Tunable and quantitative serial dilution on multi-channel miniaturized microfluidic electrochemical platform

Accepted Manuscript Title: Your article is registered as a regular item and is being processed for inclusion in a regular issue of the journal. If this is NOT correct and your article belongs to a Special Issue/Collection please contact [email protected] immediately prior to returning your corrections.–>Tunable and quantitative serial dilution on multi-channel miniaturized microfluidic electrochemical platform Authors: Hao Wan, Heyu Yin PII: DOI: Reference:

S0925-4005(18)31429-1 https://doi.org/10.1016/j.snb.2018.08.003 SNB 25150

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

21-5-2018 30-7-2018 1-8-2018

Please cite this article as: Wan H, Yin H, Tunable and quantitative serial dilution on multi-channel miniaturized microfluidic electrochemical platform, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tunable and quantitative serial dilution on multi-channel miniaturized microfluidic electrochemical platform Hao Wan12*, Heyu Yin2 1

Key Laboratory for Biomedical Engineering of Ministry of Education, Biosensor National Special

Laboratory, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, China, Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, 48823, USA.

SC R

Corresponding author: [email protected]

IP T

2

M

A

N

U

Graphical abstract

ED

Tunable and quantitative serial dilution on a multichannel microfluidic electrochemical platform

PT

Highlights for review

This work implements tunable and quantitative serial dilution on a multichannel microfluidic electrochemical platform.



The electrochemical platform can continuously share and dilute samples from prior channels, which effectively reduces sample cost with only one outlet required.

A

CC E





The miniaturized electrochemical platform was established with microfabrication process and tested to validate its performance.

Abstract:

Electrochemical technique has been widely applied in trace analysis due to high sensitivity, low detection limit, low time cost and simple instrumentation. Precise concentration gradients of biomolecules and chemicals play significant roles in electrochemical measurements for disease diagnosis, environmental monitoring and healthcare. Conventional approaches to generating gradients are generally complicated and time-cost with unpredictable and uncontrollable profiles over time and space. This paper presents a multi-channel miniaturized microfluidic electrochemical platform featuring tunable and quantitative serial dilution by integrating with microfluidic device and

IP T

multichannel electrochemical chip. By continuously sharing and diluting samples from prior channels,

this platform can effectively reduce sample cost with only one outlet required. To implement serial

and quantitative dilution on the microfluidic device, the microfluidic structure was analogous to an

SC R

electrical circuit and designed accordingly. The function of tunable and quantitative serial dilution was validated with COMSOL simulation. Both the microfluidic device and the multichannel electrochemical chip were fabrication by standard photolithography microfabrication. This

U

miniaturized electrochemical platform was achieved by the alignment and bonding of the microfluidic device and the electrochemical chip. The platform with four independent channels was tested in

N

potassium ferricynide/ferrocynide to validate the performance and serial dilution functionality in

A

electrochemical sensing. Experimental results were demonstrated and well matched simulation results. The platform featuring tunable and quantitative serial dilution provides a promising approach to

M

generating precise and tunable gradients that can enable fast calibration in electrochemical sensing.

ED

Key words: Lab on chip, tunable serial dilution, electrochemical chip, microfluidic device, multi-

A

CC E

PT

channel

2

1

Introduction

Various biomolecules and chemicals are required to be precisely detected in many applications such as disease prevention and diagnosis, environmental monitoring and healthcare [1,2]. During quantitative measurements of these biomolecules and chemicals, accurate concentration gradient plays a significant role for sensor calibration and sample quantification. However, conventional in vitro approaches such as biological hydrogel gradients [3,4], micropipette-generated gradients [5] and Zigmond chambers [6] are not ideal to generate accurate, quantitative and user-defined concentration

IP T

gradients with tailored spatial and temporal profiles [7]. Chemical gradients by mixing and diluting with different compositions are extremely complicated and time-cost with unpredictable or uncontrollable profiles over time and space [1,8]. Also, low efficient repetition work is needed in order

SC R

to generate new concentration gradients, which largely limits the productivity and timeliness in

applications. An efficient, tunable and quantitative approach that can produce precise concentration gradients is of great significance.

