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Microarrays are widely utilized in bioanalysis. Electrochemical biosensing techniques are often applied in microarray-based assays because of their simplicity, low cost, and high sensitivity. In such systems, the electrodes and sensing elements are arranged in arrays, and the target analytes are detected electrochemically. These sensors can be utilized for high-throughput bioanalysis and the electrochemical imaging of biosamples, including proteins, oligonucleotides, and cells. In this chapter, we summarize recent progress on these topics. We categorize electrochemical biosensing techniques for array detection into four groups: scanning electrochemical microscopy, electrode arrays, electrochemiluminescence, and bipolar electrodes. For each technique, we summarize the key principles and discuss the advantages, disadvantages, and bioanalysis applications. Finally, we present conclusions and perspectives about future directions in this field., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The respiratory activity of cultured cells can be electrochemically monitored using scanning electrochemical microscopy (SECM) with high spatial resolution. However, in SECM, the electrode takes a long time to scan, limiting simultaneous measurements with large biological samples such as cell spheroids. Therefore, for rapid electrochemical imaging, a novel strategy is needed. Herein, we report electrochemiluminescence (ECL) imaging of spheroid respiratory activity for the first time using sequential potential steps. L-012, a luminol analog, was used as an ECL luminophore, and H 2 O 2 , a sensitizer for ECL of L-012, was generated by the electrochemical reduction of dissolved O 2 . The ECL imaging visualized spheroid respiratory activity-evidenced by ECL suppression-corresponding to O 2 distribution around the spheroids. This method enabled the time-lapse imaging of respiratory activity in multiple spheroids with good spatial resolution comparable to that of SECM. Our work provides a promising high-throughput imaging strategy for elucidating spheroid cellular dynamics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cell aggregates have attracted much attention owing to their potential applications in tissue engineering and drug screening. To evaluate cellular respiration of individual cell aggregates in these applications, noninvasive and on-chip high-throughput analytical tools are necessary. Electrochemical methods for detecting oxygen concentrations are useful because of their noninvasiveness. However, these conventional methods may be unsuitable for high-throughput detection because it is difficult to prepare many electrodes on a small chip owing to the limitation of area for connecting electrodes. Alternatively, a bipolar electrode (BPE) system offers clear advantages. In this system, electrochemical reactions are induced at both ends of a BPE without complex wiring. In this study, we present a BPE array for detecting the respiratory activity of cell aggregates. Oxygen concentrations near cell aggregates at cathodic poles of BPEs were converted to electrochemiluminescence (ECL) signals of [Ru(bpy) 3 ] 2+ /tripropylamine at anodic poles of BPEs. To separate ECL chemicals from cell aggregates, we fabricated a closed BPE device containing analytical and reporter chambers. As a proof of concept, 32 BPEs were controlled wirelessly using a pair of driving electrodes, and the respiratory activities of individual MCF-7 cell aggregates as a cancer model were successfully detected by monitoring ECL signals. Compared with conventional electrode arrays for cell analysis, the wiring of the current device was simple because the multiple BPEs functioned with only a single power supply. To the best of our knowledge, this is the first study of on-chip analysis of cellular activity using a BPE system.

The light-driven splitting of water to oxygen (O 2 ) is catalyzed by a protein-bound tetra-manganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster in Photosystem II. In the current study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to perform two-dimensional imaging of light-induced O 2 evolution from spinach leaves. The employed Bio-LSI chip consists of 400 sensor electrodes with a pitch of 250 μm for fast electrochemical imaging. Spinach leaves were illuminated to varying intensities of white light (400-700 nm) which induced oxygen evolution and subsequent electrochemical images were collected using the Bio-LSI chip. Bio-LSI images clearly showed the dose-dependent effects of the light-induced oxygen release from spinach leaves which was then significantly suppressed in the presence of urea-type herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Our results clearly suggest that light-induced oxygen evolution can be monitored using the chip and suggesting that the Bio-LSI is a promising tool for real-time imaging. To the best of our knowledge, this report is the first to describe electrochemical imaging of light-induced O 2 evolution using LSI-based amperometric sensors in plants.

