Vol. 35, Supplement A (2019)

1.

Electrorotation is a kind of electrokinetic force which caused by the interaction of rotating electric field and the dipoles induced in cells exposed to the field, which result in the induced torque to cells. Since the rotation rate depends on the membrane capacitance of cells, electrorotation is a major candidate for characterizing dielectric properties of cell membrane in single cell manner without injuring cells. In this study, we describe a method for characterizing comprehensively dielectric properties of single-cells based on electrorotation by using a three-dimensional grid electrode device. The device consisted of two substrates with patterns of interdigitated microband-array (IDA) electrode. These substrates were placed together with a PET film (30 m in thickness) as a spacer between the electrodes to form the fluidic channel and the grid-cross of IDA electrodes. It enables to induce a rotating electric field in each micro-grid by applying AC signal with phase differences of 90 degree between their applied sinusoids to four microband electrodes of two substrates. For demonstrating a usefulness of the device based on measuring rotation rate of cells as a single-cell analysis, we performed identifying two types of cells with similar shapes (Jurkat and THP-1) and detect the cells expressing forcedly membrane receptor.

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2.

Nonenzymatic glucose sensors are attracting attention from the viewpoint of easy to handle and life as compared with enzyme glucose sensors. In our previous study, a mesoporous platinum, which has a lot of high index surface, shows high sensitivity for electrochemical glucose sensing. Various solutions such as NaOH, H₂SO₄, Phosphate Buffer, etc. can be used as an electrolyte for sensing because a nonenzymatic glucose sensor does not use an enzyme, which is known to work properly in the biological environment. In this study, NaOH was used as an electrolyte and compared with H₂SO₄ conducted in the past study. As a result, in NaOH solution the sensing performance is improved by 40 times that of H₂SO₄. In addition, L-ascorbic acid (AA) and 4-acetamidophenol (AP), which are contained in real samples and are known as interfering species for glucose sensing, were mixed into glucose solution. Then, it was found that the effect of interfering species is greatly reduced than in the case of H₂SO₄.

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3.

Recently, there are increasing cases and applications in which respiratory activity of animal and microbial cells needs to be measured using samples and reagents of very small volumes. To this end, miniaturized Clark-type oxygen electrode with an oxygen-permeable membrane that separates the internal electrolyte solution from the external analyte solution is useful. However, previous oxygen electrodes need expertise to handle particularly in their activation before use. To solve this problem, water that is necessary for the reduction of oxygen was introduced through the oxygen-permeable membrane into the electrolyte layer in the form of water vapor. The fabricated oxygen electrode showed clear responses to changes in dissolve oxygen concentration. In addition to the above-mentioned problem, evaporation of water from the electrolyte solution of a very small volume posed a problem. To solve this problem, sorbitol was added to the electrolyte solution, which was actually effective to suppress evaporation of water and stabilize the output current.

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5.

Planar typed electrochemical sensing system is newly developed for monitoring of drinking water quality. The electrochemical chip is consisted of polypyrrole coated Au, polyaniline coated Au, and Au electrodes as working electrode, reference electrode, and counter electrode, respectively. Based on the fundamental of electrochemistry, the signal of the potentiostat is the sum of half reactions of working electrode and reference electrode. Generally, the standard potential is determined by the fixed reaction of reference electrode. In this study, both reactions of reference and working electrodes are used for multiple-electrochemical sensing. pH and electroconductivity are measured on the redox of polyaniline. Here, it is reported that the newly developed bacteria sensing. Plasma membrane of bacteria is coated with sugar sequence. This sugar sequence contains hydroxyl groups in chemical structure. Therefore, bacteria behalves as anion in electric field. Polypyrrole captures anion at the positive potential. Bacteria are associated with the polypyrrole. Here, the polarization of polar molecule such as citrate, enzyme, protein, and fatty acid, is caused in electric field. Thus, the dielectric constant of interface of working electrode will be altered. This change is detected as the capacitance in Nyquist plot. This sensing system is demonstrated in drinking water in rural area of Hokkaido. The trend data will be introduced in this study.

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6.

