2018, Vol.23 No.

3, 244-250

Article ID 1007-1202(2018)03-0244-07
DOI https://doi.org/10.1007/s11859-018-1317-y

Generation of Linear and Parabolic

Concentration Gradients by Using a
Christmas Tree-Shaped Microfluidic
Network

□ SHEN Qilong, ZHOU Qiongwei, LU Zhigang, 0 Introduction

ZHANG Nangang†
School of Electrical and Electronic Engineering, Wuhan
Textile University, Wuhan 430200, Hubei, China
© Wuhan University and Springer-Verlag GmbH Germany 2018
Abstract: This paper describes a simple method of generating
concentration gradients with linear and parabolic profiles by using
a Christmas tree-shaped microfluidic network. The microfluidic
gradient generator consists of two parts: a Christmas tree-shaped
network for gradient generation and a broad microchannel for
detection. A two-dimensional model was built to analyze the flow
field and the mass transfer in the microfluidic network. The simu-
lating results show that a series of linear and parabolic gradient
profiles were generated via adjusting relative flow rate ratios of
the two source solutions ( RL2 ≥ 0.995 and RP2 ≥ 0.999) , which
could match well with the experimental results ( RL2 ≥ 0.987 and
RP2 ≥ 0.996) . The proposed method is promising for the genera-
tion of linear and parabolic concentration gradient profiles, with
the potential in chemical and biological applications such as com-
binatorial chemistry synthesis, stem cell differentiation or cyto-
toxicity assays.
Key words: tree-shaped network; concentration gradient; linear
profile; parabolic profile
CLC number: O 351
Received date: 2017-12-07
Foundation item: Supported by the National Natural Science Foundation of
China(81372358, 81527801, 51303140, and 81602489), the Natural Science
Foundation of Hubei Province (2014CFA029), the Colleges of Hubei Province
Outstanding Youth Science and Technology Innovation Team (T201305), and
the Applied Foundational Research Program of Wuhan Municipal Science and
Technology Bureau (2015060101010056)
Biography: SHEN Qilong, male, Master candidate, research direction: design
and preparation of microfluidic chip. E-mail:1271616978@qq.com
† To whom correspondence should be addressed. E-mail: ngzhang@wtu.edu.cn
Microfluidics has gained great attention and been
proven useful for a variety of chemical and biological
applications. As one of the most important functions,
concentration gradient plays an essential role in these
applications including chemotaxis[1-5] and cytotoxic-
ity [6-10] assays. However, there are only a few techniques
capable of generating and maintaining them with
well-defined profiles easily in solution. Techniques using
chemotaxis chambers[11, 12] or pipets[13] are limited in
their capability to generate gradients of different shapes
and maintain gradients over long periods of time. Stable
gradient profiles can be generated by using solutions
with different concentrations flowing at a constant flow
rate through a microchannel [14, 15]. This method is com-
pact and simple but can only generate characteristic sig-
moidal profile resulting from diffusion between two
streams, which has no ability to be adjusted for different
applications[16,17]. In some cases, it requires impractically
long microchannels to generate a gradient profile with
sufficient width. In addition, the shape of the gradient
profile depends on the position along the channel and on
the flow rate [18].
These drawbacks were effectively overcome in the
devices introduced in Refs. [19] and [20]. The key part
of their devices is a gradient-making network of miro-
channels that typically has a recognizable pyramidal
shape. While allowing for precise spatial control of the
gradients, the concentration profiles in these devices are
flow rate dependent, which could affect results in
SHEN Qilong et al : Generation of Linear and Parabolic Concentration …
cell-base assays[4]. An alternative gradient-making net-
work allowing generation of complex concentration pro-
files was proposed by Irimia et al [21]. However, it was
only shown to be operational at a very low flow rate (20
μm/s).
Recently, another approach was presented to gener-
ate linear or arbitrary concentration gradients profiles by
mixing two input streams by varying volumetric propor-
tions [8, 22,23]. Also, a series of dilution microfluidic de-
vices were proposed to generate logarithmic and linear
step-wise concentrations via adjusting the flow rates of
two converging fluids at the channel junctions of the
ladder network in Refs.[24] and [25]. The gradient pro-
files are not significantly affected by flow velocity, as
their design is predicated on the control of volumetric
flow rate. To design the proper ladder networks, a cali-
bration of channel lengths and widths was necessary to
define the exact flow resistances through computational
fluid dynamic (CFD) simulation[26]. However, this
CFD-assisted design would be a major barrier to wide-
spread use of these previously proposed dilution micro-
devices[27].
