Redox cycling-based electrochemical CMOS imaging sensor for real time and selective imaging of redox analytes

Journal Pre-proof Redox cycling-based electrochemical CMOS imaging sensor for real time and selective imaging of redox analytes Hiroya Abe, Hiroshi Yabu, Ryota Kunikata, Atsushi Suda, Masahki Matsudaira, Tomokazu Matsue

PII:

S0925-4005(19)31444-3

DOI:

https://doi.org/10.1016/j.snb.2019.127245

Reference:

SNB 127245

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

3 July 2019

Revised Date:

13 September 2019

Accepted Date:

7 October 2019

Please cite this article as: Abe H, Yabu H, Kunikata R, Suda A, Matsudaira M, Matsue T, Redox cycling-based electrochemical CMOS imaging sensor for real time and selective imaging of redox analytes, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127245

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Redox cycling-based electrochemical CMOS imaging sensor for real time and selective imaging of redox analytes

Hiroya Abe*1, 2,3, Hiroshi Yabu2, Ryota Kunikata4, Atsushi Suda4, Masahki Matsudaira5 and Tomokazu Matsue*3 1. Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, 2-1-1 Katahira, Aoba,

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Sendai 980-8577, Japan

2. WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan

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3. Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan

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4. Japan Aviation Electronics Industry, Ltd. 1-1, Musashino 3-chome, Akishima-shi, Tokyo, Japan.

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5. Micro System Integration Center, Tohoku University 519-1176 Aramaki-aza Aoba, Aoba-ku,

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Sendai, Japan.

Corresponding Authors:

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Dr. Hiroya Abe: [email protected] WPI-AIMR, Tohoku University, Japan

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Katahira 2-1-1, Aoba-ku, Sendai, 980-8577, Japan

Prof. Dr. Tomokazu Matsue: [email protected] Graduate School of Environmental Studies, Tohoku University, Japan 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan

Highlights: 

Integration of redox cycling-based electrodes and CMOS electrochemical imaging sensor

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Tracking a diffusion of redox analytes such as ferrocene methanol and dopamine

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Selective imaging of dopamine in the presence of ascorbic acid

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Tracking a dopamine release from PC12 spheroids in the presence of ascorbic acid

Abstract

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In this study, we have developed a novel electrochemical device (IDEA-Bio-LSI) incorporating interdigitated electrodes array (IDEA) and a LSI-based amperometric device (Bio-LSI) for high speed (4 – 200 ms) and selective imaging of an analyte diffusion and cellar activities such as dopamine

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release. The amplification factor (ηamp) and capture efficiency (CE) of IDEA of the device were 2.17 and 0.767, respectively. Compared with previously reported IDE based imaging sensor, the acquisition

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speed of the present device to acquire one image was improved up to 50 - 250 times. In addition, the

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dopamine release from PC12 spheroids in the presence of ascorbic acid was successfully obtained by using the IDEA-Bio-LSI. Therefore, IDEA-Bio-LSI can apply to rapid analyte diffusion biological

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events such as release of dopamine release.

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Introduction

Electrochemical imaging for tracking the diffusion of analytes and biological events has

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emerged as an increasingly important. In particular, dopamine (DA), a neurotransmitter, is crucial in a variety of biological and chemical processes in a mammalian central nervous system.(Marder et al., 2014; Schultz et al., 1997) Monitoring dopamine released from the neural tissues, such as 3D-cultured neural cells, a neural network and a brain, is crucial to screen drugs against neural cells and reveal the mechanism of intercellular communication. The electrochemical technique is a major method to monitor dopamine because that has the advantages of higher sensitivity and selectivity. In the

electrochemical method, various measuring methods have been developed including differential pulse voltammetry (DPV) (Kim et al., 2010; Marcenac and Gonon, 1985), fast scan cyclic voltammetry (FSCV) (Heien et al., 2005; Wightman et al., 1991) and chronoamperometry (Doherty and Gratton, 1992; Sabeti et al., 2003). DPV offers excellent sensitivity and selectivity but it has some drawbacks on poor temporal and spatial resolution because it takes long time (usually several minutes) to complete a single voltage sweep. FSCV realized excellent sensitivity and selectivity, besides, high temporal resolution (100 ms or less). McCarty and coworkers have reported the 4 channels microelectrode array

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to monitor the different neurochemical events in the brain slice by using FSCV(Zachek et al., 2010). Recently, Cima and coworkers have reported 16 channel FSCV instrumentation which is adaptable to implant environment(Schwerdt et al., 2017). Meanwhile, more numerous electrodes are required for

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mapping neurotransmitter release signals in the neural tissues. Additionally, the oxidation potential of DA overlaps with that of many other substances in the central nervous system, especially ascorbic acid

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(AA). Since the concentration of DA is much lower (< 10 μM)(Schwerdt et al., 2017) than that of AA (100-1000 μM)(Zhang et al., 2007), its accurate detection is a challenging issue.