U

Over the past decades, lab-on-a-chip (LoC) platforms have attracted great interests in many applications for biosensing and chemical sensing due to their outstanding performance in measurement

N

time, portability, flexibility and cost [9,10]. Microfluidic device leads to well-defined laminar flow,

A

controllable diffusion and easy implementation for massively parallel analysis [11]. Therefore, by utilizing the merits of microfluidic devices, quantitative, reproducible and well-profiled concentration

M

gradients can be easily achieved. Many related work such as serial mixing [12,13], logarithmic diluter [14,15] and diffusion based gradients [16] has been reported to implement controllable gradients.

ED

On the other hand, many sensing technologies such as optical [17,18], colorimetric [19] and electrochemical [20–22] have been utilized with microfluidic devices for precise and quantitative

PT

measurements . Among these approaches, electrochemical method offers superior capability in high sensitivity, good selectivity, low power consumption and easy miniaturization [23–25]. Also, electrochemical sensors can be miniaturized with the integration of three-electrode system by standard

CC E

microfabrication processes [26], which are very compatible with microfluidic device fabrication. By integrating microfluidic device with electrochemical sensors, this electrochemical platform can provide an effective approach for electrochemical sensing of different substances in various

A

applications.

Though microfluidic devices enable controllable and predictable gradients for sample analysis, most

work only implements linear and logarithmic gradient profiles due to the limitations of their microfluidic device design [15][27]. Seldom works provide effective approaches to generate tunable gradients. Kangsun et al. reported a generalized serial dilution module that could achieve precise linear and non-linear concentration gradients [8]. However, independent fluidic passages were designed without sharing any fluids that lead to multiple outlets and waste of samples. Herein, we present a 3

multi-channel miniaturized microfluidic electrochemical platform with tunable and quantitative serial dilution. By continuously sharing and diluting samples from prior channels, this platform can effectively reduce sample cost with only one outlet required. The electrochemical platform was integrated with an elaborately designed microfluidic device and an electrochemical chip. An analogous model was constructed for microfluidic design, and the featuring of tunable and quantitative dilution of the proposed structure was verified by simulations. The electrochemical platform was built with microfabrication and tested in potassium ferricynide/ferrocynide to validate the performance and

2

IP T

tunable serial dilution in electrochemical sensing.

Experimental Design of the electrochemical platform

SC R

2.1

The electrochemical platform is comprised of a microfluidic device and a four-channel electrochemical chip as shown in Fig. 1. The four-channel chip enables independent electrochemical measurements in each channel. Each channel contains a working electrode (WE), a counter electrode

U

(CE) and a reference electrode (RE) that can conveniently implement electrochemical measurements.

N

The microfluidic device consists a sample inlet and a buffer inlet, in which the fluid in the buffer inlet mixes with samples for dilution. The buffer inlet divides into three branches and connects to different

A

channels of the electrochemical chip. Four channels are connected by microfluidic passages to share

M

samples from prior channels. By adjusting parameters of the microfluidic device, tunable and quantitative serial dilution can be achieved in different channels, thus enabling the measurement of

A

CC E

PT

ED

samples with different concentrations in only one platform.

Figure 1 The schematic of the electrochemical platform with tunable and quantitative serial dilution 2.2

Schematic design of the microfluidic device

To achieve serial dilution in the microfluidics, inlets for samples and buffer solutions, one outlet and mixers are required. For a four-channel device, the buffer solution needs to be split into three branches to mix with samples, thus diluting samples into three different concentrations. A schematic of the 4

multi-channel microfluidic for serial dilution is shown in Fig. 2. Two inlets for sample and buffer solution, one outlet for waste disposal, three mixers for fluidic mixing and four chambers are included in the microfluidic structure. Microfluidic mixers should enable a sufficient mixing between sample and buffer solution. Three branches are split from the buffer inlet, and different branches flow into different mixers for mixing and diluting. Different branches should be elaborately designed to achieve

N

U

SC R

IP T

the desired serial dilution ratio in different working chambers.

