cellular structures in microfluidic channels provides a promising approach.
As a simple method for the arrangement, porous membranes have been integrated
into the microfluidic channels to separate layers of the channels. This membrane-
integration approach has been widely adopted to fabricate various organ-on-a-chip
systems, since most organs have epithelial tissues in vivo. The simplest way to
integrate a porous membrane into the microfluidic device is to utilize commercially
available cell culture inserts (Fig. 9.16a) [70, 71]. The device can be fabricated by
standard soft lithography techniques and have compartments with a holder for the
cell culture insert. These compartments support two culture modes: exposing tissue
to medium flow, and shielding tissue from the underlying flow by the porous
membrane of the cell culture insert. The latter mode is especially effective for the
culture of skin tissue since the air-liquid interface is necessary for culturing and
maintaining the epidermal layer. Owing to this advantage, co-cultures of skin biopsy
and liver spheroid [70], and co-culture of reconstructed skin-equivalents and hair
follicle [71] have been demonstrated. By co-culturing tissues, crosstalk between
tissues can be established and thus improvements in their morphologies were
Fig. 9.16 Organ-on-a-chip system based on membrane-integrated microfluidic channels. (a) Skin
and liver co-culture device integrated with cell culture inserts. (b) An intestinal model constructed in
the device with microchannels separated by porous membrane. (c) Gut-on-a-chip system utilizing
flexible porous membrane. (Images are reprinted with (a) permission from [70], ©2013 Royal
Society of Chemistry, (b) permission from [72], ©2013 Royal Society of Chemistry, and (c)
permission from [85], ©2012 Royal Society of Chemistry)
achieved. However, the extensibility of the membrane-integration using cell culture

inserts is not high since the design of the microfluidic device is limited by the
available size and shape of the cell culture inserts. In particular, it is difficult to set
microfluidic channels on the top surface of the cell culture inserts due to the plastic
walls of the membrane holder.
Bare porous membranes are used to realize more arbitrary designs than cell
culture inserts. In this approach, the porous membranes are sandwiched by silicone
rubber substrates with microfabricated channels. By seeding Caco-2 intestinal cells
on the top surface of a semipermeable membrane separating the upper and lower
microchannels, an intestinal model device was fabricated (Fig. 9.16b) [72]. The stir-
bar-based micropumps in the device allowed for the perfusion of medium and the
application of shear force on the cells. Furthermore, the polarized transport of
rhodamine 123 from the basolateral side (lower side) to the apical side (upper
side) was observed by optical fibers embedded in the device. This intestinal model
device was extended to multi-organ-on-a-chip systems by adding liver and lung
chambers inoculated with hepatic and lung cancer cells, respectively [73]. Orally
administered or biologically active anticancer drugs were tested on the system.
Furthermore, the device named “HuMiX (human-microbial crosstalk)” used to

emulate the gastrointestinal human-microbe interface was developed by extending
an intestinal model [74]. The device had three layers of microfluidic channels. The
first and second layers were separated by a nanoporous membrane whose top surface
was inoculated with microbes, and the second and third layers were separated by a
microporous membrane whose top surface was inoculated with Caco-2 cells. Owing
to the layered channels, a gradient of oxygen and biomolecules was achieved.
Besides the intestine, the membrane integrated in the microfluidic device can be
applied for the construction of other organs such as the kidney [75, 76], blood-brain
barrier [77], placenta [78], and skin [79, 80]. Owing to the microchannels separated
by a porous membrane, renal inner medullary collecting duct cells on the membrane
have been stimulated by fluid shear stress, and hormonal stimulation, osmotic
gradient, and optimal culture conditions have been investigated [75, 76]. Similarly,
the influence of the flow condition on the barrier function of the blood-brain barrier
[77] and the formation of microvilli in the placenta [78] have also been investigated.
Furthermore, transdermal drug permeation has been demonstrated by applying drugs
on a skin model on a porous membrane and sampling the medium from the
microchannel under the membrane [79].
A combination of several of the approaches stated above has also been proposed.
For example, an on-chip model composed of intestinal, liver, and cancer cells has
been successfully used in an assay for an anticancer drug [81]. In this device, the
intestinal compartment was fabricated with a collagen-coated porous membrane, and
the liver compartment was designed to trap hepatic cells cultured on carrier beads via
a vertical microchannel. Similarly, a multi-organ-on-a-chip system composed of
intestinal, skin, liver, and kidney equivalents has been reported [82]. In this device,
the kidney compartment was constructed by seeding RPTEC/TERT-1, a proximal
tubule cell line, on the membrane separating the upper and lower microchannels as
stated above. The intestinal and skin compartments were established by integrating
cell culture inserts into the microchannel. According to these studies, more func-
tional organ-on-a-chip systems can be fabricated by combining several approaches
such as the integration of cell culture insert, bare porous membranes, and trapping
structures.
However, it is difficult to apply mechanical forces to the membrane, such as
tension and compression, via the cell culture insert or bare membrane. To overcome
this limitation, a microfluidic device integrated with a porous stretchable membrane
has been proposed (Fig. 9.16c) [83–87]. The device consists of upper and lower
microchannels separated by a polydimethylsiloxane (PDMS) porous membrane and
vacuum chambers on both sides of the microchannels. By vacuuming the chambers,
the porous PDMS membrane is able to be stretched. Using this device, a lung-on-a-
chip was proposed [83, 84]. The alveolar epithelial cells and pulmonary endothelial
cells were cultured on the upper and lower surfaces of the membrane, respectively.
By applying cyclic strain, inflammatory responses of the endothelial cells to the
nanoparticles were increased compared to the static condition. Moreover, as a result
of the inflammatory responses, the endothelial capture of neutrophils and their
transmigration from the endothelial layer to the epithelial layer were induced
[83]. Since similar results were obtained by experiments using whole mouse lung,
this model could be used as alternatives to animal experiments. Furthermore, gut-on-
a-chip were also reported using the similar device [85–87]. Human intestinal epi-
thelial cells (Caco-2) were inoculated onto the PDMS porous membrane and cul-
tured under fluid shear stress and cyclic strain. These stimulations promoted the
formation of intestinal villi. Moreover, microbes were cultured on the differentiated
epithelium and the influence of the cyclic stretch was investigated. As stated above, a
microfluidic device integrated with a porous stretchable membrane is a promising
tool for emulating in vivo conditions since real organs normally experience mechan-
ical strain.
9.4.3 Organ-on-a-Chip Based on ECM Gel Channels
In order to investigate cell-ECM interactions in a 3D environment and/or

maintaining large tissues with a nutrient supply, many studies have focused on
fabricating microchannels in an ECM gel. In addition to the methods using cellular
fibers and tubes (see Sect. 9.3.2), various methods have been proposed to fabricate
the microchannels. The simplest method is to extract needles or wires embedded in
the collagen gel (Fig. 9.17a) [88–90]. After fabricating the microchannels, endothe-
lial cells were seeded in the microchannels to fabricate blood vessel models. The
blood vessel models were used to investigate the influence of fluid shear stress on
cell viability, barrier function [89], and angiogenesis [90].
However, it is difficult to fabricate complex blood vessels using this method. For
the fabrication of complex blood vessels, soft lithography techniques have been
employed [91]. Briefly, microstructures on collagen gels were molded from PDMS
stamps. The microstructured collagen gel was sealed with flat collagen gel supported
by jigs, followed by an inoculation of endothelial cells. The blood vessel model was
cultured with gravity driven flow of the medium and used for permeability tests and
angiogenesis studies.
To make more physiologically relevant blood vessel models, neovascularization
of endothelial cells have been harnessed (Fig. 9.17b) [92, 93]. The microfluidic
device for neovascularization had microchannels partitioned by microposts: the
center channel was filled with fibrin gel to be seeded with endothelial cells and
vascularized by the endothelial cells, medium channels flanking the center channel,
and stromal cell channels next to the medium channels to induce neovascularization
of the endothelial cells. Using this blood vessel model, physiological features of
blood vessels were observed such as NO synthesis depending on the fluid shear
stress and inflammatory response.
Recently, 3D printing technology has been utilized for the construction of
microchannels with arbitrary 3D shapes in ECM gels (Fig. 9.17c) [94–97]. In this
approach, ECM gel and sacrificial structures were printed by a 3D printer. The
sacrificial structures were made of gelatin or Pluronic F-127 that were liquefied and
removed by temperature changes. After seeding endothelial cells in the resulting
Fig. 9.17 Microchannels fabricated in ECM gel. (a) Blood vessel model fabricated by extracting
needle and seeding endothelial cells. (b) Blood vessel model fabricated by neovascularization of
endothelial cells. (c) Blood vessel model printed by 3D printer. (Images are reprinted with (a)
permission from [88], ©2006 Elsevier, (b) permission from [92], ©2013 Royal Society of Chem-
istry, and (c) permission from [95], ©2014 John Wiley and Sons)
microchannels, long-term cultures (6 weeks) and permeability tests were performed

[97], indicating that this system can be used as both a nutrient supply pathway for 3D
tissues as well as a blood vessel model for physiological studies and drug screening.
Although many organ-on-a-chip applications using microchannels in ECM gel
are blood vessel models, it is presumed that the microchannels in the ECM gel are
also suitable for modeling organs with a lumen structure, such as an intestinal model
on a geometrically engineered hollow channel in a porous protein scaffold [98]. The
geometrically engineered hollow channel was fabricated by removing a nylon screw
embedded in the silk scaffold. The channel was seeded with intestinal epithelial cells
after a collagen gel containing intestinal myofibroblast was delivered into the silk
scaffold. This intestinal model exhibited high accumulation of mucous secretions

from the epithelium compared to a 2D culture, O2 concentration similar to those
observed in vivo, and co-culture of bacteria on the epithelium.
9.4.4 Organ-on-a-Chip Utilizing Anchored Cellular Tissue
Various cellular tissues composed of cells and ECM gel shrink and/or dynamically
contract during cultivation. To maintain structure and function of these tissues
against the shrinkage and contraction, anchor structures have been used. By com-
bining the anchor structures with microfluidic devices, mechanical evaluation for
tissue functions were achieved. Typical example is a heart-on-a-chip. The heart-on-
a-chip was fabricated by gelling fibrin containing cardiomyocytes in a rectangular
casting mold that had two flexible posts on the both edges (Fig. 9.18a) [99, 100]. The
flexible post worked as anchor to keep the shape of cardiac tissue, and the force of
the tissue was estimated by the deformation of the post [99]. Moreover, toxicity test
of a drug (doxorubicin) was achieved by applying the drug and measuring the time
course change of the force. Furthermore, microchannels were fabricated in the tissue
by dissolving alginate fibers embedded in the tissue by sodium citrate or alginate
lyase [100]. Since the microchannels were connected to the post with a hollow
channel, medium perfusion was achieved, resulting in improved tissue morphology.
However, it was difficult to precisely control the tissue shape due to shrinkage by
this method. In addition, cell orientation was also difficult to be controlled since it
depends on the shape of the tissue. To overcome these limitations, a heart-on-a-chip
composed of fiber-shaped cardiac tissue whose ends were fixed to anchors was
proposed (Fig. 9.18b) [49] (see Sect. 9.2.2). Owing to high cell density and anchors,
the fiber shape was maintained, and the fiber shape aligned cardiomyocytes. More-
over, by setting anchors on a PDMS substrate with cantilevers, the force measure-
ment was performed. Using this system, the efficacy of drugs was tested by
measuring the change of contractile force and frequency.
Skin-on-a-chip systems were also fabricated by adopting anchor structure
[38, 39]. Perfusable vessel-like channels were fabricated by dissolving sacrificial
structures made of alginate gel and seeding endothelial cells [101]. Owing to
movable posts with a hollow channel connected to the channels, perfusability was
maintained even after the skin tissue had shrunk. Transdermal drug tests have been
demonstrated by another skin-on-a-chip system (Fig. 9.18c) [54] (see Sect. 9.2.3).
The skin tissue also had perfusable vessel-like channels fabricated by removing
nylon wires embedded in the tissue and seeding endothelial cells. Since the vessel-
like channels were fixed to the device channels by anchors, both the perfusability and
the plane shape of the skin tissue were maintained. Owing to the perfusability and
plane shape, transdermal drugs were able to be applied onto the surface of the skin
tissue, and the drugs permeated into the vessel-like channels were successfully
collected. From this result, this skin-on-a-chip system might be a useful tool for
the development of cosmetics and drugs that are topically applied.
Fig. 9.18 Organ-on-a-chip systems using anchor structures. (a) Engineered heart tissue anchored
to two posts. (b) Fiber-shaped cardiac tissue on a chip. (c) Skin-on-a-chip for percutaneous
absorption test. (Images are reprinted with (a) permission from [99], ©2010 Wolters Kluwer Health,
Inc., (b) permission from [49], ©2016 Royal Society of Chemistry, and (c) permission from [54],
©2017 Elsevier)
9.5 Summary
Microfluidic devices can construct various-shaped cellular structures with high

throughput and high uniformity, while also allowing for the manipulation of the
cellular structures for assembly. Therefore, using the cellular structures as building
modules, macroscopic cellular tissues can be constructed. Furthermore, the integra-
tion of the cellular tissues with microfluidic devices to create organ-on-a-chip
systems can be used for the analysis of pharmacokinetics and toxicology under
physiological conditions. The studies introduced in the chapter will provide prom-
ising methods and tools to contribute to future improvements in this field. Further-
more, technical advances in the integration of cellular structures and microfluidic
devices will help design novel devices such as biosensors and biological robots. As
discussed, microfluidic technology will be widely studied with potential to explore
new research fields.
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Chapter 10
Nanobiodevices for Cancer Diagnostics
and Stem Cell Therapeutics
Daisuke Onoshima, Hiroshi Yukawa, and Yoshinobu Baba
Abstract This chapter describes the potential of multifunctional nanobiodevices in

combination with quantum dots (QDs) to meet the requirements of diagnostic and
theranostic systems. Nanobiodevices are primed to be powerful tools to provide the
basis for the detection of small amounts of samples and simple operation. QDs can
be utilized in these devices as bio-probes or labels for biological imaging of single
molecules and cells. They have developed into new formats of biosensing to push
the limits of detection. QDs has been also demonstrated to construct a
multifunctional nanoplatform for stem cell transplantation and labeling with diag-
nostic and therapeutic modalities. The potential clinical applications of QDs have
been expanded by the development of considerably low cytotoxicity QDs that do not
include cadmium or selenium, as well as the development of longwave fluorescence
D. Onoshima (*)
Institute of Innovation for Future Society, Nagoya University, Nagoya, Japan
ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Nagoya, Japan
H. Yukawa (*)
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University,
Nagoya, Japan
Y. Baba
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University,
Nagoya, Japan
Health Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), Takamatsu, Japan
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan,
Republic of China

276 D. Onoshima et al.
QDs with strong permeability into the body. It provides the promising applications
and further perspectives on future regenerative medicine.
Keywords Nanobiodevice · Quantum dot · Cancer diagnosis · Regenerative

medicine
10.1 Introduction
Nanobiodevice is a piece of contrivance, equipment, machine, or component in the

overlapping multidisciplinary activities of nanotechnology and biotechnology. The
research efforts have been focused on the development of novel nanodevices for
future personalized medicine and evidence based healthcare, which includes the
following grand challenges: (1) multifunctional nanobiodevices for single platform
bioimaging and targeting therapy; (2) ultra-high sensitive, highly specific, low
invasive, and reliable multiplexed detection of disease at their inception; (3) remote
disease monitoring through the ability of nanotechnology to provide on-line sensing
and information relay; (4) construction of tissue-growth facilitating structured cell
sheets for organ repair and replacement; (5) application of biological nanostructures
in bioelectronics and environment-friendly nanofabrication processes; (6) single cell
interventions and diagnostics including stem cell growth and differentiation and
cellular level genomics and proteomics; and (7) stem cell differentiation and site-
specific delivery. The field of multifunctional nanodevices has continued to witness
strong growth during a decade. Peer reviewed publications on this field have been
continuously increasing and the diversity of applications continues to expand. There
is a continued healthy and necessary focus on the physical science fundamentals for
both multifunctional nanodevices and an abundance of multidisciplinary biomedical
applications are being built upon these fundamentals.
This chapter covers fundamentals and basic researches of nanobiodevices relating
to multifunctional quantum dots (QDs)-based cancer diagnostics and stem cell
therapeutics for regenerative medicine. A field of recent diagnostics and therapeutics
has been advanced with QDs. QDs have developed into new formats of biomolecular
sensing to push the limits of detection in biology and medicine. QDs can be also
utilized as bio-probes or labels for biological imaging of living cells and tissues.
More recently, QDs has been demonstrated to construct a multifunctional
nanoplatform, where the QDs serve not only as an imaging agent, but also a
nanoscaffold for diagnostic and therapeutic modalities. This chapter highlights the
promising applications of multi-functionalized QDs as advanced nanosensors for
diagnosing cancer and as innovative fluorescence probes for in vitro or in vivo stem
cell imaging in regenerative medicine.
The development of semiconductor QDs has been heading toward an interdisci-
plinary field with bioassays and bioimaging technologies [1–3]. It offers significant
advantages over those of the previous methods, which have relied on conventional
organic fluorophores or fluorescent proteins. The unique and useful optical
10 Nanobiodevices for Cancer Diagnostics and Stem Cell Therapeutics 277
properties of QDs have been explored such as broad absorption spectra, narrow
emission spectra, high quantum yields, long fluorescent lifetime, size-tunable
photoluminescence, and exceptional photostability with a strong resistance to
photobleaching [4–7]. The optoelectronic characteristics of QDs arise through the
systematic transformation in the density distribution of the electronic energy levels
as a function of the size of the QDs [8]. As a result, the nature of the QD surface
plays a key role in their optical behavior. The ability to modify the surface of QDs
with biomolecules or other polymers through various conjugation methods under-
pins their versatility.
QDs have been incorporated into various biological assays for the reversible
detection and quantification of biomolecules. Such assays often focus on the use of
distance-dependent fluorescence resonance energy transfer (FRET) [9, 10]
andmultiplexed bioanalysis with multiple colors [11, 12]. These techniques can be
introduced for applications such as immunoassays [13, 14], molecular diagnosis
[15, 16], clinical assays [17–19], and cellular analysis [2, 20]. Many kinds of
QD-based biological assays have been demonstrated in bulk solution or on solid
substrates [21]. A more competitive platform is microfluidic device. The device is
able to handle the extremely small quantities of fluid with multiple analytical
samples transported through the microfabricated channels [22]. It can provide
opportunities for further removal or replacement of QD-based sensing
components [23].
When applying QDs to biological imaging and cellular studies, the toxic nature of
cadmium-containing QDs remained a major concern. However, with the recent
advances in the development of surface modifications of QDs and cadmium-free
QDs, the potential toxicity of cadmiumis no longer a problem for in vitro and in vivo
imaging studies. The cellular uptake of QDs can be modulated by their size [24],
shape [25–27], and surface functionalization [28]. In fact, the use of multicolor QDs
for stem cell imaging is probably the most important and clinically relevant appli-
cation for regenerative medicine in the immediate future. Despite the enormous
potential of QDs in therapeutics, the fundamental information on the interaction
between QDs and therapeutic cells [29] is relatively limited. This review thus aims to
outline the beneficial properties presented by QDs, along with important advances in
their biological applications for cancer diagnostics and stemcell therapeutics. Par-
ticularly, the observation of the QD-mediated cellular responses, such as cellular
uptake and intracellular behavior of QDs, will provide insights into the nanoparticle
design and the therapeutic efficacy for regenerative medicine.
10.2 Quantum Dots-Based Sensing and Detection
10.2.1 Quantum Dots
QDs stand out as one of the most exciting research tools in chemistry, physics, and
biology (Fig. 10.1). These inorganic fluorescent nanocrystals typically comprise
periodic groups of CdSe and CdTe or InP and InAs semiconductor materials. For
Fig. 10.1 Schematic of a CdSe (Core)

major negatively charged
QD. (Reprinted with
permission from Ref. [137]) ZnS (Shell)
Polymer coating
Functional group
15 ~ 20 nm
the semiconductor nanocrystals, the energy levels are quantized due to the quantum-
confinement effect [30, 31], and their spacing can be controlled by the crystal sizes.
This effect leads to the superior optical properties of QDs, such as narrow, symmet-
ric, and size-tunable emission spectra. Also, the broad excitation spectra of QDs
enable multicolor fluorescent applications. Compared with organic fluorophores or
fluorescent proteins, QDs show 10–100 times brighter fluorescence and 100–1000
times higher fluorescence stability against photobleaching. These benefits of QDs
facilitate long-term monitoring of intermolecular and intramolecular interactions in
living cells and tissues. Consequently, much interest has been focused on the
exploration of QDs for biomedical applications.
Among the array of synthetic routes devised for the preparation of biocompatible
QDs, the coating of the CdSe core with the ZnS layer is indispensable [2, 3]. Passiv-
ation by the ZnS layer protects the CdSe core from oxidation, and reduces the
toxicity of the CdSe core from leaching out to the surrounding solutions. It also
enhances the photoluminescence yield. Although the synthesis of QDs has been
performed directly in aqueous solution, QDs themselves have little specificity for the
aqueous nature of the biological environment [32, 33]. For example, the ZnS-coated
QDs are only soluble in nonpolar organic solvents. Altering the surface properties of
QDs mainly relies on the conjugation with biological molecules such as aptamers
[34], antibodies [35], oligonucleotides [36], and peptides [37] to gain the biological
affinity [38].
As a result of the conjugations of QDs, they have achieved optimal stability,
monodispersity, crystallinity, solubility, and biocompatibility in the fields of diag-
nostic and therapeutic research. For example, within a certain range of concentra-
tions, CdSe/ZnS shows good biocompatibility with human amniotic mesenchymal
stem cells [39]. The effect of QD size and poly(ethylenimine) coating on the labeling
efficiency with stem cells has been also reported [40]. Furthermore, a peptide-coated
QDs can mimic cellular transport mechanism in stem cells through endosomal
escape [41].
10.2.2 Fluorescence Resonance Energy Transfer
In terms of the fluorescence application to monitor binding interactions or confor-

mational changes of biomolecules, FRET phenomenon, the non-radiative energy
A Intercalater (YOYO-3) B Nuclease-

(Em: 631 nm) tolerant
Control FRET probe
Biotinylated
oligomer
Excitation
(440 nm)
DNA
Streptavidin
FRET
Emission (QD)
(605 nm) Nuclease-tolerant FRET probe
Fig. 10.2 A nuclease-tolerant FRET probe. (a) Schematic of the FRET between the QD donor and
the DNA-intercalating dye (YOYO-3) acceptor. The QD emission overlaps the absorption spectrum
of the dye, suggesting that an efficient FRET between the QD donor and the YOYO-3 dye acceptor
can take place. (b) An electrophoresis analysis of the FRET probe for the effect on DNA digestion.
A yellow circle shows the undigested FRET probe by a restriction endonuclease (EcoRI).
(Reprinted with permission from Ref. [137])
transfer between an excited state fluorophore (donor) and another fluorophore

