Densified Electrochemical Sensors Based on Local Redox Cycling

Mar 12, 2014 - between Vertically Separated Electrodes in Substrate. Generation/Chip Collection and Extended Feedback Modes. Kosuke Ino,*. ,†...
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Densified Electrochemical Sensors Based on Local Redox Cycling between Vertically Separated Electrodes in Substrate Generation/Chip Collection and Extended Feedback Modes Kosuke Ino,*,† Yusuke Kanno,† Taku Nishijo,† Hirokazu Komaki,† Yuta Yamada,† Shinya Yoshida,§ Yasufumi Takahashi,§ Hitoshi Shiku,† and Tomokazu Matsue*,†,§ †

Graduate School of Environmental Studies and §WPI Advanced Institute for Materials Research, Tohoku University, Sendai, Japan 980-8579 S Supporting Information *

ABSTRACT: A new local redox cycling-based electrochemical (LRC-EC) device integrated with many electrochemical sensors has been developed into a small chip device. The LRC-EC chip device was successfully applied for detection of alkaline phosphatase and horseradish peroxidase activity in substrate generation/chip collection (SG/CC) and extended feedback modes, respectively. The new imaging approach with extended feedback mode was particularly effective for sharpening of the image, because this mode uses feedback signals and minimizes the undesired influence of diffusion. The LRC-EC chip device is considered to be a useful tool for bioanalysis.

fabricate an n × n array of crossing points with only 2n bonding pads for external connection. When appropriate potentials are applied at these electrodes, redox cycling can be induced at desired crossing points that act as individual electrochemical sensors. We have previously applied the LRC-EC system to detection of enzyme activity,16,17 gene-expression analysis,14,15 cell differentiation analysis,11−13 DNA detection,18 and dynamic analyses of droplets12 using several types of LRC-EC chip devices, such as a device with comb-type interdigitated array (IDA)11−13,16 and disk−ring14 and ring−ring15 electrodes. However, it was very difficult to densify the sensor points in the previous LRC-EC chip devices,11−13,16 because the IDA electrodes for the sensors were fabricated two-dimensionally (Figure S1, Supporting Information). To solve this problem, a three-dimensional configuration of generator and collector electrodes has been adopted to achieve a higher density of electrochemical sensors (Figure S1, Supporting Information). This structure has two advantages when compared with typical planar electrodes. First, the gap between the microband electrodes is defined by the thickness of the insulation layer, which can easily be thinner than a lithographically defined pattern. Second, the area required for the generator and collector electrodes can be minimized due to the simple three-dimensional structure, which makes the entire device very compact. Some groups have previously reported a three-dimensional configuration of generator and collector electrodes. Niwa et al.19 fabricated

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ioimaging is a crucial technology in modern biology, and among the various bioimaging methods, electrochemical imaging has been used to characterize biomaterials by taking advantage of its simplicity, lower detection limit, and selectivity. Scanning electrochemical microscopy (SECM), which is a type of scanning probe microscopy, has frequently been used for bioimaging and to characterize localized chemical reactions by scanning a sample with a probe electrode.1,2 In contrast, microelectrode array devices have also been developed to obtain higher temporal resolution toward real-time or easy-to-use bioimaging. Microelectrode arrays can realize two-dimensional amperometric imaging of redox species in tissues3 and have thus received considerable attention for high-throughput analysis of biosamples, such as DNA,4 proteins,4 and cells.5,6 The electrochemical signals can be processed by conventional electronics in a very cheap and fast manner, and miniaturized electrochemical transducers can easily be integrated in a microsystem by employing conventional microfabrication technologies, so that various types of amperometric microelectrode arrays have been designed and applied to chemical and biological analyses.3−10 However, it is difficult to collect electrochemical responses at many individual sensors with an electrode array device where electrodes are simply fabricated, because space for electrodes including sensors, lead connections, and bond pads is insufficient on a small chip device. To solve this problem, we have proposed a novel method based on redox cycling and designated this method as a local redox cycling-based electrochemical (LRC-EC) system.11−18 Two arrays of microelectrodes are orthogonally arranged in the LRC-EC system to © 2014 American Chemical Society