Fig. 2. A schematic of the multichannel microfluidic device for serial dilution. Methods for tunable and quantitative serial dilution

A

2.3

M

Tunable serial dilution in different working chambers is implemented by carefully adjusting the volumetric flow rate in different buffer branches. Thus, the tunable dilution ratio can be achieved after

ED

mixing the sample and the buffer solution with different volume ratio. Since the volumetric flow rate in microfluidics is controlled by the hydrodynamic resistance of microchannels, the hydrodynamic resistance can be analogous to the resistance in an electric circuit [28]. The volumetric flow rate acts

PT

as the current, and the pressure difference acts as the voltage. Since inflows in buffer inlet and buffer inlet are realized by a syringe pump with constant inflow rate, the inflows act as current sources. Fig. 3

CC E

shows the analogous circuit for the overall microfluidic system presented in Fig. 2. According to the Kirchhoff’s circuit law, the current relationship can be described as: (1)

ik+4Rk+4=ik+3Rk+3+ikRk, k=2, 3;

(2)

A

ik+1 =ik+ik+4, k=1, 2, 3;

where Rk indicates the hydrodynamic resistance in different microfluidic sections, and ik is the current flowing through Rk. To dilute solutions into a specific ratio, the current must be accurately designed based on the dilution ratio αi in four different working chambers: α1 = 1;

α2 = i1/(i1+i5);

α3 = i1/(i2+i6);

α4 = i1/(i3+i7)

Thus, the relationships between currents and dilution ratio can be described as: 5

(3)

i2=(1/α2)i1;

i3=(1/α3)i1;

i4=(1/α4)i1;

i5=(1/α2-1)i1;

i6=(1/α3-1/α2)i1; i7=(1/α4-1/α3)i1. (4)

The resistances of three branches can be directly corrlated to dilution ratios based on Equation 2. By adjusting the length of three brances to change the resistance, the tunable dilution ratio in different working chambers can be easily achieved. It is worth noting that this design strategy can also be applied in more channels with tunable and quantitative serial dilution. In that case, more working chambers and branches from the buffer inlet should be designed accordingly. The principle to implemeting tuanalbe serial dilution in more channels can be still analogous to an electric circuit, and

IP T

the relationship between different length of different branches can be deduced using the Kirchhoff’s

ED

M

A

N

U

SC R

circuit law.

Fig. 3. The analogous circuit for the multichannel microfluidic device. 2.4

Design and fabrication of the microfluidic device and electrochemical chip

PT

The microfluidic device provides four channels for serial dilution and multichannel measurements. According to the schematic in Fig. 2, two inlets, one outlet, working chambers and mixers should be

CC E

contained in the device. Mixers enable efficient mixing between samples and buffer solutions. Three branches were divided from the buffer inlet to implement tunable serial dilution. The length of three branches is determined to implement different dilution ratios according to Equation 2. Therefore, the structure of the microfluidic device is shown in Fig. 4. Serpentine mixers were designed for mixing,

A

and different length of three branches was designed to achieve serial dilution in different channels. Four-channel electrochemical sensors should be realized to integrate with the microfluidic device. In

each channel, three electrodes were integrated to miniaturize the sensor size. Disk electrodes were designed with 400 µm diameter of the WE. The diameter of the whole disk sensor is 800 µm that can be well integrated with the working chamber with a diameter of 1 mm. Four sensors were placed in line to match the structure of the microfluidic device. 6

IP T

SC R

Fig. 4 The structure design of the microfluidic device (blue) and the multichannel electrochemical chip (yellow)

Standard photolithography was used to fabricate the microfluidic device. Briefly, a 2 inch silicon

U

wafer was used as the substrate for the microfluidic mold fabrication. Photoresist SU-8 2075 was spincoated on the wafer, and the wafer was baked on a hot plat. UV exposure was utilized to pattern the

N

microfluidic structure on the photoresist. After subsequent baking, the wafer was immersed in SU-8

A

developer for developing. The wafer should be rinsed using fresh SU-8 developer and IPA after the immersion. The microfluidic mold was obtained after cleaning with IPA, deionized water and N2. The

M

mold was then fixed in a petri dish using a double-side tape. About 30 mL PDMS mixture (base and curing agent 10:1) was poured into the petri dish and placed in vacuum for degasing. The PDMS

ED

mixture was baked at 80°C for 1 hour for curing. Thus, the PDMS-made microfluidic device can be obtained by slicing and hole-punching.