(acceptor) through long-range dipole–dipole coupling, has been recognized as a
quite useful detection scheme in various bioanalyses [42]. The utility of this detec-
tion scheme is explained that it generates fluorescence signals sensitive to the
changes in 1–10 nm range. More specifically, the FRET-based transduction using
QDs as donors has become a popular approach for assay development. Their broad
absorption spectrum in the UV region can be used advantageously to avoid the direct
excitation of acceptor dyes. As a result of the introduction of multiple pathways for
energy transfer, the FRET efficiency can be enhanced by the QDs. The transduction
strategy can be also arranged using QDs and dyes. For example, many QDs have the
opportunity to interact with a single acceptor dye, or multiple dyes can interact with a
single QD.
QD-based FRET probes have also been reported, including QD conjugated
hybridization probes for the preliminary screening of siRNA sequences [43] and
an ultrasensitive DNA nanosensor with a singleQD [36, 44],which benefit from the
use of the unique photophysical properties of QDs and their conjugates. Recent work
in our laboratory [45] has demonstrated the feasibility of using QDs as a nuclease
tolerant FRET probe. The streptavidin-coated QD binds to the biotinylated DNA end
(Fig. 10.2a), and the FRET-inducing electrostatic coupling and the structural
changes to the QD-DNA conjugates can be detected (Fig. 10.2b) based on the
conjugation of the QDs with YOYO-3 dye-intercalated DNA. The QD-DNA con-
jugate and the tolerance properties induced by dye binding are thought to be notable
due to the utilization of QD conjugated functional materials in living cells or tissues.
Transduction via FRET also makes it possible to employ multiple methods for data
analyses. For example, ratiometric detection is often used for differences in assay
preparation among multiple analyses and instrumental drift. The narrow symmetrical
emission profile of QDs facilitates the deconvolution of acceptor signals for the
recovery of absolute signals [46].
10.2.3 Microfluidic Devices
Microfluidic system is based on the microfabricated structures to control or manip-

ulate fluids constrained in a small space [22, 23]. The potential to be used in
biological applications is a major driving force behind the rapid development of
this system [48–51]. They are primed to be powerful tools to provide the basis for the
detection of biomolecules with small amounts of reagents and simple operation
[47]. In addition, the large surface area-to-volume ratio and mass transport by
nondiffusive means offers the transduction of analytes within seconds to minutes.
Microfluidic devices have been known to facilitate single-molecule measure-
ments in combination with QDs [51]. Such approaches are useful to determine
patterns and distributions that may otherwise be masked by ensemble averages
[52]. Single-molecule studies are usually performed by monitoring a fixed volume
of solution. Analytes are allowed to diffuse into this fixed volume in order to be
detected. A continuous-flow system, as found in capillaries and microfluidics,
ensures that multiple analytes are moved across the detection volume, increasing
the probability of detection. For example, a mismatch repair protein complex that
slides while maintaining continuous contact with DNA can be visualized by QDs in
real time [53]. The DNA-binding proteins are engineered with epitope tags and
labeled with antibody coupled QDs. The motion of the proteins is observed on the
single DNA molecule that is anchored and elongated in a microfluidic device. Using
similar protein labeling methods with QDs, several kinds of specific sequences
related to genetic or epigenetic changes of genomic DNA molecules have been
detected for optical mapping applications [54]. Moreover, the mapping method is
successfully applied to the detection of DNA methylation at a single molecule
level [55].
On the other hand, recent advances in a microfluidic system developed by our
group (Fig. 10.3a and b) allow for the visual observation of nucleolytic cleavage for
the direct screening and detection of a specific sequence in genomic DNA [56]. By
using DNA molecular tagging with QD, it is possible to track the motion and
position of a single DNA molecule (Fig. 10.3c and d) gaining access to a restriction
enzyme on the microchannel surface. This kind of experimental approach will help
enable applications for ultra-sensitive, high-speed and small-volume bioanalytical
measurements.
A B
QD
DNA
C D 60
X-coordinate
0.0 s 40
(µm) 20
0.3 s
0
0.6 s 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time laps (s)
0.9 s 20 Time (s)
Y-coordinate
10
(µm)
1.2 s 0
-10
1.5 s -20
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
1.8 s Time laps(s)
Time (s)
Intensity (a.u.)
Fluorescence
2.1 s 200
100
2.4 s
0
2.7 s 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time laps (s)
Time (s)
Fig. 10.3 A microfluidic molecular tracking system. (a) Schematic design for the time-series
analysis of ongoing processes in the enzymatic reaction through the detection of single-molecular
movements. (b) Layout of the molecular tracking chip. Enzymatic reactions are done in a conven-
tional microfluidic device, consisting of a polydimethylsiloxane (PDMS) microstructure and a
syringe pump. A single QD was connected to one DNA molecule as a tracking dye. Enzymes are
immobilized on the bottom surface of a microchannel, and QD-tagged DNA molecules are flown in
the channel. The fluorescent images during the event are captured by a total internal reflection
fluorescence (TIRF) microscope. (c) Video monitoring in action at the enzyme area. The flow keeps
the motion of DNA molecule constant, and a restriction enzyme (Apa I) on the surface changes the
motion. (d) A trajectory of QD-tagged DNA represented as x coordinate, y coordinate and
fluorescence intensity. Dashed line shows the control with no enzyme immobilized. (Reprinted
with permission from Ref. [137])
10.3 Cancer Diagnosis by Quantum Dots

10.3.1 Protein and Nucleic Acid
Early diagnosis of cancers is at the forefront of cancer research. Cancer biomarker-

based in vitro assays are useful for the screening and diagnosis of cancer, as well as
for monitoring the response to therapy. Many proteins are considered as useful
diagnostic markers of various cancers [58–60]. They are also the targets for basic
biomedical research. Since the protein biomarkers are often present at very low
concentrations, analytical methods with low detection limits are required for the
early diagnosis of cancers [60]. QDs have proven to be applicable for the sensitive
detection of cancer biomarkers such as ovarian cancer [61], breast cancer [62], and
prostate cancer [63]. More recently, two tumor biomarkers, α-fetoprotein and
carcinoembryonic antigen in human serum, have simultaneously been detected by
a QD based nanosensor [64].
Nucleic acids are another type of tumor marker for various cancers. QDs are also
very useful in the detection of nucleic acids, especially in a multiplexed format. For
example, a multicolor optical coating for biological assays comprises embedding
different-sized QDs into polymeric microbeads at precisely controlled ratios. These
were designed as a DNA hybridization system using oligonucleotide probes and
triple-color encoded beads. The coding signals can identify different DNA
sequences [65]. Moreover, a solid-phase FRET assay using immobilized QDs as
donors can detect two target sequences simultaneously [21]. This assay showed that
over 50% of the analyte’s signal readout was obtained even in bovine serum and
with a large excess of non-complementary genomic DNA as background noise.
More recently, a single-quantum dot-based nanosensor for specific miRNA
detection has been developed [66]. Among the numerous analytical approaches for
miRNA detection, most suffer from problems such as non-specificity and low
sensitivity. To overcome these problems, two-stage exponential amplification reac-
tions and single-QD-based nanosensors were fabricated. The detection limit of 0.1
aM miRNA was achieved, and even single-nucleotide differences between miRNA
family members could be distinguished.
10.3.2 Cancer Cells
Sensing the interaction between drug-carrying vehicles and cell membrane is the
primary requisite for a successful diagnostic process, where the diseased cells are
first located, following by subsequent cellular uptake and release of a therapeutic
agent into the cytosol or nucleus of cells [68]. Especially for cancer research and
therapy, QDs have been utilized as imaging probes [2, 3] for the recognition of
specific cell types and tissues in the clinical settings [67]. For example, a static
immunostaining of cellular targets with QDs has been demonstrated and shown to be
both brighter and more photostable than comparable organic fluorophores [17]. In
particular, QD-peptide conjugates can specifically target the tumor vasculature in
mice [37]. A PEG coating has reduced the reticuloendothelial clearance in the
in vitro histological results. An ABC triblock copolymer-coated QD probe has
also been developed to target and image prostate cancer [35]. For in vivo studies,
the tumor site can be actively probed by the antibody-conjugated QDs and imaged in
living animals. The large size and immunogenicity of antibodies sometimes affect
their pharmacological behavior. Alternatives are small ligands such as peptides [38]
or aptamers [34] that are employed to conjugate QDs.
A QD nanoprobe has been developed to sense glioma cells based on the
overexpression of the extracellular matrix glycoprotein, tenascin-C [69]. Tenascin-
C is involved in tissue remodeling, and plays a role in the invasion of glioblastoma
into the surrounding brain tissue. The QDs were targeted to tenascin-C by a single-
stranded DNA aptamer. Non-cadmium-containing QDs have also been shown to be
able to label cells fluorescently [70]. For example, phospholipid micelle-
encapsulated silicon QDs could be conjugated to transferrin and taken up by
pancreatic cancer cells. The concentration of silicon QDs applied to these cells
was not toxic, because there was 95% cell viability after 24 h.
Since the cellular uptake of exogenous material generally occurs through inter-
nalization mechanisms [71], QDs are passively uptaken via non-specific endocytosis
along the migratory pathway of human mammary epithelial tumor cells. Moreover,
unmodified QDs have been used as an alternative marker over gold nanoparticles for
phagokinetic tracking to monitor cell motility as a potential assay for cancer metas-
tasis [72]. The use of a transfection agent such as liposomes or micelles can assist the
delivery of QDs. Polymer- or ligand-modification of the QDs is usually more
specific and efficient than non-specific endocytosis alone [73, 74]. A general obser-
vation is that the endocytosed QDs are often trapped in endosomes and lysosomes.
They can be visualized as punctate fluorescence staining. This plays an important
role in tracking the spatiotemporal behavior of the cells.
10.4 Stem Cell Labeling by Quantum Dots
10.4.1 Stem Cell Labeling
Regenerative medicine is expected to overcome the shortage of donated organs,

donor site morbidity and immune reactions. Many kinds of stem cells, such as
induced pluripotent stem (iPS) cells [75, 76], embryonic stem (ES) cells [77, 78],
and some kinds of somatic stem cells, including bone marrow-derived stem cells
[79, 80] and adipose tissue-derived stem cells [81, 82], have been discovered and
may be useful in numerous applications for regenerative medicine [84–86]. To
ensure the safety and maximum therapeutic effects of regenerative medicine, ana-
lyses of the behavior, accumulation and condition of transplanted stem cells in vivo
have become increasingly important [86, 87]. However, the conventional in vivo
imaging modalities used in clinical practice are not sufficient for the analysis of
transplanted stem cells [88, 89]. Fluorescent imaging (FI) with the prominent
fluorescence properties of QDs is expected to detect the transplanted stem cells
in vivo with higher sensitivity in comparison to other imaging modalities
[90, 91]. Thus, “stem cell labeling technology using QDs” and “in vivo fluorescent
imaging technology for visualizing transplanted stem cells labeled with QDs” are
essential for analyzing the transplanted stem cells.
There are generally thought to be two methods that can be used to label stem cells
with QDs; the conjugation of QDs with a stem cell surface [92] and the transduction
of QDs into a stem cell [90, 93]. The conjugation of QDs with the cell surface is
associated with some problems, such as a reduced accumulation rate of stem cells in
tissues/organs and the separation of QDs from the stem cell surface during circula-
tion in the body. The transduction of QDs into the stem cell may overcome these
problems; however, the cytotoxicity of the QDs is very high and the transduction
efficiency is very low when using physical stimulus methods, such as ultrasonic
transduction [91] and electroporation [74, 94]. In contrast, the labeling of stem cells
with QDs using chemical modification methods, such as cationic liposomes, cell
penetrating peptides, and high molecular nano-carrier (polymer micelles) was
reported to be useful [96–98], because these molecules have been used in clinical
applications for the transduction of DNA and proteins into cells.
10.4.2 Cationic Liposomes
QDs are unable to label stem cells with high efficiency because of their low rate of
interaction with the cell membrane, so chemical modifications using cationic lipo-
somes are expected to be useful for the stem cell labeling application of QDs
[98, 99]. Some cationic liposomes such Lipofectamine® (Life technologies),
COATSOME® (NOF corporation), and ScreenFect™A (Wako) are commercially
available, and are mainly used for gene transfection. These cationic liposomes can
interact with negatively-charged QDs (especially COOH-conjugated QDs), then the
cationic liposomes rapidly enclose the negatively-charged QDs through electrostatic
forces [93, 100].
In fact, when cationic liposomes and negatively-charged QDs were mixed at the
optimal mixture ratio in culture medium and then were added to stem cells in the
culture medium. TheQDs could be transduced into the stem cells through endocy-
tosis within a few hours, and were maintained in the cytoplasm near the nucleus by
escaping the exocytotic mechanism [101] (Fig. 10.4a). The transduction efficiency
was very high, and no severe cytotoxicity was identified [102, 103] (Fig. 10.4b and
c). In addition, QD labeling with cationic liposomes did not affect the stem cell
characteristics, such as their potential for self-renewal and their multilineage poten-
tial [93] (Fig. 10.4d). This labeling method is simple and relatively rapid, and so
appears to be useful for stem cell labeling with QDs.
However, high concentrations of cationic liposomes induce the death of stem
cells. The cell membrane is generally negatively-charged, so the cell membrane
structure is unstable and destroyed by the interaction with strong positively-charged
cationic liposomes. The validation of the optimal concentration of cationic lipo-
somes is important and necessary to make it possible to label various kinds of stem
cells without adverse effects [99].
A C
10000
Number of cells
Phase Fluorescence 8000
6000
4000 0nM
0.08nM
2000 0.4nM
200 µm 200 µm 0.8nM
0
0 2 4 6
Time (day)
B ** D
** Adipocyte
120
100
Cell viability (%)
80
20 µm 20 µm
60
Osteocyte
40
20
0
0 0.08 0.4 0.8 2 4 20 µm 20 µm
Concentration of QDs655 (nM)
Fig. 10.4 Quantum dots labeling using poly-cationic liposome (Lipofectamine®) for imaging of
adipose tissue-derived stem cells. (a) Morphology of ASCs and fluorescence of the QDs655
(0.8 nM) observed by phase-contrast microscopy. (b) Cell viability of ASCs labeled with
QDs655 using Lipofectamine®. (c) Proliferation rate of ASCs labeled with QDs655 using
Lipofectamine®. (d) Adipogenic and osteogenic differentiation of ASCs labeled with QDs655
(0.4 nM). (Reprinted with permission from Ref. [137])
10.4.3 Cell-Penetrating Peptides
Cell-penetrating peptides (CPPs), also known as protein transduction domains

(PTDs), are expected to be useful for the stemcell labeling application of QDs,
because several transduction domains can deliver a large size-independent variety of
molecules into cells, including proteins, peptides, antisense oligonucleotides and
large metal beads [104]. Representative CPPs include the Tat protein of human
immunodeficiency virus (HIV-1) [105, 106], VP22 protein of herpes simplex virus
[107] and Antennapedia (Antp) homeoprotein of Drosophila [108], and all of these
CPPs possess arginine- and lysine-rich sequences. In addition, poly-arginine
(PolyR), especially 8–11-arginine peptides, and poly-lysine have been shown to
exhibit an even greater efficiency in the delivery of several peptides and proteins
[109, 110] (Fig. 10.5a).
Most studies have reported that these CPPs were conjugated with negatively-
charged QDs (CPP-QDs) through chemical or electrostatic forces, and the condition
of the QD surface was dramatically changed to make it positively-charged. In a case
A C *
120
Octa-arginine (R) 100
Cell viability (%)

Peptide name 8R 80
Sequence RRRRRRRR 60
Molecular weight 1267.54 40
20
Arginine (R) 0
0 0.4 2 8 24
Concentration of QDs655 (nM)
B Phase Fluorescence D
18000
QDs : R8 = 1 : 0
15000
Number of cells
12000
9000
200 µm 200 µm
6000 0nM
0.8nM
QDs : R8 = 1 : 1000 3000 4.0nM
8.0nM
0
0 2 4 6
Time (day)
200 µm 200 µm
E Adipocytes
QDs : R8 = 1 : 10000
200 µm 200 µm 20 µm 20 µm
Osteocytes
QDs : R8 = 1 : 100000
200 µm 200 µm 20 µm 20 µm
Fig. 10.5 Quantumdots labeling using cell penetrating peptides (octa-arginine: R8) for imaging of
adipose tissue-derived stem cells. (a) Constitutional formula of arginine molecule and the informa-
tion of normal chain R8. (b) Optimal formation of QDs and R8 for labeling ASCs. (c) Cell viability
of ASCs labeled with QDs655 using R8. (d) Proliferation rate of ASCs labeled with QDs655 using
R8. (e) Adipogenic and osteogenic differentiation of ASCs labeled with QDs655 (0.4 nM) using
R8. (Reprinted with permission from Ref. [137])
of chemical binding, the CPPs and QDs were mainly chemically-bonded by the
amide bonding of the amino group of CPPs with the carboxyl group of the negatively
charged QDs [103, 109]. Chemically-bound CPPs–QDs are very stable; however,
the synthesis and purification of CPPs–QDs involves a great deal of time and effort.
On the other hand, in the case of electrostatic binding, the CPPs and QDs were
conjugated by the positively-charged amino acids of CPPs binding the negatively-
charged functional groups of the QDs [111, 112]. These electrostatic-bound CPPs–
QDs were stable in cell culture medium, and the production of CPPs–QDs is simple
and quick in comparison to chemical binding methods.
Indeed, in that case, CPPs–QDs could be transduced into stem cells through
micropinocytosis within several hours, and were maintained in the cytoplasm near
the nucleus by escaping exocytosis (Fig. 10.5b). Macropinocytosis occurs indepen-
dent of clathrin-mediated and caveolin-mediated endocytosis, and the size of parti-
cles that can be uptake is more than 1 μm, and the process requires dynamin GTPase
activity [113]. Similar to the complexation with cationic liposomes, the transduction
efficiency for micropinocytosis was very high, and there was no severe cytotoxicity
observed (Fig. 10.5c). The CPPs–QDs did not affect the stemcell characteristics such
as the self-renewal or multilineage potential [114, 115] (Fig. 10.5d and e). In
addition, the CPPs do not generally induce the death of stem cells at high concen-
trations, as occurs when cationic liposomes are used for transfection [90]. Thus, this
A Phase QDs655 GFP
iPS cells
(non-labeling)
500µm 500µm 500µm
iPS cells
labeled with
QDs655
500µm 500µm 500µm
B
a b c
d e f
Fig. 10.6 Maintenance of undifferentiated state and multipotency of iPS cells labeled with QDs
using R8. (a) Morphologies and fluorescence images of 1 day after EB formation of non-labeled iPS
cells and iPS cells labeled with QDs using R8 (phase contrast, red fluorescence derived from
QDs655, and green fluorescence derived from GFP). (b) Teratoma formation of iPS cells labeled
with QDs using R8 after 4 weeks of the injection of labeled iPS cells to the nude mouse. a: Obtained
histological sections were stained with hematoxylin and eosin. b: Nervelike structures, c: cartilage-
like structures, d: gut epithelium-like structures, e: adipose-like structures, f: glomerulus of the
kidney-like structures. (Reprinted with permission from Ref. [137])
labeling is also expected to be utilized for the stem cell labeling of QDs. Moreover, a
previous study showed that iPS cells could be labeled with QDs using CPPs at high
efficiency, and iPS cells labeled with QDs maintained their undifferentiated state and
pluripotency [116] (Fig. 10.6a and b). Therefore, CPPs are expected to be useful
molecules for stem cell labeling with QDs. However, validation of the optimal
concentration is important and will necessary to determine the best way to label
various kinds of stem cells without adverse effects [117].
10.5 In vivo Fluorescence Imaging of Transplanted Stem

Cells Labeled with Quantum Dots
10.5.1 In Vivo Fluorescence Imaging
In vivo fluorescence imaging systems, which can detect and analyze the fluorescence
or emission from the body, have been developed for small animals such as mice and
rats [87]. The Maestro™ (PerkinElmer) Clairvivo OPT (SHIMADZU) and IVIS
Imaging System (PerkinElmer) are representative instruments (Fig. 10.7a). These
systems generally have an integrated ultrasensitive cooled CCD (charge-coupled
device) camera, and can detect the weak fluorescence and emission in vivo. The
number of photons from the fluorescence or emission can be counted, and the
fluorescence or emission intensity can then be quantitatively determined. In addition,
these fluorescence or emission data can be combined the data from other modalities,
such as X-ray CT (computed tomography) [97], MRI (magnetic resonance imaging)
[114, 115], SPECT (single photon emission computed tomography) and PET (pos-
itron emission computed tomography) [118, 119].
These systems can be used for in vivo fluorescence imaging of transplanted stem
cells labeled with QDs. However, there are some major problems that prevent the
successful in vivo fluorescence imaging of transplanted stem cells at high resolution,
which include the strong scattering, absorption and autofluorescence derived from
the whole body. In order to overcome these problems, fluorescence probes that can
absorb the excitation light and emit strong fluorescence in the near-infrared region
(about 700–900 nm) in the “Biological OpticalWindow” to decrease the scattering,
absorption and autofluorescence derived from the body, are strongly desired [121–
123] (Fig. 10.7b). Some QDs showing strong fluorescence in the near-infrared
region have been developed and are commercially available, so QDs are expected
to be useful for in vivo fluorescence imaging of transplanted stem cells (Fig. 10.7c).
Moreover, the autofluorescence derived from normal food must be considered
[123]. In fact, autofluorescence of mice given normal feed was detected from the
gastrointestinal tract by excitation in the red or near-infrared region. To diminish the
impact of this effect on the results of the in vivo fluorescence imaging, the mice were
given feed not including fluorescent components (alfalfa-free feed) for at least
1 week. The autofluorescence intensity of the alfalfa-free feed was reported to be
about ten-fold lower than that of normal feed (Fig. 10.7d).
A B
Near-Infrared
Region (NIR) 120
QD655
Absorption coefficient
Fluorescence ( a.u.)
100
80
60
40
20
0
500 600 700 800 900 1000 500 600 700 800 900
Wavelength (nm) Wavelength (nm)
C
a b c
D a b c
Epi-fluorescence
10.0E-05
Normal Alfalfa-free
8.00E-05
Total efficiency (cm2)
4.0
6.00E-05
x10-5
4.00E-05
2.0
2.00E-05
Efficiency
0.00E+00
Normal Alfalfa-free
Fig. 10.7 Biological window in near-infrared region and comparison of the fluorescence intensity
between normal with alfalfa-free feed. (a) Biological window in near-infrared region (about
700–900 nm) (b) Fluorescence spectra of QDs655, QDs705, and QDs800 in the culture medium.
(c) In vivo imaging fluorescence system. a: The Maestro™ (PerkinElmer), b: Clairvivo OPT
(SHIMADZU), c: IVIS Imaging System (PerkinElmer) (d) Comparison of the fluorescence inten-
sity between normal with alfalfa-free feed. a: In vivo fluorescence image of mouse fed on normal
feed. b: Fluorescence images of normal and alfalfa-free feed. c: Fluorescence intensity of normal
and alfalfa-free feed. (Reprinted with permission from Ref. [137])
10.5.2 Subcutaneous Transplantation
In vivo fluorescence imaging of subcutaneously transplanted stem cells labeled with

QDs into mice has been frequently reported [87, 124]. In fact, the fluorescence
A
ASCs labeled 1h day 1 day 2 day 5 day 7
with QDs655
3.0 x 105 cells