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According to the report described by Niwa and coworkers, a redox cycling for selective detection of DA have been reported by using interdigitated electrodes (IDEs)(Niwa, 1995; Niwa et al.,

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1991). IDEs amplify the amperometric signal from DA but does not amplify the signal of AA, because DA is one of the reversible redox species which induces the redox cycling on IDEs, but AA is an

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irreversible redox species which does not induce the redox cycling on IDEs (Figure 1). During the redox cycling, DA is oxidized to form dopamine-quinone at generator electrode before diffusing to a

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nearby collector electrode where it is reduced back to DA. For closely spaced generator and collector electrodes, the species will transport between the electrodes multiple times (redox cycling) before diffusing out into the bulk of the solution, which leads to the amplification of the current. From these backgrounds, the arrayed IDEs or other electrochemical devices, which can induce the redox cycling, are required for a selective and simultaneous imaging of dopamine. We(Ino et al., 2012a), (Kanno et al., 2015b), Wolfrum and coworkers(Kätelhön et al., 2014) have designated the

novel methodology as local redox cycling- based electrochemical (LRC-EC) detection. In the system, two arrays of band microelectrodes are orthogonally set to form an n × n array of crossing points with only 2n bonding pads for external connection. By setting the potentials at the row and column electrodes to appropriate values and inducing local redox cycling at a particular crossing point, the local redox cycling-based signals can be acquired from an individual crossing point.(Ino et al., 2012b, 2011a) The detection system has been applied for the expression of reporter proteins in a cell culture array and evaluation of alkaline phosphatase activity 3D-cultured embryoid bodies. However, this

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LRC-EC system is not suitable for real-time imaging for rapid events because the scan rate of switching the row and column electrodes was well slow; i.e. 60 s/image for 32 × 32 electrodes(Ino et al., 2011b), 10.3 to 90.4 s/image for 16 × 16 electrodes(Ino et al., 2012b).

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For more sensors to be incorporated into a chip device, state-of-art semiconductor technology has been applied(Bellin et al., 2016; Hattori et al., 2012; Kim et al., 2013; Mizutani et al., 2017;

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Wydallis et al., 2015). These chip devices, especially a large-scale integration (LSI), are useful for the evaluation of dopaminergic cells. We previously developed a complementary metal-oxide

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semiconductor (CMOS)-based amperometric device containing 400 sensors at excellent time resolution (4 to 200 ms/image)(Abe et al., 2018, 2016, 2015; Inoue et al., 2015, 2012; Kanno et al.,

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2017, 2015a) and the spatial resolution (250 µm) of the device was suitable for an analysis of the neural activity. Each sensor in this device, designated Bio-LSI, contains an operational amplifier with a

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switched-capacitor type I−V converter for in-pixel signal amplification.(Inoue et al., 2015) The BioLSI is capable of rapid and quantitative electrochemical imaging of dopamine release from 3D-

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cultured PC12 cells (rat pheochromocytoma cells) which are often used as a model of dopaminergic cells.(Abe et al., 2015) In Bio-LSI, potentiostat is incorporated in an external circuit. Bio-LSI can apply two different voltages to arbitrary electrodes, similar to a common bipotentiostat. These potentials at arbitrary electrode can be controlled by changing switches in each unit cell.(Inoue et al., 2015)

For high speed and selective imaging of analytes and cellar activities such as dopamine release, in this study, we developed a novel electrochemical device incorporating interdigitated electrodes array (IDEA) and the Bio-LSI. We (Abe et al., 2015; Inoue et al., 2015) and other researchers (Kim et al., 2013) have been reported on the dopamine sensing by applying oxidation potential at each electrode. This conventional method cannot induce the redox cycling because the distance between each electrode was too long to induce the redox cycling. Therefore, Bio-LSI was redesigned the electrode pattern to enable in order to induce the redox cycling. At first, we developed

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a 180 IDEA-Bio-LSI consist of 180 IDEs arrayed on the Bio-LSI for a characterization of a selectivity and an amplification efficiency. To extend the device for more detail analysis, the detection points were increased to 360 from 180 IDEs by designing the device. From these renovations of the IDEs and

Experimental IDEAs-Bio-LSI Fabrication

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Bio-LSI, the real time imaging was successfully obtained.

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The fundamental design and fabrication process of the LSI circuit and electrodes are the same as that of the previously reported Bio-LSI.(Inoue et al., 2015) Briefly, Pt interdigitated electrodes

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arrays, used as sensors, were prepared on Al pads via photolithography and sputtering. SU-8 (SU-8 3005, MicroChem, USA) microwells (70 × 100 µm; depth ~5 µm) were fabricated on the Pt electrodes

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to define the surface area of electrodes. The 180-IDEA-Bio-LSI and 360-IDEA-Bio-LSI chips were then affixed to connector pads on a ceramic substrate (Japan Aviation Electronics Industry, Ltd., Japan) was bonded to the substrate to retain the sample solution at the sensor points. The 180-IDEA-Bio-LSI consists of 180 generator electrodes, 180 collector electrodes and 180 detection points (Figure S1). Likewise, the 360-IDEA-Bio-LSI consists of 40 generator electrodes, 360 collector electrodes and 360 detection points (Figure S2). Herein, the number of collector electrodes compatible with detection

point because the electrochemical images was configured by using the current from collector electrodes.

Electrochemical Detection of Ferrocenemethanol and Dopamine In order to determine potentials for redox cycling, cyclic voltammetry (CV) was performed in a standard three electrode cell connected to the potentiostat (Model 2325, BAS Inc., Japan). A Pt disk electrode (diameter: 3 mm) was the working electrode, an Ag/AgCl (sat. KCl) was the reference

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electrode, a Pt wire was the counter electrode. All measurements were performed in HBSS, and scanned 0.0 to +0.6 V at 50 mV/s).