Received: January 31, 2014 Accepted: March 12, 2014 Published: March 12, 2014 4016

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Figure 1. General outline of LRC-EC chip device. The LRC-EC chip device consisted of 16 upper and 16 lower electrodes. A multichannel potentiostat is connected to these electrodes through connector pads and a switching system, all of which are controlled with a computer. In substrate generation/ chip collection (SG/CC) mode, local redox cycling between the electrodes is induced only at designated cross points.

products were detected via redox cycling between electrodes and enzymes above the crossing points of the LRC-EC chip device. We refer to this detection system as the extended feedback mode. This is the first report of a feedback mode for an LRC-EC system.

vertically separated IDA electrodes, where upper and lower electrodes were separated by a 0.5 μm SiO2 layer. Zhu et al.20 have previously reported that upper and lower electrodes were separated by a polyimide layer. In the present study, vertically separated electrodes were incorporated into a LRC-EC chip device to densify many electrochemical sensors. The general architecture for the present study is presented in Figure 1. The LRC-EC chip device with vertically separated electrodes was applied in generation/collection and feedback modes. The present device realizes the redox cyclingbased addressable electrochemical measurements by use of vertically separated electrodes, while the devices reported by some groups utilized vertically separated electrodes only for redox cycling-based signal amplification and not for addressable measurements. To characterize the LRC-EC chip device with vertically separated electrodes, embryoid bodies (EBs) from embryonic stem (ES) cells were electrochemically evaluated by their alkaline phosphatase (ALP) activity. The enzymatic products were detected on the LRC-EC device after the ALP-catalyzed reaction, which is similar to the substrate generation/tip collection mode with SECM.1 We also refer to this detection system as substrate generation/chip collection (SG/CC) mode.11−17 The enzymatic products were detected via redox cycling between electrodes at the crossing points of the LRC-EC chip device. Furthermore, a novel detection system of the LRC-EC chip device was developed that used feedback signals, which is similar to the feedback mode used in SECM.1 The detection system was applied to addressable imaging of horseradish peroxidase (HRP) activity, where the H2O2 fuel for HRP-catalyzed reaction was produced on the electrodes of the LRC-EC device and the enzymatic products after HRP-catalyzed reaction with fuel were detected on the electrodes of the device. Thus, the enzymatic

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MATERIALS AND METHODS Device Fabrication. The fabrication process of the LRC-EC chip device is schematically illustrated in Figure 2 and is described in detail in the Supporting Information. Scanning ion conductance microscopy (SICM) was used to acquire three-dimensional images of the LRC-EC chip device. SICM instrumentation and protocols have been described previously.21,22 Experimental Setup for Electrochemical Measurement. The experimental setup for electrochemical measurements was described in our previous paper.11 Briefly, the connector pads of the LRC-EC chip device were connected to a multichannel potentiostat (HA-1010 mM4, Hokuto Denko) though a switching matrix (NI PXI-2529, National Instruments) (Figure 1). The potentiostat and switching matrix were controlled by a program written with LabVIEW (National Instruments). Ag/AgCl (saturated KCl) and Pt electrodes were used as reference and counter electrodes, respectively. The electrodes were inserted into the sample solution. All electrochemical detection was performed in a Faraday cage. Device Characterization by Redox Cycling between Electrodes in SG/CC Mode. Electrochemical performance of the LRC-EC chip device for redox cycling between electrodes was characterized by cyclic voltammetry and amperometry in 1.0 mM ferrocenemethanol (FcCH2OH) in phosphate-buffered saline solution (PBS). For single-mode cyclic voltammetry, the upper and lower electrodes were scanned between 0.00 and 0.60 V at 20 mV/s; for dual-mode cyclic voltammetry, the lower electrodes were scanned between 0.00 and 0.60 V at 20 mV/s 4017

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Figure 2. Schematic diagram of LRC-EC device fabrication process. (1) A glass substrate is used for device fabrication. (2) Pt (or Au) lower electrodes are fabricated. (3) A polyimide layer is deposited on the lower electrodes. (4) Pt upper electrodes are fabricated. (5) The polyimide layer is removed by a reactive ion etching (RIE) process to reveal the lower electrodes. The polyimide layer under the upper electrodes is preserved because the upper electrodes act as a mask during the process. (6) Finally, SU-8 microwells are fabricated at individual crossing points. Lower and upper electrodes are used as column and row electrodes, respectively.