1.0 x 105 cells
0.5 x 105 cells
B C
400
Total Signal /Exp. (ms)
3.0 105 cells

300 1.0 105 cells
0.5 105 cells QDs525 QDs565
200
QDs605 QDs655
QDs705 QDs800
100
0
1 2 5 7
Time (day)
Fig. 10.8 Detection and multiplex imaging capability of QDs in subcutaneous transplantation. A:
In vivo fluorescence images of mouse subcutaneously transplanted stem cells (0.5, 1.0 and
3.0 105 cells) labeled with QDs655 (0.8 nM) using R8 into the backs of the mice after 1 h,
1, 2, 5 and 7 days. B: Bar graph of the changes of fluorescence intensity at the number of stem cells
labeled with QDs655 using R8 for 7 days. C: In vivo fluorescence Image of mouse subcutaneously
transplanted stem cells labeled with QDs525, 565, 605, 655, 705 and 800 into the back of the mice
after 10 min with a single excitation light source. (Reprinted with permission from Ref. [137])
intensity derived from QDs could be detected and analyzed quantitatively by using
an in vivo fluorescence imaging system after the subcutaneous transplantation of
different numbers of stem cells labeled with QDs into the back or other sites of mice.
Subcutaneously transplanted stem cells labeled with QDs could be detected clearly
at the level of several thousand cells over a period of about 1 week, and the
fluorescence was QD dose-dependent [90] (Fig. 10.8a and b). However, the stem
cells labeled with QDs could proliferate rapidly, so the sensitivity of detection was
decrease in inverse proportion to the proliferation of the stem cells.
Moreover, the multiplex imaging of subcutaneously transplanted stem cells has
been reported by utilizing the fluorescence characteristics of QDs [90, 125]. In fact,
stem cells were labeled with different kinds of QDs emitting fluorescent light from
525 to 800 nm, then the stem cells could be detected in different colors using
excitation of the same wavelength at high resolution [125] (Fig. 10.8c). The avail-
ability of QDs emitting fluorescence in the near-infrared region is relatively low for
subcutaneous transplantation, because visible light can enter through the skin to
some extent. This technology is expected to be useful for in vivo fluorescence
imaging of different cell populations included in regenerative tissues and organs at
the same time.
10.5.3 Intravenous and Other Transplantation
Stem cell therapy via intravenous transplantation has been expected to be useful in
clinical applications for some diseases of the lungs, liver and pancreas [127–
129]. Adipose tissue-derived stem cells [ASCs], bone marrow-derived stem cells,
hematopoietic stem cells and progenitor cells derived from these stem cells have all
been used for stem cell therapy [84–86, 129]. In vivo fluorescence imaging of
intravenously transplanted stem cells labeled with QDs may make it possible to
trace the transplanted stem cells in vivo and analyze their rate of accumulation into
specific tissues or organs [87, 88].
In a previous study, adipose tissue-derived stem cells labeled with QDs were
intravenously transplanted into mouse models of emphysema or acute liver disease,
and then the location and the rate of accumulation of stem cells in major organs, such
as the heart, kidneys, lungs, liver and spleen, were investigated [123, 130]. In the
emphysema model, the transplanted ASCs were observed in the lungs at 1 and 4 h
after transplantation, and more ASCs remained in the lungs with emphysema
compared with the lungs from normal mice [130] (Fig. 10.9a). In the model of
acute liver disease, the transplanted ASCs were found to accumulate more in the
liver when there was simultaneous administration of heparin compared to when the
ASCs were transplanted alone [123] (Fig. 10.9b, c, and d).
In these cases, QDs with near-infrared fluorescence were useful for the in vivo
fluorescence imaging of transplanted stem cells, because near-infrared fluorescence
can be seen through the skin and organs at high efficiency. In fact, intravenously
transplanted ASCs labeled with QDs800 (fluorescence peak at 800 nm) could be
detected in the lungs and liver even without laparotomy; whereas transplanted ASCs
labeled with QDs655 (fluorescence peak at 800 nm) could not be detected [123]
(Fig. 10.9b, c, and d).
On the other hand, bone marrow-derived stem cells labeled with QDs were
transplanted in the ipsilateral striatum of a rat model of cerebral infarct, then the
impact of the timing and stem cell dose on the therapeutic effects were determined by
in vivo fluorescence imaging of transplanted stem cells labeled with QDs [131]. Neu-
ral stem cells labeled with QDs were transplanted into the striatum contralateral to
the ischemic hemisphere, then the therapeutic effects were confirmed by in vivo
imaging of transplanted neural stem cells labeled with QDs. The findings of that
study suggest that the in vivo fluorescence imaging of transplanted stem cells labeled
with QDs may assume increasing importance in association with the development of
stem cell therapy for regenerative medicine.
A
Normal ASCs labeled
with QDs800
B QDs655
a ASCs only b ASCs + Heparin
Before After Before After
C QDs800
a ASCs only b ASCs + Heparin
Before After Before After
QDs800
D
a ASCs only b ASCs + Heparin c Ratio of Fluorescent Intensity (RFI) =
Fluorescent intensity of the organ
100 (%)
Total fluorescent intensity of 5 organs
Heart Lung Heart Lung

**
100
80 ASCs only
Spleen Kidney Kidney Spleen ASCs + Heparin
RFI (%)
60
**
Liver 40
Liver
20
0
Heart Lung Kidney Spleen Liver
Fig. 10.9 In vivo and ex vivo imaging of stem cells labeled with QDs using R8 after intravenous
injection. (a) In vivo fluorescence images of mouse transplanted stem cells labeled with QDs800
using R8 through the tail vain into mouse after 10 min. (b) In vivo fluorescence images of mice with
acute liver failure after transplantation of stem cells labeled with QDs655 using R8 in combination
without heparin (a) or with heparin (b) without laparotomy. (c) In vivo fluorescence images of mice
with acute liver failure after transplantation of stem cells labeled with QDs800 using R8 in
10.6 Recent Progress and Views
10.6.1 Overcoming of Problems Caused by Using

Quantum Dots
Three major problems caused by using QDs, such as assurance of the safety,
realization of high functionality, and barrier of national regulation, appear to be
resolved for the clinical application to stem cell therapy and regenerative medicine.
Firstly, the assurance of safety is thought to be the most important problem. Much
safer elements against several kinds of cells including iPS cells or bodies should be
selected as the constituent elements of QDs to overcome this problem. Secondly, the
realization of high functionality enables us to expand the extent of the application of
QDs such as the detection of state changes of stem cells. Finally, the actual situation
of the barrier of national regulation on regenerative medicine by using stem cells
including iPS cells should be considered in each country.
CdSe and CdTe semiconductor materials have been generally used as the core of
QDs, however these elements were reported to show the cytotoxicity at low con-
centration. Thus, the novel QDs based on silicon or AgInS2 cores not including Cd,
Se, and Te elements have been already developed, and these QDs were reported to
have very low cytotoxicity [112, 132, 133]. On the other hand, QD-based sensing
mechanisms such as FRET and multiple colors are thought to be useful for the
detection of the state changes of stem cells. However, there are little information and
reports on these technologies, thus the early development of these technologies is
strongly expected [9–12]. Moreover, clinical research environment have been
improved in recent years. New three laws associated with regenerative medicine
have been established especially in Japan, so the barrier of national regulation on
regenerative medicine has become lower all over the world. Collectively, QDs will
actually be able to apply for stem cell therapy and regenerative medicine in the future
with the further improvement and development.
10.6.2 Conclusions and Future Directions
Organic fluorophores have been widely used as fluorescent probes in conventional

biometrics; however the research on QDs for biometrics has expanded greatly in
recent years. The recent findings suggest that QDs can be used to detect and analyze
⁄
Fig. 10.9 (continued) combination without heparin (a) or with heparin (b) without laparotomy. (d)
Ex vivo fluorescence images of five organs in mice with acute liver failure after transplantation of
stem cells labeled with QDs800 using R8 without heparin (a) or with heparin (b), and ratio of the
fluorescence intensity (RFI) of five organs (c). (Reprinted with permission from Ref. [137])
objects which conventional biometrics cannot detect. In this review, we described

the fluorescence properties of QDs, and then introduced QD-based biometrics
technologies, the “Bio-sensing” technology, Stem cell labeling technology and In
vivo fluorescence imaging technology for transplanted stem cells. These technolo-
gies will likely contribute to regenerative medicine, especially stem cell therapy,
which requires high-sensitivity imaging at the cellular level.
The potential clinical applications of QDs have been expanded by the develop-
ment of considerably low cytotoxicity QDs that do not include cadmium (Cd) or
selenium (Se) [112], as well as the development of the second near infrared region
(1000–1500 nm) fluorescent QDs with strong permeability into the body
[134, 135]. In fact, clinical trials based on the data obtained from basic research
have been started in the U.S.A [136]., and research and development are becoming
increasingly important. Moreover, the evolution of QDs including the development
of hybrid materials of QDs and other functional molecules probably enables the
diagnosis and therapy of stem cells in vivo at the same time, an approach termed
“in vivo theranostics”.
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Chapter 11
Nanopore Device for Single-Molecule
Sensing Method and Its Application
Masateru Taniguchi and Takahito Ohshiro
Abstract Nanopore analysis is very promising for single-molecule sensing plat-

form. The feature of nanopore platform is a simple, high-throughput single-mole-
cule/particle detection of a wide range of analytes at low-cost at single-molecule
level. The single-molecule sensing ability of nanopore device have been utilized for
single-molecule DNA sequencing, protein, peptide and carbohydrates detection, and
so on. Due to recent progress on the improvement of selectivity, molecular control,
fabrication technique, the nanopore platform became “smarter”. Therefore, the
applicability of nanopore–sensing methodology are expanding not only for basic
research fields but also for medical applications, such as disease diagnosis, drug
screening, virus detection.
Herein this chapter, background of nanopore studies (Sect. 11.1), principle of
nanopore sensing (Sect. 11.2), the nanopore-fabrication technique (Sect. 11.3),
application studies of nanopore sensing (Sect. 11.4), the selective and accuracy
improvement studies of nanopore-sensing (Sect. 11.5), recent novel nanopore plat-
form studies (Sect. 11.6), and the summary and future of the nanopore-sensing
method (Sect. 11.7) are introduced.
Keywords Nanopore · Single-molecule analysis · Bio-nanopore · Solid-sate

nanopore
11.1 Introduction
Development of methodology of identifying molecules and/or detecting / monitoring

molecular behaviors at the single-molecular level is one of the important research
issues for chemistry and biology. As one of the promising methodologies, a
nanopores based single molecule analysis method has been extensively studied
[7, 37, 49]. A nanopore is a pore with nanometer-size diameter, which is comparable
M. Taniguchi (*) · T. Ohshiro

Osaka University, Ibaraki, Osaka, Japan
e-mail: [email protected]; [email protected]

302 M. Taniguchi and T. Ohshiro
Fig. 11.1 Bio-nanopore and Solid-state nanopore: The nanopores are classified into “biological
nanopores (Bio-nanopore)” (a) and “Solid-state nanopores” (b). (a) Bio-nanopore is produced by
insertion of transmembrane channel-proteins in lipid membranes (See left figure). (b) Solid-state
nanopore is drilled by etching, ion-beam, electron-beam lithography in an insulating thin-
membrane, such as silicon, silicon oxide, silicon nitride, graphene and so on. Ideally, a single-
molecule can translocate these formed nanopores across the membranes one by one. By using the
nanopore-sensing, single-molecule biopolymers, such as DNA, RNA, peptide, protein, carbohy-
drates and sugar complexes, and so on, can be detectable
to a single-molecule size and it can be created by a pore-forming protein in lipid

bilayers, “bio-nanopore”, [17, 37] or as a through-hole in synthetic materials, such as
silicon-nitrate or graphene, “solid-state-nanopore” [26, 74] (Fig. 11.1). These
nanopores can interact with comparable-size sample molecules, and their phenom-
ena induces some kinds of physical disturbances around the pore, resulting in
detection of sample-molecular behaviors as characteristic analyte signals. The fea-
ture of nanopore based sensor is a simple, high-throughput single-molecule/particle
detection of a wide range of analytes at low-cost. Therefore, the methodology
become attracted not only for basic research fields but also for medical applications.
11.2 Background and Principle of Nanopore Sensing
Among nanopore based sensors, electric resistance-measurement type sensors have

been well-known. As the first conventional electrical sensing method, a single-
particle sensor using pores (Coulter Counter) was developed by W. H. Coulter in
1953 [22]. This is a method of measuring conductivity of pore by a change in ion
current during analyte-particle translocation (Fig. 11.2). The sensing principle is as
follow: This method consists of two chambers partitioned by an electrically insulated
membrane having a single pore. Each of chambers is filled with an electrolyte
solution and arranged with Ag/AgCl electrodes, respectively, which apply
11 Nanopore Device for Single-Molecule Sensing Method and Its Application 303
Fig. 11.2 Principles of nanopore electrical sensing. An insulated thin membrane partitions an
electrolyte solution into two chambers and two Ag/AgCl electrodes are placed into each of the
chamber solutions to provide dc-voltage and record electrical current through the pore. The
dc-electric field causes the electrolyte ions in the solution to move only through the pore by
electrophoresis. (a) In the “open” state, the stable background current (I0) flows. (b and c) When
a sample molecule (Sample A or B) penetrates into its pores, the ion-current flow is prevented, and
current-drop signals are observed (See middle and right figures). In the “close” state, the decrement
of current signal (ΔIa for Sample A and ΔIb for Sample B) are closely related to the excluded
volume of molecules (Va, for Sample A, and Vb for Sample B). The duration time (Ta and Tb) is
time length of the sample translocation across nanopore. The difference is influenced by the sample
characters such as molecular shape, orientation and the interaction between sample and side-wall of
nanopore. From these information of signals can serve to identify sample molecules in a mixed
sample solution
dc-electric field between chambers across the pores. The dc-electric field causes the
electrolyte ions in chamber solution to move only through the pore by electropho-
resis and this ion-current flow is monitored. When a single particle penetrates into its
pores, the excluded volume of that particle decreases the electrical conductivity of
the pore and decreases the ionic current. From the information on the magnitude,
transit time, and passage frequency of the ion current-change signal of the pore
generated at this time, it is possible to detect/identify the sample particle translocated
through the pore. As an analysis for identification and counting of blood cells and
cultured cells, this resistive measurement method is routinely used and become
indispensable in the clinical uses instead of conventional time-consuming visual-
type cell-examinations. Essentially, the analyzable-size of sample by using this
sensing method depends on the pore-size so that the nanopore preparation/
fabrication technique is a crucial for single-molecule nanopore detection. By recent

development of the nanopore preparation/fabrication technique, the use of nanopore
become reproducible and reliable so that the nanopore electric resistance-
measurement methods became a powerful methodology for “single-molecule” sens-
ing method. The detail is described in Sect. 11.3.
11.3 Fabrication/Preparation of Nanopore for Nanopore

Analysis
The key for development of nanopore sensors is the fabrication / production tech-
nique of the nanopores because the nanopore-shape and its robustness significantly
influence the reliability and stability of signal detection. In this section, the recent
nanopore fabrication studies are described. The nanopores are roughly classified into
two categories. One is “biological nanopores (bio-nanopore)”, which is derived from
cell channel proteins, and the other is “solid-state nanopores, which are artificially
created by micro/nano-fabrication technology. Each of the techniques and their
features are introduced in below.
Bio-nanopore sensors (Table 11.1) relies on the use of transmembrane channel-
proteins, “porins”, which are embedded in lipid membranes. These channel proteins
typically have pores with nanometer-scale diameter, which can passively diffuse
smaller-pore-size biomolecules across the membranes. In nanopore experiments,
α-hemolysin channel protein (αHL), which is a channel protein of bacteria that
causes lysis of red blood cells has, has been extensively used. Recently, the use of
Mycobacterium smegmatis porin A (MspA), as bio-nanopore, is also growing
because of the better ability of ion-current blockage, which is determined by the
volume of the narrowest region of the pore protein. The most outstanding feature of
bio-nanopore is that the uniform pores-structure can be produced by the self-
organizing ability of the biomolecule. This feature serves reproducible atomic-
Table 11.1 Characteristics of bionanopores

Material Diameter (nm) Analytes Spatial resolution Refs.
α-hemolysin 1.5 ssDNA Single base molecules *1
ssRNA Single RNA molecules *2
microRNA Single microRNA *3
Protein Single protein *4
MspA 1.2 ssDNA Single base molecules *5
5mC, 5hmC Single base molecules *6
Aerolysin 1.0 Protein Single protein *7
Oligosacchaaride Single molecules *8
N. farcinica < 2.0 Peptide Single peptide *9
ClyA 3.3 ssDNA, dsDNA Single DNA *10
*1: [21]; *2: [23], [5], [4]; *3: [134], [112], [122]; *4: [92], [79]; *5: [71]; *6: [63]; *7: [86]; *8:
[29]; *9: [101]; *10: [32]
precision geometries for bio-nanopore and reproducible current-signal by the

bio-nanopore sensing. Although there is small degree of freedom in choosing the
pore-size, the protein engineering technique can tune the pore-diameter, structures
and charge-state by single-point mutations, resulting in improvement of detection
abilities with the nanopore and/ or translocation behaviors of analytes [15, 50]. There-
fore, in bio-nanopore sensing, the optimal nanopore is determined by the character of
target molecules.
Solid-state nanopores (Table 11.2) are typically made of thin supporting mem-
brane of insulated materials by top-down technique, mainly, semiconductor fabri-
cation processing techniques. Recent development on these process techniques
realize the high-reproducible mass-production of these solid-state nanopore struc-
tures. Solid-state nanopore production has been typically implemented by fabricat-
ing / sculpting a pore in an insulating thin-film materials by electron beam (EB) or an
ion beam (FIB) [16, 65, 107]. They successfully demonstrated the fabrication of a
pore with single-nanometer diameters. As synthetic materials of solid-state
nanopore, silicon-nitrite (SiN), silicon-oxide, and aluminum oxide have been exten-
sively studied. In addition, solid-state nanopores is recently reported using HfO2,
which is stronger than SiN and has a better chemical resistance [62]. In general, in
order to achieving sufficient spatial resolution for the identification of a small
molecules, thinner non-conductive film materials, i.e., 2D material membranes, are
ideal because the ionic current blockade is generally determined by the volume of the
molecule present in the nanopore. As the candidate of 2D materials, single and
several-atomic layer material-based solid-state nanopores has also been explored.
Among them, the interest for “graphene” based nanopore is increasing [33, 73, 106,
118, 129]. Graphene, a single layer of carbon atoms, is one of the ideal materials for
detection of small molecules by solid-state nanopore because of atomically thinness
Table 11.2 Characteristics of solid-state nanopore

Material Diameter (nm) Analytes Refs.
SiN 3.0 RNA + drug *1
3.7 dsDNA+ɤ-PNA *2
SiN/SiO2/SiN 20 Histone nucleosome *3
HfO2/SiNx < 2 (HfO2) ssDNA, dsDNA *4
Graphene/SiO2 22 (graphene: 1 layer) λ-DNA *5
Graphene/SiN 5 (graphene: 1-2 layer) 10 kb DNA *6
Al2O3/graphene/Al2O3 9 dsDNA, dsDNA+protein *7
TiO2/graphene/ SiN /SiO2 8 (graphene: 3-15 layer) 15 kb DNA *8
Chemical modified 5-15 ssDNA *9
graphene
MoS2/SiNx 5 (MoS2: 1-3 layer) λ-DNA *10
DNA origami/SiN 15 dsDNA *11
Chemical modified SiN 25 Protein *12
Chemical modified Au/SiN 20-25(Au) Histagged protein IgG *13
antibody
*1: [99]; *2: [100]; *3: [104]; *4: [62]; *5: [97]; *6: [33]; *7: [118]; *8: [73]; *9: [98]; *10: [68];
*11: [11]; *12: [57]; *13: [128]
and impenetrability of ions. However, the surface of graphene is hydrophobic so that

hydrophilic target molecules, such as DNA, protein, are not suitable for graphene-
nanopore based sensing. To overcome this issue of the hydrophobic interactions
between these biomolecules and the surface, hydrophilic thin membrane based solid-
state nanopores by using material, such as chemically modified graphene [98], MoS2
(6.5 Å-thick layers) [68], have been studied. As the other nanopore fabrication
method, a bottom-up technique, such as self-folding and self-assembly methods, is
also utilized. Among them, nanopore formation by using DNA origami technique,
which is nanoscale folding method of DNA to create non-arbitrary two- and three-
dimensional shapes at the nanoscale nanopore, have been extensively studied
[11, 46, 47, 87, 127, 128]. Compared to top-down fabrication-technique, the self-
assembly property and nanometer precision engineering of DNA origami
nanostructures are attractive for creating hetero-structure and hybrid nanopores.
In general, compared to bio-nanopores, the structure of the solid-state pore is
robust and stable in various external conditions, i.e., pH, temperature, and electrolyte
concentration. The feature serves to measure various analyte and the wide range of
experimental condition, also serve to modify the surface of solid-sate nanopore,
which can give specific interaction with analyte and/or prevent physical absorption
of analyte and contaminated molecules. Therefore, solid-state nanopores are poten-
tially suitable for a multi-target sensing platform. Until now, the multi-detection on
several kinds of biopolymers target have been reported [90, 100] (Table 11.2). The
detail contents on the multi-target sensing are described in Sect. 11.4. In addition, by
nano-fabrication technology, solid-state nanopore can be easily integrated with
various type of nanostructures, such as nano-fluid for molecular precise guidance,
nano-gap electrode for sensing, and other detection sensing methods, such as optical
detection, and force measurements and so on. The detail contents are described in
Sect. 11.5.
11.4 Application of Nanopore Based Single-Biomolecule

Sensors
11.4.1 Single-Molecule Nanopore Sequencer
Application area for nanopore-based sensors are recently growing for various bio-
medical analysis because of the simple, high-throughput single-molecule detection
ability and versatility at low-cost. One of the most attractive applications is a single-
molecule DNA sequencing technology [13]. The first standard method for DNA
sequencing is developed by Fred Sanger in the 1970s [95]. Based on the Sanger-
method based sequencer, the first draft sequences of the human genome, i.e., Human
Genome Project, were completed in 2003 and 1000 Genomes Project has reported
sequencing the entire genomes of 179 individuals [61, 111, 119]. However, those
DNA sequencing technologies were still slow and expensive to be used routinely for
individual medication. Since then, new types of sequencers are being developed one
after the other from companies such as Roche, Illumina, Life Technology and so
on. There is a race to develop new sequencer technologies that are capable of
sequencing individual genomes for a cost of $1000 or less [12]. In the race, as the
new sequencer, various technologies to sequence DNA at the single-molecule level
have been proposed. This category consists of various techniques, including
nanopore sequencing [13], single-molecule sequencing by synthesis (SBS) [39],
single-molecule real-time SBS [28], electron microscopy [10] and so on.
Among the new sequencer technologies being explored, nanopore-sensor based
DNA sequencer is the most promising methodology for personal-genome uses
[30, 31, 67, 123] (Fig. 11.3). Compared to other sequencers, the advantages of
nanopore sequencing is potentially label-free, long reads, high throughput, and
PCR-free. These features simplify the genome-reading process, resulting in
Fig. 11.3 Nanopore-based DNA sequencing. (a) Schematics of Bio-nanopore DNA sensing.
Bio-nanopore are introduced in lipid bilayers. In the ion-current nanopore sensor, the passage of
molecules induced the current blockage. (b) In sequencing, each of nucleotide blocks the pore
differently giving a different amplitude so that this information can determine DNA sequence
information. Ideally, this method can sequence long polynucleotides. (c) Typical ionic current-time
profile through a bio-nanopore (aminocyclodextrin-modified α-haemolysin) for individual mono-
nucleotides (dAMP, dCMP, dGMP, dTMP). Each mono-nucleotide causes a unique current
blockage in nanopore conductance, resulting in identification of mono-nucleotide species. (d)
Histogram of the ion-current blockage current. (Adapted with permission from [117]. Copyright
2011 Nature Publishing Group)
reduction of the cost of DNA sequencing for various research applications. First
nanopore sequencing methodology was proposed around the 1990s [24]. The con-
cept is as follow: In the ion-current nanopore sensor, when the pore is blocked due to
the passage of molecules, the current flow is also blocked. In sequencing, each of
nucleotide blocks the pore differently giving a different amplitude and duration of
the current blockade so that this information is converted into DNA sequence
information [21, 27, 71, 117] (Fig. 11.3). As the first report, the channel protein
α-hemolysin was utilized for identification of homopolymers, such as poly
(deoxyadenylic acid) (polydA) and poly(deoxycytidylic acid) (polydC) [2, 25]. It
took 20 years to achieve the identification of single base molecules in DNA after this
report of a DNA sequencer based on bionanopores. In order to obtain this ion-current
signal at single-nucleotide resolution, reduction of the translocation speed of ssDNA
molecules is one of the important issues. As one of the solution, this was addressed
by modifying the inner nanopore surface with a cyclodextrin that had a smaller
diameter than that of the nanopore [21]. In addition to this approach, DNA sequenc-
ing has been realized with α-hemolysin and a complex of Mycobacterium smegmatis
porin A (MspA) with Phi29 DNA polymerase, which denatures dsDNA to form
ssDNA and makes the ssDNA flow slowly into the nanopore [63, 71]. In addition,
since nanopore sequencing produces fast results at low-cost, it is a powerful tool for
“on-site diagnosis” of infectious agents. Indeed, nanopore sequencer successfully
analyzed Ebola samples on-site in Africa [55]. The possibility of “on-site sequenc-
ing” by nanopore sequencer would potentially give a big impact on medical, clinical
and health-care applications [14, 70, 121].
11.4.2 DNA/RNA Nanopore-Sensing
Bionanopore based sensing can be also applied for RNA detection and its quantifi-
cation [77, 122, 125, 126] and the detection of aberrant DNA methylation, i.e.,
N6-methyladenosine, and N5-methylcytosine, which is very important for cancer
diagnosis and treatment. Until now, the RNA four base molecules identification is
achieved by fixing the base molecules of RNA in the interior of the nanopore [4]. In
order to sequence and/or identify RNA sample, fast translocation inside nanopore
should be addressed for detection of single-base resolution. As one of the
approaches, studies by combining an enzyme (PNPase cuts RNA one base molecule
at a time) with the head of ssRNA and modifing the α-hemolysin with amino-
cyclodextrin adapters is reported, and the modification enabled translocation of the
single base molecules of the one molecule units [5]. Separately, the identification
and/or detection of miRNA by hybridizing target miRNA with complementary
oligonucleotide probes or PNA probes is being investigated [112]. In this sensing,
the probe should be first translocated into a nanopore, and then, target miRNA is
pulled by the probe inside the nanopore, resulting in selective detection of the target
RNA molecules. The structural DNA/RNA variants, which are self-assembled
nanostructures form of nucleotide such as hybridization-form and stem RNA loop-
structure, can be also detected [45]. For instance, in the case of double-stranded
DNA sensing, cytolysin A (ClyA), which has a minimum pore diameter of 3.3 nm
and thus is larger than that of B-form dsDNA, is utilized for sensing and then found
that the obtained signals have been shown to pass both ssDNA and dsDNA selec-
tively [32]. In this way, the optimal selection of bio-nanopore especially with the
optimal nanopore size is a crucial element for clear nanopore-sensing.
11.4.3 Protein/Peptide Nanopore-Sensing
The nanopore based peptide/protein sensing have been also and interested and
explored. Since a protein expression and its concentration changes in the cell is
closely related to biological perturbations such as disease or drug treatment, the
development of single-molecule protein sensing and quantitative analysis technique
can address these comprehensive proteomics analyses at single cell level. In protein/
peptide analysis, most of these target molecules have various conformational vari-
ants so that the conformational control during sensing is a key issue. In general, the
confinement inside nanopore can serve unfolding, denaturing, and linear transloca-
tion through a sub-nanopore structure. Using this phenomena, selective translocation
and detection of the thermal unfolding of proteins with an ionic current blockade
have been explored. In early studies, nanopore-sensing method exhibit the possibil-
ity of the protein identification and conformational change detections [3, 103,
133]. As one of the approach to detect specific target by nanopore-sensing, molec-
ular recognition methods have been used. For instance, poly-γ-D-glutamic acid
(γ-DPGA) detection by nanopore method is achieved by γ-DPGA monoclonal
antibody modified inside the nanopore [120]. Similarly, the molecular-recognition,
such as biotin/streptavidin, protein-G/immunoglobulin (IgG), and an antibody, have
been also applied for nanopore sensing. However, only the use of molecule recog-
nition agent for nanopore-sensing is not enough for the single-molecule selective
detection. As another approach to selective detection of target molecules, enzyme-
tethered nanopore methods are utilized, where the enzyme have the function to
unfold and translocate native proteins through a nanopore sensor using the protein
unfoldase. For instance, as the enzyme, ClpX was used for unfolding proteins (S1,
S2 35, and S2 148) [79] and detection of a current blockade suggested that the
unfolded protein was selectively translocated. In another study, a protein
(V5 C109 thioredoxin) was unfolded by placing an oligo-(dC)30 tag on it, with
the force of the tag dragging the protein into the nanopore and then the protein
translocation behaviors are detected as current-blockage changes [92]. By using this
method, a distinction between unphosphorylated, monophosphorylated and
diphosphorylated thioredoxin variants were also identified [93]. Selective transloca-
tion of peptides has also been explored using the Nocardia farcinica channel, which
has a minimum pore diameter of 2 nm or less, and it was revealed that a peptide
molecule (oligoarginine) adheres to the nanopore entrance when the electrophoresis
voltage is small, while it can be translocated upon the application of a large