Performance of the IDEA-Bio-LSI was evaluated by monitoring the electrochemical signals

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associated with ferrocenemethanol (FcCH2OH, Sigma Aldrich, USA) and dopamine (DA, Sigma Aldrich, USA) oxidation. Briefly, the Bio-LSI was filled with 1.5 mL of 0.1 M KCl or a buffer solution

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(Hanks’ balanced salt solution, Gibco, USA). An Ag/AgCl (sat. KCl) reference (RE-1CP; BSA Inc., Japan) and a Pt counter electrode were used for all electrochemical operations. To detect FcCH2OH, a

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drop of 0.1M KCl was placed on the device, and then 0.0 and 0.5 V were applied to the generator and collector electrodes, respectively, followed by successive addition of FcCH2OH solutions while

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monitoring the current transients. To detect dopamine, generator and collector electrodes were applied to +0.1 and +0.6 V, respectively. Electrochemical signals gained from each electrode were recorded

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every 100 or 200 ms.

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Current Simulation

To obtain the simulation current of FcCH2OH in single and dual mode, redox currents were

calculated using COMSOL Multiphysics software (ver. 5.3a, COMSOL, Inc., USA). Briefly, 3D models of electrodes containing microwells were constructed. The size and depth of the microwells were 70 × 100 µm and 5 µm, respectively, and each 4 bar electrode were defined as generator and collector electrodes. In the simulation, the electrochemical reaction was assumed to be a one-electron

process. The diffusion coefficient of FcCH2OH was set to 7.0 × 10-10 m2/s.(Cannes et al., 2003) The initial concentration of FcCH2OH in the space was set at 0.2 mM. The concentration of FcCH2OH at the generator electrodes boundary was 0 mM because the electrode potential was set to be sufficiently positive for the electrochemical reaction. In the contrast, the concentration of FcCH2OH at the collector electrodes boundary was 0.2 mM because the electrode potential was set to be sufficiently negative for the electrochemical reaction. The FcCH2OH diffusion flux was zero at insulating surface. The FcCH2OH diffusion flux on the electrodes was calculated to evaluate the current associated with

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FcCH2OH oxidation and reduction at the electrodes.

Cell Culture

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PC12 cells were donated by the Cell Resource Center for Biomedical Research at Tohoku University. PC12 cells were 3D-cultured using the hanging-drop method to prepare PC12 spheroids.

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Briefly, PC12 cells were suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS), and 20 μL droplets containing 8000 cells were

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placed on the cover of a culture dish, which was then inverted so that the droplets hung from the dish cover. The cells were then cultured in a humidified incubator at 37 °C with 5% CO2 for 6 days to allow

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for the formation of PC12 spheroids.

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Electrochemical Imaging of Injection of ferrocenemethanol (FcCH2OH and K+-Stimulated Dopamine (DA) Release from PC12 Spheroids using 360-IDEA-Bio-LSI

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Prior to stimulation, the reference and counter electrodes were inserted into a sample solution

on the device. Two outer electrodes (20 × 2) and other electrodes (20 × 18) were used as generator and collector electrodes, respectively (Figure S2), and electrochemical images consisting of 400 electrochemical signals were obtained every 100 ms. The electrochemical imaging was done by injection of a 0.15 mL FcCH2OH (1 mM) in 0.1 M KCl into a 360 IDEA-Bio-LSI filled with a 1.35 mL solution of 0.1 M KCl. The imaging of DA was performed by injecting a buffer containing PC12

spheroids into the IDEA-Bio-LSI filled with a Hank's balanced salt solution (HBSS) solution containing high concentration of (final concentration of 105 mM). The high-K+ HBSS solution stimulate the DA release from the PC12 spheroids. During the electrochemical imaging, the PC12 spheroids were also observed under a microscope (Stemi 2000, ZEISS) to acquire visual images of the

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spheroids on the device.

Results and discussion Device Fabrication For inducing the redox cycling, IDEs consisting of two electrodes with different potentials have been widely used. One set of IDE is used as a generator and the other a collector. The distance between the generator and collector electrodes affects the efficiency of the redox cycling. In our devices, both of the width of oxidation and reduction electrode was 5 µm. The gap between oxidation and reduction electrode was 5 µm. The length of the microband electrode was 70 µm. The 180 IDEs

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were arranged on the Bio-LSI device (180-IDEA-Bio-LSI, Figure 1c and d). Redox cycling can be induced individually onto each the IDE. For the redox cycling, the potentials of generator electrode were set at +0.5 V for FcCH2OH and +0.6 V for DA and those of collector electrodes at 0.0 V for

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FcCH2OH and +0.1 V for DA (Figure 1e).

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Figure 1. (a) Schematic images of proposal device. (b) redox cycling for reversible (i) such as

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FcCH2OH and DA and irreversible (ii) analyte such as AA, respectively. (c and d) Digital and (e) schematic images of 180 IDEAs-Bio-LSI device. IDEs consisting of generator and collector

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electrodes, respectively. The cross section at the dash line corresponds to Figure 1b.