Boron-doped diamond (BDD) has its excellent properties, e.g., physical and chemical stability, wide potential window, low background current and biocompatibility, and it is expected to be applied to an electrode material exhibiting sensitive detection of various electoactive substances. Although sensitive detection of an analyte can be achieved, however, selective detection from a sample containing various electroactive species is generally difficult at a bare BDD electrode. Thus, in this study, in order to detect electroactive analytes simultaneously from a multicomponent sample, we performed multivariate analysis of cyclic voltammetry (CV) data obtained at BDD electrode. Multivariate analysis of CVs for a sample containing dopamine, noradrenaline and adrenaline resulted in successful simultaneous determination of each compound. In addition, the multiple correlation coefficients of the calibration curves were found to be better for the CV obtained at BDD electrodes than at glassy carbon (GC) electrode.

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7.

Unobtrusive sensors are indispensable for “smart daily healthcare” in the coming IoT era to alleviate the strain on the social cost in the aging society. In this study, we have developed a simple sensor for urinary protein without requiring any addition of reagent, intending to install in home toilets. The sensor can quantify urine protein by detecting glutamate generated by protease, using glutamate oxidase (GluOx) immobilized on a Prussian blue (PB) modified electrode. Using the fabricated sensor, we demonstrated the detection of 5 mg/ml albumin after hydrolyzing by 1.5 mg/ml proteinase K (ProK) with 1% sodium dodecyl sulfate (SDS) at 55 °C for 21 h. The current at 0.0 V vs. Ag/AgCl changed -29.8 μA per adding hydrolyzed solution of 3.26 mM albumin. The release rate of glutamate (including glutamic acid) from albumin by ProK was revealed to be only 3.24% by L-Glutamate Assay Kit YAMASA NEO (YAMASA Corporation). To improve the hydrolysis rate of albumin, we used a mixture protease solution of endopeptidase and exopeptidase. By using 26.0 U/ml ProK and 182 U/mL carboxypeptidase A (CPA) mixture protease solution, 10.3% of glutamate was released from albumin at 37 °C for 1 h without SDS. Now we examined the immobilization of multiple proteases on a solid phase support such as silica particle. By integrating the proteases onto the sensor electrode, we will realize “only to dip” urine protein sensor without additional reagent usable in home toilet connected personal health recording system.

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特別講演1

Enzymatic bioelectrocatalysis (EBEC) is a reaction coupling a redox enzyme reaction and an electrode reaction. There are two types of the coupling: mediated electron transfer(MET)-type and direct-electron transfer(DET)-type. EBEC can be applied to biosensors, biofuel cells, and bio-reactors. Some recent topics in our laboratory on EBEC are introduced, especially by focusing on the application to biosensors. First of all, the significance of the steady-state diffusion-controlled amperometric response at microbiosensors is described with several examples. In addition, the utilization of ultra-fast MET-type EBEC in biosensors, introduction of potentiometric coulometry for highly sensitive detection, and construction of DET- EBEC system and its analysis are described.

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8.

We introduce a dielectrophoresis (DEP)-based manipulation with complete control that allows a fabrication of single-cell arrays instantaneously and a retrieval of target cells from the cell-based arrays by using the device containing the microwell-array electrode on microband electrodes. The device with a simple construction was composed of two substrates of microband array electrodes with orthogonal arrangement through a fluidic channel (30 m in height). A positive dielectrophoresis (p-DEP) was induced by applying AC signals (5 MHz, 3 Vpp) with opposite phase to the microband electrodes of both top and bottom substrate, respectively, resulting in the quick formation of the single-cell array trapped cells one by one into the microwells. Thereafter, for staying only target cells, the AC signals to microband electrodes except the electrodes above and below the microwell occupied with the target cell was awiched off. The present system enables us to retrieve and isolate single target cells from the microwells by switching off the signals applied to corresponding electrodes.

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9.

We developed a highly sensitive and rapid electrochemical method to detect endotoxin based on Limulus amebocyte lysate (LAL) assay using redox cycling with nanogap electrodes for electrochemical signal amplification. We used Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP), which we have developed before as an electrochemical substrate for LAL assay. At the last step of endotoxin induced LAL cascade reaction, pAP is liberated from the substrate, which can be detected electrochemically with the efficient signal amplification by redox cycling between the two electrodes of a nanogap. First, we demonstrated that pAP can be quantitatively detected in the model solution. Next, the endotoxin assay using fabricated nanogap device was performed. This method detected endotoxin as low as 0.2 and 0.5 endotoxin units L-1 (EU/L) for 1 h and 30 min of LAL reaction time using LGR-pAP substrate, respectively. Our nanogap device can detect endotoxin rapidly and simply with high sensitivity. This novel method is considered to be useful for sensitive and simple sensor to detect protease activity.