electrophoresis voltage [101].
Nanopore based sensor is also potentially applicable for protein/peptide sequenc-
ing [75, 84, 135]. In order to realize it, there are mainly two technical challenges.
One is to unfold tertiary and secondary structures of target protein to allow the
denatured molecules to thread through the nanopore sensor. Another is unidirec-
tional translocation of the denatured polypeptide through the nanopore. As one of the
solution for these issue, the enzyme-tethered nanopore technology can address the
issues. It is well-known that some of the enzyme protein can denature protein
structures. Therefore, by tethering these enzymes to bio-nanopore by protein-
engineering or to solid-state nanopore by chemical modification, enzyme-assisted
unfolding and translocation have been reported [79, 92]. As another solution, it is
expected that the incorporation of unfolding functional unit with nanopore by nano-
fabrication technology and nanopore functionalization serve to fine controls of
translocation inside nanopore. The detail contents on the functional nanostructure
integrated nanopore are described in Emerging nanopore of Sect. 11.6.
11.4.4 Carbohydrates and Sugar Complexes Nanopore-

Sensing
Carbohydrates and sugar complexes are also a potential target of nanopore sensors.
In general, carbohydrate groups are involved in various biological processes such as
growth control, apoptosis, cell differentiation and proliferation, as well as physio-
logical disorders like tumor, autoimmune diseases and inflammation. Therefore, the
detection and its analysis at single-molecule level are expected to understand these
important biological phenomena and its mechanisms. Until now, several studies
show the nanopore-sensor can detect the carbohydrates molecules. For instance, the
maltoporin (Lam B) ion channel binds sugar molecules [60] and has the ability to
discriminate between different sugar species [6]. Besides, in a nanopore device
based on aerolysin, which has a minimum diameter of 1 nm, the selective translo-
cation of oligosaccharides (glycosaminoglycans) has been demonstrated for identi-
fication of saccharides with the different degrees of polymerization using an ionic
current blockade and the dwell time [29].
11.5 Selectivity and Accuracy of Nanopore Electrical

Sensing
Selectivity of nanopore-sensing method is a crucial parameter for the further devel-

opment of nanopore-sensing. In general, nanopore based sensor can detect the
difference in the volume of molecules translocating through nanopore so that it is
difficult to selectively detect and/or discriminate target molecule in the analyte

solution containing other molecules with the size similar to target ones. In order to
overcome this issue, molecular recognition assisted nanopore can be used for the
improvement of target sensing. For instance, chemical modification of solid-sate
nanopore suggest can be a powerful method for this issue [57]. In this study,
SiN-based solid-state nanopores covalently tethered on their surfaces to
phenylalanine-glycine (FG) nucleoporins of 98 kDaA (Nup98) or FG nucleoporin
of 153 kDa (Nup153) exhibited selective transportation of bovine serum albumin
(BSA) and importin beta (Impβ), while both proteins passed through the unmodified
nanopores at the same frequency. In a second example, selective detection is
performed using solid-state nanopores modified with receptor molecules, When
the IgG antibody were labeled with histidine-tag (His-tag) prior to the detection
immunoglobulin G (IgG) antibody was selectively detected by using solid-state
nanopore covered with nitrilotriacetic acid (NTA) receptor, which can selectively
bind His-tage moiety in protein [128]. In both cases, the target analytes are identified
not by the current-blockade intensity but by the current-blockade duration-time
difference, which is due to the intermolecular interaction between receptor and target
molecules. This approach can be applicable for any other target molecules and give
the wide-range of application. However, it requires longer detection-time so that the
throughput can became low. In addition, by using strong intermolecular interactions,
high selectivity is achieved, but the multi-detection capacity can become lost. This
trend-off relation between selectivity and throughput, and selectivity and multi-
sensing should be optimized for application.
Electrical detection in nanopore devices involves the identification of
picoampere-level electrical currents, and their high precision and accuracy of
nanopore sensing is directly related to their ability to measure an electric current at
a high signal-to-noise ratio. In order to improve the signal-to-noise ratio, it is
necessary to reduce the translocation speed of the analytes [31, 58, 124]. The reason
is described in following. In general, typical biopolymer analyte, such as DNA and
protein, are electrically charged in electrolyte solution. Therefore, when an electro-
phoresis voltage is applied across a nanopore, the electrochemical bias voltage
generates highly localized electrophoretic forces that are used to drive the target
biopolymers in the electrolyte medium through the nanopore. Since the electropho-
retic speed of negatively charged biomolecules is proportional to the strength of the
electric field, single molecules pass through nanopores at very fast speeds. For
instance, when an electrophoresis voltage of 0.1 V is applied, the typical thickness
of a nanopore membrane is less than 50 nm, and the diameters of nanopores are less
than 10 nm, an applied electric field is 0.1 V/50 nm ¼ 20 kV/cm inside the nanopore
[26, 33, 73, 94, 96, 97, 108]. For instance, in the electrical field, single DNA
molecules pass through nanopores at very fast speeds of 105 s/base [117, 132]. It
is expected that one method for decreasing the translocation speed is to decrease the
electrophoretic voltage. However, there are no technologies currently available for
the generation of 1 μV with the low voltage noises for reducing the translocation
speed. Therefore, as an alternative approach, the single-base resolution at this
translocation speed requires an electronic sensing system at extremely high
bandwidth, and the concomitant electronic noise poses serious limitations in elec-
trically discriminating between bases. However, the physical limit for measuring
pA-level electric currents is due to the Johnson noise [54, 80], which has a maximum
speed of approximately 1 MHz. At present, the maximum measuring speed of
all-round measuring instruments available on the market is 250 kHz. Therefore, to
be able to measure electric currents with high precision and single-molecule resolu-
tion, it is necessary to develop a method for increasing the speed of current
measurements and decreasing the translocation speed of single molecules.
In bionanopores, a speed-control method has been developed mainly on the basis
of the chemical modification of the nanopores by probe molecules and/or the use of
enzymes [21, 71, 79, 92]. The enzyme or probe functional conditions are determined
by the environment, such as the temperature, ion concentration, and so on, so that the
margin for these environmental conditions can be limited. In addition, the through-
put per bio-nanopore device can be low because molecular recognition based on
intermolecular interactions takes time to detect analytes.
In solid-state nanopores, there are several methods for controlling the transloca-
tion speed. The passive methods use the viscosity [31], the temperature gradient
[8, 42, 110], or the ion concentration gradient of the analyte solution [20, 34, 40, 43,
125, 126]. The viscosity of the solution is adjusted by adding glycerol, which has a
higher viscosity than water [31]. When using this method, a five-fold reduction in the
translocation speed has been obtained. Temperature and ion concentration gradients
exist on both sides of the membrane; when the ion concentration gradient was
adjusted, a three-fold reduction in the translocation speed was obtained [40].
Chemical modification of solid-state nanopore also serves to reduce a translocation
speed inside nanopore. For instance, chemical modification of solid-state nanopores
using molecules that can interact with DNA has resulted in translocations speeds
13 25 times slower than that observed without modification [59]. As the sophis-
ticated molecule control method, the incorporation of functional unite for single-
molecule fluid dynamics into solid-state nanopore by nano-fabrication technology
have been recently emerging. The detail contents on the single-molecule speed-
control nanopore technologies are described in Emerging Nanopore: Field-effect
transistors (FETs) integrated Nanopore, and Speed Control Solid-State Nanopore
(Sect. 11.6).
11.6 Emerging Nanopore
Besides the conventional ion-current blockage nanopore sensing shown in the

previous sections, the incorporation of other functional nanostructures and/or other
detection with the nanopore sensing have been recently interested. Of these studies,
several promising method of nanopore-sensing, such as Nanogap nanopore, Field-
effect transistors (FETs) integrated Nanopore, Speed Controllable Solid-State
Nanopore, Optical-sensing Nanopore and Force-sensing Nanopore, are introduced
as Emerging nanopore in this section.
11.6.1 Nanogap Nanopore
Nanogap nanopores are solid-state nanopores with the added capability of detecting
specific molecular electronic structures. Nanogap nanopore-sensing have been pro-
posed as a promising technique for single-molecule detection [102, 136, 137]. When
applying bias voltage between the gap-electrodes integrated inside nanopore, the
quantum-mechanical phenomenon of electron tunneling via a single-molecule is
induced, which is closely related to a potential barrier via sample molecule between
the nano-gape electrodes. The obtained conductance via single molecules are deter-
mined by the electronic structures of molecules, i.e., highest occupied molecular
orbital (HOMO) or lowest unoccupied molecular orbital (LUMO) energy levels.
Therefore, the observed electron-tunneling intensity represents the characteristic
conductance of sample molecules during the translocation of gap. The ideal structure
of nanogap nanopores has not yet been developed, but as a proof of the concept,
which identifies single molecules via tunneling currents flowing between
nanoelectrodes, the single-molecule sensing ability has been proven using scanning
tunneling microscopy (STM) [18, 19, 51, 81, 109, 135] and mechanically control-
lable break junctions (MCBJ) [82, 84, 85, 114–116] (Table 11.3). Based on this
idea, single-molecule tunnel-current based sequencing has been proposed
(Fig. 11.4a, b and c). This is that, by nanogap-electrode embedded nanopore, the
sequential detection of electron tunneling during translocating DNA strand can
determine the DNA sequence. It is found that the electron tunneling phenomena is
very sensitive to molecular orientations so that the confinement of nanopore struc-
tures can improve the stability of the molecular orientation inside nanopore. In
addition, further incorporation of other functional electrodes along nanopore are
proposed for precise control of molecular translocation and orientation (Fig. 11.4d)
[53, 115, 116]. When applying local electric field by the incorporated electrodes to
charged molecules inside nanopores, the trap and release behaviors on the pore-wall
can be controlled at single-molecular level. Based on this idea, IBM group proposed
that nanoelectromechanical device, “DNA transistor”, can control the orientation
Table 11.3 Characteristics of Bionanopore, Solid-state Nanopore, Nanogap Nanopore

Bio-nanopore Solid-state nanopore Nanogap nanopore
Detection probe Ionic-current Ionic-current Tunneling-current
Analyte dsDNA dsDNA ssDNA
ssDNA ssDNA RNA
RNA RNA Peptide
Protein Protein Amino-acid
Speed control Chemical Chemical Chemical
method modification modification modification
Electrophoresis Electrophoresis Electrophoresis
Electroosmosis Electroosmosis
Temperature gradient Temperature gradient Temperature gradient
Salts gradient Salts gradient Salts gradient
Fig. 11.4 (a) Schematics of nanogap nanopore sensing for single-molecule sequencing. When
DNA are translocating between nanogap-electrodes inside nanopore, electron tunneling via a
single-nucleotide is induced. The current intensity is closely related to a potential barrier via sample
molecule between the biased nano-gape electrodes. (b) The sequential reading of electron tunneling
during nucleotide translocation can determine the nucleotide sequence. (c) Typical current-time
profile of UGAGGUA oligo-nucleotide. The observed electron-tunneling intensity represents the
characteristic conductance of sample molecules during the translocation of gap. (Adapted with
permission from [82]: Copyright 2012 Nature Publishing Group) (d) Schematics of DNA transistor.
“DNA transistor” device have metal-dielectric structures (layers) inside the solid-state nanopore. By
controlling the electric field between the metal layers, DNA can be trapped and released in the
nanopore. By cyclically turning on and off these voltages, the DNA strand can move through the
nanopore
and translocation speed of the single-stranded DNA inside the nanopore

[38, 88]. The detail study related transistor integrated nanopore are described in
Field-effect transistors (FETs) integrated Nanopore in the following section. Chem-
ical modification on the sensing site of nanopore and/or nano-gapes can also be
proposed for controlling the molecular orientation and improvement of sample
identification. Several studies reported that chemical modifications of sensing
tip/electrodes with a scanning tunneling microscope can improve the molecule
identification, which is due to the interaction and its “recognition tunneling” between
sample molecules and modified molecules [18, 19, 51, 66, 81, 135]. In early works,
each of base recognition are done by each of the complementary base modified
electrode [18, 81]. In the recent work, by using a 4(5)-substitutedimidazole-2-
carboxamide(ICA) modified electrode, the nanogap method by using the recognition
tunneling can be applicable for selective bases and amino-acid detections [19, 135].
11.6.2 Field-Effect Transistors (FETs) Integrated Nanopore
Field-effect transistors (FETs) integrated nanopore are also interested because

FET-based nanopores can potentially create large electric currents by amplifying
small changes in the potentials caused by sample translocation. Thus, these devices
present one possible approach to overcoming the difficulty of rapidly measuring
pA-level electric currents generated when single DNA molecules quickly pass
through a nanopore. In early works, a design of nanopore capacitor made in a
metal-oxide-semiconductor (MOS) [44] and a semiconductor-oxide-semiconductor
membrane [64] were proposed. Their simulation results demonstrated that such a
device can potentially sequence DNA at single base level. Recently, a progress of
nano-fabrication technique and nano-materials study have accelerated FET nanopore
[76, 89, 91, 113]. For instance, a fabrication of array of ionic field effect transistor
(IFET) nanopores were demonstrated by combining electron beam lithography and
atomic layer deposition [76]. By using the nanopore with sub-10 nm diameter on
TiO2 covered Si3N4, the ionic transport of KCl electrolyte can be efficiently manip-
ulated by the embedded electrode of TiN within the nanopore. As another recent
study on FET nanopore, nanopores with a diameter of 10 nm formed on Si
nanowires with diameters of 30 50 nm can detect both changes in the ionic
currents flowing through the nanopores and the channel conductance intensity of
the nanowires when dsDNA passes through the nanopores [130]. Nanopores with a
diameter of 10 nm fabricated on graphene nanoribbons also exhibit both ionic
current blockade and changes in graphene conductance [113]. Importantly, these
FET nanopore show large electric current changes on the order of 100 nA, which is
greater than approximately 1000 times as large as those obtained with existing
nanopore devices, and allow high-speed electric current measurements. To date,
single base molecule resolution has been achieved at a measurement speed of
hundreds of kHz. With these emerging nanopore technologies, however, it may be
possible to realize single base molecule resolution at electric current measurement
speeds of more than 1 MHz.
11.6.3 Speed Controllable Solid-State Nanopore
Single-molecule speed-control nanopore technologies have been recently explored

by the incorporation of speed control unit into solid-state nanopores because solid-
state nanopore can be potentially integrated with functional nanostructures by nano-
fabrication technology [1, 41, 131]. In general, the fluid dynamics of single mole-
cules within nanopore is influenced by the electrophoresis and electroosmotic flow.
By voltage-application of gate electrode inside solid-state nanopore, the
electroosmotic-flow can modulate the translocation speed of target molecules
because, in the case of SiO2 nanopore, cations accumulate on the surface of SiO2
inside nanopore so that the motion of liquid are induced by an applied potential.
When a negative voltage is applied to the gate electrode, stronger upward electro-
osmotic flow was induced, resulting in decrement of the translocation speed of the
DNA molecules. In contrast, when a positive voltage is applied to the gate electrode,
anions accumulate on the SiO2 surface, thus resulting in a downward electroosmotic
flow an increase in the translocation speed of the DNA molecules. Another speed
control method of interest uses the difference in the pressures applied to both sides of
the membrane. Using this method, an eight-fold reduction in the translocation speed
was obtained [69]. The fluid dynamics of single molecules within nanopores has not
yet been fully clarified: however, understanding the dynamics of single molecules
passing through nanopores will provide important insights for improving the preci-
sion of single molecule identification using nanopores.
11.6.4 Optical-Sensing Nanopore
Optical detection method can be conjugated with solid-state nanopore platform

(Fig. 11.5a) [35, 105]. In this method, optical probe tethered molecules are usually
prepared and, as the optical detectors, fluorescence spectroscopy and/or total internal
reflection fluorescence (TIRF) microscopy, are utilized for sample detection, and the
optical detection site is set around the electrical sensing nanopore. When the labelled
target molecule flow into nanopore, single-molecule optical signals are detected by
these optical detectors, and simultaneously ionic-current blockage-signals are
detected by current measurements. In addition, if patterning metallic membrane
and/or photonic nanostructures around nanopore, the single-molecule just
translocating nanopore are illuminated so the emitted optical signals are selectively
enhanced inside the nanopore [9, 78]. The nanopore optical enhancement method,
such as “plasmonic nanopore”, can significantly suppress background signals emit-
ted by molecules outside-nanopore so the ratio of signal to noise would be improved.
These low-noise single-molecule optical nanopore method is very promising for
various single-molecule biomolecule detections in real bio-samples. For instance,
fluorescently labeled DNA nucleotide molecules are detected during translocating
nanopore by fluorescence measurement at the nanopore opening, and identify the
sample species at single-molecule level. Based on this optical method, a single
molecule DNA optical sequencing technique have been proposed (Fig. 11.5b)
[36, 52, 72]. In this method, the original DNA is converted to an expanded sequence
by substituting every base in the sequence with a specific ordered oligonucleotides
conjugated fluorescent probe. The converted DNA is hybridized with the comple-
mentary “molecular beacons” of two colors. When nanopores sequentially unzip the
sample beacons during the translocation, un-quenched fluorophore at each unzipping
event giving rise to a series of optical signals, resulting in the reading the original
sequence. These results suggest possibility of single-molecule optical based DNA
sequencing using nanopores.
Fig. 11.5 (a) Schematics of optical sensing nanopore: Nanopore platform with conjugated with
optical detection. First of all, optical probe tethered molecules are prepared. Next, the optical
detection site is set around the electrical sensing nanopore. When the probe conjugated molecule
translocate through the nanopore, the probe emits the optical signals. The emitted optical sample
signals are detected by optical microscopy and simultaneously ionic-current blockage-signals are
detected by current measurements. (b) Schematics of Single molecule DNA optical sequencing
technique. First, the original DNA sequence is converted to an expanded form by substituting each
and every base in the DNA sequence with a specific ordered pair of oligonucleotides. Second, the
converted DNA is hybridized with complementary molecular beacons of two colors. Finally,
fluorophore conjugated with the DNA sequence is sequentially detected as their optical signals,
resulting in the reading the original sequence (c) Schematics of Nanopore force sensing: Nanopore
sensing platform integrated with optical trapping systems. In this nanopore platform, the applying
electrical field around the nanopore can control the exerted force on analyte (F0 ) and the bead
conjugated with sample molecule are pulled by the force of optical trapping (F). In this set-up, the
observed ion-current time-profiles represent the characteristic single-molecule force signals during
the translocation of molecular behaviors such as denaturing, refolding, and so on. The behaviors are
strongly influenced by the inter/intra-molecular interaction between molecules and/or between
molecule and wall inside a nanopore
11.6.5 Force-Sensing Nanopore
Single-molecule force-detection nanopore methods have also emerged as a powerful

tool to investigate the inter/intra-molecular forces and motions of biomolecules. By
conjugation of nanopore platform with force measurement techniques such as
magnetic tweezers, optical trapping, and atomic force microscopy, some of nanopore
force sensing platform have been reported [48, 56]. Since nanopore platform can
control the exerted electrophoretic force on charged single analyte by the applying
electrical field across the nanopore, the detected time-signals represent “single-
molecule force signals”, which are closely related to the intermolecular interaction
and/or interaction between pore and analytes during the translocation (Fig. 11.5c).
For instance, when the one-end of DNA tethered to polystyrene beads are trapped by
optical tweezer and the other end of the DNA are pulled by the voltage-driven force
of negatively charged DNA translocating through nanopore, the observed
ion-current time-profiles represent the characteristic single-molecule motion behav-
iors, i.e., self-folding of hair-pin structure, hybridization, dehybrization of DNA
duplex and so on. Such “single-molecule force signals” under the nanopore-
electrical field driven force can provide plenty of structural information for the
interpretation of biological phenomena, i.g., the enzymatic activity and biological
molecules replication errors and misfolding in structure, which are closely related to
disease, such as Parkinson’s, Alzheimer’s disease, and cancers. Therefore, nanopore
based single-molecule force detection method would be widely applicable for
various biological research field and medical diagnosis.
11.7 Future of Nanopore Sensor Method
Nanopore sensor have characteristic features, such as high-throughput, single-mol-

ecule measurement/capturing capability, and sample-versatility, so that the develop-
ment of the nanopore platform is expanding the application interests as shown in
Sect. 11.3. Of these applications, nanopore-based single-molecule sequencing would
make a big impact on biomedical application such as personal medicine and drug
screening. In addition, beyond conventional ion-current blockade detection, by
incorporation of various emerging detection methods into nanopore platform, the
nanopore-platform can detect various kinds of single-molecule information, and
become “smarter”, as shown in Sect. 11.4. The development of “smarter” nanopore
platform are supported by the development of nano-fabrication technique, which
provide the ability of mass-production, and highly-parallelization of sensors in the
platform. The development of the fabrication technique would also realize more
compact and miniature nanopore platforms, which can produce tremendous single-
molecule related data at low cost. These features are very suitable and promising for
portable sensors for the Age of Internet of Things (IoT) and Big-Data. Therefore, the
development of nanopore platform fields would be still challenging and expanding
on various applications, i.g., future clinical, health-care application fields such as
on-site diagnosis and personal medicine.
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Chapter 12
Paper Microfluidics for POC Testing
in Low-Resource Settings
Elain Fu
Abstract Paper microfluidics is a subarea of microfluidics in which porous mate-

rials are used to create devices. Advantages of paper microfluidics include fluid
transport via capillary forces, so that external pumping equipment is not necessary,
and the use of less expensive materials than those commonly used in conventional
microfluidic devices. Paper microfluidics enables the development of fully dispos-
able devices that are appropriate for use in even the lowest-resource settings, and the
potential for high impact improvement to human health. In this chapter, we first
discuss the paper microfluidic device fabrication processes of materials selection,
fluidic boundary definition, and reagent patterning. Next, we discuss tools develop-
ment for manipulating fluids in paper microfluidic devices. Then, we describe
specific medical applications with discussion of three promising paper microfluidic
devices. Finally, we close with a general discussion of challenges in the translation
of paper microfluidic devices from the lab to the field.
Keywords Paper microfluidics · Porous materials · Point-of-care testing
12.1 Introduction to Paper Microfluidics
12.1.1 Paper Microfluidics as a Subfield of Microfluidics
Paper microfluidics is a subarea of microfluidics in which porous materials are used

to create devices. As in the case of conventional microfluidics-based sensor devel-
opment, one can leverage key micro-scale properties. The effects of viscosity are
more prominent than those of inertia, such that the dimensionless Reynolds number
is smaller than one. The result is flow in the laminar regime with predictable fluid
transport and mass transfer (e.g., diffusion-based mixing across adjacent streams).
Surface tension and capillary forces can also be prominent. This is especially true in
E. Fu (*)
Oregon State University, Corvallis, OR, USA

