Mini-pillar microarray for individually electrochemical sensing in microdroplets

Journal Pre-proof Mini-pillar microarray for individually electrochemical sensing in microdroplets Yongchao Song, Tailin Xu, Jidong Xiu, Xueji Zhang PII:

S0956-5663(19)30924-8

DOI:

https://doi.org/10.1016/j.bios.2019.111845

Reference:

BIOS 111845

To appear in:

Biosensors and Bioelectronics

Received Date: 2 July 2019 Revised Date:

30 October 2019

Accepted Date: 1 November 2019

Please cite this article as: Song, Y., Xu, T., Xiu, J., Zhang, X., Mini-pillar microarray for individually electrochemical sensing in microdroplets, Biosensors and Bioelectronics (2019), doi: https:// doi.org/10.1016/j.bios.2019.111845. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

Credit Author Statement Tailin Xu designed the project. Yongchao Song carried out the experiments, analyzed the data and wrote the manuscript. Jidong Xiu provided software and devices help. Tailin Xu and Xueji Zhang supervised the project, modified the manuscript and suppled the funding.

Mini-Pillar Microarray for Individually Electrochemical Sensing in Microdroplets Yongchao Song,† Tailin Xu,*,† Jidong Xiu,† Xueji Zhang*,‡ †Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing 100083, P. R. China. ‡

School of Biomedical Engineering, Shenzhen University Health Science Center,

Shenzhen, Guangdong 518060, P.R. China

1

Abstract High throughput and high sensitivity are two important aspects in multiple biomarker recognition, drug discovery and relevant biochemical sensing. Here, we integrate mini-pillar microarray with the circuit components toward high-throughput individual

electrochemical

sensing

in

microdroplets.

On

such

droplet-microarray-based electrochemical platform, the high adhesion of the mini-pillar can hold a microdroplet (hundreds nanoliter to a few microliter) regardless of rotation and deformation. Each pillar as a unit has a three-electrode to achieve individual electrochemical sensing, and electrodes are integrated on one side to achieve the sequential electrochemical read-out. Qualitative and quantitative electrochemical assessments of multiple glucose concentrations in individual microdroplets are also achieved. Such mini-pillar-based individual electrochemical platform shows great potential in high-throughput and high-sensitive biomolecular recognitions, provides an opportunity to develop miniaturized sensing platform for emerging biological and pathological applications. Keywords：Microdroplets array; Individually Sensing; Mini-pillar Electrochemical Platform

2

1. Introduction Array sensing technologies are essential in drug discovery, toxicology, and cell biology.(Epstein and Walt 2003; Kittler et al. 2004; Miranda et al. 2010) The initial array sensing technologies always utilize 96-, 384-or more well plates as the carrier to restrict the samples in microcavities for parallelizable high-throughput sensing, which may limit by solution manipulation and information extraction.(Diaz-Mochon et al. 2007; Mark et al. 2010; Walling and Shepard 2011) In contrast, open-channel droplet-based platforms offer more benefits in terms of easy sample manipulation, low-sample consumption, and rapid information extraction.(Brutin and Starov 2018; Dong et al. 2018; Feng et al. 2018; He et al. 2018; Parolo and Merkoci 2013; Shang et al. 2017; Song et al. 2018; Tian et al. 2013; Ueda and Levkin 2013; Xu et al. 2017; Zhan et al. 2018) Mini-pillar-based open-channel platforms are more suitable for forming stable droplet arrays regardless of the extremely physical and chemical properties, which have achieved 3D cell cultures and high-throughput drug screenings.(Lee et al. 2016a; Lee et al. 2018; Li et al. 2018) Most mini-pillar platforms extract information in droplets by colorimetry or fluorescent ,(Kang et al. 2016; Lee et al. 2014) the requirements of certain color reaction or additional fluorescent labeling may limit their applications in universal molecular recognitions and detections. Electrochemical biosensors, as one of the most common methods, have been widely used to detect biomolecules for biological applications.(Fan et al. 2019; Heller and Feldman 2008; Labib et al. 2016; Privett et al. 2010; Pumera et al. 2010; Saveant 3

