Vol. 39, Supplement A (2023)

17.

We report the sensing interface modification with Co₃O₄ toward improving the CO sensing of the short-current typed solid electrolyte sensor based on c-axis oriented La_9.66Si_5.3B_0.7O_26.14 (c-LSBO). The Co nitrate-based solution with different concentration was deposited on the sensing-interface of the c-LSBO sensor, and the dependence of CO sensing property on the Co₃O₄ was investigated. The CO sensing property strongly depended on the amount of Co₃O₄ introduced at the sensing-interface. When the concentration of the cobalt nitrate precursor solution was 5.7×10^-4 mol/kg, the maximum short-circuit current value of 12.8 A/cm², which was 1.8 times higher that of the unmodified sample, was observed. These results indicate that the sensing performance of a short-circuit current type CO sensor can be improved by modifying the sensing interface with Co₃O₄, which has high CO adsorption/oxidation activity.

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18.

T-cells are a type of lymphocyte that plays a role in the mammalian immune defense system and are activated by binding to antigen molecules expressing on the membranes of virus-infected cells and cancer cells. The activated T-cells attack cells that express antigen molecules or secret cytokine molecules to attract other immune cells. Each T-cell has a different ability to secret cytokine molecules. Retrieval of the single activated T-cells according to their ability to secrete the cytokines, would be useful for deciphering the secretome analysis of single T-cells, developing master T-cell lines for cytokine production, and preparing T-cell therapies. In this study, we have characterized the electric properties of activated T-cells (Jurkat cells) using a simultaneous electrorotation method reported in previous studies. This method allows obtaining an electrorotation rate of more than 1,000 cells in a single experiment in a non-label manner. Jurkat cells activated with ionomycin and phorbol 12-Myristate 13-Acetate began to rotate at 19.2±0.56 radian s^-1 (36 cells) when an AC voltage of 200 kHz, 3 Vpp was applied to the electrorotation device. This rotation rate was significantly faster than that of non-activated Jurkat cells (16.5±0.35 radian s^-1, 59 cells). Scanning electron microscopy images revealed that the membrane roughness of the activated Jurkat cells was flatter than that of non-activated Jurkat cells. This difference in the membrane roughness is believed to be responsible for the difference in the electrorotation rate. Thus, this simultaneous electrorotation method enabled the discrimination of immune-activated and non-activated T-cells without fluorescent labeling.

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Cell-replacement therapies using human induced pluripotent stem cells (hiPSCs) with characteristics of self-renewal and pluripotency have attracted much attention in regenerative medicine fields. However, since residual undifferentiated cells after the differentiation induction could form tumors in transplanted patients, the safety of their implantation needs to be improved. In this study, to monitor the differentiation stage of hiPSCs, we developed a field effect transistor (FET) biosensor to electrically detect a glycoprotein biomarker podocalyxin, which is secreted from hiPSCs into cell culture supernatants. By functionalizing the FET surface with lectin (rBC2LCN), the intrinsic charges of the podocalyxin were directly detected by the FETs. Furthermore, the FETs could provide monitoring of the decrease in the podocalyxin concentration present in culture supernatants associated with the differentiation process. Thus, the FET-based assay is a promising technique to simply and rapidly inspect the differentiation status of hiPSCs used for transplantation.

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20.

In this presentation, we developed the estimation method of the reduction activity of quinone by NAD(P)H dehydrogenase presented in zebrafish embryos to apply it to the rapid evaluation system of the toxicity of chemical reagents. The Fish Embryo Acute Toxicity test has been adopted to estimate the toxicity of chemical reagents. However, the time required to estimate the toxicity is relatively long (several days) and the distinguishment of life and death by the morphological change under a microscopy frequently bring about the false recognition. We have been developed the estimation method of the toxicity of chemical reagents based on the oxygen consumption of the embryos by the respiration. We focused on the enzyme activity as a new indicator of toxicity to improve the reliability of the estimation. NAD(P)H:quinone oxidoreductase(NQO) that is one of NAD(P)H dehydrogenases promotes the reduction of benzoquinone (BQ) to hydroquinone (HQ). HQ generated by the enzyme in embryos can be measured by the electrode positioned at the outside of embryos, because both oxidized and reduced forms of quinone are permeable to the cell membranes. However, the addition of higher concentration gave rise to the damage to the cellular activity, while the lower concentration gave insufficient electrochemical signals. Thus, another redox couple of ferricyanide/ferrocyanide added in the outer solution was used to detect HQ generated and released by the embryos. The ferrocyanide generated by the reduction of ferricyanide induced by the HQ oxidation was detected by the electrode array around the embryos.

