Banerjee, T (2017) - COMSOL-Based Design and Validation of Dilution Algorithm With Continuous-Flow Lab-on-Chip
Banerjee, T (2017) - COMSOL-Based Design and Validation of Dilution Algorithm With Continuous-Flow Lab-on-Chip
Banerjee, T (2017) - COMSOL-Based Design and Validation of Dilution Algorithm With Continuous-Flow Lab-on-Chip
DOI 10.1007/s41403-017-0022-z
ORIGINAL ARTICLE
Received: 23 December 2016 / Accepted: 20 May 2017 / Published online: 6 June 2017
Ó Indian National Academy of Engineering 2017
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56 INAE Lett (2017) 2:55–63
(TWM) algorithm (Thies et al. 2008), which has been Since, the dilution steps used in these algorithms, consist of
studied extensively in the context to DMF. We have only (1:1) mix-split operations, the final CF, thus achieved,
designed Y-shaped serpentine microchannels, where mix- contains a term in the denominator, which can be expressed
ing of fluids is performed by convective/diffusive transport as an integer-power of 2. Hence, for the sake of conve-
through the channels. No valve or metering technique is nience, each CF is rounded-off as an n-bit binary fraction
used for volume control. Also, instead of emitting waste number 2xn , where n is a positive integer greater than 0
droplets/units following each mix-split step as in the BS- denoting the chosen level of accuracy, and x is a positive
method, we inject fluid with velocities in geometric pro- integer such that 0 B x B 2n.
gression to the inlets of the proposed fluidic-network and/or
by adjusting the dimension of the cross-section of the inlet Prior Art
tube. We observe that the target concentration factor (CF)
obtained via COMSOL simulation matches with the Several earlier techniques for producing concentration
expected value within the allowable error range. Moreover, gradients/dilution of fluids with CFM biochips and the
this analysis leads to the design of a universal and pro- design of the underlying flow network were reported in the
grammable continuous-flow network, which can be used to literature (O’Neill et al. 2006; Lee et al. 2009; Walker et al.
implement any arbitrary dilution, as required, for auto- 2010; Friedrich et al. 2012; Weibull et al. 2013; Wang et al.
mated sample preparation. 2016). However, the design, integration, and monitoring of
continuous-flow systems pose a challenge as a number of
flow parameters, such as fluid resistance, pressure, velocity,
Automated Sample Preparation electric field strength may vary along the flow path and can
affect the flow. In addition to these parameters, various
Preliminaries physical characteristics of fluids and channel dimensions
govern the behavior and robustness of the entire system
One of the crucial components in any biochemical analysis (Nguyen et al. 2013). Also, the management of inter-
is sample preparation. In sample preparation, the amount of sample contamination in CFM chips is difficult. Mixing of
raw sample in a fluid-mixture is known as concentration fluids using microchannels with different geometries have
factor (CF). We assume that the raw sample has 100% been reported (Elabbasi et al. 2012; Das et al. 2014; Bruce,
concentration factor, and the buffer fluid has CF = 0. The http://courses.washington.edu/microflo/; Karthikeyan et al.
value of CF can be any real number within the closed 2014). Theoretical modeling and simulations studies on
interval [0, 1] (Thies et al. 2008; Roy et al. 2010). Thus, for fluid mixing also appeared in the literature (Dhondi and
the dilution problem, a target CF will lie between the open Pereira 2009; Karst et al. 2013; Wang et al. 2016).