2008; Suginta et al. 2013; Wang 2005, 2008; Xu et al. 2016; Xu et al. 2014; Zhu et al. 2015) Recently, electrode arrays have provided an effective strategy to integrate multiplex individual electrochemical units for high-throughput detection, which tremendously reduces the experimental errors and accelerates analysis rates.(Ge et al. 2012; Goel 2018; Lam et al. 2013; Pumera and Escarpa 2009; Takeda et al. 2011; Zhang et al. 2017) Typical electrode arrays are integrating the individual electrodes to achieve the multiple biomarker recognitions and signals recording in the same sample solution,(Furst et al. 2013; Lee et al. 2016b; Qi et al. 2017; Radha Shanmugam et al. 2017; Uludag et al. 2014) which may result in the interferences from signal transmissions. Several groups employed the deep microwells or porous superwettable membranes to separate each electrochemical unit,(Song et al. 2019; Xu et al. 2018) and achieved the trace sample analysis and high-throughput individual sensing without interference,(Ino et al. 2014a; Ino et al. 2014b; Ino et al. 2011; Katelhon et al. 2014; Lin et al. 2009; Yang et al. 2009) which provided the innovative strategy to fabricate both electrodes array and solution individual electrochemical sensor to meet emerging high-throughput electrochemical applications. Herein, we introduced a mini-pillar microarray-based platform for high-throughput and individual electrochemical sensing. Such electrochemical platform comprises a mini-pillar platform for droplets manipulation and a circuit component for electrochemical signals transmission. On the cylindrical mini-pillar, embedded three-electrode can extract electrochemical signals in open-channel droplets, and all individual three-electrodes were integrated to one side, which provided more 4

conveniences for sequential multi-signals read-out. We successfully accomplished high-sensitive multiple glucose sensing in individual microdroplets. The mini-pillar sensor provides the convenient detection system construction without additional mediator introduction such as gold nanoparticle and carbon nanotube and reduce the time and reagent-consuming tremendously (SI Table 1). Such mini-pillar microarray with individual sensing units eliminates signal confusion and cross-contamination, holds great potentials in demanding high-throughput biological applications. 2. Materials and instruments Ethanol (>99.8%, GR), Dye, D-glucose, Ascorbic acid, Uric acid and phosphate buffered

solution

were

purchased

from

Sigma-Aldrich.

Potassium

ferricyanide/ferrocyanide and potassium chloride were purchased from Sinopharm Chemical Reagent Co. Ltd, China. The glucose oxidase and dopamine were obtained from J&K Chemical and the serum was purchased from Sangon Biotech (Shanghai, China). The polydimethylsiloxane (PDMS) was purchased from Dow Europe GmbH. The photomask with array was custom made from Beijing Zhongjingkeyi Technology Co., Ltd, China. All chemicals were analytical-grade reagents and were used without any further purification and prepared by dilution using ultrapure water (Milli-Q, 18.2 MΩ•cm). All the electrochemical mensuration was accomplished with a CHI-660D electrochemical workstation (CHI instruments, shanghai, China). 3. Results and discussion 3.1. Fabrication of mini-pillar-based high-throughput electrochemical platform The high-throughput electrochemical platform composes of a mini-pillar 5

microarray for individual droplets management and a circuit component for signal transmission. The mini-pillar array was fabricated by simply template pouring as shown in Figure 1a. As a concept-of-proof for high-throughput electrochemical sensing, we chose stainless steel sheet (SI Figure 1a) with 4×4 array through-holes (Hole diameter: 2 mm; Thickness: 1.65 mm) as pouring template for a confirmatory test. In short, the PDMS prepolymer was first mixed with curing agent, and eliminated bubbles by the vacuum drying oven. Then, the prepolymer was poured on the template, and transferred the petri dish at 80 ℃ for 4 h for solidification. After stripping, we obtained the 4×4 mini-pillar array with the height of about 1.65 mm and diameter of about 2 mm.

Figure 1. Design of the mini-pillar-based platform toward electrochemical sensing. a) Fabrication of the mini-pillar array platform including pouring and stripping. b) Assembly of the electrodes in the mini-pillar to get the individual electrochemical unit. c) Illustration of mini-pillar microarray platform with droplets management for 6

individual electrochemical detection. Figure 1b illustrated the simple procedures of integrating the mini-pillar with circuit component. First, the mini-pillar was glued on the flexible integrated circuit board (SI Figure 1b) by the adhesive of prepolymer and cyclohexane (ratio of 1:1). Then, the electrode wires (Three-electrodes: Pt as working electrode, Pt as counter electrode, and Ag as reference electrode) passed through the mini-pillar and the holes of circuit board with the guidance of the needle, and welded with the integrated circuit board (SI Figure 3). Thus, we fabricated the flexible mini-pillar-based high-throughput electrochemical platform. As demonstrated in Figure 1c, the mini-pillars with individual electrochemical unit can anchor microdroplets and achieve multiple, individual, and high-throughput electrochemical sensing.