PT

Photolithography was also used to fabricate the multichannel electrochemical chip on a 4 inch glass wafer, and the fabrication procedures were briefly introduced. The glass wafer was first cleaned using acetone, IPA and deionized water. A prebake at 115°C was performed for 2 min to remove moisture

CC E

on the wafer. Photoresist S1813 was spin-coated on the wafer with 4000 rpm for 45 s. After softbaking on a hot plat at 115°C for 1min, UV exposure was conducted for electrode patterning. The wafer was then developed by the developer 352 and cleaned using deionized water and N2. Thermal evaporation was employed for metal deposition. Ti with a thickness of 3 nm and Au with a thickness

A

of 50 nm were deposited on the patterned wafer. The wafer was then immersed in acetone for 2 hours, and an ultrasonic cleaning was used to achieve full lift-off. After rinsing with acetone, IPA and deionized water, the multichannel electrochemical chip was obtained for further study.

7

3

Results and discussion 3.1

Simulation of microfluidic components

COMSOL 5.2 laminar flow module was used to design and simulate the microfluidic device. Factors including the working chamber shape, channel connection and mixer were optimized to ensure efficient fluidic exchange and mixing. In the simulation model, the size of the chambers was 1000 µm in diameter, the inlet velocity was 0.02 m/s, the outlet pressure was zero and the fluidic material was water. Different working chamber shapes including circle, ellipse and square were studied to evaluate

IP T

their impacts to the velocity distribution. The surface velocity profile of the working chamber for

different shapes and channel connections is shown in Fig. 1S (a)-(c). By calculating the low fluid exchange area (less than 5% of the inlet velocity), the circle-shaped working chamber was chosen for

SC R

further microfluidic design, which shows smallest low fluidic exchange area that enables fastest fluidic exchange. The channel connection was also optimized with different placement angle (Fig. 1S (a) and (d)), and a channel placement angle of 0° was chosen for further design that could facilitate rapid

U

fluidic exchange in the WE area. Since the sample should be sufficiently mixed with the buffer solution for subsequent measurements, a mixer should implement effective fluidic mixing in the

N

microfluidic device. A serpentine mixer was designed and simulated using COMSOL, and the

A

concentration profile after mixing is shown in Fig. 2S. The variation of sample concentration in the outlet is around ±4%, indicating good mixing performance of this mixer. Thus, this mixer was used in

Simulation of tunable and quantitative serial dilution

ED

3.2

M

the microfluidic device to implement efficient mixing between the sample and the buffer solution.

To validate the effectiveness of the analogous circuit for microfluidic design, two microfluidic structures were designed and simulated. A microfluidic structure that implements two-fold serial

PT

dilution in four channels was designed. In this case, the dilution ratios in four channels are 1, 1/2, 1/4

CC E

and 1/8. According to the above principles, the currents should be: i2=2i1; i3=4i1; i4=8i1; i5=i1;

i6=2i1; i7=4i1.

(5)

The resistance relationship of the three branches is thus: (6)

R7=1/2R6+R3;

(7)

A

R6=1/2R5+R2;

Considering the linear relationship between hydrodynamic resistance and the microchannel length, the lengths of the three branches (R5, R6 and R7) were set as 5.01 mm, 11.98 mm and 16.3 mm that meet the equations. In the initial conditions of the simulation, the flow velocity of buffer inlet and sample inlet was set to 0.2 and 1.4 mm/s, respectively. The concentration was set to 6 mM for the sample and 0 for the buffer solution. The simulated concentration profile is shown in Fig. 5(a). Apparently, 8

different channels contain diluted samples with different concentrations due to the mixing between the sample and the buffers in different branches. The concentration profile of each working chamber is shown in Fig. 5(b). With the designed branch lengths, the average concentration in the four working chambers was simulated to be 6.00 mM, 2.92 mM, 1.48 mM and 0.74 mM. Compared to the desired dilution ratios, the relative error is 2.67%, 1.33% and 1.33%, denoting well-matched simulated results to the theoretical results. Since two-fold dilution can be easily implemented by conventional microfluidic devices [29], another

IP T

microfluidic structure was designed and simulated, in which the dilution ratios in different channels

were 1, 2/3, 1/3 and 1/6. Based on the above principles, the currents should be i2=1.5i1; i3=3i1; i4=6i1; i5=0.5i1; i6=1.5i1; i7=3i1. The resistance relationship of the three branches is thus:

SC R

R6=1/3R5+R2; R7=1/2R6+R3;

(5)

(6).