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10.

For prevention of stress-related diseases at initial stage, development of simple detection method is important to measure the concentration of biomarkers correlated with stress. Secretory immunoglobulin A (s-IgA) is known as one of stress markers, and it is abundantly present in the mucus. Therefore, s-IgA containing mucus samples such as saliva or sweat expect to be used as a specimen for non-invasive stress test. To quickly detect s-IgA, the field effect transistor (FET) biosensor is a promising measurement device which can detect intrinsic charges of the target proteins specially adsorbed on the sensing surface. In this study, jacalin, which is one of small lectin, was selected as a receptor in anticipation of an increase in charges that can be detected within the Debye length. In addition, jacalin has four binding sites that specifically recognize glycans present on the surface of s-IgA, expecting the increase of the specifically-adsorbed s-IgA involved in the FET detection. The specificity of the jacalin-immobilized FET biosensor to s-IgA was evaluated, and its usefulness toward a stress check of s-IgA from sweat was examined.

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Plenary Lecture

A method of integrating alternating current (AC) electrokinetics and electrochemical impedance spectroscopy (EIS) was proposed, designed, and demonstrated to overcome drawbacks such as slow reaction rates and long detection times brought by diffusion. AC electroosmosis (ACEO) and positive dielectrophoresis (pDEP), which was induced by a biased AC electric field, can rapidly gather and catch the analyte onto the surface of an EIS working electrode within a few minutes. An immunochip was designed for rapid, sensitive, and label-free detection based on the proposed method. Binding of the antibody with the antigen caused a change in the electron transfer resistance (Ret), which was calculated and measured in real time. The impedance change that was measured achieved a plateau for only 90 s, after concentration of AC electrokinetics, and a detection limit that reached 200 pg mL-1. Compared with the conventional incubation method, the electrokinetic-enhanced method resulted in a reaction time that was approximately 100 times faster and a detection limit that was reduced by 30 times. The results of this rapid and label-free method suggest that it can be further used in the detection of DNA hybridizations and viral infections.

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Keynote Lecture

Graphene has been proposed as a good gas sensing material because of its two-dimension morphology and outstanding electrical behaviors. However, interferences between different gaseous molecules have become major obstacles for graphene-based sensing technologies toward applications. To address this issue, we present an artificial-stacked bilayer graphene architecture for plasma-enhanced surface modification to improve specific affinity to different targets. Utilizing the double-layer structure and a low-damage plasma, in this work, O2 and N2 plasma modifications are achieved. To analyze the modification results, Raman and XPS are used. The experimental results show active functional groups have been successfully modified on the graphene surface without degrading graphene electrical properties. This work presents potentials of graphene to be used as a multi-target gas sensing material.

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11.

The development of biosensors is highly dependent on the immobilization of various bio-recognition elements. Among various surface modifications, covalent immobilization, specifically the covalent linkage between solid substrate and bio-recognition elements via self-assembly monolayers (SAMs) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/ N-hydroxysuccinimide (EDC/NHS), has been applied extensively due to its controllability, predictability, long-term stability, and high bond strength. Hence, the exploration for the optimum parameters will have a significant impact on the stability of the adsorption reaction. This study intends to identify the kinetics of an EDC/NHS reaction on an 11-mercapto- undecanoic acid (11-MUA)-immobilized gold surface. Surface-enhanced infrared absorption spectroscopy (SEIRAS) is commonly used in a number of spectro- electrochemical and bioanalytical approaches. Attenuated total reflection (ATR) was the mode that was chosen because it allows a high signal-to-noise ratio for in situ monitoring adsorption/desorption of species at the solid interface. The reaction was conducted under different buffer solutions and different pH values. To confirm the effects of the EDC/NHS reaction corresponding to the pH values and the ionic composition in buffer solutions, electrochemical techniques were further utilized. According to the high correlation between the results of SEIRAS and electrochemistry, it strongly demonstrates that the EDC/NHS reaction would be obviously interfered by phosphate ions in neutral pH conditions.

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12.