interval (0, 1), whereas buffer (CF = 0) and raw sample
(CF = 1) are generally provided as input-fluids. In DMF, Bit-Scanning (BS) or twoWayMix (TWM)
the (1:1) mix-split model is very well known (Thies et al. Algorithm
2008; Roy et al. 2010). In this model, mixing of two unit
volume of droplets is followed by balanced splitting of the Thies et al. (2008) first proposed a mixing algorithm based
composite droplet into two equal unit-volume droplets. In on bit-scanning (BS)/Min-Mix method. Dilution is a special
the BS/TWM algorithm (Thies et al. 2008), the process of case of mixing where a sample is mixed with a buffer in a
diluting a raw sample can be envisaged using a directed certain ratio. As stated earlier, to implement dilution, the
acyclic graph, known as mix-split tree/graph. A traversal of given target CF is first approximated with a user’s defined
this mix-split tree/graph provides the desired sequence of n-bit binary fraction, where n is a positive integer that
(1:1) mix-split operations that are to be performed with a determines the maximum allowable error 2nþ1 1
in CF. Mix-
digital microfluidic biochip (DMFB) in order to achieve the ing is performed based on the (1:1) model, where one unit
target CF. The accuracy of the target CF is determined by of sample is mixed with one unit of buffer, and only one
the depth of the mixing graph, which is a positive integer n unit of the resulting mixture is used discarding the
specified by the user. In BS/TWM algorithm (Thies et al. remaining unit as waste. The algorithm twoWayMix (TWM)
2008), the error limit (a.k.a. accuracy level) in the target works on scanning the bits of the binary fraction from
1
CF is 2nþ1 which is obtained assuming that the number of right-to-left to decide whether a droplet (unit-volume) of
allowed (1:1) mix-split operations is at most n. Note that a sample (if the current bit = 1), or of buffer (if the current
sequence of ten mix-split steps provide a good approxi- bit = 0) is to be mixed with one unit of fluid that was
mation of a target CF with an error less than produced at the preceding step (see Fig. 1). Thus, except
1
211 ’ 0:000488281, which is very small (Roy et al. 2010). the last step where two units of fluid with target CF are
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INAE Lett (2017) 2:55–63 57
obtained, one fluid-unit is wasted at each mix-split step. algorithm used for DMF. We provide a solution in this
This algorithm requires the minimum number of (1:1) mix- paper by controlling the injection velocities of fluids and by
split steps needed to produce a target concentration with modifying the inlet dimensions. The proposed technique
accuracy level n, where reuse of waste-droplets is not mimics the underlying mix-split tree/graph that describes
performed. Figure 1 shows the mix-split sequencing steps the sequence of mix-split operations, but unlike the pre-
313 vious DMF-based methods, it does not need any ‘‘split
to generate the target concentration CF = ð1024 Þ10 =
(0.0100111001)2, where 0’s (1’s) appearing from right-to- step’’ or cause waste of fluids.
left govern the injection of buffer (sample) droplets in the
mixing graph. Note that all extra 0’s appearing on the right
end of the binary fraction are deleted. Also, corresponding Problem Description
to the least significant 1-bit, both buffer and sample dro-
plets are injected (see Fig. 1). In the proposed work, we Input A supply of sample fluid (100% concentration, i.e.,
present a method for diluting a fluid sample using CFM CF = 1), and buffer fluid (0% concentration, i.e., CF = 0),
LoC, in tune with the BS/TWM algorithm. In other words, and a positive integer n denoting the accuracy level.
given any target CF, we will inject sample or buffer Output A mix-sequence that will generate the target
1
through the inlets of the continuous-flow network follow- concentration CF = T not exceeding allowable error 2nþ1
ing the same sequence as mandated by the bits present in when implemented with a CFM biochip.
the binary fraction representing the CF.
Proposed Fluidic-Network
Motivation
We propose a continuous-flow fluidic network for imple-
In the field of CFM LoC, one of the most important issues menting the dilution algorithm, where each inlet will have
is to control the fluid-injection velocity. Also, for ease of a pre-determined cross-section and fluid injection velocity
implementation, we consider a scenario where no valve- regardless of the target CF. We consider two types of
control is required. In order to achieve a target-CF with geometric layout for the fluidic network, the dimensions of
CFM LoC, we need to calculate the volume of raw sample which are shown Fig. 2 (Model 1) and Fig. 3 (Model 2). In
and buffer fluids to be injected per unit time into the both models, we use a passive serpentine mixer (Lee et al.