Figure 2. Characterizations of the mini-pillar-based electrochemical platform. Investigation of the microdroplet adhesion of the mini-pillar array by rotating 45°(a), 7

90°(b), and 180°(c). The mini-pillar platform steadily fixed microdroplets regardless of d) stretching, e) bending and f) twisting. g) Integrated mini-pillar array electrochemical platform. h) Maximum volume of microdroplets on different diameters of mini-pillar (1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mm). i) Investigation of the resistance to various liquids (water, juice, coffee, cola, dye, serum, 1 M HCl and 1M NaOH). 3.2. Characterizations of the mini-pillar-based electrochemical platform. The mini-pillar microarray-based platform shows excellent properties to achieve individual droplet management, which is systematically characterized in Figure 2. The influences of mechanical deformation on such platform for microdroplets management were evaluated. The mini-pillars are considerable enough for anchoring microdroplets against rotation of 45°, 90° and 180° (Figure 2a-2c) and the mini-pillars keep anchoring the microdroplets regardless of stretching (Figure 2d), bending (Figure 2e) and twisting (Figure 2f). In addition, the mini-pillars can anchor a broad range of complex liquids such as juice, coffee, cola, dye, serum, HCl (1M) and NaOH (1M) (Figure 2i), revealing the brilliant stability of such platform toward complex biological detections. 3.3 Optimization of the mini-pillar-based electrochemical platform. The adhesion performance of such mini-pillars was evaluated as shown in SI Figure 2. The results in SI Figure 2a and 2b suggested that the maximum adhesion forces had no significant differences with curing agent ratio of 5:1, 8:1, 10:1, 15:1 and 20:1. In contrast, the maximum adhesion forces gradually increased with the 8

increasing diameters of the mini-pillars as demonstrated in SI Figure 2c and 2d. The maximum microdroplet loading capacity of different mini-pillars were also investigated in Figure 2h, maximum loading volumes increase from 6.7, 15.7, 27.8, 36.5, 54.1, 70.8 to 90.0 µL along with the increasing of diameters from 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 to 4.0 mm, respectively. 3.4. Electrochemical performances of the mini-pillar-based platform. The electrochemical performances of the mini-pillar array were systematically characterized as shown in SI Figure 4. The different scan rates varied from 50, 75, 100, 125, 150, 175 to 200 mV/s were applied for cyclic voltammetry scanning with 10 µL 10 mM phosphate buffered solution (PBS, pH 7.4) containing 0.1 M KCl and 10 mM potassium ferricyanide/ ferrocyanide. The anodic and the cathodic peak currents were nearly symmetric, and the peak potential increased the linearly with scan rates with a correlation coefficient of 0.9965, indicating that the redox process on the mini-pillar was a surface controlled electrochemical process (SI Figure 4a). In addition, we evaluated the influence of droplet volumes on such mini-pillar platform toward electrochemical measurement. Droplets (Volumes: 5, 10, 15, 20 and 25 µL) that contained 10 mM phosphate buffered solution, 0.1 M KCl and 10 mM potassium ferricyanide/ ferrocyanide were carefully dropped on the mini-pillars for electrochemical measurement (Scan rate: 100 mV/s). As shown in SI Figure 4b, the cyclic voltammetry curves were almost coincident with the increasing of volumes, revealing the stability of such mini-pillar platform toward electrochemical sensing regardless of the droplet sizes. Then potassium ferricyanide/ferrocyanide droplets (10 9

µL) with different concentrations (1 to 25 mM) were added on individual mini-pillar units. As shown in SI Figure 4c, all the mini-pillar units can obtain complete cyclic voltammetry curves, and the electrochemical signals increased gradually as the increasing of potassium ferricyanide/ferrocyanide concentrations. Thus, the mini-pillar microarray-based platform is suitable for high-throughput electrochemical sensing.

Figure 3. a) Schematic illustration of mini-pillar-based electrochemical platform for glucose detection. b) Amperometric responses of such mini-pillar-based droplets (10 µL) microarray with various glucose concentrations (1-40 mM). 3.5. High-throughput glucose sensing on mini-pillar-based electrochemical platform. As a concept-of-proof sensing, the glucose was chosen to verify the versatility and feasibility of 4 x 4 mini-pillar microarray-based electrochemical platform toward high-throughput and quantitative biochemical detection in patterned microdroplets as shown in Figure 3a. The detection mechanism was based on classical electrochemical detection of glucose (Wang 2008). In brief, 10 µL glucose oxidase (1 kU/mL) was first added on mini-pillar array, and then evaporated for modifying on the working 10