Discrimination of microorganisms is important in food-contamination testing. We have proposed a rapid and simple method for discrimination of microorganisms termed “colony fingerprinting”, rather conventional observation method, which is time-consuming and labor-intensive. In colony fingerprinting, the colony images, referred to as “colony fingerprints (CFPs)” are obtained using a wide-field of view lens-less imaging system composed of a CMOS image sensor and LED light. The lens-less imaging system is capable of visualization of microcolonies with the size of sub-millimeter scale. Subsequently, image features are extracted from CFPs with bioimage informatics methodologies. The extracted image features were analyzed with several machine learning approaches including support vector machine (SVM), random forest (RF), and artificial neural network (ANN). Based on the colony fingerprinting, we successfully demonstrated discrimination of bacteria and fungi with high accuracy. These results indicate that the colony fingerprinting is a powerful tool for discrimination of food-borne pathogens.

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13.

Cell chip devices have a potential to perform high-throughput analysis for the functions of single-cells. In previous work, we developed a single-cell microarray chip for high-throughput screening and analysis of antigen-specific single B-cells. Using the technology of cell microarray chip, we have developed a new system for the high-sensitive and rapid diagnosis of malaria. The cell microarray chip is made from polystyrene polymers with over 20,000 microchambers, which can accommodate 130 erythrocytes. As the result, over 2,700,000 erythrocytes were dispersed on a microarray. We could detect malaria as low as 0.0001% with spreading of human erythrocytes on the microarray by staining of malaria nuclei with fluorescent dye within 15 min. Furthermore, using the same cell microarray chip, we tried to make a system to detect circulating tumor cells (CTCs) in blood, which are related to metastasis cancer. As a model of CTCs detection, we could detect rare carcinoma cells spiked into human whole blood with anti-cytokeratin monoclonal antibodies. And verification of carcinoma cells in the microchambers was performed by double staining with the fluorescence-labeled monoclonal antibodies. Therefore, this cell chip system has high potential to detect malaria-infected erythrocytes and CTCs with sensitivity and accuracy.

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14.

Short functional peptides are promising materials for use as targeting recognition probes for biosensing because of their diverse functionalities and with certain peptides, the potential for specific binding to particular materials including organic and inorganic. Herein, focusing on peptides as ligands that retain partial features of protein function, peptide arrays have been constructed for direct interaction analyses with microorganisms. Peptides with binding affinity to certain microorganisms were selected among linear amino acid sequences derived from cell surface receptors or complementarity determining regions of monoclonal antibodies. The peptide modified to pore sensor offer affinity analysis at single-particle sensitivity that promises wide applications in bioanalyses including bacterial and viral screening to infectious disease diagnosis.

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15.

We have developed the electrochemical sensing system for analysis of wine. The planar typed electrochemical sensor chip with four electrodes was newly fabricated. Working electrode was modified with polyaniline (PANI) to measure pH of wine. PANI also has a function of electron mediator between electrode and organic matters of solution. It was demonstrated the electrochemical oxidation of organic matters in wine. It was also noticed that PANI prevented the non-specific adsorption in wine. Therefore, the electrochemical sensor chip can be used repeatedly. Every test requires one drop of wine (50 µl) without any additional reagents and takes 18-s for one measurement. White wine (Sauvignon blanc), non-filtered white wine (Kerner), and apple juice (for cidre) were evaluated by electrochemical sensing system in brewery of winery in winter of Hokkaido. Even though the practical condition was out of control (low temperature), the system worked well and showed the amount of organic matters and alcohol in samples. We also reported the comparison between white wine, rose wine, and red wine.

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16.

Living cells exchange several metal ions and chemicals with the surroundings. Array electrochemical ion image sensors can measure their extracellular concentrations, and obtain information on the exchanging of ions. We have been developing several ion image sensors with polyvinyl chloride (PVC) membrane to reveal the living metabolism. However, the PVC membrane was unsuitable to culture weak-proliferating-ability cells. Here, we report a new CCD array potassium ion image sensor using an organic-inorganic hybrid silica membrane, which is said to be biocompatible. The membrane was prepared by sol-gel method of tetraethoxy silane (TEOS) and diethoxy dimethyl silane (DEDMS) including valinomycin and potassium tetrakis (4-chlorophenyl) borate. The potassium ion image sensor with the optimal membrane responded to K⁺ (53.5 mV/decade) at 10^-2 M to 10^-4 M. The sensor had no response to pH in neutral region. The other cations, Na⁺, Ca²⁺, Mg²⁺, NH₄⁺ did not affect the K⁺ response so long as their ordinal concentrations of cells.

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