mixing chamber. Our motivation is to design a universal 2011; Ober et al. 2015) with turning radius 6.5 lm as
fluidic architecture/model that can be used to produce an shown in Figs. 2 and 3 (Kane et al. 2008); additionally, in
arbitrary target concentration CF = T (0\T \1) with Model 2, we use special rectangular boxes to augment the
accuracy level n using CFM in tune with the BS/TWM inlets as shown in Fig. 3. We choose channel-dimensions
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Fig. 2 Geometric layout of the serpentine fluidic mixer for Model 1; width of all inlets = 25 lm
and fluid-injection velocities following certain rules (de- Reynolds number (r) (Howard 2007) is a dimension-
scribed later), and study the flow dynamics in 2D for dif- less parameter, used to characterize the nature of fluid
ferent input concentration profiles using COMSOL motion. It is the ratio of the inertial force to the viscous
Multiphysics Software (https://www.comsol.co.in/). force, i.e.,
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INAE Lett (2017) 2:55–63 59
R ¼ Reynolds number Table 1 shows the required inlet boundary width of the
Inertial force rectangular area, applied velocities, and equivalent
¼ velocities appearing at the arms of Y-shaped serpentine
Viscous force ð1Þ
fluidic mixer microchannel. Since we have performed 2D
qu‘
¼ COMSOL simulation, the length of each inlet tube is
l
assumed to be unity, and the width is varied as shown in
where, q represents the density of fluid, l denotes fluid Table 1.
dynamic viscosity, ‘ stands for fluidic channel length, and u
is the speed of fluid-flow. A high value of r indicates
Simulation Framework
turbulent flow, whereas a low value indicates the presence
of laminar flow. In our proposed model, we choose these
We have studied the flow dynamics for different input
parameters (q; l; ‘ and u) in such a way that r is lower
concentration profiles using COMSOL Multiphysics Soft-
than 2000 so that laminar flow for miscible fluids is
ware. Figures 4 and 5 represent the flow dynamics for
guaranteed by the model (Howard 2007). 313
target concentration CF = ð1024 Þ10 = (0.0100111001)2 for
We deploy a cascade of Y-shaped junctions each of
which merges into a serpentine channel of rectangular n = 10, based on Model 1 and Model 2, respectively.
cross-section filled with inter-digitized orthogonal obsta- Initially, the channels are assumed to be filled with air. The
cles (Figs. 2, 3). The detailed dimensions of the channels fluids may have different parameters such as density, vis-
are shown in the figures. The inflow of sample/buffer is cosity, electrical and thermal conductivity, diffusion coef-
passed into the serpentine mixing chamber through dif- ficients, and surface tension. We have used single-phase
ferent microchannels via several inlets oriented with an laminar flow (SPF) to study the parameters of fluids such as
angle of 1208 to each other (Figs. 2, 3). The mixing of fluid velocity profile, fluid pressure profile, and transport of
fluids is performed by convective/diffusive transport diluted species (TDS) to study the concentration profile of
through the serpentine channels. Our method does not the fluids along the channels.
need any valve-control, and no waste management is SPF solves the Navier–Stokes equation (Howard 2007)
needed as in the BS/TWM algorithm (Thies et al. 2008). In that governs the motion of fluids, and is analogous to
our Model 1, we increase fluid-injection velocities in Newton’s second law of motion for fluids. In the case of an
geometric progression at each subsequent inlet so that the incompressible Newtonian fluid, this equation is:
volume of fluid inflow is doubled at each step. At the two ou
q þ u ru ¼ rp þ r
inlets of the first stage, the velocity is set to 0.1 mm/s ot
each, and then it is multiplied by a factor of two at every
T 2
following inlet. l ru þ ðruÞ lðr uÞI þ F
3
The number of required Y-shaped serpentine channels is
ð2Þ
n and the total number of inlets is (n ? 1), where n is the
number of bits in the binary fraction used to approximate the where u is the fluid velocity, p is the fluid pressure, q is the
target CF. Hence, in Model 1, we need n fluid-injection fluid density, and l is the dynamic viscosity. The left-hand-
velocities in geometric progression (in the first stage, we use side term represents inertial forces; on the right-hand side,
two equal velocities). However, Model 2 allows us to reduce the term (-rp) represents pressure-force; viscous-force is
the number of required fluid-injection velocities by changing represented by r l ru þ ðruÞT 23 lðr uÞI , and