In this paper, we presented a Christmas tree-
shaped microfluidic network for generating linear and
parabolic concentration gradients profiles which could
be precisely controlled via adjusting relative flow rates
of the two source solutions in our Christmas tree-shaped
network. The whole process has been simulated using
COMSOL model software, which could match well with
experimentally validated data.
1 Experimental
1.1 Device Design
Figure 1 shows a photograph of the Christmas
tree-shaped microfluidic network used for generating
gradients. Similar to the microdevices introduced by in
Refs.[19] and [20], the microfluidic device consists of
two parts: a Christmas tree-shaped network for gradient
generation and a broad microchannel for observation. In
the gradient-making network, the oblique serpentine
channel (width 90 μm; height 40 μm; length 11.3 mm)
are connecting with the vertical serpentine channels
(width, 90 μm; height, 40 μm; length, 32.9 mm). And the
gradient-making network has two inlets. Both of them
were connected to syringes that contained solutions with
different concentrations. The two solutions were injected
simultaneously into the microfluidic network through the
245
individual inlets. They were repeatedly split, mixed, and
re-converged as the fluid streams flowed down the net-
work. The gradient-making network generated a series of
solutions with different concentrations at the terminals of
the microfluidic network. Then all the branches were
brought together into a broad channel (width 900 μm;
height 40 μm; length 8 mm), and a gradient was estab-
lished across this channel, perpendicular to the flow
direction.
Fig. 1 Optical microscopy image of the Christmas tree-like
microfluidic network visualized with red and green food
coloring solution
1.2 Microfabrication
A Polydimethylsiloxane(PDMS, Sylgard 184, Dow
Corning, Midland, USA) and glass microfluidic gradient
generator were fabricated using soft lithographic tech-
nique [28]. SU-8 2050 (MicroChem, Newton, MA, USA)
positive relief structures were prepared on 3-inch silicon
wafer (Luoyang Single Crystal Silicon, Henan, China).
PDMS pre-polymers (in the ratio of A:B=10:1) were
poured over the wafer and cured overnight at 75 ℃ in an
oven. PDMS layer was then cut and peeled from the silicon
wafer, and access holes for inlets and outlets were punched
on the PDMS slab. Surfaces of the PDMS slab and a mi-
croscope slide were treated with oxygen plasma (PDC-32G,
Harrick Plasma, NY, USA) to activate the surfaces for ir-
reversible sealing before they were bonded together, as
described previously. Then the microdevice was baked at
80 ℃ for 24 h in an oven to strengthen the bonding.
1.3 Imaging and Data Analysis
Food coloring (Safeway Inc., Pleasanton, CA, USA)
was used to visualize concentration profiles in bright-
field. Imaging was performed with a high-speed CCD
camera (DP72, Olympus, Tokyo, Japan) mounted on an
inverted microscope (IX71, Olympus, Tokyo, Japan).
 246
Images were captured at the observation region using IPP
software (Media Cybernetics Inc., Silver Spring, MD,
USA). In order to quantify concentration gradients, all
24-bit RGB images obtained in bright-field were trans-
formed into 8-bit gray images using ImageJ software
(National Institute of Mental Health, Bethesda, Maryland,
USA). Then the concentration of food coloring in mi-
cro-channels at observation region of microdevice was
evaluated by measuring the intensity of gray at that loca-
tion in the 8-bit gray image.
1.4 Numerical Simulating
An equivalent 2D model was established from the
Christmas tree-shaped network. Due to the low Reynolds
numbers(Re) of microfluidic operation (Re＜0.2), the
entire microfluidic system was governed by incom-
pressible Navier-Stokes equations and convection- diffu-
sion equation. Fluid flow and mass transfer within the
Christmas tree-shaped microfluidic network were mod-
eled using COMSOL Multiphysics 3.5 software
(COMSOL Inc., Burlington, MA, USA).
2 Result and Discussion
2.1 Laminar Flow-Based Concentration
Gradients Generation
Re can be described as Re = duρ/μ, where d, u, ρ
and μ are length scale, flow rate, density and dynamic
viscosity of the carrier fluid, respectively[29]. Laminar
flow occurs at low Re(＜2000), and the fluid dynamics
are dominated by viscous drag rather than by inertia. In
the microfluidic network described here, due to the low
Re of microfluidic operation Re(＜0.2), the flow streams
from different inlets easily formed laminar flow in the
microchannels. While two source solutions flowed
through the microfluidic network, they were repeatedly
split, mixed, and re-converged, and a series of solutions
with different concentrations were generated at the ter-
minals of the microfluidic network.