Therefore, the lengths of three branches were adjusted to 5.01 mm, 12.82 mm and 16.72 mm. The inlet

U

velocity of the buffer solution was set to 1.0 mm/s, and the other initial conditions remained

N

unchanged. The simulated concentration profile of the microfluidic structure is shown in Fig. 4(c), and the concentration in four channels is shown in Fig. 5(d). Compared to Fig. 5(a), the concentration

A

profile in Fig. 5(c) is apparently different due to adjusted branch lengths and inflow velocity of the

M

buffer solution. The averaged concentrations in four working chambers were simulated to be 6 mM, 3.89 mM, 1.97 mM and 0.99 mM. Compared to the desired dilution ratios, the relative error is 2.75%,

ED

1.50% and 1.00%. This result indicates that the microfluidic system can achieve desired the dilution ratios in different chambers by carefully designing the length of all branches. It is worth noting that

A

CC E

PT

this design can be extended to more chambers to achieve different dilution ratios in each chamber.

9

IP T SC R U

Fig. 5 Concentration profile of the multi-channel microfluidic device with two different dilution ratios.

N

(b) and (d) present the concentration distribution in each working chamber with two different

Device assembly and electrochemical measurements

M

3.3

A

structures.

To validate the simulation results, a microfluidic device according to Equation 5 and 6 was fabricated

ED

and assembled with a multichannel electrochemical chip to construct the platform. Plasma treating was utilized to assemble the microfluidic device and the multichannel electrochemical chip. The device surface and the chip surface were exposed to O2 plasma with a power of 100 mW for 100 s. After

PT

alignment and bonding between the device and the chip, the assembled platform was baked in an oven at 80°C for 30 min to achieve very solid bonding. To implement multichannel measurements, a PCB

CC E

was also designed for convenient electrical connection. The assembled electrochemical platform on PCB is shown in Fig. 6(a). This platform features compact structure design integrating with a microfluidic device, an electrochemical chip and a PCB that enables tunable serial dilution and multichannel measurements. The zoomed electrode arrays are shown in Fig. 6(b), in which the gold

A

electrode arrays well fit in the working chambers. The branch structure of the microfluidic device is shown in Fig. 6(c), and well-defined microfluidic channels can be observed.

10

IP T

Fig. 6 (a) The assembled electrochemical platform; (b) zoomed electrode arrays in microfluidic

SC R

chambers; (c) zoomed microfluidic channels

The electrochemical platform was tested in K3[Fe(CN)6]/ K4[Fe(CN)6] to evaluate its electrochemical performance and verify the function of quantitative serial dilution. To verify the electrochemical

U

performance of four electrochemical channels, each channel was tested with different scan rates (SR) using cyclic voltammetry. Fig. 7(a) shows the voltammograms of four channels in 1 mM K3[Fe(CN)6]/

N

K4[Fe(CN)6] with a SR of 50 mV/s. Well-defined peak-shaped voltammograms can be clearly

A

observed, and the oxidation peak of [Fe(CN)6]3- and reduction peak of [Fe(CN)6]4- are presented. Though four sensors in different channels were simultaneously fabricated under the same fabrication

M

process and conditions, four sensors present slightly different electrochemical performance. The plausible reason can be ascribed to slight deviation due to different locations of four sensors that lead

ED

to different sensor surface. To further validate the sensor performance, all channels were tested with different SRs. All channels presented similar response to different SRs, and the cyclic voltammograms of channel 1 in 4 mM K3[Fe(CN)6]/ K4[Fe(CN)6] are shown in Fig. 7(b). With the increasing of SR

PT

from 10 mV/s to 50 mV/s, both oxidation peak and reduction peak increase accordingly. The peak currents of oxidation and reduction reaction were extracted, and the plot of peak currents vs. SR is

CC E

shown in Fig. 7(c). The amplitude of reduction currents is very close to that of oxidation currents, indicating good reversibility of the reaction and good performance of the electrochemical sensors for

A

multichannel measurements.