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21.

Antibiotics are effective in inhibiting the activity of pathogenic microorganisms. For microorganisms with respiratory activity, it is possible to evaluate the effect of antibiotics by measuring the change in the respiratory activity using an oxygen electrode. However, for multiplexed measurement of the effect of antibiotics of different concentrations using integrated oxygen electrodes is necessary. To solve the problem, arrays of Clark-type oxygen electrodes were fabricated based on bipolar electrochemistry. Dissolved oxygen concentration was measured on cathodic poles from the intensity of electrochemiluminescence (ECL) generated on anodic poles of bipolar electrodes. With suspensions of P. aeruginosa and E.coli as samples, ECL intensity or dissolved oxygen concentration depended clearly on their density. Efficacy of an antibiotic (gentamicin, cefmetazole, oxacillin) was also examined using suspensions of the same concentrations. Clear difference in ECL intensity was observed depending on the density of gentamicin, cefmetazole, and oxacillin, demonstrating that this device can be used for drug sensitivity measurement.

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23.

The potentiostat is used to operate three-electrode systems and situations in which it is used are expanding rapidly. We have already reported methods to control the potential in bipolar electrochemical systems using polymeric ion-selective membranes. The method can be a basis to create devices with electrochemical circuits. In this study, a disposable was fabricated by combining a potential control unit with a polymeric ion-selective membrane with an electrochemical cell. The potential difference generated from the potential control unit was applied between the working electrode and the reference electrode in the main chamber. Reasonable expected responses were observed when the working electrode potential was changed by changing conditions related to the main and control chambers.

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24.

Graphene is a hydrophobic material and thus its surface is not suitable for supporting substrate of supported lipid bilayer (SLB). Here we created ε-poly-l-lysine-modified monolayer graphene (ε-PL/MG) and confirmed that the ε-PL modification increases the hydrophilicity and SLBs were successfully formed on the ε-PL/MG and the SLB on ε-PL/MG exhibits fluidity by using fluorescence recovery after photobleaching (FRAP). We also confirmed that ε-PL/MG electrode and the monolayer graphene electrode provided comparable electrochemical properties by cyclic voltammetry (CV) and electrical impedance spectroscopy (EIS).

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25.

Taste sensors are equipping lipid/polymer membranes as a receptor. It can quantify tastes by utilizing changes in membrane potentials caused by the electrostatic and hydrophobic interactions between the membranes and taste substances. The saltiness enhancement effect is a phenomenon in which the saltiness taste is intensified by adding specific substances to dietary salt (NaCl); however, evaluating enhanced saltness with taste sensors has not been possible. This was because the conventional sensor evaluates the saltiness by only detecting the anions in sample solution. In this study, introducing of a novel lipid/polymer membrane that recognizes Na+ ions and quinine simultaneously; accordingly, the taste sensor enabled to evaluate the saltiness enhanced by quinine was achieved.

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特別講演2

In microphysiolopgical system (MPS), the fusion of cell culture technology and cellular function-analysis are essential. MPS is expected to realize more accurate pharmacokinetic evaluation and drug screening. In the future, it is required to quantitatively validate and compare how precisely the actual in vivo phenomenon is reproduced by individual MPS. Technological innovations related to organoid culture systems and organ-on-a-chips are making remarkable progress as alternatives to animal testing systems. Since the 2000s, our group has produced three-dimensional culture-based cell chips and worked on electrochemical evaluation of cell functions. In this presentation, I will introduce research examples such as drug screening in vascular MPS and highly sensitive immunoassays using protease cascade reactions.

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26.