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Characterization of the IDEA-Bio-LSI Device

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Chronoamperometry was applied for evaluation of the electrochemical performance of 180 IDEA-Bio-LSI device. A cyclic voltammetry (CV) measurement of FcCH2OH in HBSS carried out in

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order to determine the potential of generator and collector electrodes. From the CV curve (Figure S2a and S2b), the anodic and cathodic peak potentials of FcCH2OH were appeared at +0.26 and +0.19 V, respectively. Therefore, the oxidation and reduction potential of FcCH2OH were set to 0.5 and 0.0 V to oxidize FcCH2OH and reduce the oxidized form of FcCH2OH, respectively. Figure S4a and b show the electrochemical responses of the 180 IDEA-Bio-LSI device for FcCH2OH at single and dual mode, respectively, when the concentration of FcCH2OH in the measurement solution was changed from 0

to 200 μM. Here, the system of dual mode is the condition that oxidation and reduction potentials apply to each electrode. The single mode is the condition that only oxidation potentials apply to only the generator electrode. The electrochemical signals at the generator electrode (single and dual mode) and the collector electrode (dual mode) rapidly responded to the addition of FcCH2OH and increased with increasing concentration of FcCH2OH. The calibration plots showed a linear response for both modes and the detection limit was found to be at least 10 µM for both modes (Figure 2). The oxidation currents in the single and dual mode at 200 µM FcCH2OH solution were 1.85 and 3.96 nA, respectively, and

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the reduction current in the dual mode was -3.03 nA.

The performance was evaluated by the amplification factor (𝜂𝑎𝑚𝑝 ) and the capture efficiency (CE), the ratio of reduction current at collector electrode to oxidation current at the generator electrode.

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The 𝜂𝑎𝑚𝑝 and CE values are defined by; 𝜂𝑎𝑚𝑝 = 𝐼𝑔𝑒𝑛.𝑑𝑢𝑎𝑙 /𝐼𝑔𝑒𝑛.𝑠𝑖𝑛𝑔𝑙𝑒

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𝐶𝐸 = 𝐼𝑐𝑜𝑙.𝑑𝑢𝑎𝑙 /𝐼𝑔𝑒𝑛.𝑑𝑢𝑎𝑙

where, 𝐼𝑔𝑒𝑛.𝑑𝑢𝑎𝑙 , 𝐼𝑔𝑒𝑛.𝑠𝑖𝑛𝑔𝑙𝑒 , and 𝐼𝑐𝑜𝑙.𝑑𝑢𝑎𝑙 are the current of generator electrodes at dual mode, the

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current at single mode and the current of collector electrodes at dual mode, respectively. The values for 𝜂𝑎𝑚𝑝 and 𝐶𝐸 of the present device were calculated to be 2.17 and 0.767, respectively.

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A theoretical value of the generator electrode during the redox cycling was calculated by using below equation(Aoki, 1989).

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|𝐼| = 𝑚𝑏𝑛𝐹𝐶𝐷[0.637 ln{2.55(1 + 𝑤 ⁄𝑤𝑔)} − 0.19⁄(1 + 𝑤 ⁄𝑤𝑔)2 ]

where m is the number of microband anodes or cathodes, b is the length of the microband electrode of

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the IDE, n is the number of electrons, C is the concentration of analyte, D is the diffusion coefficients of the analyte, and w and wg are the widths of anode or cathode electrode and the gap between the anode or cathode electrode, respectively. The calculated value was 3.7 nA, which reasonably accorded with the experimental value. For more precise discussion, we carried out the simulation using the finite element method (COMSOL Multiphysics), incorporating the effect of height of microband electrodes. The current

obtained from the simulation for the generator electrode in single mode was calculated to be 1.71 nA, and those of the generator and collector electrodes in dual mode were 4.05 nA and -3.05 nA, respectively. These values corresponded well with currents experimentally observed for generator electrode (1.85 nA for single mode; 3.96 nA for dual mode) and collector (-3.03 nA for dual mode) electrode, indicating that the device was successfully as designed.

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Figure 2. Calibration curves of generator electrodes in single (green) and dual (red) mode and collector

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electrodes (blue) in dual mode as a function of FcCH2OH concentration.

Estimation of selectivity for DA detection

Since AA usually interfares the electrochemical oxidation of DA, the influence of AA on the selective detection of DA was evaluated by chronoamperometry (Figure 3a). Before characterization of redox cycling for DA, CV was carried out in order to determine set potential for redox cycling (Figure S2c). The 1st and 2nd oxidation peak potentials of DA in HBSS were appeared at +0.24 and +0.48 V, respectively. The reduction peak potential was appeared at +0.13 V. Therefore, the oxidation and reduction potential were set to +0.6 and +0.1 V to oxidize DA and reduce the oxidized form of DA, respectively. AA has the oxidation peak potential at +0.29 V (Figure S2d), which indicates that

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the AA was oxidized with DA at same oxidation potential. However, reduction peak potential of AA was not observed because oxidized AA was immediately decomposed to 2,3-diketogulonate (Goluch et al., 2009). Therefore, although DA was reduced on the collector electrode, AA was not reduced on

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the collector electrode, which indicate that the only DA can induce the redox cycling.

When AA (500 uM) was added at about 70 s, the oxidation current at the generator electrode

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immediately increased but no reduction reaction was observed at the collector electrode due to irreversible oxidation of AA.(Perone and Kretlow, 1966) As DA (500 uM) was added at about 165 s,

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simultaneous increases in oxidation and reduction currents were observed at generator and collector

the presence of AA.