Electrochemical Imaging of Horseradish Peroxidase Membrane in Extended Feedback Mode. An HRP membrane was prepared by mixing 150 mg of bovine serum albumin (BSA), 967 μL of PBS, 33 μL of 25% glutaraldehyde (GA), and 30 mg of HRP. The HRP membrane was cut and immersed in PBS containing 1.0 mM FcCH2OH on the device. The detection scheme is described in the following section and in Figure S7 (Supporting Information).

while the potential of the upper electrodes was kept at 0.00 V (Figure S2, Supporting Information). For single-mode amperometry, the upper and lower electrodes were stepped from 0.00 to 0.50 V; for dual-mode amperometry, the lower electrodes were stepped from 0.00 to 0.50 V while the potential of the upper electrodes was kept at 0.00 V (Figure S2, Supporting Information). Electrochemical Imaging of Alkaline Phosphatase Activity on Embryoid Bodies in SG/CC Mode. To fabricate EBs, mouse ES cells (129/SvEv; DS Pharma Biomedical. Co., Ltd., Japan) were cultured in StemMedium (DS Pharma Biomedical. Co., Ltd.) with 1000 units/mL mouse leukemia inhibitory factor (LIF) and 1 mM β-mercaptoethanol. EBs were formed by the hanging drop method.23−25 Briefly, ES cells were suspended in StemMedium supplemented with 15% fetal bovine serum (FBS). The cell suspension was introduced onto the cover of a culture dish to form droplets (20 μL) containing the ES cells (200 cells/drop). The droplets were then hung from the dish cover and incubated for 2 days to form EBs. The scheme for electrochemical imaging of ALP activity on EBs is shown in Figures S3 and S4 (Supporting Information). Device Characterization by Redox Cycling between Enzymes and Electrodes in Extended Feedback Mode. Electrochemical performance of the LRC-EC device for redox cycling between enzymes and electrodes was characterized by cyclic voltammetry and amperometry in 1.0 mM FcCH2OH and 5 mg/mL HRP in PBS. In this case, the Au electrodes were used as lower electrodes (Figure S5, Supporting Information) and were scanned between 0.00 and −0.60 V at 20 mV/s, while the potential of the upper electrodes was kept at 0.00 V (Figure S6, Supporting Information). Amperometry with the LRC-EC chip device was conducted in 1.0 mM FcCH2OH and 5 mg/mL HRP in PBS. The lower electrodes were stepped from 0.00 to −0.60 V, while the potential of the upper electrodes was kept at 0.00 V (Figure S6, Supporting Information).

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RESULTS Device Fabrication. Figure 2 shows a schematic diagram of the fabrication process for the LRC-EC chip device. Briefly, the LRC-EC chip device consists of four layers. The lower Pt (or Au) electrodes (column electrodes) and upper Pt electrodes (row electrodes) are separated by a polyimide layer (Figure 2). Figure 3A shows an optical image of the entire device and a micrograph of the 256 electrochemical sensors that were successfully packed within a small area (0.23 mm2) with only 32 connector pads. The microwells are 22 μm in diameter and are separated from the adjacent wells by a distance of 30 μm (center-to-center). The SICM image and crosssectional profile of the microwell indicates the heights of the polyimide layer and the microwell were approximately 3.4 and 6 μm, respectively (Figure 3B,C). Device Characterization by Redox Cycling between Electrodes in SG/CC Mode. Electrochemical performance for redox cycling between electrodes in SG/CC mode was investigated by use of FcCH2OH. The connection between the potentiostat and these electrodes through a switching matrix is presented in Figure S2 (Supporting Information) for cyclic voltammetric and amperometric measurements in single and dual modes.11 For single mode, the potentials for the upper and lower electrodes are synchronized and swept in the same way. For dual mode, the potential of the lower electrodes is swept while the potential of the upper electrodes is kept at 0.00 V. 4018

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Figure 3. Images of LRC-EC chip device. (A) Photographic image of the entire device, which consists of 256 sensor points with only 32 connector pads. (B) Three-dimensional SICM image and (C) cross-sectional SICM profile of a microwell.