11

IP T

Fig. 7 The electrochemical performance of four channels on the electrochemical platform using CV

scanning: (a) performance comparison of four channels; (b) CV results of channel 1 with different SRs;

3.4

SC R

(c) the calibration of peak currents during oxidation and reduction. Serial dilution

The quantitative serial dilution of this electrochemical platform should be verified. Since different

U

channels present slightly different electrochemical performance, each channel should be individually calibrated. Thus, all channels were tested in K3[Fe(CN)6]/ K4[Fe(CN)6] (0.1 M KCl) with

N

concentrations of 1 mM, 2 mM, 4 mM and 6 mM. The CV response of channel 1 in different

A

concentrations is shown in Fig. 8(a). Well-defined voltammograms can be apparently observed, and both reduction peak current and oxidation peak current increase with the increasing of concentrations.

M

Peak currents of both oxidation and reduction reaction were extracted for calibration as shown in Fig. 8(b). Both calibrations indicate very good sensitivity and linearity for electrochemical sensing. The

ED

calibration of oxidation peak current was used for quantitative measurements of [Fe(CN) 6]4-. To implement the serial dilution of the electrochemical platform, the inflow rate of sample solution (6 mM) and buffer solution (0 mM) is 12 µL/min and 60 µL/min, respectively. Since serial dilution was

PT

implemented in channel 2, 3 and 4, the three channels were measured and calculated using the above calibration equations. The calculated concentrations in the three channels and the comparison with

CC E

theoretical values and simulation values are shown in Table 1. The test values are quite close to both simulation values and theoretical values, and the relative errors compared to theoretical values are quite small. These results validate that the performance of the designed electrochemical platform well matches the simulation results. Also, tunable and quantitative serial dilution in different channels can

A

be easily achieved by adjusting the length of different branches.

12

IP T

Fig. 8 The electrochemical response of channel 1 for potassium ferricynide/ferrocynide test with

SC R

different concentrations (a) and the calibration of peak currents during reduction and oxidation (b).

dilution

3.89 1.97 0.99

M

4.00 2.00 1.00

N

Concentration (mM)

Relative error No dilution -2.75% -1.50% -1.00%

Test values Concentration (mM)

Relative error

3.72 1.82 1.12

-7.00% -9.00% 12.0%

ED

Channel 1 Channel 2 Channel 3 Channel 4

Simulation values

A

Theoretical values Concentration (mM)

U

Table 1 The theoretical values, simulated values and calculated values in four channels after serial

PT

Conclusion

This work achieves tunable and quantitative serial dilution based on a multi-channel miniaturized electrochemical platform. The structure of the microfluidic channels was well designed and validated

CC E

using simulation results. The tunable and quantitative serial dilution of the platform was also achieved by adjusting the length of different branches. Effective microfluidic model was constructed, and two simulation structures were demonstrated to implement tunable serial dilution with different ratios. An electrochemical platform featuring quantitative serial dilution was fabricated based the simulation

A

structure, and electrochemical measurements were performed in potassium ferricynide/ferrocynide. The results validate the good electrochemical performance and quantitative serial dilution in different channels. The platform featuring tunable and quantitative serial dilution provides a promising approach to generate precise and tunable gradients that can enable fast calibration in electrochemical sensing. Moreover, in cell-based biosensing applications, gradient drug and toxin with different concentrations should be prepared to evaluate the efficacy and toxicity to cells [30-32]. The function 13

of tunable and serial dilution of the platform also provides an effective way to implementing convenient gradient dilution for biosensing.

Conflicts of interest There are no conflicts of interest to declare.

Acknowledgment

IP T

This work is supported by the Fundamental Research Funds for the Central Universities (No. 2018QNA5018, 2018FZA5018).

[1]

SC R

References

T. M. Keenan, and A. Folch, Biomolecular gradients in cell culture systems, Lab Chip. 8 (2008) 34–57.

[2]

W. Saadi, S. W. Rhee, F. Lin, B. Vahidi, B. G. Chung, and N. L. Jeon, Generation of stable

U

concentration gradients in 2D and 3D environments using a microfluidic ladder chamber,

S. Sant, M. J. Hancock, J. P. Donnelly, D. Iyer, and A. Khademhosseini, Biomimetic gradient

A

[3]

N

Biomed. Microdevices. 9 (2007) 627–635.