electrode surface. Subsequently, 2.5% glutaraldehyde as crosslinker was also dropped and evaporated on mini-pillars. The unlinked glutaraldehyde and glucose oxidase were removed by washing with ultra-pure water. Thus, the high-throughput glucose electrochemical platform had been modified. After modification, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 40 mM glucose in PBS buffer (10 µL, 10 mM, pH 7.4) were separately added to individual mini-pillar electrochemical units. The electrochemical detection carried out using chronoamperometry. In the presence of glucose oxidase, the glucose was oxidized to gluconic acid and generated hydrogen peroxide, then hydrogen peroxide was subsequently catalyzed at the working electrode with the current fluctuation. With the glucose decomposition, all the current fluctuations (∆I) were recorded and integrated as shown in Figure 3b. ∆I was proportional to the glucose concentrations ranging from 1 to 40 mM with the correlation coefficient of 0.9954 (Figure 3b). Comparing the parallel experiments in 3 mM and 5 mM glucose sensing, the mini-pillar sensor exhibited the high reproducibility with the relative standard deviations (RSD) of 6.32 % and 7.91 % as shown in SI Figure 5a,5b. Additionally, the long-term stability of mini-pillar glucose sensor was investigated. The mini-pillar sensor toward 5 mM glucose sensing was carried out every day in a week and the electrochemical signal have slight changed with the RSD of 8.39 % as shown in SI Figure 5c, revealing the excellent stability of such mini-pillar glucose sensor. Then we explored the selectivity of mini-pillar glucose sensor as shown in SI Figure 5d. The electrochemical signal has negligible change with the interfering electroactive substances (ascorbic acid, uric acid and 11

dopamine) addition, indicating high selectivity toward glucose detection. The electrochemical responses to 3, 5 and 8 mM glucose in serum samples were consistent with in PBS, which indicated the low influence on glucose detection (SI Figure 6a). Subsequently, we detected the four human serum samples on the mini-pillar sensor with the glucose concentration of 2.96 mM (Sample 1), 5.73 mM (Sample 2), 6.30 mM (Sample 3) and 7.01 mM (Sample 4), which are consistent with the glucometer results (SI Figure 6b). Therefore, the mini-pillar microarray-based electrochemical platform demonstrated excellent performance for multiple glucose sensing, which provided the infinite opportunity to human blood glucose investigation and health monitoring. 4. Conclusions In conclusion, we have demonstrated a mini-pillar microarray-based platform for high-throughput

and

individual

electrochemical

sensing.

The

proposed

electrochemical platform integrates the mini-pillar microarray for high-throughput droplets management and circuit components for electrochemical signal transmission. The mini-pillar microarray with high adhesion shows the excellent capability of anchoring microliter droplets in spite of mechanical deformation and rotation. In addition, by embedding the electrode wires, each mini-pillar can hold a few microliter microdroplet as an individual miniaturized sensing unit for electrochemical signal acquisition and electrodes are integrated on one side to achieve high-throughput and simultaneous sensing. Such mini-pillar units have achieved the simultaneous and quantitative detection of multiple glucose with excellent reproducibility and stability 12

and successfully detected the human serum sample, provide the great potential to develop miniaturized sensing platform for human blood glucose investigation and health monitoring. Acknowledgement We

acknowledge

funding

from

China

Postdoctoral

Science

Foundation

(2019M650479), National Natural Science Foundation of China (21804007, 21890742), Beijing Natural Science Foundation (2184109), Fundamental Research Funds for Central Universities (FRF-TP-17-066A1, FRF-BR-18-009B), and National Postdoctoral Innovative Talents Support Program of China (BX20180036). Conflicts of interest There are no conflicts to declare. Appendix A. Supplementary data Supplementary data to this article can be found online Reference Brutin, D., Starov, V., 2018. Chem. Soc. Rev. 47(2), 558-585. Diaz-Mochon, J.J., Tourniaire, G., Bradley, M., 2007. Chem. Soc. Rev. 36(3), 449-457. Dong, R., Zhang, T., Feng, X., 2018. Chem. Rev. 118(13), 6189-6235. Epstein, J.R., Walt, D.R., 2003. Chem. Soc. Rev. 32(4), 203-214. Fan, L., Yan, Y.R., Guo, B., Zhao, M., Li, J., Bian, X.T., Wu, H.P., Cheng, W., Ding, S.J., 2019. Sens. Actuator B-Chem. 296, 126697. Feng, W., Ueda, E., Levkin, P.A., 2018. Adv. Mater. 30(20), 1706111. 13

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Highlights 1. A mini-pillar-array electrochemical sensor is designed. 2. The

mini-pillar-based

electrochemical

sensor

anchor

the

microliter droplets for high throughput and individual sensing. 3. The mini-pillar-based electrochemical sensor achieve qualitative and quantitative electrochemical sensing of multiple glucose concentrations in array microdroplets.

There are no conflicts to declare.

All authors have read and approved the content, and agree to submit for consideration for publication in the journal.