326 E. Fu
paper microfluidics, where the dimensionless capillary number, the ratio of viscous
forces to surface tension, is very small, ~1 106 for many paper systems1.
Paper microfluidics shares several advantages with conventional microfluidics
[1]. These include high surface area to volume ratios that enable fast surface
reaction-based processes. Another shared advantage is the short diffusion distances
(and times) that enable efficient mass transfer. For paper microfluidic systems, the
diffusion distances set by the material pore diameter are typically 10 times smaller
than in conventional microfluidic channels (i.e., a 10 μm characteristic pore diameter
versus a 100 μm channel height). The micro-scale devices are compatible with
microliter-sized sample volumes, and enable a rapid time to result, reagent savings,
and reduced waste. Finally, parallelization enables the probing of multiple reaction
conditions, as well as high-throughput processing in a reasonable amount of time.
Paper microfluidics has several advantages over conventional microfluidics. First,
lateral flow test development over the past three decades has produced a strong
knowledge base that is useful for paper microfluidic device development. Second,
porous materials are generally lower cost than other materials that have traditionally
been used in microfluidic devices (e.g., silicon and glass). And most importantly,
fluid transport within porous materials occurs via capillary forces, so external
pumping systems are not necessary. This enables the development of fully dispos-
able devices that are appropriate for use in even the lowest-resource settings.
12.1.2 Capillary Flow in Paper Microfluidic Devices
Flow within a capillary tube is driven by capillary pressure [2],
2 ∙ γ ∙ cosθ
Pc ¼ ,
R
where γ is the fluid surface tension, R is the capillary tube radius, and θ is the contact
angle of the liquid-air interface with the solid. The Hagen-Poiseuille equation [3]
describes fluid flow in the tube,
π ∙ R4 ∙ ΔP
Q¼ ,
8∙η∙L
where Q is the volumetric flow rate, η is the dynamic fluid viscosity, L is the fluid
column length, and the pressure difference across L, ΔP, is equal to PC. From these
expressions, a relationship between the position of the fluid front L and time t, the
Lucas-Washburn equation [4, 5] can be derived,
1
The estimate is based on a surface tension of 7.2 102 N/m, a velocity of 1 104 m/s, and a
viscosity of 1 103 Pa•s.
12 Paper Microfluidics for POC Testing in Low-Resource Settings 327
1
γ ∙ R ∙ cosθ 2
L¼ t :
2∙η
Fluid is pulled into the tube by the capillary force, while viscous resistance coun-
teracts the capillary force. The viscous resistance increases with the fluid column
length within the capillary tube, such that the velocity of the fluid front decreases as
the fluid penetrates into the capillary.
Given that a group of parallel cylindrical capillaries of varying radii can be used
to describe a strip of porous material, the fluid front within the strip will follow
Lucas-Washburn flow with R equal to the mean of the capillary radii, Rmean [4]. (See
Mendez et al. [6] for a thorough discussion of this topic.) The pore structure of
common porous materials used in paper microfluidics can be far from uniform, both
in terms of the shape of the “pores”, as well as the distribution of pore sizes.
Nitrocellulose, most often used in lateral flow-based devices, has a sponge-like
structure [7]. In contrast, cellulose consists of a dense network of randomly oriented
fibers. Chopped glass fiber also consists of an intricate network of fibers. The
wicking behavior of some common porous materials is described below.
12.1.3 The Role of Paper Microfluidics in Point-of-Care

Testing (POCT)
A main goal of the field of POCT is to translate current high-resource laboratory-

based testing into technologies that can be used in POC settings by the patient. There
are many POC settings and these vary in terms of their resource levels [8]. POC
settings can range from the higher-resource hospital emergency rooms, to clinics and
physician’s offices, to the lower-resource settings in the developed world, such as
schools, workplaces, and homes, to the very lowest resource settings of the devel-
oping world. For the lowest-resource settings, there are a number of challenges that
must be addressed for a technology to succeed in those settings [8–10]. These
include a limited contact time with patients, a limited training of test providers, a
lack of laboratory facilities (i.e., testing environments with uncontrolled tempera-
tures and humidity levels), limited local infrastructure including a lack of cold chain
for refrigeration of reagents, a lack of support for maintenance on instrumentation,
and a need for a low cost per test. Finally, the performance specifications of the
application must be met. These requirements are summarized by the ASSURED
standards: affordable, sensitive, specific, user-friendly, rapid (and robust),
equipment-free, and deliverable to the user [11].
The current standard bioassay format that is used in the lowest-resource settings is
the lateral flow test (LFT) [12]. A well-known example is the home pregnancy
dipstick test. It is affordable, and can be very low cost to produce [13]. The test is
user-friendly and rapid. Most tests require the user to apply the sample (and a buffer)
and then look for the appearance of color in the ‘test’ and ‘control’ lines within a
328 E. Fu
period of 10–20 min. The test is often equipment free. An external pumping system
is not required since fluid is transported via capillary forces in the nitrocellulose and
cellulose substrates. There is also no need for instrumentation for detection of the
signal. The test can be based on the interpretation of colored line development via
human visual perception. And the test is ‘deliverable to the user’ with typical shelf
lives of 1 or 2 years at ambient temperature [14]. Trade offs for LFT simplicity
include that conventional LFTs are limited to a single delivery step of premixed
sample and conjugate that can translate to limited sensitivity and specificity. Addi-
tionally, LFTs generally have a qualitative output when not used with some sort of
an instrument, and they are difficult to multiplex for the simultaneous detection of
multiple targets. Thus, although LFTs conform to many of the ASSURED standards,
conventional LFT performance would not enable clinical relevance for a number of
compelling heath conditions. Thus, there is a need to improve POCT in the lowest-
resource settings, and paper microfluidics is a potential high impact solution.
One area of focus in paper microfluidics device development has been enabling
testing for multiple targets from a single sample. To address this, the Whitesides
group has developed paper networks for multi-analyte detection. Their 2007 article,
in which they demonstrated a device consisting of a diverging network for the
simultaneous detection of glucose and protein from a small volume of urine, started
a resurgence of interest in paper-based devices [15]. An image series of the color-
imetric results from their bi-plexed paper-based device for increasing analyte con-
centrations is shown in Fig. 12.1a. Following this work, there have been numerous
demonstrations of multi-analyte detection in both 2- and 3-dimensional [16] formats
in porous materials [17].
A second area of focus in paper microfluidics has been on providing quantitative
output using optical (e.g., colorimetric, fluorescence, etc.) or electrochemical signals
[18, 19]. These efforts range from developing equipment-free quantitative readout
[20] to minimally-instrumented strategies based on simple readers [21]. One
approach to achieving equipment-free quantitative readout is to use differences in
colorimetric signal intensities or hues to indicate analyte level [20, 22]. The use of
multiple indicators to produce distinct intensity- and hue-based signals is shown in
Fig. 12.1b [22]. Another approach that has potential for equipment-free quantitative
readout is to use the spatial distribution of signal in the detection region to indicate
analyte level [20, 23]. The visible signal develops along the length of the detection
region in the direction of flow and in proportion to the concentration of analyte in the
sample [23]. A variation of this is the “ladder-bar” readout, in which the level of
analyte is quantified by enumerating the number of discrete detection sub-regions in
which signal developed [20, 24]. A demonstration of this method using an enzyme-
based system is shown in Fig. 12.1c [24]. Complementary to this, much progress has
been made in the area of developing analysis algorithms and simple companion
reader technology to extract high quality optical [25–27], or electrochemical signals
[21] that can inform on the level of target analyte within a sample.
A third area of focus in paper microfluidics device development, complementary
to the previous areas, is on using paper networks to perform automated multi-step
sample processing for high sensitivity testing [28]. A common theme across high-
Fig. 12.1 Three areas in which paper microfluidics can improve on the performance of standard
conventional lateral flow tests are in enabling multi-analyte detection, quantitative output, and
higher sensitivity analysis. (a) 2D multi-analyte paper-based device for the simultaneous detection
of glucose and protein in a urine sample. (Reproduced with permission from Ref. [15]). (b) Paper-
based device that produces visual quantitative output based on intensity and hue through the use of
multiple colorimetric indicators. (Reproduced with permission from Ref. [22]). (c) Paper-based
device with “ladder-bar” readout where the number of bands in which signal develops increases
with increasing concentration of analyte. (Reproduced with permission from Ref. [24]). (d)
Prototype amplified malaria immunoassay in an automated paper-based device. The operator
adds sample and water to pads on the card and folds the card to start the assay. The amplified
assay detects clinically-relevant levels of malaria antigen in a mock sample. The limit of detection
of the amplified assay was found to be four-times lower than without the rinse and amplification
steps. (Reproduced with permission from Ref. [30])
performance laboratory-based tests is processing of the sample to maximize signal

and minimize noise. For example, laboratory-based ELISA, or enzyme-linked
immunosorbent assay, requires multiple sequential sample processing steps to
achieve high sensitivity. The assay takes place in a well, coated with an antibody
molecule that specifically binds to the target analyte of interest. The multiple steps of
the assay include sample introduction and incubation, one or more steps to label the
binding event, and a step to chemically amplify the signal due to the original label for
high sensitivity detection. Also key, are the multiple wash steps used to minimize the
nonspecific binding events in the system. Thus, a promising strategy for improving
the performance of LFTs in low-resource settings is to implement additional sample
processing to improve device performance, while still retaining the ease of use of
LFTs [29]. An example of this is the amplified assay shown in Fig. 12.1d [30]. After
application of the appropriate fluids on the pads of the device, the operator folds the
330 E. Fu
device closed and waits until the test is complete. A concentration series indicates
that the amplified assay is able to detect malaria antigen in a clinically relevant range
[30]. The ability to manipulate fluids and the reagents within them in paper networks
is critical to enabling automated processing for high performance. So, there is a need
for robust paper microfluidic tools that serve as the analogs of pump controls and
valves of conventional microfluidics [28], and compatible paper microfluidic device
fabrication methods.
12.2 Paper Microfluidic Device Fabrication
Device fabrication can be divided into three processes: materials selection, fluidic
boundary definition, and reagent patterning.
12.2.1 Porous Materials Selection
For materials selection, the properties of the material, such as the capacity and flow
rate, are important considerations. In addition to cellulose, there are other porous
substrates with properties that can be advantageous in a device, including nitrocel-
lulose substrates, which are widely used in LFTs, fabrics which can be useful in
wearable sensor applications, and glass fiber substrates, which with their high flow
rates, can be useful when rapid fluid flow is desired. Flow rates can vary substantially
between different porous materials. For the nitrocellulose membranes used in LFTs
(characteristic pore sizes from 3 to 17 μm), capillary flow times range from 75 to
240 s per 4 cm (Millipore Hi-Flow membranes) and variability in capillary flow
times can be up to 25%. For cellulose materials, the commonly used Whatman #1
filter paper has a capillary flow time of ~70 s per 2 cm, while for glass fiber, capillary
flow times can be considerably shorter, e.g., 4 s and 15 s per 2 cm for Ahlstrom 8951
and 8950, respectively. The fluid capacities of different porous materials can also
vary substantially (e.g., ~12 μL/cm2 for Millipore Hi-Flow 135, ~16 μL/cm2 for
Whatman #1 filter paper, ~80 μL/cm2 for thicker cellulose substrates [31], and
~60 μL/cm2 for Ahlstrom 8951 glass fiber). Thus, for a given application, target
processing volumes and average flow rates can be attained by judicious choice of
material or a combination of materials.
12.2.2 Defining Channels in Porous Materials
Fluidic boundary definition is a critical process in the fabrication of a paper

microfluidic device. Since the Whitesides group first described the use of optical
lithography [15, 32] to pattern fluidic boundaries in cellulose, many methods of
defining channels have been developed [33, 34], such as depositing hydrophobic
PDMS barriers using a plotter [35], etching using inkjet technology [36] or a plasma
[37], laser-etching hydrophobic paper [38], and contact stamping ink barriers
[39]. One of the most commonly used fabrication methods has been printing wax
directly onto substrates using a wax printer, followed by heating of the substrate to
create functional wax barriers in the substrate. The main advantages of wax printing
are its simplicity and reasonable cost with wax printers retailing for less than $600
(USD). The most commonly used porous substrate in direct wax printing is cellulose
filter paper [40, 41], although nitrocellulose [42] has also been demonstrated to be
compatible with direct wax printing. A severe limitation of direct wax printing is that
it is only compatible with substrates that can be mechanically processed by the wax
printer. Substrates that are too thick, coarse, fragile, flexible, or deviate significantly
from the canonical 8.5 inch 11 inch sheet dimensions are not recommended for
use in the printer and could result in damage to the printer or substrate. Typical wax
printers are limited to substrates with basis weight between 60 g/m2 and 220 g/m2
and dimensions between 76 mm 127 mm and 216 mm 610 mm. Thus,
non-standard porous substrates that might have useful properties in paper
microfluidics are not always compatible with the method of direct wax printing.
Several alternate wax-based patterning methods have been demonstrated includ-
ing patterning using a wax pen [41], wax screen-printing [43], and wax dipping
using an iron mold to selectively exclude wax in desired regions [44]. An extension
of direct wax printing in porous materials is wax transfer printing [31, 45]. In this
method, a transparency film is used as an intermediate template that can be mechan-
ically processed by the wax printer and subsequently used to transfer the printed wax
pattern onto a porous substrate by placing the two into contact and heating. Using the
wax transfer method, fragile and flexible porous materials such as tissue and fabric
can be straightforwardly patterned with wax [31]. Further, multiple wax transfer
cycles can be used to pattern thicker materials (e.g., cellulose substrates with a large
fluid capacity) [31]. Several wax-based methods are schematically highlighted in
Figs. 12.2a–d.
Complementary to methods that define channels through patterning are methods
based on physically cutting out structures with a blade [46] or laser [47]. The latter
are particularly useful for creating hybrid devices composed of multiple materials.
Advantages of these methods are simplicity and a relatively low cost of supporting
equipment. Further, in the case of laser cutting systems, the power and speed of the
laser beam can be adjusted to etch materials rather than cut through the materials.
This can be useful when fabricating devices containing porous materials on a
backing (e.g., nitrocellulose is often cast on a polyester backing), since the porous
material can be etched away, while keeping the backing intact [47]. A potential
disadvantage of cutting out structures is that they can be difficult to handle without
attaching them to a support layer, which then adds a step to the device assembly
process. These methods are highlighted in Figs. 12.2e–f.
One factor that may affect the choice of which channel definition method to use is
the target size of the porous channel. Typical paper microfluidic devices have a
channel thickness of ~100 to several hundred μm and channel widths and lengths of
332 E. Fu
Fig. 12.2 Examples of fabrication methods for defining channels in porous materials. (a) The most
common method is direct wax printing, in which a printer is used to deposit wax onto the porous
substrate, and then the wax-coated substrate is heated to create robust hydrophobic boundaries.
(Reproduced with permission from Ref. [40]). (b) For substrates that are not compatible with
processing through the wax printer, an intermediate template, such as a polyester sheet, can be used
to transfer the patterned wax to the substrate in a method called wax transfer printing. (Reproduced
with permission from Ref. [31]). (c) Alternatives that do not require a wax printer include creating
boundaries using a wax “pencil”, or tracing wax boundaries onto a pre-printed ink pattern.
(Reproduced with permission from Ref. [41]). (d) A substrate can also be patterned by applying
melted wax through a screen or mask in wax screen printing. (Reproduced with permission from
Ref. [43]). (e) Fluidic channels can be defined in porous material by cutting the porous material
using a laser. Further, partial etching of materials can be useful for device assembly. (Reproduced
with permission from Ref. [47]). (f) A knife blade can also be used to define the channels in a porous
material. (Reproduced with permission from Ref. [46])
at least several mm. The mm-scale lateral dimensions are compatible with the
majority of the above channel definition methods. However, in cases for which
smaller dimensions are required, direct wax patterning (with demonstrated channel
widths of ~600 μm [40] and ~300 μm [48]) and optical lithography [15] are the
methods that would have the highest lateral patterning resolution. Additional factors
that may affect the choice of channel definition method for a particular application
are cost, ease of use, and the need for compatibility with other device fabrication
processes such as reagent patterning and the incorporation of fluidic controls.
12.2.3 Patterning Reagents in Porous Materials
A second critical process in the fabrication of microfluidic devices is patterning

reagents within the device to perform the required biochemical reactions in the field.
Current patterning methods vary greatly in terms of cost and versatility.
On one end of the spectrum, manual pipetting is a low-cost method for reagent
deposition [49, 50], but is constrained to mm-scale circular patterns. In addition, the
spot uniformity can be highly dependent on operator skill and the method is limited
to low-throughput situations. Further, in the case of lateral flow immunoassays (i.e.,
nonspecific adsorption of antibodies onto nitrocellulose [7]), producing different
uniform reagent concentrations on the substrate is not straightforward.
At the other end of the spectrum, expensive, automated, liquid dispensing
systems can produce high-resolution (~200 μm) patterns of protein in a variety of
shapes and over a range of concentrations. In the context of LFTs, the most common
reagent patterning system “stripes” antibodies in a narrow region across the nitro-
cellulose strip [12]. Other useful configurations of patterned reagents include
extended regions to obtain information on the signal binding profile [51] or for
continuous distance-based readout [20], and multiple discrete capture regions for
distance-based readout [52] or multi-analyte patterning [53]. Additionally, pattern-
ing nanoliter drops to control rehydrated reagent concentration as a function of space
and time has been demonstrated [54] and applied to an amplified assay [55]. Liquid
dispensing instrumentation [56] has been used to produce spots [55, 57, 58], stripes
[59, 60], and larger, mm-scale rectangular regions [51]. Disadvantages of these
instruments include a high cost, between $30 k and $120 k (USD), the need for
continued maintenance, and the need for operator training.
Alternative cost-effective stamping methods have been developed to complement
manual pipetting of reagents. In one example, highlighted in Fig. 12.3a, cellulose
stamps were used to pattern dye and protein onto cellulose substrates in high volume
[61], but with reduced edge definition due to the imbibition of fluid into adjacent
non-stamped substrate regions. In another example, highlighted in Fig. 12.3b, com-
posite porous glass fiber stamps that included buffer-containing regions to restrict
imbibition of the protein solution in the substrate were used to produce mm-scale
rectilinear patterns of antibody on nitrocellulose [62].
Although physical adsorption works well for creating high-density regions of
antibodies on nitrocellulose substrates, it may not be as effective for other molecule
and substrate combinations. The latter is true for antibodies and cellulose, and a
number of methods for creating high-density regions of immobilized antibody on
cellulose have been demonstrated. These include crosslinking with glutaraldehyde
334 E. Fu
A fluid reservoir
wood B
i ii handle mp
Sta
plexiglass
hole
Substrate
tape 1 2
paper stamping area
pipette tip
photoresist iii
Protein
Buffer
iv
3
2 cm
vi
2 cm
Fig. 12.3 Examples of fabrication methods for patterning reagents in porous materials. (a)
Cellulose stamps, configured in a layered network, were used to pattern reagents onto cellulose in
a high-throughput parallel process. (Reproduced with permission from Ref. [61]). (b) Schematic of
a related method in which glass fiber stamps were used to pattern antibodies onto nitrocellulose. The
stamps incorporated buffer-filled pads adjacent to the protein-filled stamps in order to improve
boundary definition. The images below the schematic show antibody stamped in different patterns
and subsequently labeled with gold nanoparticles. (Reproduced with permission from Ref. [62])
[63], covalent bonding using periodate oxidation [63], diazonium-based covalent

functionalization [64], and photoreactive methods [65].
Adding a layer of complexity to the patterning of reagents in a paper microfluidic
device is the need for the preservation of these reagents until rehydration with
sample. As mentioned above, LFTs provide a benchmark for preservation timescales
of 1 or 2 years at ambient temperature [14]. Methods of drying reagents, such as
antibodies [66], for preservation include lyophilization and vacuum drying. One of
the most commonly used additives for the preservation of proteins is the disaccha-
ride trehalose, and it is known to stabilize protein structure [67]. As an example, in
the demonstration of a paper microfluidic enzyme-amplified assay, horseradish
peroxidase (HRP) was preserved by vacuum drying the enzyme with a mixture of
trehalose, iron, and EDTA on glass fiber. The activity of the enzyme after rehydra-
tion (and previous dry storage at elevated temperature for 5 months) was ~80% that
of fresh enzyme [68]. In a related study, SU-8 epoxy resin [69] was demonstrated to
have a positive stabilizing effect on HRP storage in cellulose.
12.2.4 Manipulating Fluids in Porous Materials
The key to enabling high performance in paper microfluidic devices is the robust and
accurate control of fluids within the devices. Thus, the paper microfluidics commu-
nity has dedicated substantial effort to increasing the capability of paper-based
devices via the development of a versatile set of methods to control fluid flow in
paper-based devices or “flow tools” [70]. Flow tools have been developed to change
the fluid velocity within a channel relative to a conventional porous channel, as well
as function as a valve by turning on or off fluid flow within a porous channel. These
flow tools use many different methods to affect fluid flow. A comprehensive review
of the area of flow tools development is presented in a review by Fu and Downs
[70]. The majority of the tools operate via manipulation of capillary flow in porous
media, including fluid surface tension, medium pore size, contact angle, or fluid
viscosity. Some of the more recent flow tools use open channels within the porous
material to manipulate the overall transport of fluid within the device. Complemen-
tary to these are tools that rely on mechanical actuation, either via the operator or
automated via an element responsive to a particular external stimulus. A summary of
some flow tools, organized by function, is presented in Table 12.1 [70].
The ability to sequentially deliver multiple fluid volumes is required for multi-
step sample processing (e.g., signal amplification [30, 71]), and potentially improved
analytical performance. Many of the fluid flow control tools that have been devel-
oped can be used to sequence fluids; four methods are highlighted in Fig. 12.4. A
first strategy for sequencing fluids uses dissolvable barriers as shown schematically
in Fig. 12.4a. Specifically, sugar barriers can be used to create delays in the transport
of the fluid within a paper network [72]. Both the extent of the sugar barrier and the
concentration of the sugar solution used to form the barrier within the porous
material can be used to produce a delay time of seconds to an hour [72, 73]. The
time delay is mainly due to an increased viscosity of the system fluid by the addition
of the sugar [72]. Coefficients of variation for the hand-dipped barriers range
between 11 and 24%. A second strategy for sequencing fluids is to use varying
path lengths to control the arrival times of multiple fluid volumes to a common
detection region [30, 73]. As shown in Fig. 12.4b, the multiple reagent volumes are
delivered to the detection region in order from right to left. This device design was
used to create the amplified assay shown in Fig. 12.1d. A third strategy for sequenc-
ing fluids is to use porous material shunts in which the porous material enables an
alternate flow path for the system fluid. Delays between 3 and ~12 min were
produced. As shown in the example in Fig. 12.4c, the shunts can be integrated
into a paper network with varying path length in order to effectively sequence fluids
[49]. In particular, the shunts enable the delivery of larger fluid volumes for a given
network design. And a fourth strategy highlighted in Fig. 12.4d, also based on path
length, uses a linear design with multiple input pads to produce the sequential
delivery of multiple well-defined fluid volumes [74].
336 E. Fu
Table 12.1 Summary of some fluid flow control tools