[4]

M

hydrogels for tissue engineering, Can. J. Chem. Eng. 88 (2010) 899–911. B. Heit, S. Tavener, E. Raharjo, and P. Kubes, An intracellular signaling hierarchy determines

[5]

ED

direction of migration in opposing chemotactic gradients., J. Cell Biol. 159 (2002) 91–102. M. Postma, and P. J. M. Haastert, Mathematics of Experimentally Generated Chemoattractant Gradients, in: Humana Press, Totowa, NJ, 2009: pp. 473–488. S. H. Zigmond, Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic

PT

[6]

factors., J. Cell Biol. 75 (1977) 606–16. C.-G. Yang, Y.-F. Wu, Z.-R. Xu, and J.-H. Wang, A radial microfluidic concentration gradient

CC E

[7]

generator with high-density channels for cell apoptosis assay, Lab Chip. 11 (2011) 3305.

[8]

K. Lee, C. Kim, B. Ahn, R. Panchapakesan, A. R. Full, L. Nordee, J. Y. Kang, and K. W. Oh,

A

Generalized serial dilution module for monotonic and arbitrary microfluidic gradient

[9]

generators, Lab Chip. 9 (2009) 709–717. M. Hosokawa, T. Hayashi, T. Mori, T. Yoshino, S. Nakasono, and T. Matsunaga, Microfluidic Device with Chemical Gradient for Single-Cell Cytotoxicity Assays, Anal. Chem. 83 (2011) 3648–3654.

[10]

G. M. Whitesides, The origins and the future of microfluidics, Nature. 442 (2006) 368–373. 14

[11]

D. Mark, S. Haeberle, G. Roth, F. Von Stetten, and R. Zengerle, Microfluidic Lab-on-a-Chip Platforms: Requirements, Characteristics and Applications, in: Springer, Dordrecht, 2010: pp. 305–376.

[12]

J. K. Chang, H. Bang, S.-J. Park, S. Chung, C. Chung, and D. C. Han, Fabrication of the PDMS microchip for serially diluting sample with buffer, Microsyst. Technol. 9 (2003) 555–558.

[13]

Y. Gao, J. Sun, W.-H. Lin, D. J. Webb, and D. Li, A compact microfluidic gradient generator

[14]

IP T

using passive pumping, Microfluid. Nanofluidics. 12 (2012) 887–895. L. Kim, M. D. Vahey, H.-Y. Lee, and J. Voldman, Microfluidic arrays for logarithmically perfused embryonic stem cell culture, Lab Chip. 6 (2006) 394.

C. Kim, K. Lee, J. H. Kim, K. S. Shin, K.-J. Lee, T. S. Kim, and J. Y. Kang, A serial dilution

SC R

[15]

microfluidic device using a ladder network generating logarithmic or linear concentrations, Lab Chip. 8 (2008) 473. [16]

K. Campbell, and A. Groisman, Generation of complex concentration profiles in microchannels

microfluidic

device

for

fluorescence

activated

cell

sorting,

J.

Micromechanics

M

Microengineering. 12 (2002) 324. [18]

N

J. Kruger, K. Singh, A. ONeill, C. Jackson, A. Morrison, and P. OBrien, Development of a

A

[17]

U

in a logarithmically small number of steps, Lab Chip. 7 (2007) 264–272.

B. Kuswandi, Nuriman, J. Huskens, and W. Verboom, Optical sensing systems for microfluidic

ED

devices: A review, Anal. Chim. Acta. 601 (2007) 141–155. [19] A. K. Ellerbee, S. T. Phillips, A. C. Siegel, K. A. Mirica, A. W. Martinez, P. Striehl, N. Jain, M. Prentiss, and G. M. Whitesides, Quantifying Colorimetric Assays in Paper-Based Microfluidic

8452.

W. Dungchai, O. Chailapakul, and C. S. Henry, Electrochemical Detection for Paper-Based

CC E

[20]

PT

Devices by Measuring the Transmission of Light through Paper, Anal. Chem. 81 (2009) 8447–

Microfluidics, Anal. Chem. 81 (2009) 5821–5826.