Electrochemical imaging is an excellent technique to characterize an activity of biomaterials, such as enzymes and cells. Large scale integration-based amperometric sensor (Bio-LSI) has been developed for the simultaneous and continuous detection of the concentration distribution of redox species generated by reactions of biomolecules. In this study, the Bio-LSI system was demonstrated to be applicable for simultaneous detection of different anaytes in multiple specimens. The multiple specimens containing human immunoglobulin G (hIgG) and mouse IgG (mIgG) were introduced into each channel of the upper substrate across the antibody lines for hIgG and mIgG on the lower substrate. Hydrogen peroxide generated by the enzyme reaction of glucose oxidase captured at intersections was simultaneously detected by 400 microelectrodes of Bio-LSI chip. The oxidation current increased with increasing the concentrations of hIgG, which can be detected in the range of 0.01-1.0 µg mL(-1) . Simultaneous detection of hIgG and mIgG in multiple specimens was achieved by using line pattern of both antibodies. Therefore, the presence of different target molecules in the multiple samples would be quantitatively and simultaneously visualized as a current image by the Bio-LSI system., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have developed a large-scale integrated (LSI) complementary metal-oxide semiconductor (CMOS)-based amperometric sensor array system called "Bio-LSI" as a platform for electrochemical bio-imaging and multi-point biosensing with 400 measurement points. In this study, we newly developed a Bio-LSI chip with a light-shield structure and a mode-selectable function with the aim of extending the application range of Bio-LSI. The light shield created by the top metal layer of the LSI chip significantly reduces the noise generated by the photocurrent, whose value is less than 1% of the previous Bio-LSI without the light shield. The mode-selectable function enables the individual operation of 400 electrodes in off, electrometer, V1, and V2 mode. The off-mode cuts the electrode from the electric circuit. The electrometer-mode reads out the electrode potential. The V1-mode and the V2-mode set the selected sensor electrode at two different independent voltages and read out the current. We demonstrated the usefulness of the mode-selectable function. First, we displayed a dot picture based on the redox reactions of 2.0 mM ferrocenemethanol at 400 electrodes by applying two different independent voltages using the V1 and V2 modes. Second, we carried out a simultaneous detection of O2 and H2O2 using the V1 and V2 modes. Third, we used the off and V1 modes for the modification of the osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) at the selected electrodes, which act as sensors for H2O2. These results confirm that the advanced version of Bio-LSI is a promising tool that can be applied to a wide range of analytical fields.

We developed a novel protease detection method based on amperometry using a p-aminophenol (pAP) conjugated substrate. We prepared Boc-Leu-Gly-Arg-pAP (LGR-pAP) as a novel substrate for a clotting enzyme, which is a protease activated by an endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. The basic study using cyclic voltammetry revealed that the oxidation peak potentials of LGR-pAP and pAP were sufficiently separated from each other (0.25 V) to conduct amperometric detection of protease activity. We combined simple amperometric detection with a screen-printed electrode chip to produce a practical protease sensor. As an application of the sensor, we demonstrated quantitative endotoxin sensing. The endotoxin activated zymogens contained in the LAL to generate pAP, which was then electrochemically detected by potential step chronoamperometry (PSCA). The observed oxidation current increased with the concentration of endotoxin in the LAL assay solution. This PSCA detection was performed with a disposable chip sensor consisting of a screen-printed electrode and a fluidic channel with a hydrophilic cover. This chip sensor successfully detected 10-1000 EU L(-1) endotoxin within 60 min. This novel amperometric measurement with a screen-printed electrode not only provides compact, low-cost, and easy-to-use sensors for on-site monitoring of endotoxin, but also shows promise for use in other in vitro protease assays for biochemical research, diagnosis, and drug development.

A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5h, and their ALP activity in a glucose-free buffer decreased after 2h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3h. The chip is expected to be useful for the evaluation of cell activities., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We have developed an LSI-based amperometric sensor called "Bio-LSI" with 400 measurement points as a platform for electrochemical bio-imaging and multi-point biosensing. The system is comprised of a 10.4 mm × 10.4 mm CMOS sensor chip with 20 × 20 unit cells, an external circuit box, a control unit for data acquisition, and a DC power box. Each unit cell of the chip contains an operational amplifier with a switched-capacitor type I-V converter for in-pixel signal amplification. We successfully realized a wide dynamic range from ±1 pA to ±100 nA with a well-organized circuit design and operating software. In particular, in-pixel signal amplification and an original program to control the signal read-out contribute to the lower detection limit and wide detection range of Bio-LSI. The spacial resolution is 250 μm and the temporal resolution is 18-125 ms/400 points, which depends on the desired current detection range. The coefficient of variance of the current for 400 points is within 5%. We also demonstrated the real-time imaging of a biological molecule using Bio-LSI. The LSI coated with an Os-HRP film was successfully applied to the monitoring of the changes of hydrogen peroxide concentration in a flow. The Os-HRP-coated LSI was spotted with glucose oxidase and used for bioelectrochemical imaging of the glucose oxidase (GOx)-catalyzed oxidation of glucose. Bio-LSI is a promising platform for a wide range of analytical fields, including diagnostics, environmental measurements and basic biochemistry.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E(1/2)) for [Fe(CN)(6)](4-) oxidation and O(2) reduction measured versus the internal Pt pseudo reference was shifted by about -0.2V, compared with the E(1/2) measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E(1/2) for PAP oxidation measured versus the Pt layer was not shifted, compared with the E(1/2) versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction.