Function Tool
Methods of changing Changes in channel width/length produce small magnitude velocity
fluid velocity changes [73]
Downstream region with increasing cross-sectional area produces
quasi-steady flow [6]
Porous channels enclosed by flexible films produce up to 10
increase in flow rate compared to capillary flow [75]
Hollow channels with hydrophilic base produce up to 7 increase in
flow rate [76, 77]
Etched trenches in paper channel produce speeding up or slowing of
fluid [78]
Double layer porous channels produce an over 6 decrease in flow
time (4.5 cm at 35% relative humidity) [79]
Open channels in omniphobic paper produce fast pressure-driven
flow [80]
Hollow channels with hydrophobic base enable pressure-driven flow
[81]
Wax coatings in porous channels decrease wettability and produce
time delays [82, 83]
UV treatments of TiO2-coated papers alter surface wettability and
produce delay times [84]
Pressed papers with reduced pore sizes produce delay times of 7.4-
fold [85]
Dissolvable species dried into channels for increased fluid viscosity
upon rehydration produce delay times [72, 73]
Porous shunts divert fluid to produce time delays [49]
Sealing paper channels with charged plastic substrates produces time
delays [86]
Methods of valving Mechanical user actions including bridging [87], push buttons [88],
folding [30], and sliding [89]
Polarization of a hydrophobic dielectric coated on paper to be
hydrophilic [90]
Temperature affects the surfactant solubility in water-filled, hydro-
phobic porous channels [91]
Corona discharge treatment switches coated paper from hydrophobic
to hydrophilic [92]
Melting of wax plug allows flow [93]
Submerged inlet length in a well shuts off flow when fluid level drops
below inlet [94]
Dissolvable bridge shuts off flow of fluid [95, 96]
Magnetic forces actuate to connect or disconnect a fluidic path [97]
Expandable polymers actuate upon a fluid trigger to connect/discon-
nect a fluidic path [50]
Rehydration of dried surfactant enables fluid to cross a hydrophobic
region [98, 99]
(continued)
Table 12.1 (continued)

Function Tool
Pseudo-valves 2D paper network [71, 73] and 3D paper network [89] with varying
path lengths
Linear design (1DPN) with wells or pads [74]
Inkjet printing of barriers produces varying path lengths [100]
SlipPAD configuration of fluid sources and channel in adjacent layers
sequences the fluids in the channel upon sliding contact between the
layers [101]
The table is adapted with permission from Ref. [70]
Fig. 12.4 Four methods of sequencing fluids in paper-based devices. (a) Dissolvable barriers
consisting of sugar can be used to create time delays from seconds to 50 min. (Reproduced with
permission from Ref. [72]). (b) Varying the path lengths of different fluid inputs into the network
can be used to sequentially deliver the fluids to a common detection region. (Reproduced with
permission from Ref. [30]). (c) Porous shunts can be integrated into a design based on varying path
lengths to achieve additional control of fluid delivery. (Reproduced with permission from Ref.
[49]). (d) Varying path lengths in a linear design with porous pads is a straightforward method of
sequencing fluids. (Reproduced with permission from Ref. [74])
A main advantage of the dissolvable barriers is a large tunable range of time

delays, while a potential disadvantage is the large amount of sugar released that may
be incompatible with downstream assay processes. A main advantage of using the
2D network with varying path lengths for sequencing fluids is simplicity, with fluid
338 E. Fu
control built into the network without additional components. However, for larger
volumes, the network size (and associated dead volume) increases. The use of shunts
can be used to increase the volumes delivered for a given size network, but at the
expense of additional materials and fabrication steps. The 1D design is the simplest
of the four and compatible with current lateral flow manufacturing processes.
Additional potential benefits of integrating flow tools into a device are simplified
operation and/or improved device robustness. The choice of which flow tool to use
depends on the requirements of the application of interest. Compatibility with core
device fabrication processes, requirements for assay time, requirements for repro-
ducibility, or environmental constraints of the setting, can all guide the choice of
flow tool.
12.3 Paper Microfluidic Devices for Medical Applications
12.3.1 Categories of Medically-Relevant Devices
There have been numerous examples of paper-based devices developed for medical
applications. Paper microfluidics has the potential to decentralize sophisticated
laboratory testing and reach populations in low-resource settings that would not
otherwise be able to access testing. One category of medical applications for paper
microfluidic devices is tumor biomarker detection. Targets include alpha-fetoprotein
[102, 103], carcinoembryonic antigen [102], prostate specific antigen [102], and
cancer antigen (CA) 15-3 [102, 104], CA 125 [105], and CA 19-9 [102, 106]. Another
category of medical applications for paper microfluidic devices is the analysis of
metabolite level to assess health status. Targets in this category include glucose
[107, 108], lactate [22, 109, 110], uric acid [22, 108], cholesterol [109, 111, 112],
creatinine [113], and markers of liver function [114–116]. A third category of targets
for paper microfluidic devices is infectious disease detection. These include E. coli
[117], methicillin-resistant S. aureus [118], hepatitis C virus [119], Plasmodium
falciparum [120], and influenza virus [121–123]. And a fourth category of targets is
composed of analytes relevant for therapy monitoring. Targets in this category
include phenylalanine for phenylketonuria patients to better manage nutritional
therapy [124], theophiline for patients with asthma and other respiratory conditions
to optimize their therapeutic dosages [125, 126], and lithium for patients with
depression [127]. A critical review by Yamada et al. [128] includes a more compre-
hensive list of medically-relevant targets in paper microfluidics.
The majority of the work in the field of paper microfluidics thus far has focused on
either the development of general tools/formats in model systems or proof-of-concept
demonstrations of specific applications using mock samples and expert operators in
well-controlled laboratory environments. This work is critical to overall progress in
the field, but is also only the first step in the process of the translation of promising
technology into medically-relevant, field-use devices. There are relatively few exam-
ples of paper microfluidic devices that have advanced to the stage of testing clinical
specimens in the field. Three informative examples, a liver function monitor, a sickle
cell disease diagnostic test, and an influenza diagnostic test, are highlighted below.
12.3.2 Paper-Based Liver Function Monitor
Liver toxicity for patients undergoing HIV and TB therapy is a significant issue. The
incidence of drug-associated liver toxicity can reach ~13% for the HIV therapeutic
nevirapine [129], and can be as great as 33% for the commonly used TB therapeutics
isoniazid, rifampin, and pyrazinamide [130, 131]. Liver function monitoring is
routine in developed countries [116]. However, access to healthcare facilities for
this testing is not available to many in low-resources settings. Thus, the ability to
monitor liver function on populations in need within low-resource settings is an
excellent potential application of paper microfluidics.
A paper-based liver function monitor was introduced in 2011 [114]. The rapid test
accepted a finger-stick sample of whole blood and produced colorimetric output for
three targets related to liver function, (i) aspartate aminotransferase (AST),
(ii) alkaline phosphatase (ALP), and (iii) total serum protein. The vertical flow
design consisted of stacked patterned cellulose and a plasma separation membrane,
encased between polyester support layers. The multiple zones for detection were
patterned using wax printing of the cellulose substrate and reagents were dried into
the zones. The plasma separation membrane (Pall Vivid GX) was composed of a
graded pore structure that effectively separated the red blood cells from input whole
blood. Device operation consisted of two user steps. First, a finger-stick sample of
whole blood was obtained using a lancet and applied to the inlet of the device. Whole
blood was wicked into the sample port, and the plasma that exited the opposite face
of the plasma separation membrane was split between the multiple detection zones.
Second, at 15 min, readout of the device was performed on the device face opposite
to the sample port, either by eye or using a reader.
A subsequent stage in the development of the liver function monitor narrowed the
targets to AST and alanine aminotransferase (ALT) and addressed four critical issues
in device translation [116]. First, three controls were added to the device to detect
improper functioning of various test components. Second, the effect of ambient
temperature on assay results was assessed and indicated that higher temperatures
resulted in accelerated assay signal development. The authors demonstrated that
appropriate choice of read time, based on the ambient temperature, could be used to
correct for temperature effects. Third, the effect of sample volume was assessed and
indicated that a minimum sample volume of 25 μL was required to achieve robust
function of the device, but that reasonable variation in sample volume above that
minimum had a negligible effect on the assay signal. Fourth, assay interference by
common substances in a blood matrix was assessed and indicated that two sub-
stances, pyruvic acid and ascorbic acid had a significant effect on the ALT signal
(greater than 10% change). And finally, the ability of human readers (N ¼ 3) to
resolve different levels of analyte using a color intensity guide was assessed using
340 E. Fu
Fig. 12.5 An excellent example of the process of paper-based device technology translation is
provided by the development of a liver function monitor for patients with HIV or TB. (a) The
simple design consisted of layers of wax-patterned cellulose and a plasma separation membrane
laminated together. Finger-stick blood was applied to one side of the device and readout of the
device consisted of noting the intensity of color that appears in detection zones on the opposite side
of the device. (b) The multiple operator steps are shown in the image series. Note that the device
required a large blood drop (i.e., larger than for standard glucose meters). A capillary tube was used
for the robust application of blood sample to the device. (c) A “Read Guide” was used by device
operators to determine the level of ALT in the sample based on the color intensity in the device
detection zone. (Reproduced with permission from Ref. [132])
clinical samples and estimated to be 90% accurate relative to gold-standard mea-

surements on paired patient samples.
Subsequent development work on the liver function monitor further addressed
issues critical to device translation, and assessed device performance in the field
[132]. The device, shown in Fig. 12.5a, was modified to include only ALT detection,
the assay chemistry was optimized for clinically-relevant color intensity ranges, and
two controls zones were incorporated into the device to indicate inadequate sample
volume, hemolysis, or nonfunctioning reagents. Operator steps, outlined in

Fig. 12.5b, were further refined, and included (i) the use of a capillary tube for the
transfer of the finger-stick blood sample to the device, and (ii) a more comprehensive
set of assay read times between 10 and 18 min. The read guide for this version of
their test is shown in Fig. 12.5c. The field evaluation was performed at a clinic in
Vietnam with local nurses as the device operators on HIV patient samples (N ¼ 600).
ALT levels in patient samples were also assessed using a lab-based automated
analyzer for comparison. The study results were promising, indicating that operator
accuracy on ALT level assessment was ~84% and agreement between the two
operators was ~96%. Disadvantages of the evaluated version of the device include
a substantially higher limit of detection compared to a gold-standard ALT detection
method, and the high-level of operator training (described as “intensive”) required to
perform and to interpret the test. Overall, the documented development of this device
is an instructive example of multiple issues that must be addressed in creating a
robust, field-use device.
12.3.3 Paper-Based Sickle Cell Diagnosis
Sickle cell disease (SCD) is a genetic disorder caused by a mutant form of hemo-
globin. Under conditions of deoxygenation, the “sickle” hemoglobin forms long
polymer chains that produce structural and functional changes in the red blood cells
of an individual with sickle cell anemia (SCA), including cell rupture or the blockage
of blood vessels [133]. SCA is most prevalent in Africa where children under 5 years
old with SCA are at substantial risk of death [134, 135]. Early detection and
treatment can substantially improve health outcomes and quality of life for individ-
uals with SCA [133], but lab-based testing is not available to many populations in
developing countries. Thus, there has been considerable interest in a test to diagnose
SCD in low-resource settings [136].
A paper microfluidic device for the detection of SCD in children and adults was
developed [137, 138]. The user steps, highlighted schematically in Fig. 12.6a,
consisted of (i) mixing 20 μL of finger-stick blood and a buffer (composed of a
reducing species and a hemolytic) in a small tube at a 1:10 volume ratio, (ii) waiting
10 min and pipetting a drop onto the paper test, and (iii) waiting 25 min and
interpreting the result. The differences in the flow of the soluble non-sickle Hb
species and the polymerized sickle Hb species resulted in distinguishable blood
patterns between HbAA (wild-type allelles), HbSA (heterozygous for the sickle cell
trait), and HbSS (homozygous for the sickle cell trait) individuals, as shown in
Fig. 12.6b. The field evaluation was performed at a hospital in Angola on mothers of
newborns (N ¼ 226) and compared with gold-standard measurements (isoelectric
focusing) on paired samples. Results indicated a sensitivity and specificity for HbS
detection of 94% and 97%, respectively, and positive predictive and negative
predictive values of 92% and 98%, respectively. Alternative readout using auto-
mated analysis of blood pattern image data indicated 100% sensitivity and specificity
342 E. Fu
Fig. 12.6 Another informative example of paper-based device technology translation is the
development of a diagnostic test for sickle cell anemia for use in low-resource settings. (a) User
steps included pipetting a finger-stick blood sample into a tube containing a buffer and mixing,
pipetting a drop of the mixture onto the device, and then interpreting the resulting blood pattern at
35 min after the start of the test. (b) Red blood cells that contain sickle hemoglobin have slowed
transport compared to red blood cells containing normal hemoglobin, and results in distinct blood
patterns for individuals with two normal alleles, one mutant and one normal allele, or two mutant
alleles. (Reproduced with permission from Ref. [138])
and would be compatible with use in some higher-resource settings. Strengths of the
paper-based test are it’s high level of performance even in variable ambient condi-
tions (note that quantification of the effects of temperature and humidity were not
attempted in this study), and its relative ease of use that is compatible with minimal
user training. Further development produced a version of the device sensitive
enough for screening newborns for SCD at the expense of additional user steps to
remove cellular debris from the sample [139, 140]. A limitation of the visual readout
version of the device is that it did not provide information on other forms of SCD,
but progress has been made on a version that makes use of automated, instrumented
readout of blood pattern image data [138].
12.3.4 Paper-Based Influenza Detection
The respiratory infection influenza causes approximately 36,000 deaths and 200,000
hospitalizations each year in the U.S., and results in over $10 billion in direct
Fig. 12.7 A third informative example is the development of an influenza diagnostic. The panel
summaries the device operating protocol. A strength of the device design is that operator steps have
been streamlined to eliminate pipetting steps that can lead to variations in input sample volume or
consistency, and downstream variability in signal. (Reproduced with permission from Ref. [123])
medical costs [141]. FDA-approved LFTs have been reported to have poor sensi-
tivities of less than 53% [142, 143]. Thus, there is a clear need for a higher sensitivity
POC test than is currently available. Potential target settings include hospital emer-
gency rooms, clinics, military units, and the home. The potential positive impact of
accurate influenza diagnosis include the use of anti-viral therapy [144], isolation of
infected persons to avoid outbreaks [145], and the reduction of antibiotic use due to
misdiagnosis [144]. Thus, there has been interest in developing improved POC tests
for influenza detection [123, 146–149].
One of these tests advanced to the stage of preliminary clinical testing in a Seattle
children’s hospital [123]. The test targeted influenza virus nucleoprotein A and B via
parallel sandwich immunoassays, followed by enzyme-based amplification. Auto-
mation of the multiple delivery steps was accomplished using a 2D paper network
with varying path lengths (based on the work of Fig. 12.4b) for the sequential
delivery of sample and label, rinse, and amplification reagents. A schematic of the
2D paper network is shown in Fig. 12.7a, and the operator steps performed by
healthcare professionals, are described in Fig. 12.7b. The prototype paper-based
device completed analyte detection from a nasal swab in approximately 35 min.
Device accuracy from the preliminary clinical study (N ¼ 25) was ~70% for the
detection of influenza A, and the device negative predictive value was 81%. Planned,
targeted improvements to device sensitivity include optimizing the device flow rate
and geometry, and automating a robust protocol for readout with a smartphone. The
main strength of this demonstration was the high level of device integration,
344 E. Fu
minimizing user steps and the potential for error. Current rapid tests that are
compatible with nasal swab samples require the user to insert the swab into a tube
of lysis buffer and then pipet a fraction of the lysis buffer-plus sample mixture into
the device. User steps for the paper-based influenza test were significantly simplified
-- the user inserted the swab into a port in the device and a well-defined volume of
sample fluid was automatically input into the device. Further, optimization of the
device ease of use was based on feedback generated from a user study focused on
making operator steps easy to understand and perform.
12.4 Challenges of Translation to the Field
Paper microfluidics holds particular promise for enabling medical testing that is not
currently available in low-resource settings. There have been a number of promising
proof-of concept demonstrations of potential medical applications of paper
microfluidic devices, but few field demonstrations. The above discussion of three
paper-based devices that have advanced to field evaluation highlights multiple issues
that must be addressed in order to create a robust field-ready device. These include
(i) optimizing user steps to make the device robust to variability in operators,
(ii) incorporating the appropriate positive and negative device controls and calibra-
tors, (iii) ensuring that the device performance is robust across the range of envi-
ronmental conditions that span the expected variation in field conditions,
(iv) ensuring the device is robust against interference from common substances in
the human matrix of interest, (v) ensuring that the device is appropriate for use by
operators who have the expected level of training for the end use scenario, and
(vi) ensuring that devices have robust performance across multiple manufacturing
lots, and after realistic delivery and storage conditions and times.
Moving forward, the overall challenge in paper microfluidics device development
for medical applications continues to be the design and implementation of devices
appropriate for the intended operator and setting. The examples above highlight the
tension between simplifying a device design and placing more burden on the
operator, versus automating a device for ease of use, but at the expense of increasing
device complexity, adding fabrication processes, and increasing cost. Following best
practices, the requirements and constraints of the application should guide device
design choices. The ideal device design for use by nurses at a high-resource clinic in
a developed country may differ substantially from the ideal device design for use by
healthcare workers at an outdoor clinic in a developing country. In particular, the
device characteristics of time to result, cost, ease of use, robustness to variations in
environmental conditions, and analytical performance, must match the requirements
of the application and the constraints of the use scenario. By addressing these
challenges of device translation to the field, the paper microfluidics community
can realize the potential of paper-based devices for many precision health
applications.
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Chapter 13
Paper-Based Microfluidics for Point-of-Care
Medical Diagnostics
Kentaro Yamada and Daniel Citterio
Abstract In the last decade, the chemistry research community has witnessed an
explosive growth of microfluidic devices made of paper (paper-based microfluidics).
Use of paper as a substrate material brings several attractive features including
extremely low cost and auxiliary pump-free liquid transportation, among others,
and application of paper-based microfluidics to on-site medical diagnosis has been
actively pursued. To meet the demand for medical diagnostic devices operable by
end-users without expert knowledge in resource-limited settings, recent studies on
paper-based microfluidics pay particular attention to simplification of user opera-
tions prior to an assay (e.g. achieving multistep enzymatic assays by single pipetting)
and resulting signal readout (e.g. achieving naked eye-based analog thermometer-
style result interpretation). One of the objectives of this chapter is to overview state-
of-the-art research progresses in simplification of user operational procedures and
development of equipment-free signal readout approaches. In addition, the basics of
paper-based microfluidics including a short history of paper-based microfluidics, a
comparison of paper-based and conventional plastic- or glass-based microfluidic
devices and general requirements for ideal point-of-care testing devices are
described. The authors believe this chapter helps researchers new to the field and
researchers with different background to learn about analytical applications exclu-
sively achieved by paper-based microfluidics and future challenges in developing
“truly” practical medical diagnostic devices.
Keywords Paper-based · Microfluidic devices · Point-of-care · Medical diagnosis
K. Yamada · D. Citterio (*)

Department of Applied Chemistry, Keio University, Yokohama, Japan

354 K. Yamada and D. Citterio
13.1 Introduction
Being driven by advancements in micro-processing technology in the semiconductor

industry, research and development of miniaturized analytical systems made from
polymeric substrates has grown rapidly in the late twentieth century. Since the
concept of micro-total analysis systems (μTAS, or “lab-on-a-chip” if synonymized)
was introduced by Manz in 1990 [1], it has gained attention as a promising analytical
platform offering a number of advantageous characteristics such as low consumption
of samples and reagents, short diffusion distances in the μm to nm scale, integrated
pre-conditioning steps for (bio)chemical assays (e.g. separation, mixing) as well as
multiplexed detection with high sensitivity and spatial resolution [2–5]. Above all,
the capability of sensitive and high-throughput detection from a tiny volume of
sample (nL to aL [2]) gave birth to a myriad of applications of μTAS predominated
by the life science field such as disease diagnostics, drug discovery, cell biology,
gene engineering and proteomics [6]. The primary conformation of traditional
microfluidic devices is a palm-sized polymeric substrate (most typically plastics
represented by polydimethylsiloxane [2, 7, 8]) having a hollow channel inside.
On the other hand, the 2010s (and the final years of the 2000s) witnessed an
explosively growing interest of researchers worldwide in microfluidic devices made
of a paper material (Fig. 13.1), of which the trend was ignited by the work published
in 2007 from the Whitesides group at Harvard University [9]. In this report, a
branched microfluidic channel was created on a sheet of chromatography paper by
photolithography. By pre-depositing the assay reagents for the detection of glucose
and protein (albumin) in separate areas of the channel, multiplexed colorimetric
Fig. 13.1 The number of publications related to paper-based microfluidics. (Data source: Web of
Science™; search criteria: “Title: paper-base*” OR “Title: patterned paper” OR “Title: paper
device*” AND “microfluidic*”; search date: February 21, 2018). Note that these topical searching
criteria do not necessarily cover “100%” of the publications on paper-based microfluidics, and
therefore, the actual number of publications is certainly larger than shown in the graph. This data is
intended to showcase the increasing number of publications related to paper-based microfluidics
13 Paper-Based Microfluidics for Point-of-Care Medical Diagnostics 355
assays of those analytical targets as well as a control test were demonstrated by a

single application of a low-volume sample (5 μL). Microfluidic devices made of
paper share some common useful capabilities with traditional microfluidics (i.e. low
consumption of reagents and samples, multiplexed detection from a single sample
application and, although not being demonstrated in the first report by Whitesides
and co-workers [9], automated sample processing prior to an assay). Despite these
similar benefits brought by patterning of microfluidic structures, paper-based
microfluidics may not be simply described as a “paper variant” of conventional
microfluidic devices. For example, the opacity of paper due to light scattering
obviously makes paper-based microfluidics less compatible with microscopic obser-
vation and highly sensitive optical detection techniques relying on light transmission
or luminescence emission. On the other hand, the whiteness of paper enables
unaided eye-based identification of color developed on this substrate with a good
contrast, making colorimetry an important detection motif for paper-based
microfluidics [10, 11]. Therefore, the medical and biological application fields
targeted by paper-based microfluidics are “complementary” to those of the tradi-
tional microfluidics, namely point-of-care (POC) diagnostics performable under
situations lacking infrastructure and/or a skilled operator, rather than being a tool
for basic life science research in central laboratories.
Besides potential application fields, the difference of device substrate materials
allows discussing dissimilar characteristics of paper-based and conventional
microfluidics from various standpoints. This chapter aims at describing the very
basics of paper-based microfluidics (physical and chemical characteristics of the
substrate material, liquid transportation principle in a microfluidic channel) as well
as providing examples of state-of-the-art applications of paper-based microfluidics
with particular attention on POC diagnostics by untrained users in resource-limited
settings. A comparison between paper-based and traditional microfluidics has been
integrated in the discussion as much as possible. Obviously, manufacturing methods
of paper-based microfluidics dominated by printing techniques vary from those of
the plastic-based counterparts (lithography). However, fabrication techniques of
paper-based microfluidics have been comprehensively overviewed in a number of
review articles [12–15], and thus, this topic is not repeatedly discussed in this
chapter.
13.2 Paper as an Analytical Platform Substrate