[21]

Z. Nie, C. A. Nijhuis, J. Gong, X. Chen, A. Kumachev, A. W. Martinez, M. Narovlyansky, and

A

G. M. Whitesides, Electrochemical sensing in paper-based microfluidic devices, Lab Chip. 10

[22]

(2010) 477–483. D. G. Rackus, M. H. Shamsi, and A. R. Wheeler, Electrochemistry, biosensors and microfluidics: a convergence of fields, Chem. Soc. Rev. 44 (2015) 5320–5340.

15

[23]

H. Wan, Q. Sun, H. Li, F. Sun, N. Hu, and P. Wang, Screen-printed gold electrode with gold nanoparticles modification for simultaneous electrochemical determination of lead and copper, Sensors Actuators B Chem. 209 (2015) 336–342.

[24]

H. Wan, D. Ha, W. Zhang, H. Zhao, X. Wang, Q. Sun, and P. Wang, Design of a novel hybrid sensor with microelectrode array and LAPS for heavy metal determination using multivariate nonlinear calibration, Sensors Actuators B Chem. 192 (2014) 755–761.

[25]

H. Wan, Q. Sun, H. Li, F. Sun, N. Hu, and P. Wang, Design of a miniaturized multisensor chip

IP T

with nanoband electrode array and light addressable potentiometric sensor for ion sensing, Anal. Methods. 7 (2015) 9190–9197.

H. Wan, H. Yin, and A. J. Mason, Rapid measurement of room temperature ionic liquid

SC R

[26]

electrochemical gas sensor using transient double potential amperometry, Sensors Actuators B Chem. 242 (2017) 658–666. [27]

C.-Y. Chen, A. M. Wo, and D.-S. Jong, A microfluidic concentration generator for dose-

K. W. Oh, K. Lee, B. Ahn, and E. P. Furlani, Design of pressure-driven microfluidic networks

N

[28]

U

response assays on ion channel pharmacology, Lab Chip. 12 (2012) 794.

[29]

A

using electric circuit analogy, Lab Chip. 12 (2012) 515–545. K. P. Kim, Y.-G. Kim, C.-H. Choi, H.-E. Kim, S.-H. Lee, W.-S. Chang, and C.-S. Lee, In situ

M

monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device, Lab Chip. 10 (2010) 3296.

ED

[30] X. Zhang, J. Fang, L. Zou, Y. Zou, L. Lang, F. Gao, et al., A novel sensitive cell-based Love Wave biosensor for marine toxin detection, Biosensors and Bioelectronics, 77(2016) 573-9.

PT

[31] N. Hu, T. Wang, Q. Wang, J. Zhou, L. Zou, K. Su, et al., High-performance beating pattern

CC E

function of human induced pluripotent stem cell-derived cardiomyocyte-based biosensors for hERG inhibition recognition, Biosensors and Bioelectronics, 67(2015) 146-53.

[32]

T. Wang, N. Hu, J. Cao, J. Wu, K. Su, P. Wang, A cardiomyocyte-based biosensor for

A

antiarrhythmic drug evaluation by simultaneously monitoring cell growth and beating, Biosensors and Bioelectronics, 49(2013) 9-13.

16

Author biographies Hao Wan received the B.S. and Ph.D. degree in Biomedical Engineering from Huazhong University of Science and Technology, Wuhan, China and Zhejiang University, Hangzhou, China in 2010 and 2015, respectively. He is currently a lecturer in the Department of Biomedical Engineering, Zhejiang University, China. His research interests are sensor microfabrication, electrochemical sensing and

IP T

instrument development in environmental monitoring.

Heyu Yin revived the B.S. degree in Engineering management from Hubei University, Wuhan, China

and Master degree in Microelectronics and Nanoelectronics from Tsinghua University, Beijing, China.

SC R

He is currently a Ph.D. student in the Department of Electrical and Computer Engineering, Michigan

State University, East Lansing, MI, USA. His research interests are electrochemical sensor and lab-on-

A

CC E

PT

ED

M

A

N

U

CMOS integration for portable biosensing and environmental monitoring application.

17