Kuntaegowdanahalli 2009
Kuntaegowdanahalli 2009
Kuntaegowdanahalli 2009
In this work we report on a simple inertial microfluidic device that achieves continuous multi-particle
separation using the principle of Dean-coupled inertial migration in spiral microchannels. The
dominant inertial forces coupled with the Dean rotational force due to the curvilinear microchannel
geometry cause particles to occupy a single equilibrium position near the inner microchannel wall. The
position at which particles equilibrate is dependent on the ratio of the inertial lift to Dean drag forces.
Using this concept, we demonstrate, for the first time, a spiral lab-on-a-chip (LOC) for size-dependant
focusing of particles at distinct equilibrium positions across the microchannel cross-section from
a multi-particle mixture. The individual particle streams can be collected with an appropriately
designed outlet system. To demonstrate this principle, a 5-loop Archimedean spiral microchannel with
a fixed width of 500 mm and a height of 130 mm was used to simultaneously and continuously separate
10 mm, 15 mm, and 20 mm polystyrene particles. The device exhibited 90% separation efficiency. The
versatility of the device was demonstrated by separating neuroblastoma and glioma cells with 80%
efficiency and high relative viability (>90%). The achieved throughput of 1 million cells/min is
substantially higher than the sorting rates reported by other microscale sorting methods and is
comparable to the rates obtained with commercial macroscale flow cytomerty techniques. The simple
planar structure and high throughput offered by this passive microfluidic approach make it attractive
for LOC devices in biomedical and environmental applications.
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to demonstrate efficient sorting of 0.8 mm, 0.9 mm and 1 mm with ap/Dh $ 0.07 (where, ap is particle diameter and Dh is the
diameter microspheres. Although these techniques operate in microchannel hydraulic diameter), the lift forces dominate and
continuous mode, the need for narrow channel geometries in are responsible for the particles equilibrating within the micro-
PFF and presence of obstructions in DLD may lead to channel channel.17–19 Herein, we demonstrate that the position at which
clogging and particle–particle interactions. Additionally, the particles of different sizes equilibrate is dependent on the ratio of
reported throughputs are insufficient for cell sorting and lift and Dean drag forces which varies with the third power of the
cytometry applications. particle diameter. By modulating the flow parameters, individual
Recently, inertial migration based particle filtration techniques particle streams can be generated for separation applications as
have been reported to achieve high throughput particle separa- shown in Fig. 1.
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tion.15–19 Under the influence of the inertial forces, neutrally In a plane Poiseuille flow, the parabolic nature of the velocity
buoyant particles flowing in a microchannel migrate to stable profile results in a shear gradient induced inertial lift force that
equilibrium positions along the channel periphery. Di Carlo drives the suspended microparticles away from the microchannel
et al.18 showed that by employing a curvilinear channel the Dean center towards the channel walls. As the particles move closer to
vortices can be used to reduce the number of equilibrium posi- the channel wall, the rotational wake around the particles is
tions to one. This single particle stream can later be extracted by disturbed due to the presence of the wall inducing a lift force on
employing bifurcated outlets. Using this technique rapid micro- the particles directed away from the wall.18,23,24 These oppositely
particle filtration can be achieved.17–22 For example, we recently directed forces exert a net lift force (FL) on the particles equili-
demonstrated complete separation of a two particle mixture brating them into focused streams around the microchannel
(7.32 mm and 1.9 mm diameter) by ensuring that the smaller perimeter. Segre and Silberberg were the first to report this effect
particle motion was affected solely by Dean forces and the larger demonstrating that a uniformly distributed suspension of
particle migration by inertial lift forces.17 neutrally buoyant particles form a narrow band at 0.6R from the
In our previous work,17 we introduced a design to achieve channel center in a circular channel of radius R.25,26 Chun and
complete separation of two different particle sizes by exploiting
the effects of Dean drag and inertial lift forces. The larger
particles equilibrated under the combined influence of the inertial
lift forces and the Dean force at the inner microchannel wall. The
inertial lift forces however did not affect migration of the smaller
particles, which were transposed to the outer microchannel wall
due to Dean drag. Since particle separation was not dependent
on the ratio of the two forces, a major limitation of the design
was its inability to separate more than two particle mixtures
irrespective of the number of outlets.