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K(d)-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36±0.33×10(-15) mol s(-1), 1.38±0.58×10(-15) mol s(-1), and 3.44±2.07×10(-15) mol s(-1), respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An electrochemical device is proposed for high-throughput electrochemical detection that consists of 32 row and 32 column electrodes on a single glass substrate. The row and column electrodes are connected to interdigitated array (IDA) electrodes to form 1024 (32 × 32) addressable sensor points in the device. Electrochemical responses from each of the 1024 sensors were successfully acquired on the device within 1 min using redox cycling at individual IDA electrodes, which ensures application of the device to comprehensive, high-throughput electrochemical detection for enzyme-linked immunosorbent assay (ELISA), reporter gene assay for monitoring gene expressions, and DNA analysis.

We report here a rapid, simple, and simultaneous immunosensing method for two tumor markers, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), by applying the negative-dielectrophoretic (n-DEP) manipulation of microparticles. Microparticles modified with different antibodies rapidly accumulated to designated areas of poly(dimethylsiloxane) (PDMS) fluidic channels modified with different antibodies within 1 min by n-DEP upon the application of AC voltage. The presence of specific antigens, AFP or CEA, permitted the irreversible capture of microparticles via the formation of immuno-complexes between the PDMS surface and the microparticles. Uncaptured microparticles redispersed after switching off the AC voltage. The fluorescent intensity from the irreversibly captured microparticles allowed us to determine the concentration of AFP and CEA in the sample. Neither the unreacted analytes nor the microparticles required separation steps, since we detected the fluorescent signals only from the microparticles captured on the PDMS surface. The detectable concentration range shifted to lower values when the amount of the antibody on the PDMS surface increased. The range for both AFP and CEA assays was 0.1-100 ng/mL, which was sufficient to cover the concentration required for the medical diagnoses. We simultaneously detected the concentrations of AFP and CEA by using a device, with two channels modified for different antibodies. Since n-DEP was used for the rapid manipulation of the microparticles toward the PDMS surface, the time required for the assay was substantially short; 1 min for forcing and 5 min for redispersion of the microparticles and sensing., ((c) 2010 Elsevier B.V. All rights reserved.)

We developed an electrochemical-sensing device for continuous monitoring extracellular hydrogen peroxide (H(2)O(2)). The device consists of an indium-tin-oxide electrode coated with osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) and a poly-dimethyl siloxane well to house the cells on the chip. Granulocyte-like differentiated HL-60 cells were accommodated in the well and stimulated with phorbol 12-myristate 13-acetate (PMA), which triggered the generation of H(2)O(2). The extracellular H(2)O(2) released from the cells was enzymatically reduced at the Os-HRP-modified electrode chip using Os(II) as an electron donor, resulting in reduction current responses by the device. The reduction current increased immediately upon PMA stimulation and this current transient was similar to that obtained by conventional chemiluminescence assays using sodium luminol. Apocynin, an inhibitor of NADPH oxidase activation, eliminated both the electrochemical and chemiluminescence signals. On the other hand, superoxide dismutase (SOD) increased the amperometric signals and catalase (CAT) decreased, whereas SOD decreased luminescence emission and CAT did not. These results were in accordance with the expected reaction mechanism, and strongly indicate that this new electrochemical-sensing device successfully detects extracellular H(2)O(2) production., ((c) 2009 Elsevier B.V. All rights reserved.)

Endocrine disruptors that act like hormones in the endocrine system might have toxic effects. Therefore, it is important to develop a portable device that can detect hormone active chemicals in samples rapidly and easily. In this study, a microfluidic device was developed for the detection of hormone active chemicals using genetically engineered yeast cells. The yeast cells were used as biosensors since they were genetically engineered to respond to the presence of hormone active chemicals by synthesizing beta-galactosidase (beta-gal). For achieving further sensitivity, we incorporated interdigitated array (IDA) electrodes (width, 1.2 microm; gap, 0.8 microm) with 40 electrode fingers into the analytical chamber of the microfluidic device. The yeast cells precultured with a hormone active chemical, 17beta-estradiol (E2), were trapped from the main channel of the device to the analytical camber by electrophoresis. After trapping in the analytical chamber, we performed electrochemical detection of beta-gal induced in the yeast cells with the IDA electrodes. Actually, electrochemical detection was performed on p-aminophenol that was converted from p-aminophenyl-beta-D-galactopyranoside with beta-gal. The electrochemical signals from the yeast cells precultured with 17beta-estradiol were successfully detected with the device. Furthermore, the inhibitory effects of antagonists such as tamoxifen were also detected electrochemically by using the device. Thus, the present microfluidic device can be used for highly sensitive detection of hormone active chemicals.