In this study, the low-potential operation (+0.05 V) of a direct electron transfer (DET) glucose biosensor strip with a small-volume (2 L) electrochemical capillary chamber is demonstrated. We constructed a DET formation in which de-bundled single-walled carbon nanotubes (SWCNTs) were placed near the reaction center pocket of flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH). The DET biosensor has the advantage of long-term stability, unlike an electron transfer mediator, which is naturally reduced to cause sensing malfunction in a mediated electron transfer biosensor, adopted in commercial devices. However, DET biosensors still have a high working potential owing to their overpotential. In this article, we have resolved this issue via a very short interval voltage application (+0.4 V) that reduced the onset potential, in other words, enhanced the current owing to an enzymatic reaction.

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27.

There is a demand for more accurate point-of-care at home from urine and other sources using inexpensive, disposable biosensors. We proposed a novel creatinine biosensor based on a chemiresistive detection method, in which the change of sensor resistance (Chemiresistor) is used as a biochemical reaction. The chemiresistor consisting of enzymes, a polymer, and Pt nanoparticles is formed on metal electrodes on a glass plate. The enzyme-catalyzed reaction produces H₂O₂ from creatinine in the chemiresistor, and H₂O₂ is then redoxed by the platinum nanoparticles, resulting in a change in the electrical conductivity of the chemiresistor. The Pt nanoparticles can be sensitive to slight chemical changes by adjusting their concentration in the polymer to show percolation effects. The resistance changes are observed for creatinine solutions ranging from 0.5 to 300 mg/dL. This result indicates that this chemical resistance biosensor can detect a wide range of concentrations of creatinine in a solution and also can be applied to various biomarkers detections using different enzyme reactions.

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28.

Monolayer graphene is attracting attention as a biosensor electrode material because of its excellent electrical conductivity and high biocompatibility. We have reported a reaction-promotion effect depending on potassium ions (K⁺) concentration in redox reactions of electroactive anion species using a monolayer graphene electrode. We have also reported a fluorescence-detecting biosensor in which monolayer graphene is modified with single-stranded DNA (aptamer) that has a molecular recognition function. On the other hand, guanine-rich sequences in single-stranded DNA are known to form a G-quadruplex and form a complex with potassium ions. In this study, we created a monolayer graphene electrode modified with DNA to form G-quadruplex chains. We then studied whether the K⁺ concentration-dependent reaction promotion effects by the DNA modification. As a result, we confirmed that modification of the DNA forming the G-quadruplex amplifies the peak current by promoting effect of the electroactive anion species.

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29.

In this study, a novel electrochemical sensor based on a boron-doped diamond (BDD) electrode was developed for the detection of protein concentrations in human urine. BDD electrodes have the advantage of a wide electrochemical window, low non-specific adsorption, and easy surface regeneration, making them ideal for simple, rapid, compact, and energy-saving devices for home detection of bio-relevant substances. Coomassie Brilliant Blue (CBB), a dye that selectively, stoichiometrically, and strongly binds to urine protein, was found to be redox active and showed a decrease in its reductive current in relation to the concentration of protein in urine samples, providing us a reproducible and linear BDD sensor response. This principle is thought to be based on the decrease in free CBB caused by the selective binding of urine protein to the redox active CBB. Our detailed studies of BDD electrodes showed their detection limit of 2.57 µg/mL and that they have a linear response that ranges from 0 to 400 µg/mL in urine samples that contain other trace compounds. We also investigate the detection of urine protein in different urine samples, mixtures of samples, and urine samples from a single donor. Our results agreed with those obtained using conventional colorimetric analysis.

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30.

The purpose of this study is to develop a highly selective drug sensor that is flexible enough for subcutaneous implantation for non-blood therapeutic drug monitoring in neonates. We have so far developed sensors based on MIP carbon paste (MIP-CP) electrodes, in which graphite particles with molecularly imprinted polymers immobilized on them are mixed with oil. Since higher mechanical strength is required for the sensor to be implanted in the body, we fabricated an MIP-carbon dough (MIP-CD) electrode by mixing MIP with thermosetting silicon resin prepolymer and thermosetting it. The electrode, which was formed by pre-applying carbon ink to the surface of a wire and then forming a vancomycin (VCM)-templated MIP-CD on it, exhibited high friction resistance. The current emitted by this electrode was sensitive to VCM, but not to teicoplanin, which has a similar structure. The wire-type sensor with the MIP-CD fixed to the electrode shows promise as a subcutaneous implantable sensor for monitoring VCM.