Figure 4. Device performance with redox cycling between electrodes in SG/CC mode. (A, B) Cyclic voltammograms and (C, D) amperograms for 1.0 mM FcCH2OH in (A, C) single mode and (B, D) dual mode are shown. Currents were acquired from (red) upper and (blue) lower electrodes. The scan rate used for cyclic voltammetry was 20 mV/s. (A) For single mode, all electrodes were scanned. (B) For dual mode, the potential of the upper electrodes was kept at 0.00 V while the lower electrodes were scanned. (C) For amperometric measurements in single mode, all electrodes were stepped from 0.00 to 0.50 V at the time indicated by the arrow. (D) For amperometriv measurements in dual mode, all lower electrodes were stepped from 0.00 to 0.50 V at the time indicated by the arrow while all upper electrodes were held at 0.00 V.

Single-mode voltammograms show the oxidation and reduction peaks (Figure 4A). In contrast, dual-mode measurements give sigmoidal voltammograms with anodic and cathodic limiting currents at the lower and upper electrodes, respectively (Figure 4B). The anodic limiting current is significantly larger than the peak current in single mode, which indicates that redox cycling of FcCH2OH/FcCH2OH+ between these electrodes proceeds effectively in the device. Amperograms of FcCH2OH also showed that signal amplification (Figure 4C,D) and steady-state currents were rapidly acquired in dual mode. We calculated the proportion of reduction current at the upper electrode to oxidation current at the lower electrode after 120 s of the potential step for collection efficiency. The collection efficiency was found to be 93%. Electrochemical Imaging of Alkaline Phosphatase Activity on Embryoid Bodies in SG/CC Mode. The ALP activity on EBs was electrochemically imaged by use of the LRCEC device in SG/CC mode. A schematic diagram and scheme for

the electrochemical imaging of ALP activity on EBs are shown in Figures S3 and S4 (Supporting Information). Briefly, EBs were introduced into a p-aminophenyl phosphate (PAPP) solution on the LRC-EC chip device. ALP in the EBs converted the PAPP to p-aminophenol (PAP), and the PAP was detected by the LRCEC chip device. Electrochemical images were based on reduction currents of the oxidation products, p-quinonimine (QI), at the upper electrodes (Figures S3 and S4, Supporting Information). Figure 5 shows that the electrochemical image effectively followed the position of EBs on the device. The current signal originated from redox cycling of PAP generated by the ALP-catalyzed reaction on EBs. The electrochemical image was acquired within 30 s. Device Characterization by Redox Cycling between Enzymes and Electrodes in Extended Feedback Mode. HRP activity can be electrochemically detected in extended feedback mode. A scheme for HRP detection in extended feedback mode is shown in Figure 6. In this mode, Au electrodes 4019

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(−0.60 V) (Figure 7C,D), and FcCH2OH+ formed by the HRPcatalyzed reaction is also reduced at the upper electrodes held at 0.00 V (Figure 7C). In contrast, no current was acquired from the upper electrodes without HRP (Figure 7D) Electrochemical Imaging of Horseradish Peroxidase Membrane in Extended Feedback Mode. A HRP membrane was placed on the LRC-EC device in PBS containing FcCH2OH, and electrochemical imaging was performed in extended feedback mode (Figure S7, Supporting Information). Reduction current of FcCH2OH+ from the individual upper electrodes is used for electrochemical imaging of HRP. Figure 8 shows the electrochemical image that follows the position of the HRP membrane in solution, which indicates that extended feedback mode can be applied for addressable detection of HRP activity. The electrochemical image was acquired within 33 s.