Firstly, one situation was investigated that the flow
rates of the two source solutions were equal. To predict
the profile of the concentration gradient at the outlet of
this Christmas tree-shaped microfluidic network, it is
necessary to know the relative ratio at which the streams
are mixed together in the long serpentine channels. The
mixing ratio is governed by the splitting ratio of the
streams at the each branching point in the networks. For
convenience, we define the part of the network that con-
tains n vertical serpentine channels as a branched system
of n-th order (B=n). Within each branched system of n-th
Wuhan University Journal of Natural Sciences 2018, Vol.23 No.3
order, we label each vertical serpentine channel (V), from
the left to the right, starting with V=0 and ending with
V=B-1 (Fig. 2).
Fig .2 Scheme of the nomenclature used for the mathemati-
cal description of the branched network
B was defined as the order of the branched system and V as the vertical channel
within the branched system
The flow resistance (R) is described by the follow-
ing equation in a rectangular channel with low aspect
ratio:
1
12  L h  192  1  nπw  
R 3
1   5  5 tanh   (1)
wh w  π n 1,3,5 n  2h  
where μ is the dynamic viscosity of the fluid, L, w and h
are the length, width and height of the channel, respec-
tively, as detailed by Walker et al[8]. In our microfluidic
network, according to Eq. (1), all vertical serpentine
channels have the same sizes and thereby have the same
resistance for the carrier fluid. In order to analyze the
splitting ratio of the streams at each branching point we
make an analogy with an equivalent electronic circuit.
The flow resistance of the vertical serpentine channels is
the same within our branched system. Therefore, the flux
of fluid through each vertical serpentine channel of a
branched system is equal. Due to the vertical mirror
symmetry of the microfluidic network, the splitting ratios
are symmetric with respect to the axis of symmetry. By
combining all boundary conditions, we selected the fol-
lowing recursive Eqs. (2) and (3) governing the splitting
ratios at all of these branching points as pre-derived in
Refs. [19] and [20].
The portion of the flow stream that turns left is
governed by
( BV  V ) / B (2)
The portion of the flow stream that turns right is
governed by
(V  1) / B (3)
Here B is the order of the branched system and V is
SHEN Qilong et al : Generation of Linear and Parabolic Concentration …
the vertical channel within the branched system. As
shown in Fig. 3, the two neighboring oblique channels
share a vertical serpentine channel of the following
branched system; and the two neighboring vertical
channels at lower order (n) accept streams from three
channels at upper order (n+1). Suppose concentrations of
the incoming streams are Cb, Cc and Cd, and those of the
combining streams are Cm (left) and Cn (right). Accord-
ing to Eqs. (2) and (3), the concentrations at the end of
the vertical serpentine channels (Cm, Cn) can be calcu-
lated by multiplying the concentration of the incoming
streams (Cb, Cc, Cd) with the corresponding numbers of
the splitting ratio ((Vb+1)/B, (B-Vc)/B, (Vc+1)/B and
(B-Vd)/B, as indicated in Fig. (3). As a result, the con-
centrations of solution in lower order channels were
Cm  Cb * (Vb  1) / B  Cc * ( B  Vc ) / B (4)
Cn  Cc * (Vc  1) / B  Cd * ( B  Vd ) / B
Fig. 3 Schematic demonstrating application of the formulas
governing the splitting ratios at the branching points
The dotted lines indicate the boundary between the two combined streams; the
resulting concentrations can be calculated by Eq.(4) (left) and Eq.(5) (right)
In the Christmas tree-shaped microfluidic network
with two inlets and five terminals, supposing one inlet
contains solution of concentration C, the other contains
blank solution of concentration 0. According to Eqs. (4)
and (5), a series of concentrations at the terminals of the
branched system were predicted to be 0, C/4, C/2, 3C/4
and C.
Secondly, the other situation was investigated that
the flow rates of the two source solutions were not equal.
In this case, the mixing ratio is still governed by the
splitting ratio of the streams at the each branching point
in the networks, but it is not applicable to use the recur-
sive Eqs. (2) and (3) to govern the splitting ratios at all of
these branching points. To predict the profile of the con-
centration gradient at the outlet of this Christmas
tree-shaped microfluidic network, we numerically simu-
lated the obtainable concentration gradient profiles. In
this case, we found empirically that all calculated con-
centration gradient profiles generated by the network
having n inlets lie on a curve described by a polynomial
of n-th order (a polynomial of n-th order is defined by
247
the following: f ( x)  a0  a1 x  a2 x 2    an x n )[20]. In
other words, parabolic concentration gradients could be
generated via adjusting relative rate ratios of the two
source solutions in the Christmas tree-shaped microflu-
idic network.