13.2.1 Basics of Paper and Comparison with Conventional
Substrates
The main component of paper is cellulose, a biopolymer consisting of a linear chain

of several hundred to over ten thousand D-glucose units [16]. The fact that cellulose
forms the plants’ cell walls makes this material the most abundant organic polymer
Table 13.1 Property comparison of paper with conventional substrate materials for microfluidics
(glass, silicon, PDMS)
Substrate material
Property Glass Silicon PDMS Paper Remarks on paper
Flexibility No No Yes Yes Less likely to be damaged dur-
ing transportation and operation
Surface-to-vol- Low Low Low High Provides high capacity for
ume ratio reagent storage
Fluid transport Active Active Active Passive Offers capillarity-based power-
free sample transport; but hard to
control flow rate
Sensitivity to No No No Yes Often requires desiccant during
moisture storage
Biocompatibility Yes Yes Yes Yes Amenable to fragile biological
substances
Disposability No No No Yes Safely disposable by
incineration
High-throughput Yes Yes No Yes Lowers manufacturing cost per
fabrication device
Optical Yes No Yes No Not compatible with micro-
transparency scopic analyses
Material Yes Yes Yes No Suffers from low assay precision
homogeneity
Price Moderate High Moderate Low Affordable to a wide range of
users
Adapted from Ref. [21] with permission from Springer
on the earth [17–19]. The cellulose fibers composing paper are primarily originated
from wood pulp in plants. Typically, paper is manufactured by passing a suspension
of cellulose fibers through a mesh to remove water, followed by pressing for further
water drainage and a subsequent drying process [20]. This manufacturing process
results in a paper sheet having porous structures formed by randomly woven
cellulosic fibers. Thanks to the abundance of natural sources and a roll-to-roll
manufacturing technique, paper has become a ubiquitous substrate material of
extremely low-cost. Table 13.1 compares the properties of paper to those of common
polymer materials applied for conventional microfluidics [21].
13.2.2 Paper-Based Diagnostic Devices Before Emergence

of Microfluidics
Among the benefits of paper shown in Table 13.1, the high surface-to-volume ratio
allowing the storage of chemical assay reagents within the porous structure has been
recognized since early times, and gave birth to “dip-and-read” simple paper strips
represented by the litmus paper introduced in the seventeenth century [22] and urine
dipsticks first emerged in 1850 [23]. Other attractive characteristics including
low-cost, high manufacturing throughput, safe disposability by incineration and
chemical inertness of pure cellulose (in the case of chromatography paper and filter
paper) led to the success of paper as a simple diagnostic platform as seen with several
commercialized relevant products. Colorimetric detection-based urine dipsticks have
remained successful paper-based diagnostic devices thanks to their ability to inspect
multiple urine constituents through simple “dip-and-read” user operation in a short
time (~ 120 sec). Albeit information provided by the naked eye-based color inspec-
tion being semi-quantitative (i.e. “approximate” concentration of target analytes),
simplicity and rapidity offer high-throughput screening of kidney, urogenital tract,
metabolic and liver diseases, as well as hemolytic disorders. Another representative
diagnostic device made of “paper” (its nitro derivative, to be exact) substrates is the
lateral flow immunoassays (LFIAs). Although not being as long-established as urine
dipsticks, it has been already three decades since the concept of LFIAs appeared in
US Patents [24, 25]. Thanks to the use of antibodies conjugated with labeling agents
(e.g. gold nanoparticles, dye-loaded particles), LFIAs allow visual detection of a
given target antigen with high specificity. Commercially-available paper-based
analytical devices relying on this technique are represented by the home pregnancy
tests for detecting human chorionic gonadotropin (hCG), and the influenza testing
kits for detecting the nucleoprotein of the influenza virus.
Since the emergence of LFIAs in the 1980s, there seems to be no landmark in the
development of paper-based analytical devices. However, a growing number of
colorimetry-based paper-made chemical assays have come into the market (e.g. the
Merckoquant test strips [26]), reflecting a high demand for simple, portable, rapid,
and disposable testing devices for various analytical targets. However, the passive
liquid transport offered by the porous structure of paper substrates has been
overlooked until very recent in the analytical chemistry research community, with
the exception of a single report on chromatographic separation of dyes on a piece of
patterned filter paper demonstrated by Müller and Clegg in 1949 [27]. In this
application, the convenience of patterning of a confined separation paper channel
lead to acceleration of the diffusion process and reduction of the reagent consump-
tion. In spite of the extra-values of a patterned paper substrate, there was no response
of the scientific community to this research field at that time. After more than half of
a century, the American chemist George Whitesides rediscovered microfluidically
patterned paper as a valuable platform to construct simple yet functional (bio)
chemical sensing devices in 2007 [9]. Despite the explosively increasing number
of publications, there is no unified nomenclature applied to this new class of
microfluidic devices. Currently, paper-based microfluidics are also referred to as
“microfluidic paper-based analytical devices (μPADs)”, “lab-on-paper”,
“paperfluidics”, and so on. In this chapter, “paper-based microfluidics” will be
consistently used as a term to describe microfluidic devices made of “paper”,
including not only pure cellulosic filter paper and chromatography paper, but also
nitrocellulose.
13.2.3 From Plastic to Paper-Based Microfluidics:

Similarities and Dissimilarities
The concept of μTAS has been envisioned as a revolutionary (bio)chemical analyt-

ical platform for its efficiency, rapidity, economical use of samples and reagents and
well-established manufacturing technologies. Despite a few examples of commer-
cialized microfluidic devices [4, 28], the current state is still far from the situation
where everyone monitors his/her health condition using microfluidics. The reason is
primarily attributed to (1) the complexity of liquid handling systems requiring
difficult start-up processes and operational expertise, and (2) the necessity of sophis-
ticated detectors for signal acquisition. The most traditional research approaches in
this field are devoted to fluidic control relying on external equipment, and hence,
microfluidics have not evolved as the versatile “lab-on-a-chip” system, but partially
remained a sophisticated “chip-in-a-lab” system [29].
Paper-based microfluidics are fabricated by patterning a “boundary” defining a
fluidic channel region onto a paper substrate. This “boundary” is typically defined
either by (1) a hydrophobic substance (e.g. solid wax [30, 31]) coating the surface of
the cellulosic fibers throughout the paper thickness; (2) a solid substance blocking
the porous structure of paper (e.g. photoresist [9], polystyrene [32]); or (3) the paper-
air interface at the periphery of a cut paper sheet [33]. Filter paper or chromatography
paper are by far the most commonly used as the substrate material because of their
pure cellulose composition with a high degree of chemical inertness. More impor-
tantly, the availability of capillary action derived from μm-sized inter-fiber pores
offers power-free fluid transport, addressing one of the significant bottlenecks of
traditional microfluidics. Last not but least, paper is light-weight (~10 mg cm2) and
affordable (~0.05 cents cm2), providing ease-of-transportation and enabling the use
of paper-based microfluidics even in remote areas and resource-limited settings.
Standard office paper, although being inexpensive and very accessible, is not widely
utilized in the field, due to its poor wetting property originating from (1) small pore
sizes (several hundred nm [34]) and (2) hydrophobic surface treatment by sizing
agents to prevent ink bleeding. In addition, additives (e.g. fillers, optical brighteners,
etc.) with unknown composition may impact the chemical reaction for detection,
further discouraging developers to use this material as a substrate for chemical
analyses.
As is clear from the structure of paper-based microfluidics, a bundle of numerous
capillaries formed by interwoven cellulosic fibers serves as the fluidic channels on
these devices, whereas the conventional microfluidics possess a hollow flow path in
a substrate material such as plastic, glass and silicone. Despite their different
configurations, the capability of sequential chemical reactions and multiplexed
assays with small consumption of samples and reagents are common advantages.
In addition, the liquid transportation in paper-based microfluidics also exhibits
laminar flow [35, 36] due to the μm-sized pore radius in the paper medium and the
liquid flow speeds typically at several mm per minute, making the Reynolds number
(Re) less than 1, as expressed by the following Eq. (13.1):
Fig. 13.2 Application examples of laminar flow on paper-based microfluidics; (a) separation of a
dye (tartrazine) from a protein (bovine serum albumin: BSA). (Reproduced from Ref. [35] with
permission from The Royal Society of Chemistry); (b) plasma separation from whole blood,
(Adapted from Ref. [37] with permission from The Royal Society of Chemistry)
ρVL
Re ¼ < 1, ð13:1Þ
μ
where ρ is the fluid density (kg m3), V the fluid velocity (m s1), L the pore
diameter (m) and μ the fluid dynamic viscosity (kg m1 s1 ¼ N s m2). Various
reports have demonstrated that the laminar flow phenomenon is applicable to
on-paper separation purposes including dye purification from a protein solution
(Fig. 13.2a) [35] and plasma separation from whole blood (Fig. 13.2b) [37].
A clear difference lies in the fact that the fluidic channels are “fully closed” in the
conventional microfluidics, whereas the paper-based counterpart possesses “open-
air” flow paths, unless covered by lamination films or adhesive tapes. Since detection
in conventional microfluidics is carried out in a sealed channel, the generation of the
detection signal is accounted for by an equilibrium between the sample phase and a
second interface hosting a receptor for the given analyte (e.g. adjacent laminar flow,
channel surface, interface of a droplet dispersed in the sample). Therefore, the signal
reflects the “concentration” of the analytical target in the sample fluid introduced to
the device. In the case of paper-based microfluidics, however, open-air flow paths
are prone to sample evaporation. Consequently, the detection mechanism is reliant
on depletion of analytes at the detection area, rather than an equilibrium state
between an aqueous sample phase and a receptor immobilized onto the paper
surface. In contrast to traditional microfluidics, the detection signal obtained from
paper-based microfluidics reflects information on the “absolute analyte amount”
transported to the detection region [38], with the presumption that sufficient
ligand/receptor is available. In applications where an equilibrium state between
two immiscible phases is essential (e.g. on-paper detection of ionic species by the
ion-selective optode system [39, 40]), an additional lamination process of the paper
substrate is necessary to prevent evaporative loss of the sample liquid during the
assay [41–43].
13.3 Applications of Paper-Based Microfluidics to POC

Diagnostics
13.3.1 Potential Target Application in the Medical

and Biological Fields
Although some divided opinions may persist, the ultimate benefits of paper over the
conventional substrate materials are its extremely low cost and ability of pump-free
liquid transportation by capillary forces, when it comes to the use as a microfluidic
device substrate. “Affordability” and the fact of “no requirement of external equip-
ment” are of significant importance for an analytical device intended for carrying out
assays in situations lacking sufficient infrastructure and economic resources, namely
medical diagnostics in remote rural areas of developing countries. Even in developed
countries, highly distributed medical screening could also be an important scenario
where the low-cost and equipment-free characteristics of paper-based microfluidics
become particularly attractive. On the other hand, more high-tech biomedical appli-
cations (e.g. detailed examinations requiring accurate and precise quantification of
clinical targets, advanced studies in a sophisticated laboratory) less likely give paper-
based microfluidics full play, because (1) the advantages of cost reduction and
equipment-free operation will not pay for the reluctance to replace traditional
systems, and (2) the sensitivity and spatial resolution of detection on paper-based
microfluidics are generally poor compared to the advanced analytical technologies
already used in fully equipped settings. Therefore, effective medical and biological
application of paper-based microfluidics should be point-of-care (POC) testing for
medical diagnostics in resource-limited settings and highly distributed medical
screening at private homes or small clinics.
13.3.2 General Challenges in Developing Paper-Based

Microfluidics
There are several landmarks in developing effective POC test devices. According to
the guidelines provided by the World Health Organization (WHO), practicable POC
devices should meet the following criteria summarized below under the
“ASSURED” acronym [44]: Affordable; Sensitive (no false negative results); Spe-
cific (no false positive results); User-friendly (easy to operate, no use of invasive
chemicals); Rapid and Robust; Equipment-free; Delivered (to the end users). On the
other hand, the U. S. Food & Drug Administration (FDA) formulates a guideline for
in vitro diagnostic (IVD) tests for home or over-the-counter use known as CLIA
(Clinical Laboratory Improvement Amendments) [45], where particular importance
is laid on simplicity of user operation both in “sample-in” (introduction of sample to
the device) and “answer-out” (interpretation of assay result) steps. Considering these
criteria, practical paper-based microfluidics should balance basic analytical perfor-
mance (accuracy, precision, limit of detection) and acceptance by targeted users
(simplicity of sample handling, ease of result interpretation, long-term storage
stability).
Despite various attractive characteristics of paper-based microfluidics and a huge
number of relevant publications exceeding one thousand, they have hardly made it
into the marketplace as of 2018. A large fraction of prototypes remains a proof-of-
concept demonstration in an academic research paper and has yet to prove its
usefulness in practical applications. The missing key to the final success of paper-
based microfluidics as practical diagnostic devices has been described in recent
review articles as: “...UED (i.e. User-friendly, Equipment-free, Delivered) are
important requirements that deserve more research to increase the commercializa-
tion of paper-based analytical devices.” [46] and “... major challenges remaining in
proof-of-concept μPADs for routine health checks can be summarized as (1) neces-
sity of complicated user operations (assUred), (2) insufficient examination on long-
term stability (assuRed), and (3) reliance on detection equipment unfamiliar for
general users (assurEd).” [47] To sum up, in academic research efforts the user
acceptance aspect (user-friendliness and equipment-free criteria, among others)
tends to be overlooked or sacrificed to the enhancement of the analytical perfor-
mance of a device. Fortunately however, the research community dealing with
paper-based microfluidics is certainly becoming aware of this issue that needs to
be addressed. The following sections describe the current state of the development of
paper-based microfluidics with simplified user operations targeting medical and
biological applications.
13.3.3 Simplification of Multi-Step Biochemical Assays
Immunoassays and nucleic acid testing are representative bioanalytical techniques

with high sensitivity and specificity. They have a great potential to prevent epi-
demics of infectious diseases by offering rapid and accurate diagnosis. Infectious
diseases (e.g. malaria, Ebola, Zika, tuberculosis, dengue) account for a significant
mortality rate in developing countries, and thus, accurate and early diagnostic
techniques are of substantial importance, because they ultimately allow adequate
treatment and prevention of further spread of the diseases. With the high demand to
implement immunoassays and nucleic acid testing into infectious disease marker
detection in remote settings, efforts have been made to reduce the operational
complexity of these analytical techniques.
13.3.3.1 Lateral Flow Immunoassays (LFIAs)
Immunoassays offer highly selective detection of an analyte of interest utilizing the

very specific antigen-antibody reaction. LFIAs, prominently represented by home
pregnancy tests and influenza test kits, are well-known diagnostic devices relying on
this bioanalytical detection technique. They provide a “yes-no” answer of the
presence of target antigens (human chorionic gonadotropin (hCG) for pregnancy
testing, virus-specific protein for influenza testing) in the sample based on visuali-
zation of a line typically derived from the gold nanoparticle labels. However, the
early diagnosis of infectious diseases with the conventional LFIAs is generally
challenging owing to their insufficient sensitivity to detect a trace amount of the
target [48, 49].
Amplification of the optical signal from the gold nanoparticle labels is achievable
by adding a gold [50, 51] or silver [52–54] salt to enhance the visibility of the
colloidal gold. However, the necessity of multi-step addition of sample and reagents
increases the complexity of user operation. A pioneering work addressing this
bottleneck utilized an automated time-lagged delivery of the assay reagents on
paper-based microfluidics coined as a two-dimensional paper network (2-DPN).
Figures 13.3a, b show two types of 2-DPN. Those devices allow a user to carry
out a signal-amplified immunoassay of the PfHRP2 malaria protein simply by
(1) adding the assay components (sample, enhancement reagent for signal amplifi-
cation, washing buffer) onto a specified paper area and by (2) folding the plastic
card. Different path lengths (Fig. 13.3a) [55] or different flow resistance of the
channels stemmed from sucrose impregnation (Fig. 13.3b) [56] result in the sequen-
tial delivery of the assay components to the detection region (indicated by a red
arrow in the figures) in the desired order. In the PfHRP2 malaria protein detection
using each of these 2-DPN, a four-fold [55] and 2.6-fold [56] signal amplification
has been achieved over the case without optical signal enhancement.
The use of the enzyme-linked immunosorbent assay (ELISA) method is another
choice to enhance the sensitivity of immunoassays. In ELISAs, the secondary
antibodies are labeled with enzymes (e.g. alkaline phosphatase, horseradish perox-
idase) in place of gold nanoparticles, and subsequent addition of a chromogenic
substrate generates an optical signal amplified in the enzymatic catalytic reaction.
Here again, the requirement of multiple reagent addition with a strictly fixed order
remains as an issue in terms of user-friendliness. In the most ideal case, the target
antigen/enzyme-labeled antibody immunocomplexes and chromogenic substrates
are sequentially transported to the test zone only by a single introduction of a sample
liquid to the device. The use of different path lengths to the test zone in a similar
manner to the 2-DPNs also works in carrying out the ELISA. The “maze-like” paper-
based microfluidics schematically shown in Fig. 13.4a [57] allowed the detection of
hCG only by immersing the bottom of the device into the sample with a detection
limit of 8.1 mIU mL1, demonstrating a slight improvement over commercial LFIA
devices for pregnancy testing (20 mIU mL1). Here, the color development of the
BCIP/NBT chromogenic substrate was catalyzed by alkaline phosphatase (ALP),
resulting in an improvement of the sensitivity of the immunoassay in spite of the
Fig. 13.3 2-DPN lateral flow immunoassay devices for automated sequential reagent delivery
based on (a) different length of flow paths to the detection region. (Adapted with permission from
Ref. [55]. Copyright 2012 American Chemical Society); (b) different flow resistance of sucrose-
impregnated channels (concentrations of impregnated sucrose solution: 0, 30, 54, 65% from legs #1
to #4) to the detection region. (Reproduced from Ref. [56] with permission from The Royal Society
of Chemistry)
much longer flow path of the sample than in the case of conventional LFIAs,
potentially causing loss of analyte by adsorption onto the nitrocellulosic substrate.
Very recently, an automated ELISA has been demonstrated on a paper-based
microfluidics resembling the traditional LFIA devices [58]. The device shown in
Fig. 13.4 Schematic illustrations of lateral flow devices for automated ELISA: (a) a “maze-like”
configuration utilizing different path lengths to the reaction area. (Adapted from Ref. [57] with
permission from The Royal Society of Chemistry); (b) a “classical format” configuration utilizing
delayed transport of chromogenic enzyme substrates from a wax-modified sample pad; (c) detailed
structure of the wax-modified sample pad and the working principle of delayed delivery of the
enzyme substrates enabling the automated ELISA from single sample application. (Adapted from
Ref. [58])
Fig. 13.4b shares a common architecture with the classical LFIA format, only with
the exception that the sample pad is replaced by filter paper patterned by a thin wax
barrier and the BCIP/NBT substrate (Fig. 13.4c, (i). The wax-modified filter paper-
based sample pad allows to form a stationary sample droplet (Fig. 13.4c, (ii). While
the sample liquid flows in the device by the capillary force, the pre-deposited BCIP/
NBT is gradually rehydrated into the bulk sample liquid (Fig. 13.4c, (iii), and is
eventually transported in a delayed manner together with the remaining sample
liquid (Fig. 13.4c, (iv) toward the downstream test zone, where sandwich
immunocomplexes are already present. The delayed delivery of BCIP/NBT enabled
a “one-step” ELISA of mouse IgG as a model analyte. Surprisingly, the achieved
limit of detection of 15.8 ng mL1 was comparable to that of a commercial microtiter
plate-based ELISA kit (10 ng mL1), which relies on a labor-intensive operational
procedure (multistep reagent addition, incubation, washing) and absorbance mea-
surement with a sophisticated microplate reader. Although the application to the
detection of infectious disease markers is yet to be demonstrated, paper-based
microfluidics for an automated ELISA are promising POC diagnostic devices
providing easy-to-use yet high-performance infectious disease tests.
Fig. 13.5 A “paper machine” enabling nucleic acid detection by pipetting and sliding. (Adapted
with permission from Ref. [62]. Copyright 2015 American Chemical Society)
13.3.3.2 Nucleic Acid Tests
The detection of nucleic acids (DNAs, RNAs) extracted from microorganisms is

another powerful technique for the diagnosis of infectious diseases. To achieve high
sensitivity, amplification of target nucleic acids of interest is often required prior to
the detection by techniques such as the reverse transcription polymerase chain
reaction (RT-PCR) [59], loop-mediated isothermal amplification (LAMP) [60], or
rolling circle amplification (RCA) [61]. Although the detection of nucleic acids on
paper-based microfluidics has been described in a number of reports, the current
situation is that the whole detection process entails multiple steps of operation and
electricity-powered heating equipment. In the most integrated and user-friendly
configuration of paper-based microfluidics for nucleic acid amplification tests
(NAATs), a “paper machine” developed by Diagnostics For All is able to carry
out the whole blood sample processing, LAMP reaction and fluorescence-based
detection in four steps of reagent deposition and handle-sliding operations
(Fig. 13.5) [62]. Complexity of user operation has been minimized with the inte-
grated “paper machine” format, however, the requirement of an incubator remained
as an issue [62]. For the field use of nucleic acid detection, the development of
battery-powered heating equipment requiring minimum maintenance (or elimination
of its necessity) would be a key factor determining the practical feasibility in
resource-limited settings. Nucleic acid detection for infectious disease diagnosis on

paper-based microfluidics has been comprehensively described in a recent review
article [63], and therefore, further detailed discussions will not be repeated here.
13.3.4 Simplification of Detection Signal Readout
Besides the simplicity of user operations prior to detection, interpretation of resulting

detection signals is a crucial step in (bio)chemical assays for POC tests. In the case
where targeted end users are non-healthcare workers (e.g. routine health check by
monitoring clinically-relevant metabolites at private homes), intuitive signaling
approaches on paper-based microfluidics are of significant importance to eliminate
the risk of misinterpretation of assay results. Colorimetric urine dipsticks, a marketed
paper-based medical screening tool, rely on visual comparison of color intensities or
hues (i.e. type of color) between the sample paper and a read guide to semi-
quantitatively determine the concentration of a given analyte in urine. However,
readout results are inherently dependent on the color recognition capacity of the user
and on the environmental light conditions. Several alternative signaling motifs have
been proposed to address this issue encountered with the traditional colorimetric
detection method. Table 13.2 summarizes representative works on paper-based
microfluidics utilizing a simplified signal detection mode for medical and biological
applications.
13.3.4.1 Analog Thermometer-Style Readout
Analog thermometers let us know the ambient temperature based on the length of an
inner liquid filling a straight glass capillary. All a user has to do is to simply identify
the position of the top of the inner liquid relative to adjacent scale marks. The
“distance-based” quantification mode on paper-based microfluidics resembling ana-
log thermometers is typically achieved relying on one of two distinct principles:
(1) analyte depletion in a microfluidic channel (Scheme 13.1a); (2) flow resistance
alteration in a microfluidic channel caused by analyte molecules (Scheme 13.1b). As
shown in Table 13.2, the thermometer-style readout mode is frequently implemented
on paper-based microfluidics, possibly because various chemistries are available to
achieve this signaling method.
In the first approach, analyte molecules in the flowing sample liquid are contin-
uously consumed by binding to receptors immobilized within the paper channel,
eventually resulting in depletion of analyte and no further generation of analyte-
receptor conjugates along the flow channel. It should be mentioned that the paper
matrix itself (e.g. functional groups present on the surface of cellulose fibers) can
potentially act as a “receptor” binding an analyte. At a fixed amount of receptor sites
present in a microfluidic paper channel, full depletion of the analyte from the flowing
sample liquid is delayed with increasing original amount of analyte, resulting in the
Table 13.2 Representative works on paper-based microfluidics utilizing simplified signal detec-
tion targeting medical applications
Signal
readout
mode Analyte Assay reagent Detectable range References
Analog ther- Theophylline Antibody (recep- 0–40 mg L1 [64]
mometer tor); glucose, GOx, 0–36.6 mg L1 [65]
(analyte HRP, 4-chloro-1-
depletion naphthol
principle) (detection)
Theophylline Antibody (recep- 0–40 mg L1 [66]
tor); glucose, GOx,
HRP, dicarboxidine
dihydrochloride
(detection)
Cholesterol ChOx, HRP, 1500–4000 mg L1 [67]
MBTH, DMA
(detection)
High-density lipo- Cholesterol ester- 250–1000 mg L1 [68]
protein cholesterol ase, ChOx, HRP,
MBTH, DMA
(detection)
Glucose GOx, HRP, DAB 11–270 mg dL1 [69]
(detection)
Lactoferrin Terbium chloride 0.05–4 mg mL1 [70]
(detection); ι-carra-
geenan
(immobilizer)
Cocaine Aptamer/ 0–400 μM [71]
glucoamylase-
doped hydrogel,
amylose, GOx,
HRP, DAB
(detection)
Cocaine Aptamer-invertase- 0–500 μM [72]
Adenosine bead conjugate, 0–400 μM
sucrose, GOx,
HRP, DAB
(detection)
DNA HNB, MgCl2 4.14103–7.88106 [73]
(detection) copies μL1 a
PEI (immobilizer)
DNA SYBR Green I 1 aM-1 fM b [74]
(detection); PEG
100000
(immobilizer)
Carcinoembryonic HRP-labeled anti- 0–40 ng mL1 [75]
antigen body, H2O2, TMB
(detection)
(continued)
Table 13.2 (continued)