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electrodes, respectively. These results showed that 180-IDEA-Bio-LSI can selectively detect DA in

Figure 3b shows the calibration curves and Fig. S3 depicts the electrochemical responses

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of the 180-IDEA-Bio-LSI device on addition of a DA (0 to 300 μM) with and without 200 µM AA. In the case of DA solution without AA, the electrochemical signals of the generator (red) and collector

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(blue) electrodes increase with the concentration of DA (Figure 3b and S5a). In the case of DA solution with 200 µM AA, the response at the generator electrode becomes large due to simultaneous oxidation of DA and AA. The presence of AA slightly lowers the response at the collector (open circle) electrode (Figure 3b and S5b). The difference in the responses is caused by the difference in oxidation reactivity. DA is reversibly oxidized and reduced at the generator electrode and the collector electrodes, respectively, inducing signal amplification due to the redox cycling. When AA is present in the

solution, DA acts as a redox mediator for oxidation of AA at the generator electrode, which in turn causes slight decreases in electrochemical signal at the collector electrode. This influence can be avoided by coating with an anion exchange membrane(Niwa et al., 1994) and by using 3D structured electrodes.(Goluch et al., 2009) In the near future, we are planning to develop Bio-LSI with nano electrodes, such as nano cavity(Kanno et al., 2015b; Wolfrum et al., 2008), ring-disk nanoelectrodes(Fu et al., 2018) and so on, which makes it possible to measure low concentration of

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dopamine with high sensitivity and high selectivity.

Figure 3. (a) Electrochemical responses of generator (red line) and collector (blue line) electrode for

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500 µM AA (70 s) and 500 µM DA (165 s) addition. (b) Calibration carves of generator (red and filled black circle) and collector (blue and open circle) electrodes in the solution w/ and w/o 200 µM AA,

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respectively.

Electrochemical Imaging of analytes using 360 IDEAs-Bio-LSI Bio-LSI originally arranged 400 electrodes (20 × 20) electrodes with the interval of 250 µm (Figure S2c). On the other hand, in the 180-IDEA-Bio-LSI device, the detection points are arranged with the intervals 353 µm (Figure S1c). This density of detection points is insufficient to obtain the

image of analytes such as dopamine released from 3D neural tissues. To densify the detection points, electrodes consisting of 40 (2 × 20) generator electrodes and 360 (18 × 20) collector electrodes are arranged at a pitch of 250 µm was fabricated and named as 360-IDEA-Bio-LSI (Figure 4). In this LSI device, nine reduction electrodes (collector) and one oxidation electrode (generator) were arranged in a measurement unit. A both of width and the gap of the IDE was 5 μm. The device possesses 20

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measurement units and, therefore, 360 collectors for detection of the reduction current.

Figure 4. (a and b) Digital and (c) schematic images of 360-IDEAs-Bio-LSI device. In this IDEs, nine

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collector electrodes correspond to one generator electrode.

In order to obtain an image of an injection FcCH2OH solution, the generator and collector electrodes were applied to +0.5 and 0.0 V, respectively. The successive electrochemical images at the collector electrodes were acquired immediately after 1 mM FcCH2OH solution was injected from the direction of the arrow is shown in Figure 5a and Movie S1 (a final concentration was 100 µM). The electrochemical signals increased rapidly in the injected area and expanded as the injected solution diffused. The current value on the generator (Figure S6) and collector (Figure 5b) electrode was changed by injecting the solution, which indicates the 360-IDEA-Bio-LSI can induce redox cycling.

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Figure 5b shows the amperometric response at the corrector electrode indicated by the blue square in Fig. 5a. The reduction current rapidly increased on injection of FcCH2OH solution and leveled off at approximately -1.85 nA after pipetting the solution. The level-off response corresponded to that at the

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rapid electrochemical imaging of the redox species.

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collector for 100 µM FcCH2OH. The above findings indicate that 360-IDEAs-Bio-LSI can be used for

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Figure 5. (a) An electrochemical image and (b) an amperogram of injection of ferrocenemethanol

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solution

An event of dopamine release from PC12 spheroids was monitored using 360-IDEA-Bio-LSI. It is known that AA concentration in the brain changes with stimulation.(Grünewald, 1993; Grünewald and Fillenz, 1984)

Therefore, it is important to design a device which has no influence on AA. In

this method, PC12 spheroids are introduced into a high-K+ buffer on the device to stimulate the spheroids semi-simultaneously. K+ stimulation depolarizes the cells and induces the release of

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dopamine by exocytosis. During this measurement, oxidation (+0.60 V vs. Ag / AgCl) and reduction (0.10 V vs. Ag / AgCl) potential are applied to generator and collector electrodes, respectively. Figures 6 shows optical microscope and electrochemical images of the spheroids, together with the

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amperograms observed at the collectors indicated in Fig. 6b. Electrochemical signal images were acquired after the stimulation, and the electrochemical response clearly increased at the position of the

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PC12 spheroids (Figure 6a and b). The images indicate that the device can monitor the event of dopamine release in spite of the presence of 200 µM AA in the measurement solution. From the

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amperograms, the responses at the generator and collector electrodes reached a peak in about 20 s after PC12 spheroids were injected. This observation corresponds well with the previous result where the

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time to reach the release peak of dopamine in PC12 spheroid is 20 s. Therefore, the signal of dopamine release from PC12 spheroids can be accurately detected with the present system.