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31.

Several antiepileptic drugs (AEDs) are used to control seizure frequency, and the therapeutic window for these drugs is relatively narrow. As a result, accurate drug concentration monitoring is essential. In this study, we developed a low-cost and disposable sensor chip for monitoring commonly used AEDs such as phenobarbital (PB) and levetiracetam (LT). The sensing material for the chip was a molecularly imprinted carbon paste, which is highly selective for the given AEDs. The chip used differential pulse voltammetry to measure AED concentrations in various matrices, including buffer saline, whole bovine blood, and bovine plasma. The results show that the MIP sensor chip is a simple and reliable alternative for AED monitoring in various matrices.

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32.

In designing detection methods, it is necessary to reduce the amount of specimen required for testing in order to reduce the burden on patients. Therefore, we are investigating electrochemical biosensors by screen printing on ceramic substrates that can be miniaturized, with the aim of creating a blood analyzer that can make measurements with a small amount of specimen. Ag/AgCl electrode that is currently widely used as a reference electrode is not easy to be miniaturized because an inner solution with a constant Cl^‒ concentration is required.　In addition, upon long-term use, other electrodes could be contaminated by silver ion eluted from Ag/AgCl electrode. In this study, we synthesized a new material that is conjugated with carbon-coated porous silica spheres (C/PSS) and prussian blue (PB) for a reference electrode. As a result, a sharp redox peak is observed by cyclic voltammetry, and open circuit potential (OCP) have been kept within 30 mV for 30 days.

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33.

Ion-selective electrodes (ISEs) are typical electrochemical sensors that enable the measurement of ion activity in solutions with high selectivity and sensitivity. Conventional ISEs consist of an ion-selective membrane (ISM), internal filling solution, and internal reference electrode. However, ISE configurations that require an internal reference electrode and internal solution hinder miniaturization. To overcome these disadvantages, direct-contact-type ISEs (DC-ISEs or coated wire electrodes), in which a metal electrode is directly covered with an ion-selective membrane, have been widely developed. DC-ISEs, in which ionophores are introduced into a plasticized polymer layer used as the ion-selective membrane, show a Nernstian response to the target ion with good selectivity. However, the lack of an internal reference electrode makes the response potential unstable. To solve these problems, solid-contact-type double-layered ISEs (SC-ISEs) have been reported, in which functional materials that act as ion-to-electron transducers are inserted between the ion-selective membrane and the electrode substrate. In this study, K⁺ ion-conducting KTiOPO₄ (KTP) was used as an SC layer to investigate its contribution to the potential stability of K⁺-ISE.

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34.

We have developed a novel electrochemical imaging method using a closed bipolar electrode (cBPE) array coupled with electrochemiluminescence (ECL) named “bipolar elec-trochemical microscopy (BEM)". In this study, aiming to advance the understanding of the relationship between redox reactions at the cBPE poles and ECL intensity, we measured the potential difference between the cBPE anode and luminophore solution (10 mM [Ru(bpy)₃]Cl₂/100 mM Tripropylamine; TPA, E_A) and cBPE cathode and sample solution (5 mM K₃[Fe(CN)₆] , E_C) with an electrometer, estimated the rate-limiting reaction from the comparison with the measurement results of the three-electrode system. E_A and E_C were changed by the balance of the current density at both poles of cBPE with the changing of the rate-limiting reaction, resulting in the change of ECL properties. The generation of ECL is related not only to the electrode area but also to the co-reactant concentration. In order to obtain the ECL intensity corresponding to the sample concentration, the anodic current density for the reduction current of the sample should exceed the oxidation peak current of TPA and be below the peak current of [Ru(bpy)₃]²⁺. These results are fundamental and im-portant information useful for the high-sensitivity detection of BEM.

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