In this paper, we demonstrate for the first time the use of Dean
coupled inertial migration to simultaneously separate multi-
particle mixtures. In a curvilinear microchannel, a combination
of Dean drag and inertial lift forces result in particle equilibra-
tion at the inner microchannel wall. The position at which the
particles equilibrate is dependent on the ratio of these two forces.
In this work we have developed a design which exploits the
particle size dependence of the ratio of the two forces to form
segregated, focused particle streams which can be extracted by
employing a suitable outlet system. Although the design reported
herein is similar to the previously reported spiral separators,17 the
working principle for achieving separation is different and is the
first demonstration of a multi-particle separation using inertial
microfluidics. Additionally, the system reported in this work is
simple, easy to fabricate, and is capable of continuously sepa-
rating particles with a high throughput over a wide dynamic
range. The proposed principle was also used to demonstrate size-
based cell separation between SH-SY5Y neuroblastoma cells and
C6 glioma cells.
Fig. 1 (a) Schematic of the spiral microparticle separator. The randomly
dispersed particles equilibrate at different equilibrium positions along the
2. Design principle inner wall (IW) of the spiral microchannel under the influence of FL and
FD. Separation between individual particle streams is enhanced by
In our recent work,17 we showed that particles flowing in a spiral opening the spiral channel into a wider straight channel before extracting
microchannel with rectangular cross-section experience the individual streams using a multiple outlet design. (b) Microchannel
a combination of inertial lift and Dean drag forces. The magni- cross-section illustrating effects of FL and FD on particles. The ratio of
tude and direction of these forces depends on the particle size and forces (FL/FD) is the determining factor in where a particle of given size
position across the microchannel cross-section. For particles (diameter) equilibrates.
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Ladd showed the preferential equilibration of particles with streams which can be independently extracted by designing
ap/Dh 0.1 in square cross-sectional channels.27 In our previous appropriate outlets.
work, we showed the equilibrium positions in square and rect-
angular microchannels at finite Re (1 < Re < 50) flows.15,16
3. Experimental methods
The net lift force acting on the particle suspended in a plane
Poiseuille flow is given by24 The microchannels were fabricated in polydimethylsiloxane
(PDMS, Sylgard 184, Dow Corning) using standard soft
FL ¼ rG2CLap4 (1) lithography methods. Briefly, a negative silicon master was
fabricated by patterning SU-8 photoresist (2075, Microchem
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where r is the density of fluid medium, G is the shear rate of the Corp.) of desired height using the conventional photolithog-
fluid given by G ¼ Umax/Dh, Umax is the maximum fluid velocity raphy process.32 Subsequently, PDMS polymer mixed in the
and CL is the lift co-efficient which is a function of the particle ratio of 10:1 with the curing agent was cast onto the fabricated
position across the channel cross-section and channel Reynolds silicon master to form a replica of the device. Following curing
number (Re). The magnitude of lift coefficient and hence the lift on a hotplate for 2 h at 80 C, the PDMS molds were peeled and
force rises from zero at the channel center to a maximum value input and output ports were cored using a 14 gauge syringe
before falling back to zero again at a distance of 0.2Dh away needle. The PDMS mold was then bonded to a 1 mm thick
from the channel wall, equilibrating the particles.24 Beyond this microscopic glass slide to complete the channel using corona
point, the lift coefficient changes sign indicating a dominance of wand treatment (BD-20AC, Electro-Technic Products Inc.).