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Previously, we reported on a study on imaging devices based on the LRC-EC electrochemical device consisting of multiple row and column electrodes. Because the scan rate for switching the row

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and column electrodes was slow, more than 1 min was usually required for acquire a full image. (Ino et al., 2011b), (Ino et al., 2012b), The temporal resolution of the LRC-EC device is not enough to image the dopamine release because the event occurred in about 20 s. On the contrary, the present acquires one image in 4 to 200 ms, improving the time resolution up to 50 - 250 times. A high performance liquid chromatography (HPLC) is also a conventional method for detecting dopamine released from neural tissues because HPLC can separate signals of dopamine and ascorbic acid.

However, HPLC cannot real-time detect these analytes and spatial image. IDEA-Bio-LSI can estimate individual PC12 spheroid in the interferences since IDEA-Bio-LSI has 400 electrodes with high temporal resolution which enable to locally image dopamine release.

Figure 6. (a) An digital (b) electrochemical image of dopamine release from PC12 spheroids after high-

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K+ stimulation. (c) Amperograms of generator (red) and collector (green and blue) electrodes. The

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colors of amperograms were associated to the colored squares in b.

Conclusions We have developed a novel electrochemical device (IDEA-Bio-LSI) incorporating interdigitated electrodes array (IDEA) and the LSI-based amperometric device (Bio-LSI) for high speed and selective imaging of analytes and cellar activities such as dopamine release. The amplification factor (ηamp) and capture efficiency (CE) of IDEA of the device were 2.17 and 0.767,

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respectively. These values corresponded well with those from simulation, which indicates that the IDEA correctly worked even though they were arrayed on the Bio-LSI. Compared with previously reported IDE based imaging sensor, the acquisition speed of the present device to acquire one image

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biological events such as release of dopamine release.

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was improved up to 50-250 times. Therefore, IDEA-Bio-LSI can apply to rapid analyte diffusion and

Notes

Acknowledgements

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The authors declare no competing financial interest.

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This work was supported in part by a Grant-in-Aid for Scientific Research (A) (No.

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16H02280, No. 17H01223 and No. 19K15598), Special Coordination Funds for Promoting Science and Technology, the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program from the Japan Science and Technology Agency, and Grant-in-Aid for JSPS Fellows (No. 16J02105) from the Japan Society for the Promotion of Science (JSPS). This work was also supported by a Grant-in-Aid of Tohoku University Institute for International Advanced Research and Education.

References Abe, H., Ino, K., Li, C.Z., Kanno, Y., Inoue, K.Y., Suda, A., Kunikata, R., Matsudaira, M., Takahashi, Y., Shiku, H., Matsue, T., 2015. Electrochemical Imaging of Dopamine Release from ThreeDimensional-Cultured PC12 Cells Using Large-Scale Integration-Based Amperometric Sensors. Anal. Chem. 87, 6364–6370. doi:10.1021/acs.analchem.5b01307 Abe, H., Iwama, T., Yabu, H., Ino, K., Inoue, K.Y., Suda, A., Kunikata, R., Matsudaira, M., Matsue, T., 2018. Simultaneous and Selective Imaging of Dopamine and Glutamate Using an Enzyme‐

Electroanalysis 30, 2841–2846. doi:10.1002/elan.201800386

ro of

modified Large‐scale Integration (LSI)‐based Amperometric Electrochemical Device.

Abe, H., Kanno, Y., Ino, K., Inoue, K.Y., Suda, A., Kunikata, R., Matsudaira, M., Shiku, H., Matsue,

-p

T., 2016. Electrochemical Imaging for Single-cell Analysis of Cell Adhesion Using a Collagencoated Large-scale Integration (LSI)-based Amperometric Device. Electrochemistry 84, 364–

re

367.

lP

Aoki, K., 1989. Theory of the steady-state current of a redox couple at interdigitated array electrodes of which pairs are insulated electrically by steps. J. Electroanal. Chem. 270, 35–41. doi:10.1016/0022-0728(89)85026-0

na

Bellin, D.L., Sakhtah, H., Zhang, Y., Price-Whelan, A., Dietrich, L.E.P., Shepard, K.L., 2016. Electrochemical camera chip for simultaneous imaging of multiple metabolites in biofilms. Nat.

ur

Commun. 7, 10535; 10.1038/ncomms10535. doi:10.1038/ncomms10535

Jo

Cannes, C., Kanoufi, F., Bard, A.J., 2003. Cyclic voltammetry and scanning electrochemical microscopy of ferrocenemethanol at monolayer and bilayer-modified gold electrodes. J. Electroanal. Chem. 547, 83–91. doi:10.1016/S0022-0728(03)00192-X

Doherty, M.D., Gratton, A., 1992. High-speed chronoamperometric measurements of mesolimbic and nigrostriatal dopamine release associated with repeated daily stress. Brain Res. 586, 295–302. doi:10.1016/0006-8993(92)91639-V

Fu, K., Han, D., Kwon, S.-R., Bohn, P.W., 2018. Asymmetric Nafion-Coated Nanopore Electrode Arrays as Redox-Cycling-Based Electrochemical Diodes. ACS Nano 12, 9177–9185. doi:10.1021/acsnano.8b03751 Goluch, E.D., Wolfrum, B., Singh, P.S., Zevenbergen, M.A.G., Lemay, S.G., 2009. Redox cycling in nanofluidic channels using interdigitated electrodes. Anal. Bioanal. Chem. 394, 447–456. doi:10.1007/s00216-008-2575-x Grünewald, R.A., 1993. Ascorbic acid in the brain. Brain Res. Rev. 18, 123–133. doi:10.1016/0165-

ro of

0173(93)90010-W

Grünewald, R.A., Fillenz, M., 1984. Release of ascorbate from a synaptosomal fraction of rat brain. Neurochem. Int. 6, 491–500. doi:10.1016/0197-0186(84)90120-7