2.2 Generation of Linear Gradients
Food coloring (green) was used to visualize con-
centration gradient profiles. De-ionized (DI) water was
added to one inlet and food coloring solution of low
concentration was added to the other inlet. Food coloring
and DI water flowed through microfluidic network at a
series of flow rate (Qw = Qd) of 30, 60, 90, 120 and 150
μL/h and imaged after equilibration was established, re-
spectively (Fig. 4(a)). All 24-bit RGB images obtained at
observation region were transformed into 8-bit gray im-
ages by using ImageJ software (Fig. 4(b)). Then the con-
(5) centration of food coloring was evaluated by measuring
the intensity of gray at that location in the 8-bit gray im-
age. The results demonstrated that the microfluidic net-
work generated nearly linear dilutions at flow rates of 30,
60, 90, 120 and 150 μL/h with fitting parameters RL2 =
0.987, 0.994, 0.997, 0.997 and 0.997, respectively in
Fig. 4(c). When all the diluted solution branches were
brought together into a broader channel, linear concen-
tration gradients were established across this channel,
perpendicular to the flow direction.
The linear fitting parameters RL2 ( RL2 ≥ 0.987) in-
dicated that this microfluidic network created linear
concentration gradient profiles over a range of flow rates.
The experimental results are very close to the theoretical
values in Fig.4(c). As long as complete diffusive mixing
is ensured in all vertical serpentine channels of the mi-
crofluidic network, the flow rate in the network does not
affect the concentration gradient profiles generated in the
broad channel.
2.3 Generation of Parabolic Gradients
We fixed the flow rate of the food coloring solution
(Qd = 60 μL/h) and varied the flow rate of the DI water
(Qw = 40, 50, 60, 72 and 90 μL/h). All gradient profiles
were imaged at the observation region after equilibration
was established, respectively (Fig. 5(a) and (b)). The
results demonstrated that the microfluidic network gen-
erated a series of parabolic concentration gradients when
Qd was fixed as 60 μL/h and Qw as a series of value 40,
50, 60, 72 and 90 μL/h with fitting parameters RP2 =
0.998, 0.996, 0.996, 0.999 and 0.999, respectively, as
shown in Fig. 5(c).
 248
Fig. 4 Generation of linear concentration profiles by food
coloring (green)
(a) Optical microscopy images (24-bit RGB) of concentration gradients at flow
rates (Qw = Qd) of 30, 60, 90, 120 and 150 μL/h; (b) Gray images (8-bit) using
transformed from optical images using ImageJ software; (c) Relative gray value
in the terminating channels showing linear ( RL2 ≥ 0.994) concentration gradient
entering the downstream broad channel at different flow rates
The parabolic fitting parameters RP2 ( RP2 ≥ 0.996)
indicated that concentration gradients with parabolic
profile could be easily generated by varying the relative
flow rate ratios of the two source solutions. Consistent
with our empirical finding, all profiles of concentration
gradient generated in the broad channel could be de-
scribed by polynomials of second order. In all cases, fit-
ting curves lines and experimentally observed concentra-
tions dots were in good agreement(Fig. 5(c)). Therefore,
the advantage of this microfluidic device over others is
that the profiles of concentration gradient can be con-
trolled over relative flow rates difference of the two
source solutions in our Christmas tree-shaped microflu-
idic network.
2.4 Numerical Simulating Result
Due to the low Re of microfluidic operation (Re＜
0.2), the entire microfluidic system is governed by in-
compressible Navier-Stokes equations and convec-
tion-diffusion equation.
First, the incompressible Navier-Stokes Eqs. (6) and
(7) were solved for the carrier liquid to obtain the steady
state flow field.
u  0 (6)
 (uu)  p   2 u (7)
Wuhan University Journal of Natural Sciences 2018, Vol.23 No.3
Fig.5 Generation of parabolic concentration profiles by food
coloring (green)
(a) Optical microscopy images (24-bit RGB) of concentration gradients when
Qd was fixed as 60 μL/h and Qw as a series of value 40, 50, 60, 72 and 90 μL/h;
(b) Gray images (8-bit) using transformed from optical images using ImageJ
software. (c) Relative gray value in the terminating channels showing parabolic
( RP2 ≥ 0.996) concentration gradient entering the downstream broad channel
where u is the velocity vector, ρ the density, μ the dy-
namic viscosity of the carrier liquid, and p denotes pres-
sure at a given point within the fluid domain.