wall-induced lift force. Fluorescently labeled polystyrene particles purchased from
The curvilinear geometry of a spiral microchannel introduces Bangs Laboratories were diluted in DI water before testing
a centrifugal acceleration component directed radially outward (0.05% volume fraction). A syringe pump (KDS101, KD
resulting in the formation of two symmetric-counter rotating Scientific Inc.) was used to drive syringes filled with the particle
vortices known as the Dean vortices in the top and bottom halves solution. To evaluate particle stream positions within the
of the microchannel.28,29 The magnitude of these Dean vortices microchannel, high speed images of the microchannel were
can be determined by a dimensionless number known as the captured using an inverted epi-fluorescence microscope (IX71,
Dean Number (De), which is given by Olympus Inc.) equipped with a 12-bit CCD camera (Retiga EXi,
rffiffiffiffiffiffi rffiffiffiffiffiffi QImaging). Using ImageJ software, Z-stacked composite
rUf Dh Dh Dh images were generated by overlaying a stack of 300 images. The
De ¼ ¼ Re (2)
m 2R 2R particle stream position was determined by analyzing the gray-
scale line scans across the channel width in the composite
For a straight microchannel De ¼ 0. Increasing the channel image.33
cross-section (Dh) or the flow rate increases De, resulting in To demonstrate separation of particle mixtures, a solution
stronger Dean forces. A particle suspended in a spiral micro- containing 10 mm (s ¼ 0.54 mm), 15 mm (s ¼ 0.98 mm), and 20 mm
channel experiences a transverse drag force due to these Dean (s ¼ 0.5 mm) diameter polystyrene particles labeled with DAPI,
vortices. The expression for the Dean drag force experienced by FITC, and TRITC fluorophores respectively was used. The
the particle can be arrived at by assuming Stokes drag.17,30,31 particle streams were viewed and captured separately using
appropriate filter cubes. The individual images were super-
FD ¼ 3pmUDeanap ¼ 5.4 104pmDe1.63ap (3) imposed to create a composite image to display the formation of
three separate focused particle streams.
where UDean is the average Dean velocity given by UDean ¼ To quantify separation efficiency, the outlet streams at each of
1.8 104De1.63. the eight outlets were collected and analyzed by flow cytometry.
Particle equilibration in rectangular microchannel cross- The BD LSR II (BD Biosciences) flow cytometer was used to
section is independent of Dh and rather depends on the shortest perform the analysis. Individual particle solutions were used as
channel dimension (microchannel height, H) due to varying controls to draw the gates on a FSC vs. SSC plot. The collected
shear rates across the channel cross-section.15,16 Hence, the samples were then run through the flow cytometer to determine
criterion for particle focusing is ap/H $ 0.07. We use this result to counts for each of the three particles in all the eight outlets.
design low aspect ratio spiral microchannels to enhance the To demonstrate cell separations, SH-SY5Y neuroblastoma
separation between individual particle streams over a wide range cells 15 mm (s ¼ 5 mm) in diameter were cultured in Opti-MEM
of particle sizes. The dependence of ratio of the lift force (FL) to medium containing 10% fetal bovine serum supplemented with
Dean drag force (FD) on particle size (FL/FD f ap3) and the shear L-glutamine. The C6 rat glioma cells 8 mm (s ¼ 3 mm) in
rate modulation in low aspect ratio microchannels have been diameter were cultured in Ham’s F-12 medium containing 15%
exploited to demonstrate separation of 10 mm, 15 mm, and 20 mm horse serum and 5% fetal bovine serum supplemented with
diameter particles. The dominant inertial lift forces align the 0.25 mg/mL amphotericin B, 100 U/mL penicillin, and 100 mg/mL
randomly distributed particles at the inlet near the inner micro- streptomycin. Both cultures were maintained at 37 C in
channel wall as the flow progresses downstream. On the other a humidified atmosphere containing 5% (v/v) CO2. Cells were
hand, the significant Dean drag force move these focused streams cultured in sterile 25 cm2 flasks (Corning). Cells were sub-culti-
farther away from the channel wall depending on the particle vated (1:4) three times a week and media was replaced every
size, with the largest particle being closest to the inner channel 48 hours. Sub-confluent monolayers were dissociated with 0.01%
wall. This results in the evolution of three distinct particle trypsin and 5.3 mM EDTA solution, re-suspended in fresh basal
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Fig. 5 Superimposed fluorescent images illustrating distribution and position of the 20 mm (pseudo-colored orange), 15 mm (pseudo-colored green), and
10 mm (pseudo-colored purple) diameter particles. The panels represent the inlet, a 500 mm wide channel section just prior to the outlet, and the
bifurcated outlet of the 130 mm high spiral microchannel at De ¼ 14.4. The randomly distributed particles at the inlet form ordered focused streams
which are then collected separately at outlets 1, 2 and 3.
Fig. 6 Flow cytometry data indicating concentration of the three particle mixture. The dot plots indicate concentration of the 10 mm, 15 mm, and 20 mm
diameter particles at (a) the inlet, and (b–d) the first three outlets of the spiral channel. The gates in the side scatter vs. forward scatter panels were drawn
using pure particle samples in order to discriminate between the three particle sizes. (e) Particle counting results illustrating particle distribution across
the first four outlets of the spiral microchannel. A 90% separation efficiency between the three particle sizes was achieved.