-p

Hattori, T., Kojima, Y., Tokunaga, K., Masaki, Y., Kato, R., Engineering, E.I., 2012. CCD-Type

970. doi:10.5162/IMCS2012/P1.3.14

re

Multi-Ion Image Sensor with Two Kinds of Plasticized Poly ( vinyl chloride ) Membranes 967–

Heien, M.L.A. V., Khan, A.S., Ariansen, J.L., Cheer, J.F., Phillips, P.E.M., Wassum, K.M., Wightman,

lP

R.M., 2005. Real-time measurement of dopamine fluctuations after cocaine in the brain of behaving rats. Proc. Natl. Acad. Sci. 102, 10023–10028. doi:10.1073/pnas.0504657102

na

Ino, K., Ishida, A., Inoue, K.Y., Suzuki, M., Koide, M., Yasukawa, T., Shiku, H., Matsue, T., 2011a. Electrorotation chip consisting of three-dimensional interdigitated array electrodes. Sensors

ur

Actuators, B Chem. 153, 468–473. doi:10.1016/j.snb.2010.11.012 Ino, K., Kanno, Y., Nishijo, T., Goto, T., Arai, T., Takahashi, Y., Shiku, H., Matsue, T., 2012a.

Jo

Electrochemical detection for dynamic analyses of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device. Chem. Commun. 48, 8505. doi:10.1039/c2cc34264b

Ino, K., Nishijo, T., Arai, T., Kanno, Y., Takahashi, Y., Shiku, H., Matsue, T., 2012b. Local redoxcycling-based electrochemical chip device with deep microwells for evaluation of embryoid bodies. Angew. Chemie - Int. Ed. 51, 6648–6652. doi:10.1002/anie.201201602

Ino, K., Saito, W., Koide, M., Umemura, T., Shiku, H., Matsue, T., 2011b. Addressable electrode array device with IDA electrodes for high-throughput detection. Lab Chip 11, 385–388. doi:10.1039/C0LC00437E Inoue, K.Y., Matsudaira, M., Kubo, R., Nakano, M., Yoshida, S., Matsuzaki, S., Suda, A., Kunikata, R., Kimura, T., Tsurumi, R., Shioya, T., Ino, K., Shiku, H., Satoh, S., Esashi, M., Matsue, T., 2012. LSI-based amperometric sensor for bio-imaging and multi-point biosensing. Lab Chip 12, 3481. doi:10.1039/c2lc40323d

ro of

Inoue, K.Y., Matsudaira, M., Nakano, M., Ino, K., Sakamoto, C., Kanno, Y., Kubo, R., Kunikata, R., Kira, A., Suda, A., Tsurumi, R., Shioya, T., Yoshida, S., Muroyama, M., Ishikawa, T., Shiku, H., Satoh, S., Esashi, M., Matsue, T., 2015. Advanced LSI-based amperometric sensor array with

-p

light-shielding structure for effective removal of photocurrent and mode selectable function for individual operation of 400 electrodes. Lab Chip 15, 848–56. doi:10.1039/c4lc01099j

re

Kanno, Y., Ino, K., Abe, H., Sakamoto, C., Onodera, T., Inoue, K.Y., Suda, A., Kunikata, R., Matsudaira, M., Shiku, H., Matsue, T., 2017. Electrochemicolor Imaging Using an LSI-Based for

Multiplexed

Cell

Assays.

lP

Device

Anal.

Chem.

89,

12778–12786.

doi:10.1021/acs.analchem.7b03042

na

Kanno, Y., Ino, K., Inoue, K.Y., Şen, M., Suda, A., Kunikata, R., Matsudaira, M., Abe, H., Li, C.Z., Shiku, H., Matsue, T., 2015a. Feedback mode-based electrochemical imaging of conductivity and

ur

topography for large substrate surfaces using an LSI-based amperometric chip device with 400 sensors. J. Electroanal. Chem. 741, 109–113. doi:10.1016/j.jelechem.2015.01.020

Jo

Kanno, Y., Ino, K., Shiku, H., Matsue, T., 2015b. A local redox cycling-based electrochemical chip device with nanocavities for multi-electrochemical evaluation of embryoid bodies. Lab Chip 15, 4404–4414. doi:10.1039/C5LC01016K

Kätelhön, E., Mayer, D., Banzet, M., Offenhäusser, A., Wolfrum, B., 2014. Nanocavity crossbar arrays for parallel electrochemical sensing on a chip. Beilstein J. Nanotechnol. 5, 1137–1143. doi:10.3762/bjnano.5.124