Signal
readout
mode Analyte Assay reagent Detectable range References
Analog ther- Cocaine Aptamer-doped 0–100 μM [76]
mometer hydrogel (valving);
(flow resis- food dye solution
tance alter- (detection)
ation Alkaline Starch (valving); 0.075–5 U mL1 [77]
principle) phosphatase food dye solution
(detection)
ABO, Rh blood Anti-A, B, D anti- – [78]
types bodies, A-, B-cells
(valving)
Hematocrit EDTA, NaCl 28–57% [79]
(stabilizer)
Time H2O2 Dedicated H2O2- 0–50 mM [80]
measurement degradable hydro-
phobic compound
(valving); food dye
(detection)
Alkaline Dedicated H2O2- 0.128–7.4 pM; [81]
phosphatase degradable hydro- 13–740 pM; 45–1480
phobic compound pM; 93–1480 pM;
(valving); food dye 342–37000 pM;
(detection) 56000–74000 pM c
K+ Hemin, DNA 25–200 μM [82]
probe, H2O2, TMB
(valving), ink solu-
tion (detection)
Counting of H2O2 Dedicated H2O2- 0–100 mM [80]
color spots degradable hydro-
phobic compound
(valving); food dye
(detection)
Adenosine Aptamer-GOx con- 1.5 μM–19.3 mM [83]
jugate, glucose,
KMnO4 (detection)
Text readout ABO, Rh blood Anti-A, B, D anti- ― [84]
types bodies, water-
insoluble red ink
(detection)
Albumin Tetrabromophenol 0–10 mg mL1 [85]
blue (detection)
GOx glucose oxidase, HRP horseradish peroxidase, ChOx cholesterol oxidase, MBTH 3-methyl-2-
benzothiazolinone hydrazone, DMA N,N-dimethylaniline, DAB diaminobenzidine, HNB
hydroxynaphthol blue, PEI polyethylenimine, PEG polyethylene glycol, TMB 3,30 ,5,5-
0
-tetramethylbenzidine, EDTA ethylenediaminetetraacetic acid
a
Initial number of genomic DNA
b
Combined with polymerase chain reaction (PCR)
c
Values dependent on the structure of the valving reagent
Scheme 13.1 Schematic illustration describing two major types of the working principle of an
“analog thermometer-style” readout motif on paper-based microfluidics: (a) analyte depletion
mode; (b) flow resistance alteration mode
observation of a distance-based signal proportional to the initial analyte content. One

of the earliest examples has demonstrated the antibody-based detection of theoph-
ylline on a paper strip in the 1980s [64–66]. There, a sample containing theophylline
(target analyte) is mixed with a solution containing horseradish peroxidase (HRP)-
labeled theophylline (competing agent) as well as glucose oxidase, followed by
wicking through a chromatography paper strip coated with theophylline-specific
antibodies, consuming the free and HRP-labeled theophylline in a competitive
manner. The larger the amount of initial theophylline in the sample, the longer in
terms of distance of the wicking fluid the depletion of the HRP-labeled theophylline
by the immobilized antibodies takes. After color development by the addition of
glucose and a colorimetric substrate for HRP, the theophylline concentration in the
sample can be estimated from the length of the colored part of the strip. In that work,
the authors pointed out the advantage that the assay result is relatively insusceptible
to the stability of the enzymatic reagents, temperature and incubation time, because
the quantification signal is not reliant on the developed color intensity, but on the
colored distance [64]. In the 1990s, the analog thermometer-style detection mode has
been expanded to the quantification of cholesterol [67] and high-density lipoprotein
cholesterol [68] in whole blood. The color band providing the distance-based signal
originates from the oxidation reaction of a chromogenic peroxidase substrate
(3-methyl-2-benzothiazolinone hydrazone and N,N-dimethylaniline) by H2O2
released from the reaction of the target analyte with the selectivity providing
cholesterol oxidase enzyme. This reaction is catalyzed by HRP evenly immobilized
along a paper strip, resulting in a gradual H2O2 depletion from the flowing liquid. In
these cases, the analyte concentration in the sample is not detected by direct
depletion of the analyte itself during the flow process, but indirectly measured
through the depletion of the intermediary generated H2O2, which amount reflects
the original analyte concentration. This mechanism is applicable to a wide range of
analytes as long as a specific oxidase enzyme is available for the analyte of interest.
Indeed, the “first” analog thermometer-style detection on the type of microfluidically
patterned paper-based analytical devices introduced in 2007 demonstrated the quan-
tification of glucose by using glucose oxidase (GOx), HRP and diaminobenzidine
(DAB) as a colorimetric peroxidase substrate (Fig. 13.6a) [69]. The most straight-
forward approaches to achieve a distance-based signal make use of the direct
consumption of target analyte molecules by a colorimetric or fluorometric indicator
specific to the given analyte [69, 70, 86], or the migration distance modulation of a
simple dye solution that indirectly represents the analytical target amount in the
sample [73, 74, 76].
Fig. 13.6 Examples of analog thermometer-style detection on paper-based microfluidics:

(a) glucose detection relying on analyte depletion within the paper channel. (Reproduced from
Ref. [69] with permission from The Royal Society of Chemistry); (b) hematocrit determination
relying on channel clogging by the red blood cells in the whole blood sample. (Reproduced from
Ref. [79] with permission from The Royal Society of Chemistry); (c) alkaline phosphatase (ALP)
detection relying on wettability change of the midstream area (zone b) of the paper channel.
(Adapted from Ref. [77] with permission from The Royal Society of Chemistry)
An important condition common to all thermometer-style detection schemes

relying on the analyte depletion mechanism is that the “consumption” of the analyte
in the microfluidic paper channel must be continuous and rapid. Signaling com-
pounds (e.g. a colored complex of analyte and indicator) must be produced within
the paper channel in a continuous manner without being washed away by the sample
liquid flow. For example, insufficient (e.g. weak binding) or slow interaction
(e.g. slow reaction kinetics) of an analyte with the immobilized receptor
(e.g. indicator, cellulosic substrate) does not result in a distance-based detection
signal. In the case of pump-driven traditional microfluidics, reduction of the flow rate
of the sample fluid allows to extend the timescale for the analyte-receptor interac-
tions [87]. However, this strategy is not readily achievable on paper-based
microfluidics, where liquid transport relies on passive wicking by capillary forces.
If a water-soluble indicator specific to the given analyte is to be used for detection, it
is absolutely crucial to prevent indicator molecules from being washed away by the
sample liquid flow [70]. Commercialized colorimetric indicators are typically avail-
able in their sulfonated form for the sake of water-solubility, since they are designed
for use in aqueous systems. A well-known strategy for immobilizing charged
reagents onto a paper substrate is to deposit them together with a polymer or a
particle having the opposite charge to the reagents to be immobilized [88–91]. The
size or the multitopic interaction of polymer molecules or particles with the paper
matrix efficiently prevents them from being transported together with the sample
liquid, enabling the electrostatic immobilization of charged indicator reagents.
The second mechanism to achieve the thermometer-style detection mode is the
use of microfluidic channel “clogging” to alter the flow rate of a colored solution.
One early example relying on this system was demonstrated on a plastic-based
microfluidic platform [92]. In this proof-of-concept work, the surface of a PDMS
microfluidic channel was modified by biotin. When a sample liquid containing
streptavidin is introduced into the device, it travels through the channel by capillary
action, while the streptavidin-biotin interaction occurs at the channel inlet, eventu-
ally stopping the sample migration by acting as a “valve” clogging the microfluidic
channel close to its inlet. Since the time for channel clogging becomes shorter with
increasing concentration of streptavidin, a flow distance-based signal inversely
proportional to the target analyte amount is obtained. Interestingly, this “channel
clogging” mechanism successfully works on paper-based microfluidics, where the
channel structure is much more complex (i.e. a bundle of numerous capillaries
formed by randomly woven cellulosic fibers). Several literatures report blood anal-
ysis (blood typing [78], semi-quantification of the hematocrit [79]) based on a
physical blocking of cellulosic paper channels. In a distance-based blood typing
assay [78], blood coagulation was utilized as a “valve” to change the flow distance of
red blood cells used as the color signal to identify the ABO blood type. In the semi-
quantitative evaluation of the hematocrit [79], defined as the volume ratio of red
blood cells to the whole blood, the analyte itself (i.e. red blood cells) caused clogging
of the microfluidic paper channel, resulting in a blood migration length inversely
proportional to the hematocrit value (Fig. 13.6b). Besides bulky blood components
(e.g. red blood cells), compounds being able to undergo an analyte-dependent
switching from a poorly water-soluble hydrophobic into a hydrophilic state can also
serve as a valve to enable a thermometer-style detection approach. As an example,
the device shown in Fig. 13.6c [77] uses starch as the valving reagent deposited
together with the sample in the sample area (zone b). In the presence of alkaline
phosphatase (ALP; analytical target) in the sample, the decomposition of poorly
water-soluble starch into highly water-soluble glucose by an auxiliary enzymatic
reaction (glucoamylase) is hindered, and thus, a dye solution deposited onto the inlet
(zone a) after sample introduction migrates only a short distance in channel c. Vice
versa, in the absence of ALP, starch is readily broken down into water-soluble
glucose units, resulting in free flow of the dye solution into channel c. Similar
kinds of “wettability changes” of a paper channel are also employed to achieve
other simplified signal detection modes (timing, counting) as will be discussed in the
following sections.
13.3.4.2 Time Measurement
In this detection mode, the quantification signal is the “time” between appointed
events (e.g. sample application and color observation on a paper spot). Up to present,
the timing-based quantification signal has been solely achieved by utilizing “wetta-
bility changes” of a part of the paper channel triggered by the presence of the
analytical target. The first proof-of-concept work demonstrated the quantification
of H2O2 on a vertically-assembled paper-based microfluidic device (Fig. 13.7a) [80],
where a H2O2-degradable hydrophobic compound (“phase switching agent”) is
pre-deposited in the middle paper layer to regulate the sample flow-through speed
depending on the H2O2 concentration. The penetrated sample liquid carries the green
food dye to the bottom layer of the device, and green coloration of the bottom paper
area is recorded as the endpoint of the time measurement. Here again, since H2O2 is a
product of oxidase enzyme reactions, the mechanism can be straightforwardly
applied to clinically-relevant metabolites converted by the respective oxidase
enzyme (e.g. glucose, lactate, alcohol). Later on, the application of this mechanism
was expanded to ALP quantification [81], where the presence of ALP generated
glucose by cleaving the phosphate-substituent off a phosphorylated glucose deriv-
ative, eventually producing H2O2 through an auxiliary enzymatic reaction
(glucose oxidase) to decompose the phase switching agent. This work has also
demonstrated that the sensitivity of the time measurement-based detection can be
tailored by the microfluidic channel length, the chemical structure of the phase
switching agent influencing the decomposition kinetics, and the number of paper
layers containing the phase switching agent. However, a bottleneck of vertically-
assembled paper-based microfluidics is their relatively complex fabrication proce-
dure (stacking of multiple layers of paper substrates and double-sided tape) requiring
precise alignment and firm attachment between paper layers. The timing-based
readout motif is also performable on paper-based microfluidics with a lateral flow
configuration (Fig. 13.7b) [82]. There, the target potassium ions (K+) in the
sample result in the formation of a DNAzyme with a peroxidase-like catalytic
Fig. 13.7 Examples of time measurement-based detection on paper-based microfluidics: (a)

schematic illustration of a three-dimensional device design and assay procedure for H2O2 quanti-
fication. (Reproduced with permission from Ref. [80]. Copyright© 2012 WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim); (b) schematic illustration of a lateral flow-type device design
and working principle of potassium ion (K+) quantification. (Reproduced from Ref. [82], Copyright
2015, with permission from Elsevier)
activity, which is deposited into zone b in the first step. Subsequently, pre-deposited
3,30 ,5,50 -tetramethylbenzidine (TMB) is oxidized into water-insoluble poly(TMB)
only in the presence of K+, resulting in increased hydrophobicity of the paper zone
b. Finally, the time-based quantification of K+ was carried out by measuring the time
required for a dye solution deposited in zone a to flow a length of 1 cm in the paper
channel (zone c).
Timing-based readout offers precise quantitative results simply by using an

ubiquitous timer as the readout “instrument”. However, one disadvantage is that
this detection mode forces a user to pay attention to the device in order not to
overlook the appointed endpoint phenomenon, which can be in the order of several
minutes for the examples described above, making high-throughput parallel assays
challenging.
13.3.4.3 Counting of Colored Spots
Branched microfluidic channels allow not only multi-analyte detection but also
semi-quantitative analysis based on “counting” of the number of colored paper
areas. This readout motif has been first proposed in parallel with the timing-based
counterpart (illustrated in Fig. 13.7a of the previous section) in the same report
[80]. Figure 13.8a shows the paper-based microfluidic device that allows semi-
quantification of H2O2 by counting the number of green colored paper areas (“detec-
tion bars” in Fig. 13.8a) after the elapse of a certain analysis time [80]. The key idea
is that each branched channel has a different amount of the H2O2-degradable
hydrophobic compound (“phase switching agent”) in the midstream. A sample
containing higher concentration of H2O2 has greater decomposition capacity of the
phase switching agent per unit of time, and therefore, is able to flow through an
increasing number of the paper channels within a fixed analysis time. In contrast to
the precise time measurement-based quantification mode, this “analog” counting-
based approach is somewhat semi-quantitative. Nevertheless, a user is less likely to
“miss” the assay endpoint in this approach as compared to the timing-based mea-
surements, by simply setting an alarm at the required time of signal readout.
Another complementary work describes the counting-based detection of H2O2 by
using KMnO4 as an indicator [83]. The device shown in Fig. 13.8b has various
amounts of KMnO4 at the terminal of branched paper channels (“detection spots” in
Fig. 13.8b, top), where KMnO4 is decolored upon reduction by H2O2 in the sample.
The number of decolored detection spots allows to semi-quantitatively determine the
concentration of H2O2 in the applied sample solution. This device has been
employed for the detection of adenosine in combination with an off-chip peroxi-
dase-based enzymatic reaction (Fig. 13.8b, bottom). As is clear from the mechanism,
KMnO4 serves not only as an indicator, but also as a “scavenger” of H2O2 enabling
this counting-based readout mode. Although the targeted application is environment
assessment and is not directly related to the medical and biological fields, colored
spot counting-based acid-base titration [93] and semi-quantification of alkali earth
metals (Ca2+, Mg2+) [94] have also been achieved on paper-based microfluidics,
where acid, base and a chelating agent serve as the scavenger of the respective
analytical target (i.e. base, acid and alkali earth metals, respectively).
Fig. 13.8 Examples of colored spot counting-based detection on paper-based microfluidics: (a)
schematic illustration of a three-dimensional device design, assay procedure and actual photograph
of the counting-based H2O2 detection. (Adapted with permission from Ref. [80]. Copyright© 2012
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (b) actual photograph of a lateral flow-type
device after deposition of various amounts of KMnO4 (top) and processed images showing the
result of application to counting-based adenosine semi-quantification. (Adapted from Ref. [83]),
Copyright 2016, with permission from Elsevier)
13.3.4.4 Direct Reading of Assay Results: “Text-Displayed Signaling”
In analogy to looking at the display of a handheld blood glucose meter or a

pH-meter, reading of text or a number from a piece of paper is with no doubt the
most straightforward and user-friendly way to interpret the result of an analytical
assay. When it comes to POC diagnostics performable under situations lacking
infrastructure and/or a skilled operator, a fast and disposable “drop-and-read” or
“dip-and-read” assay requiring no further user action than the application of the
as-collected specimen is probably the ultimate goal that research and development
should aim for. However, as compared to the other signaling approaches described
above (analog thermometer-style, time measurement, counting), the “text readout”
mode is scarcely implemented into paper-based tests targeting medical applications.
In addition, the examples introduced in this section do not involve a sample fluid
transport in a microfluidically patterned paper channel, which is deemed to be an
“essence” of paper-based microfluidics. However, two examples of paper-based
Fig. 13.9 Examples of text-displaying of assay results on paper-based devices: (a) ABO and Rh
blood typing. (Reproduced with permission from Ref. [84]. Copyright© 2012 WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim); (b) semi-quantitative urinary albumin detection. (Adapted with
permission Ref. [85]. Copyright 2017 American Chemical Society)
devices employing the “text readout” motif are introduced here, considering the
potential importance of this signaling approach allowing the most straightforward
assay result interpretation. The first demonstration of a text-based signaling mode
was related to ABO and Rh blood typing on a sheet of patterned paper towel
[84]. The paper substrate underwent inkjet-printing of a colorless hydrophobic
reagent (alkyl ketene dimer: AKD) to pattern a frame of alphabetic character- or
symbol-shaped hydrophilic regions, followed by pre-deposition of anti-A, B, or D
antibodies and a water-insoluble red ink. After exposure to a whole blood sample
and subsequent saline washing, the sample blood type was displayed in red text
consisting of coagulated blood and the red ink (Fig. 13.9a). Thanks to the highly
blood type-specific agglutination reaction stemmed from the antigen-antibody reac-
tion, this paper device achieved error-free ABO and Rh blood typing of 99 samples,
including 2 weak blood samples having poor interaction capacity with the blood
agglutination antibodies [84].
As is clear from the mechanism, this text-based signaling approach is limited to
the blood typing application and is not straightforwardly adaptable to general (bio)
chemical analysis. A recent study expanded the application of the text-based signal-
ing on paper devices by using a classical colorimetric indicator for the detection of a
given analyte. The working principle relies on “color screening” of a colorimetric
indicator inkjet-deposited on paper in text form by a color-printed transparency film
serving as a “mask” (Fig. 13.9b) [85]. Each text character is only visible through the
mask when the analyte concentration is high enough to result in colorimetric
response of the indicator whose color is more intense than the overlaid mask. This
idea was applied to semi-quantitative detection of urinary albumin with combined
use of tetrabromophenol blue (TBPB) as an indicator. The text-based readout result
could be semi-quantitatively determined by reading the highest result symbol (,
Tr., 1+, 2+, 3+ or 4+) among the visible ones. Although the result of a comparison
study with a commercial colorimetric dipstick, the gold standard for urinary albumin
screening, showed similar analytical performance (accuracy rate, chance of false-
positive and false-negative readouts), the text readout mode offers more intuitive
result interpretation and better compatibility with users having color vision anoma-
lies than traditional colorimetric dipsticks, where the readout relies on a sample color
comparison with a read guide.
13.4 Conclusions and Outlook
The field of paper-based microfluidics showed an amazing growth since photolith-

ographically patterned paper was rediscovered as a promising analytical platform in
2007. This new type of microfluidic devices offers several exclusive benefits includ-
ing an extremely low cost, power-free sample liquid transport by capillary forces and
safe disposability by incineration, among others. With these attractive characteristics
and recent advancements in simplification of user operations (sample processing
prior to analysis, result readout), paper-based microfluidics have a great potential to
become highly functional yet affordable analytical tools enabling medical diagnos-
tics and screening tests without the need of infrastructure and skilled operators. On
the other hand, recent trends of research and development of paper-based
microfluidics is also directed to more “sophisticated” applications such as cell
analysis [95], tumor biomarker detection [96] and wearable sensors [97]. For these
“sophisticated” applications of paper-based microfluidics, the demonstration of clear
advantages over already available “high-tech” analytical systems will be a crucial
factor to overcome user reluctance to replace established methods. For all targeted
applications of paper-based microfluidics, be it a simple urine home test or an
advanced tumor marker assay performed at POC, in-depth studies of long-term
storage stability are absolutely necessary. Finally, collaboration between academia
and industry is required to establish high-volume production techniques and market
distribution pathways.
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Bioanalysis

Series Editor: Tuan Vo-Dinh

Manabu Tokeshi Editor

Advanced Materials, Methods, and Devices

ISSN 2364-1118 ISSN 2364-1126 (electronic)

Library of Congress Control Number: 2019935498

© Springer Nature Singapore Pte Ltd. 2019

Sapporo, Japan Manabu Tokeshi

1 Acoustoﬂuidic Blood Component Sample Preparation

9 In Vitro Tissue Construction for Organ-on-a-Chip

Maria Antfolk and Thomas Laurell

Abstract Recent developments of bulk acoustoﬂuidic technology (BAW – bulk

M. Antfolk · T. Laurell (*)

© Springer Nature Singapore Pte Ltd. 2019 1

Keywords Acoustoﬂuidics · Acoustophoresis · Cell separation plasmapheresis ·

Acoustoﬂuidics uses ultrasound to separate and handle cells either in continuous

When a particle is suspended in an ultrasound standing wave ﬁeld, the particle is

1.1.1.1 Primary Acoustic Radiation Force

Fig. 1.2 Schematic of

Fig. 1.3 Schematic of one-dimensional and two-dimensional acoustophoretic focusing

1.1.1.2 Two-Dimensional Focusing

Early acoustophoresis based particle separation experiments relied on focusing the

Fig. 1.4 Schematic of the

1.1.1.3 Acoustic Streaming

An acoustic standing wave in a microchannel will not only exercise a primary

1. One dimensionally actuated Rayleigh streaming

1.1.1.4 Size-Insensitive Separation

Most successful acoustophoresis separation experiments are dependent mostly on

In light of this, Augustsson et al. [12] recently reported on an innovative method,

1.1.1.5 Medium Switching

Another acoustoﬂuidic phenomenon that must be accounted for is the medium

1.2 Blood Components

Fig. 1.7 Schematic of Platelet/ WBC separation

As platelets display a very modest acoustophoretic mobility, several groups have

1.2.2 WBC Sub-populations

A more challenging blood component separation is fractionation of different white

1.2.2.1 Integrated Acoustic Device for Monocyte Subpopulation

1.2.3 Plasma and Plasma Proteins

Fig. 1.9 Simple acoustic

1.2.4 RBC Separation

1.2.5 Hematocrit Measurements

Hematocrit measurements are commonly made by cell counting or centrifugation

1.3 Circulating Tumor Cells

1.3.1 Cancer Cell Separation Based on a Positive Acoustic

86 2.3% with a WBC contamination of 0.57 0.14%, and in a high-purity mode

Fig. 1.11 Schematic of

1.3.2 Cancer Cell Separation Based on a Negative Acoustic

1.3.3 Cancer Cell Concentration

Fig. 1.12 Acoustophoresis BAW concentration device operated in a recirculating mode.

linked to centrifugation, i.e. no formation of a cell pellet or adsorption to centrifu-

1.3.4 Integrated Acoustic Devices for CTC Processing

1.4.2 Active Bacteria Processing

E. coli achieving a focusability of 0.95 0.35. Simulations and experiment showed

1. Lenshof A, Magnusson C, Laurell T (2012) Acoustoﬂuidics 8: applications of acoustophoresis

8. Yoshioka K, Kawashima Y (1955) Acoustic radiation pressure on a compressible sphere.

Masatoshi Maeki and Manabu Tokeshi

Abstract Protein crystallization and its three-dimensional structure analysis is

Keywords Protein crystallography · Droplet microﬂuidics · Normally-closed

M. Maeki (*) · M. Tokeshi

© Springer Nature Singapore Pte Ltd. 2019 27