Figure 5. Electrochemical imaging of EBs in SG/CC mode: (A) optical and (B) electrochemical images of EBs. The electrochemical image consisted of 256 pixels.

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DISCUSSION The sensor electrodes of the present LRC-EC device were arranged three-dimensionally to achieve dense packing of many sensors; the sensor density of the present LRC-EC device was significantly improved (44 times) compared with that of the previous device.11 Redox cycling on the device is the key feature that enables addressable electrochemical measurements of a large number of sensor points with a small number of connection bond pads. Redox cycling has been used for amplification of the electrochemical signal. The efficiency of redox cycling increases with decreasing electrode distance; therefore, nanofluidic electrochemical devices were applied for single-molecule detection by redox cycling.29−31 These nanofluidic redox cycling sensors are very powerful tools because a single molecule can be detected. In contrast, the present device is suitable for introducing large biosamples such as spheroids because there is an open space above the sensors. Also carbon IDA nanoelectrodes have been reported.32 The present LRC-EC device uses redox cycling not only for signal amplification but more importantly for a reduction of the number of connection bond pads, thereby increasing the density of sensor points within a limited area. In the present study, the gap between microband electrodes was defined by the thickness of the polyimide layer (3.4 μm), which enables the fabrication of a robust and reproducible electrode configuration to amplify the electrochemical signals.

were used instead of Pt electrodes on the LRC-EC chip device as lower electrodes to produce H2O2 from the reduction of O2 (Figure S5, Supporting Information). The electrochemically generated H2O2 acts as an oxidant for the HRP-catalyzed reaction (Figure 6). When the solution contains FcCH2OH, HRP catalyzes the oxidation of FcCH2OH by H2O2 to yield FcCH2OH+.26−28 FcCH2OH+ is then reduced to FcCH2OH on the upper electrodes. Extended feedback mode measurements are based on redox cycling of FcCH2OH/FcCH2OH+ between HRP and the upper electrodes coupled with electrochemically generated H2O2 at the lower electrodes for addressable detection (Figure 6). Cyclic voltammetry and amperometry were performed to investigate the electrochemical performance in extended feedback mode. The connection between potentiostat and electrodes through a switching matrix is shown in Figure S6 (Supporting Information). Voltammograms with or without HRP show reduction of oxygen at the lower electrodes occurred at around −0.50 V (Figure 7A,B). The upper electrodes held at 0.0 V also show a reduction peak at the same potential when HRP is added (Figure 7A). The peak is due to the reduction of FcCH2OH+ formed by HRP-catalyzed oxidation with H2O2 at the lower electrodes. In contrast, no current was acquired from the upper electrodes without HRP (Figure 7B). Amperograms measured in PBS containing FcCH2OH with HRP also indicate that O2 is reduced on the lower electrodes

Figure 6. Scheme for detection of HRP activity in extended feedback mode. A solution containing FcCH2OH is introduced onto the LRC-EC chip device. (1) O2 is reduced to H2O2 on the lower electrode. (2) FcCH2OH then reacts with H2O2 on HRP to produce FcCH2OH+. FcCH2OH+ is reduced on the upper electrodes. The reduction current of FcCH2OH+ is acquired for electrochemical imaging of HRP. 4020

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Figure 7. Device performance with redox cycling between enzymes and electrodes in extended feedback mode. (A, B) Cyclic voltammograms and (C, D) amperograms for (A, C) 1.0 mM FcCH2OH and 5 mg/mL HRP in PBS and for (B, D) 1.0 mM FcCH2OH in PBS. The responses were obtained from all (blue) lower and (red) upper electrodes. For cyclic voltammetry, all lower electrodes were scanned while all upper electrodes were kept at 0.00 V. The scan rate was 20 mV/s. For amperometric measurements, all lower electrodes were stepped from 0.00 to −0.60 V at the time indicated by the arrow while all upper electrodes were kept at 0.00 V.