Then the flow field was fixed and the
time-dependent simulation of the convection-diffusion
equation (8) was performed.
( Dc)  u  c (8)
where u is the velocity vector of the carrier liquid, D is
the diffusion coefficient, and c the concentration of the
solute within the fluid domain.
DI water was chosen as the modeled fluid with a
dynamic viscosity of 1×10–3 Pa·s and a density of 1×103
kg/m3 as well as a fluorescent dye (Rhodamine 6G, Mw =
479.01) solution as the modeled species with a diffusive
coefficient of 2.8×10–10 m2/s. As the boundary conditions,
a fully developed laminar flow condition was imposed
on the inlets with a mean velocity corresponding to the
inlet flow rate. No-slip boundary conditions and zero
pressure conditions were set on the walls and at the out-
let, respectively. The modeled solutions were introduced
into our microfluidic network from the two inlets with
concentration of 0 and 1.44×10–8 mol/m3, respectively.
In the first group of numerical simulations, the re-
SHEN Qilong et al : Generation of Linear and Parabolic Concentration …
sults demonstrated that the microfluidic network gener-
ated very linear dilutions at flow rates (Qw = Qd) of 30,
60, 90, 120 and 150 μL/h with fitting parameters RL2 =
0.999, 0.999, 0.998, 0.997 and 0.995, respectively. Fig-
ure 6(a) shows the concentration profile generated when
the DI water and Rhodamine 6G solutions are introduced
into the modeled network with the flow rate of 60 μL/h.
Therefore, according to simulation results (RL2 ≥ 0.995),
a series of nearly linear profiles of concentration gradi-
ents could be easily generated when the flow rates of the
two source solutions are equal (Fig. 6(b)).
Fig. 6 Numerical simulating of the concentration distribution
with linear concentration gradient
(a) Computed concentration distribution in the Christmas tree-like microfluidic
network at time of t＝200 s when the flow rates of the two inlets are equal (Qw=
Qd = 60 μL/h) using COMSOL simulation; (b) Concentration in the terminating
channels showing linear gradient (RL2 ≥ 0.995) profiles at a series of flow
rates (Qw = Qd) of 30, 60, 90, 120 and 150 μL/h by COMSOL simulation
In the second group of numerical simulations, Qd
was fixed as 60 μL/h and Qw as a series of value 40, 50,
60, 72 and 90 μL/h, the results demonstrated that the mi-
crofluidic network generated a series of parabolic con-
centration gradients with fitting parameters RP2 =0.999,
0.999, 0.999, 0.999 and 0.999, respectively. Figure 7(a)
shows the concentration profile generated when the DI
water and Rhodamine 6G solutions are introduced into
the modeled network with the flow rates of 60 μL/h and
90 μL/h, respectively. As a result, according to simula-
tion results (RP2 ≥ 0.999), a series of nearly parabolic
profiles of concentration gradients could be generated
via adjusting the relative flow rate ratios of the two
source solutions (Fig. 7(b)).
249
In summary, the gradient simulations indicate that
by varying flow rates of DI water and modeled species
solution, both linear and parabolic concentration gradient
profiles can be easily built up. Furthermore, the shape of
gradient profile can be controlled over relative flow rates
of the two source solutions of different concentrations
using our Christmas tree-shaped microfluidic network.
Fig. 7 Numerical simulating of the concentration distribution
with parabolic concentration gradient
(a) Computed concentration distribution in the Christmas tree-like microfluidic
network at time of t＝200 s when the flow rates of the two inlets are not equal
(Qw=90 μL/h, Qd= 60 μL/h) using COMSOL simulation; (b) Concentration in
the terminating channels showing parabolic (RP2 ≥ 0.999) gradient profiles
when Qd was fixed as 60 μL/h and Qw as a series of value 40, 50, 60, 72 and 90
μL/h by COMSOL simulation
3 Conclusion
A microfluidic device with Christmas tree-shaped
microfluidic network was designed and fabricated. The
network design has been analytically modeled and ex-
perimentally validated using an optical detection system.
COMSOL-Multiphysics simulation and flow experiment
were performed to analyze the flow field and mass
transfer within this microfluidic network. The approach
is experimentally simple and highly adaptable. Gradients
of different shapes (linear and parabolic), types (smooth
and step), and kinds (static and dynamic) can be obtained
by varying the relative flow rate ratios of the two source
solutions. The simply structured microfluidic network
presented in this study would be widely used for bio-
logical or chemical applications, such as high throughput
cell-based screening, long-term cell growth study and
 250
chemotaxis studies in the future.
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