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Fig. 7 Bright-field and epifluorescent images illustrating the distribution of the bigger 15 mm diameter SH-SY5Y cells (pseudo-colored green) and the
smaller 8 mm diameter C6 glioma cells at the inlet and the first two outlets of the spiral microchannel (scale bar 100 mm). Also shown in the figure are the
cell counting results clearly indicating 80% separation efficiency between the two cells types, with the SH-SY5Y cells collected at outlet 1 and the C6
glioma cells at outlet 2.
Based on the size of these cells, the mixture was passed through than the sorting rates achievable by other microscale sorting
a 120 mm high microchannel at De ¼ 11.8 to collect the bigger methods. In fact, the throughput of our system is quite compa-
15 mm diameter cells from outlet 1 and the smaller 8 mm rable to the sorting rates obtained with commercial flow
diameter cells from outlet 2. Fig. 7 shows the bright-field and cytometry techniques, which have a maximum throughput of
epifluorescent images illustrating the distribution of the bigger 2.4 million cells/min.36 Since only a 0.05% volume fraction was
SH-SY5Y cells (fluorescently labeled) and the smaller C6 glioma used in our experiments, the throughput may be increased even
cells (unlabeled) at the inlet and at the first two outlets of the further, nearly an order of magnitude, by simply increasing the
spiral microchannel. The inlet solution consisted of the two cell cell volume fraction and fluid flow rates (volume fraction values
mixture with equal cell concentrations (500,000 2 cells/mL). of 0.1 to 0.3% have been reported by others34,35).
Nearly 90% of the cells were collected at outlets 1 and 2, indi-
cating a high degree of cell focusing. The cell separation effi-
5. Conclusions
ciency was >80% for both the bigger SH-SY5Y cells at outlet
1 and the smaller C6 glioma cells at outlet 2, but was limited by the In this work, we introduced an inertial microfluidic system for
large variations in the cell sizes (s ¼ 5 mm). A potential concern passive simultaneous separation of a microparticle mixture. The
when separating cells is the possibility of damage to the cells due effect of Dean coupled inertial migration in spiral microchannels
to the high shear forces. Cell viability following separation was was exploited to generate focused streams of individual particles at
confirmed by bringing both SH-SY5Y neuroblastoma and C6 distinct positions within the microchannels based on their size.
glioma cells back into culture using the procedure described in the Tests with individual particles were conducted to identify the effects
methods section with >90% cell recovery after 24 hrs. of microchannel height and the Dean number on particle stream
As Pamme6 discusses in a recent review, continuous flow position. Based on the results obtained from individual particle
separation techniques are becoming popular on the microscale experiments, a 130 mm high spiral channel was employed to sepa-
due to their ability to achieve high sample throughputs. rate a mixture of 10 mm, 15 mm, and 20 mm diameter particles. The
The average throughput of many of these microfluidic systems is separation efficiency of the device was calculated to be around 90%.
2,000 cells/min,6 although higher throughputs have been recently The device was also used to show a high throughput (1 milion
reported. For example, Fu et al.34 demonstrated a throughput of cells/min) separation of neural cells with high viability, demon-
1,200–7,200 cells/min while separating Escherichia coli cells strating application of this technique in microscale cell sorting. The
expressing green fluorescent protein from a background of passive separation principle and the planar nature of the described
non-fluorescent E. coli cells in microfluidic channels using fluo- design will permit easy integration with existing LOC systems
rescently-activated sorting. A similar throughput (5,400 cells/ requiring high-throughput separations.
min) was demonstrated using the PFF approach by Takagi et al.35
who separated erythrocytes from diluted blood.
The developed spiral microparticle separator is ideal for
Acknowledgements
achieving high throughput separations due to the fact that This work was supported by the University of Cincinnati Insti-
inertial and Dean drag forces acting on particles increase with tute for Nanoscale Science and Technology and the National
increasing flow rates. For the flow rates tested (in the mL/min Institute of Occupational Safety and Health (NIOSH) Health
range), a sorting rate of 1 million cells/min was achieved in our Pilot Research Project Training Program of the University of
system. This throughput is substantially higher (more than 100) Cincinnati Education and Research Center (T42/OH008432-04).
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