Electrochemical performance for redox cycling between electrodes in SG/CC mode was investigated via voltammetric and amperometric measurements with FcCH2OH. Single-mode voltammetric measurements indicated that electrochemical signals at the lower electrodes were smaller than those at the upper electrodes, even though the lower electrodes were larger in size. Consumption of FcCH2OH at the upper electrodes reduced the flux of the species and decreased the electrochemical signal at the lower electrodes. In dual mode, the magnitude of the limiting current was significantly higher than the peak current in single mode due to redox cycling of FcCH2OH between upper and lower electrodes (Figure 4). Signal amplification by redox cycling was also apparent in the dual-mode amperometric measurements when the potentials of upper and lower electrodes were set at 0.00 and 0.50 V, respectively. Steady-state current at the lower electrodes is approximately 10 times larger in dual mode than that in single mode (Figure 4). These currents in the chronoamperometric measurement were compared with those from simulation. Detailed information on the simulation is given in Supporting Information. Briefly, diffusion-based simulation of a redox compound in the LRC-EC device was used to obtain electrochemical signals from sensors. Figure 4 and Figure S8 (Supporting Information) show that the shapes of experimental chronoamperograms were similar to those from the diffusion-based simulation. These results show that charging currents are much smaller than currents from the upper and lower electrodes when 1.0 mM FcCH2OH is used under the present conditions. To reduce the undesired influence of charging currents, electrochemical imaging with LRC-EC was conducted with only the collector current. Since the potential of collector electrodes was set at a fixed value, the collector current does not show the influence of charging currents.11 Simulation results in dual mode also showed that current densities at the

Figure 8. Electrochemical imaging of HRP membrane in extended feedback mode: (A) optical and (B) electrochemical images of HRP membrane.

Although simple devices with microelectrode arrays have been fabricated and used for multipoint measurements and bioimaging,3 it is difficult to locate a large number of electrodes with corresponding bond pads on a small chip due to the limited space. In contrast, a large number of microelectrodes can be integrated in complementary metal oxide semiconductor (CMOS)-based platforms.33−35 We have previously reported a CMOS-based chip device (Bio-LSI) comprising 400 sensors with a pitch of 250 μm for real-time bioimaging.33,34 Although the Bio-LSI system can be applied for sensitive bioassays, it is difficult to pack more electrochemical sensors into the BioLSI system due to the large size of the operational amplifiers in each unit. In contrast, a dense array of electrochemical sensors can be easily achieved with the LRC-EC system because its structure is much simpler than that of the CMOS-based platform. Furthermore, electrochemical signals in the LRC-EC system can be amplified by redox cycling. Therefore, the LRC-EC system is useful for electrochemical imaging in electrochemical chip devices. 4021