Kim, B.N., Herbst, A.D., Kim, S.J., Minch, B.A., Lindau, M., 2013. Parallel recording of neurotransmitters release from chromaffin cells using a 10×10 CMOS IC potentiostat array with on-chip working electrodes. Biosens. Bioelectron. 41, 736–744. doi:10.1016/j.bios.2012.09.058 Kim, Y.R., Bong, S., Kang, Y.J., Yang, Y., Mahajan, R.K., Kim, J.S., Kim, H., 2010. Electrochemical detection of dopamine in the presence of ascorbic acid using graphene modified electrodes. Biosens. Bioelectron. 25, 2366–2369. doi:10.1016/j.bios.2010.02.031 Marcenac, F., Gonon, F., 1985. Fast in vivo monitoring of dopamine release in the rat brain with

ro of

differential pulse amperometry. Anal. Chem. 57, 1778–9. doi:10.1021/ac00267a060

Marder, E., O’Leary, T., Shruti, S., 2014. Neuromodulation of Circuits with Variable Parameters: Single Neurons and Small Circuits Reveal Principles of State-Dependent and Robust

-p

Neuromodulation. Annu. Rev. Neurosci. 37, 329–346. doi:10.1146/annurev-neuro-071013013958

re

Mizutani, S., Okumura, Y., Horio, T., Iwata, T., Okumura, K., Takahashi, K., Murakami, Y., Dasai, F., Ishida, M., Sawada, K., 2017. Development of Glutamate Sensor for Neurotransmitter

lP

Imaging. Sensors Mater. 29, 253–260.

Niwa, O., 1995. Electroanalysis with interdigitated array microelectrodes. Electroanalysis.

na

doi:10.1002/elan.1140070702

Niwa, O., Morita, M., Tabei, H., 1991. Highly sensitive and selective voltammetric detection of

ur

dopamine with vertically separated interdigitated array electrodes. Electroanalysis 3, 163–168. doi:10.1002/elan.1140030305

Jo

Niwa, O., Morita, M., Tabei, N., 1994. Highly Selective Electrochemical Detection of Dopamine Using lnterdigitated Array Electrodes Modified with Nafion / Polyester lonomer Layered Film. Electroanalysis 6, 237–243. doi:DOI 10.1002/elan.1140060310

Perone, S.P., Kretlow, W.J., 1966. Application of Controlled Potential Techniques to Study of Rapid Succeeding Chemical Reaction Coupled to Electro-Oxidation of Ascorbic Acid. Anal. Chem. 38, 1760–1763. doi:10.1021/ac60244a034

Sabeti, J., Gerhardt, G.A., Zahniser, N.R., 2003. Chloral hydrate and ethanol, but not urethane, alter the clearance of exogenous dopamine recorded by chronoamperometry in striatum of unrestrained rats. Neurosci. Lett. 343, 9–12. doi:10.1016/S0304-3940(03)00301-X Schultz, W., Dayan, P., Montague, P.R., 1997. Neural Substrate of Prediction and. Science (80-. ). 275, 1593–1599. doi:10.1126/science.275.5306.1593 Schwerdt, H.N., Kim, M.J., Amemori, S., Homma, D., Yoshida, T., Shimazu, H., Yerramreddy, H., Karasan, E., Langer, R., Graybiel, A.M., Cima, M.J., 2017. Subcellular probes for neurochemical

ro of

recording from multiple brain sites. Lab Chip 17, 1104–1115. doi:10.1039/C6LC01398H

Wightman, R.M., Jankowski, J. a, Kennedy, R.T., Kawagoe, K.T., Schroeder, T.J., Leszczyszyn, D.J., Near, J. a, Diliberto, E.J., Viveros, O.H., 1991. Temporally resolved catecholamine spikes

-p

correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. U. S. A. 88, 10754–10758. doi:10.1073/pnas.88.23.10754

re

Wolfrum, B., Zevenbergen, M., Lemay, S., 2008. Nanofluidic redox cycling amplification for the selective detection of catechol. Anal. Chem. 80, 972–977. doi:10.1021/ac7016647

lP

Wydallis, J.B., Feeny, R.M., Wilson, W., Kern, T., Chen, T., Tobet, S., Reynolds, M.M., Henry, C.S., 2015. Spatiotemporal norepinephrine mapping using a high-density CMOS microelectrode array.

na

Lab Chip 15, 4075–4082. doi:10.1039/C5LC00778J Zachek, M.K., Takmakov, P., Park, J., Wightman, R.M., McCarty, G.S., 2010. Simultaneous

ur

monitoring of dopamine concentration at spatially different brain locations in vivo. Biosens. Bioelectron. 25, 1179–1185. doi:10.1016/j.bios.2009.10.008

Jo

Zhang, M., Liu, K., Xiang, L., Lin, Y., Su, L., Mao, L., 2007. Carbon nanotube-modified carbon fiber microelectrodes for in vivo voltammetric measurement of ascorbic acid in rat brain. Anal. Chem. 79, 6559–6565. doi:10.1021/ac0705871

Biography

Hiroya, Abe Assistant Professor

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CV/ biography Hiroya Abe received the B.S. degree in applied chemistry, chemical engineering, and biomolecular engineering from Tohoku University in Japan in 2014. He received the M. S. and Ph. D. degrees in electrochemistry from Tohoku University in 2016 and 2018, respectively. He was a research fellow (DC1) of the Japan Society for Promotion of Science from 2016 to 2018. He was a research associate in the WPI-AIMR

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at the Tohoku University from 2018 to 2019. Currently, He is an assistant professor in the WPI-AIMR and the Frontier Research Institute for Interdisciplinary Sciences (FRIS) at the Tohoku University. His research interests focus electrochemical sensors for imaging neurotransmitters from neural tissues. Furthermore, his other

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researches are electrochemical catalysts for energy and environment based on bio-inspired materials, polymer

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materials for bio-scaffold and surface modification, and so on.