the dimensions of fluid-inlet channels. We keep the length of F denotes the external forces applied to the fluid. These
each inlet same, but double the width of the inlet channel equations are solved together with the continuity equation
when needed so as to supply the required fluid-volume per (Howard 2007):
unit time through an inlet-branch. We have used rectangular
boxes as extensions at the inlets (Fig. 3) to implement this. oq
þ r ðquÞ ¼ 0 ð3Þ
Note that a larger value of n will necessitate the use of more ot
fluid-inlets and high injection velocities or wider input The two properties, namely ‘‘conservation of
channels. Unfortunately, in these cases, flow-control and momentum’’ and ‘‘conservation of mass’’ for incom-
homogeneous mixing may be harder to achieve in practice. pressible fluids are captured by the Navier-Stokes
Thus, a compromise is needed. In our experiments, we have equation (2) and the continuity equation (3), respec-
chosen n to lie between 5 and 10; such a choice provides tively. The relative concentration of the sample is rep-
sufficient accuracy in CF from users’ perspective as well as resented by Fick’s laws (Crank 1975). Using Fick’s
supports manageable flow dynamics. laws, TDS can provide the solution of the convection-
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Table 1 Generation of ten fluid-velocities using two injection-ve- rci = Ri, where Di is the diffusion coefficient of fluid
locities and five inlet-widths i in the solution, the conserved quantity ci is the con-
Required inlet Applied velocity at Equivalent velocity centration of the fluid i, and Ri is the reaction rate of the
boundary width inlet boundary in mm/s at inlet-arm in mm/s fluids. We have used ethanol (diffusivity 1.24 9 10-9
in lm m2/s) (Goranovic and Bruus 2003) and glycerol (diffu-
6.25 0.8 0.1 sivity 0.94 9 10-9 m2/s) as sample, and water as buffer.
12.5 0.8 0.2 The boundary conditions are specified on the surfaces
25 0.8 0.4 that define the computational domain. The typical boundary
50 0.8 0.8 conditions affect the values of velocity, pressure, and
100 0.8 1.6
concentration. Meshing is another important part of the
6.25 25.6 3.2
simulation. We have used the same meshing for both of our
fluidic-network models. We set the predefined element size
12.5 25.6 6.4
at extra-fine while calibrating fluid dynamics. Time-dis-
25 25.6 12.8
cretization is needed to simulate time-dependent flows.
50 25.6 25.6
Generally, smaller time steps are needed in order to achieve
100 25.6 51.2
more accurate results. A time range of 0–25 s is considered
that provides saturation for diffusive mixing in the
diffusion equation. In our model, there exists advection microchannels, and simulation is performed with a time-
(i.e., net transport of whole solution). Hence, we have step of 1 s. In Table 2, we describe the details of the com-
used TDS with transport equation: r(-Di rci) ? u putational platform used in our simulation.
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INAE Lett (2017) 2:55–63 61
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have reported COMSOL simulation of the mixing pro- Conference on CFD in Minerals and Process Industries CSIRO,
cess needed for preparing various dilutions of two dif- Melbourne, Australia, pp. 1–6
Diffusivity of Glycerol. http://www.hypertextbookshop.com/biofilm
ferent fluids and validated the correctness of our book/working_version/artifacts/tables/Module_004/Table4-1_
approach. The proposed technique can be conveniently DiffCoeffH2O.htm. Accessed 01 June 2016
used for the design and simulation of other sample- Elabbasi N, Liu XO, Brown S (2012) Modeling of laminar flow static
preparation tasks with CFM biochips. Implementation of mixers. COMSOL News
Friedrich D, Please CP, Melvin T (2012) Design of novel microfluidic
3D simulation of the proposed method using COMSOL concentration gradient generators suitable for linear and expo-
Multiphysics software will be taken up as future work. nential concentration ranges. Chem Eng J 193–194:296–303
Ghoshal S, Mitra D, Roy S, Dutta Majumder D (2010) Biosensors and
Acknowledgements One of the authors, T. Banerjee, is registered biochips for nanomedical applicationa: a review. Sens Trans-
for PhD degree at the Department of Computer Science and Engi- ducers 113(2):1–17
neering, University of Calcutta, and she would like to thank the Goranovic G, Bruus H (2003) Simulations in microfluidics. In:
Department of Science and Technology (DST), Government of India, Geschke O, Klank H, Telleman P (eds) Microsystem engineering
for supporting her research under the Indo-Taiwan Collaborative of lab-on-a-chip devices. Wiley-VCH Verlag GmbH & Co.