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upper and lower electrodes after 120 s of potential step were −14 and 9.7 A/m2, respectively (Figure S8, Supporting Information). Current densities in dual mode at the upper and lower electrodes after 120 s of potential step in the experimental results were −11 and 8.2 A/m2, respectively (Figure 4), which are similar to those in the simulation. All the LRC-EC devices were checked with FcCH2OH beforehand, and only devices that showed variation of currents from sensors within ±10% were used for measurements. Devices meeting the requirement were approximately 30% of all devices. Alkaline phosphatase is one useful biomarker to check the differentiation level of ES cells.36 ALP activity on EBs from ES cells was electrochemically detected and imaged in SG/CC mode (Figure 5). The PAP concentration on the EBs can be approximated from the maximum response shown in Figure 5, which was 300 μM and similar to our previous results.11 These result shows that the present LRC-EC device in SG/CC mode can be applied for quantitative assays. SG/CC mode provides highly sensitive imaging because the enzymatic products are accumulated in the sensor area. It should be noted that the present device consists of small densely packed sensors, so that the diffusion layer of PAP formed by the ALP-catalyzed reaction can be imaged (Figure S9, Supporting Information). In contrast, the previous device with large pixels showed the ALP activity of a single EB as a whole. Although the resolution of electrochemical images is significantly improved with the present device, the image of the EB itself in SG/CC mode was unclear compared that achieved by direct imaging with an optical apparatus due to diffusion of enzymatic products (Figure 5). Therefore, achievement of high-resolution electrochemical imaging with the present device will require a feedback process to minimize the undesired influence of diffusion. Electrochemical images were acquired within approximately 30 s with the present device (Figure 5). To image dynamic processes of biosamples, it will be necessary to improve its temporal resolution. For electrochemical imaging, the waiting time for stabilization of the currents from the row electrodes was 1000−2000 ms after stepping the potential of the column electrode (Figure 4; Figures S4 and S8, Supporting Information), which is the limiting factor for temporal resolution.16 If the distance between generator and collector electrodes is shortened, temporal resolution can be improved by shortening the waiting time for stabilization of currents from collector electrodes. We have proposed the extended feedback mode as a novel detection system with the LRC-EC system. Clear electrochemical imaging of HRP based on redox cycling between enzymes and electrodes has been demonstrated in extended feedback mode. Figure 6 shows the principle for extended feedback mode operation. O2 is reduced at the lower electrodes to yield H2O2, which then participates in the feedback process for redox cycling between enzymes and electrodes. Figure 7C shows that signals of FcCH2OH+ from the upper electrodes are decreased gradually. H2O2 production seems to be the ratelimiting reaction under the present conditions because the production of H2O2 by O2 reduction leads to gradual consumption of O2 on the lower electrodes, thereby causing a decrease in the production of FcCH2OH+ by the HRP-catalyzed reaction. FcCH2OH+ was also collected on the lower electrodes because the potential of the lower electrodes was −0.60 V. Electrochemical images obtained in extended feedback mode are based on reduction currents for FcCH2OH+ at desired crossing points on the upper electrodes (Figure 6; Figure S7, Supporting Information).

Figure 8 shows that the electrochemical image in extended feedback mode was clearer than that in SG/CC mode. H2O2 generation proceeds only at the designated lower electrodes set at −0.6 V; therefore, formation of FcCH2OH+ by the HRPcatalyzed reaction also occurs only in a limited area. The space restriction of HRP-catalyzed reaction prevents FcCH2OH+ from spreading, which enables a high-resolution image to be obtained in extended feedback mode compared with that available in SG/CC mode. Figures 5 and 8 show that there was no significant cross-talk due to diffusion of redox mediators. Although it is possible to pack more electrochemical sensors into the present system, it is necessary to consider cross-talk among sensors. We have previously reported that the LRC-EC system in SG/CC mode can be applied for quantitative assays.11,14,16 In the present study, quantitative performance of extended feedback mode of the present device has not been checked. We will investigate its quantitative performance in future studies. In conclusion, a novel LRC-EC chip device having a densely packed electrode array with vertical separation was fabricated, and electrochemical performance of the device was evaluated by voltammetry and amperometry. The LRC-EC chip device demonstrates significant improvement of electrochemical image resolution and was successfully applied for electrochemical imaging of ALP and HRP in SG/CC and extended feedback modes, respectively. This is the first report on extended feedback mode operation of the LRC-EC system. Extended feedback mode eliminated the undesired influence of diffusion, so that clear images could be obtained. In comparison with SECM imaging, electrochemical imaging with the LRC-EC system is suitable for continuous imaging of biomaterials because LRC-EC enables rapid acquisition of an image. We are currently planning to further improve the density of electrochemical sensors in the LRC-EC system for high-resolution bioimaging applicable to single cells.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text with detailed information on device fabrication and simulation of current due to redox compound diffusion, and nine figures showing generator and connector electrodes, connections of electrodes to potentiostat, diagrams of electrochemical imaging, LRC-EC chip device, simulation of current due to redox compound diffusion, and comparison of past and present devices. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected]. *E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (A) (25248032) and a Grant-in-Aid for Young Scientists (B) (23760745) from the Japan Society for the Promotion of Science (JSPS). This research was partly supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, from the Japan Science and Technology Agency. 4022

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dx.doi.org/10.1021/ac500435d | Anal. Chem. 2014, 86, 4016−4023