Research Project (2014–17). The work of B. B. Bhattacharya was KGaA, Weinheim, FRG. doi:10.1002/3527601651.ch5
supported, in part, with the grant provided by INAE Chair Howard AS (2007) Introduction to fluid dynamics for microfluidic
Professorship. flows. In: Lee H, Ham D, Westervelt RM (eds) CMOS
biotechnology. Springer, New York, pp 5–30
Kane SA, Hoffmann A, Baumga P, Seckler R, Reichardt G, Horsley AD,
Schuler B, Bakajin O (2008) Microfluidic mixers for the investigation
References of rapid protein folding kinetics using synchrotron radiation circular
dichroism spectroscopy. Anal Chem 80:9534–9541
Bruce AF (Professor Emeritus of Chemical Engineering, University Karst C, Storey B, Geddes JB (2013) Laminar flow of two miscible
of Washington). http://courses.washington.edu/microflo/. fluids in a simple network. Phy Fluids. doi:10.1063/1.4794726
Accessed 01 June 2016 Karthikeyan K, Sudharsan N, Sujatha L (2014) Design of high
Chakrabarty K, Thewes R (2007) Guest editors’ introduction: performance micromixer for lab-on-a-chip (LoC) applications.
biochips and integrated biosensor platforms. IEEE Design Test COMSOL Conference, Bangalore
24(1):8–9 Lee K, Kim C, Ahn B (2009) Generalized serial dilution module for
COMSOL (2016) Multiphysics Software. https://www.comsol.co.in/ monotonic arbitrary microfluidic gradient generation. Lab Chip
Crank J (1975) The mathematics of diffusion. Clarendon Press 9:709–717
Oxford, Ely House, London W.I. Lee CY, Chang CL, Wang YN, Fu LM (2011) Microfluidic mixing: a
Das SS, Patawari BK, Patowari PK, Halder S (2014) Computational review. Int J Mol Sci 12:3263–3287
analysis for mixixng of fluids flowing through micro-channels of Mitra D, Roy S, Bhattacharjee S, Chakrabarty K, Bhattacharya BB
different geometries. AIMTDR, pp. 236:1–236:6 (2014) On-chip sample preparation for multiple targets using
Dhondi S, Pereira GG (2009) Complex fluid mixing in microfluidic digital microfluidics. IEEE Trans Comput Aided Des Integr
devices: theory and simulations. Seventh International Circuits Syst 33(8):1134–1144
123
INAE Lett (2017) 2:55–63 63
Nguyen NT, Shaegh SA, Kashanineja N, Phan DT (2013) Design, application-specific digital microfluidic biochips. ACM Trans
fabrication and characterization of drug delivery systems based Design Autom Electron Syst 20(3):45:1–45:33
on lab-on-a-chip technology. Adv Drug Deliv Rev Thies W, Urbanski JP, Thorsen T, Amarasinghe SP (2008) Abstrac-
65:1403–1419 tion layers for scalable microfluidic biocomputing. Nat Comput
Ober TJ, Foresti D, Lewis JA (2015) Active mixing of complex fluids 7(2):255–275
at the microscale. PNAS 112(40):12293–12298 Walker GM, Monterio-Riviere N, Rouse J, Neill OT (2010) A linear
O’Neill AT, Monteiro-Riviere N, Walker GM (2006) A serial dilution dilution microfluidic device for cytotoxicity assays. Lab Chip
microfluidic device for cytotoxicity assays. In: International 7:709–717
conference of the IEEE engineering in medicine and biology Wang J, Brisk P, Grover H (2016) Random design of microfluidics.
society, New York, 2006, pp 2836–2839 Lab Chip 16(21):4212–4219
Roy S, Bhattacharya BB, Chakrabarty K (2010) Optimization of Weibull E, Matsui S, Sakai M (2013) Microfluidic device for
dilution and mixing of biochemical samples using digital generating a stepwise concentration gradient on a microwell
microfluidic biochips. IEEE Trans Comput Aided Des Integr slide for cell analysis. Biomicrofluidics 7(6):064115:1–064115:1
Circuits Syst 29(11):1696–1708
Roy S, Chakrabarty PP, Chakrabarty K, Bhattacharya BB (2015)
Layout-aware mixture preparation of biochemical fluids on
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