Vol. 35, Supplement B (2019)

17.

We have been developing polyaniline (PANI) -based sensors for detection of ammonia (NH₃) gas at room temperature. Although many NH₃ gas sensors using PANI have been reported so far, it was difficult to detect NH₃ gas in the concentration range of sub-ppm-level. In order to detect very low concentration of NH₃ gas, preparation of a PANI film with a porous structure and a high specific surface area is effective. In this study, as an attempt to increase the effective surface area for gas adsorption, a PANI film consisted of microspheres with the structure of a polystyrene (PSt) core and a PANI shell (PANI-PSt) was prepared. A film just deposited with PANI was also prepared for comparison. Calibration curves of both the PANI-PSt film and the PANI deposited film showed good linearity in the concentration range of 50 to 250 ppm of NH3 gas. Moreover, the PANI-PSt film sensor achieved 10 times higher sensitivity than the PANI deposited film.

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18.

The absorption peak of Co(Ⅱ) tetraphenylporphyrin (CoTPP) changes from 410 nm to 435 nm when nitric oxide (NO) is coordinated to central cobalt metal, and we can fabricate NO gas sensors based on this optical property. However, CoTPP molecules tend to aggregate by π-π stacking interaction, which leads to decrease in the sensor response because it prevents interaction between CoTPP molecules and NO gas. In this research, we composited CoTPP in a polymer matrix to suppress aggregation of CoTPP molecules. The reversibility of the sensor response toward NO gas based on the composite film depended on temperature, and the estimated recovery of the ethyl cellulose (EC) composite film sensor was above 95 % at 100 °C. Furthermore, the sensor response depended on the CoTPP concentration in the composite film. The magnitude of the sensor response obtained for the composite films with low CoTPP concentration was much higher than that of the CoTPP film without polymer matrix. This was because the aggregation of CoTPP molecules could be suppressed in the polymer matrix. In particular, EC composite film had the highest sensor response values, and the detection of 100 ppb of NO gas was possible.

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The number of diabetic patients is increasing worldwide, and there is high demand for glucose sensors for these patients to be able to measure their blood glucose concentration. Since nonenzymatic glucose sensors have advantages such as long life and easy handling, the researches using various materials have been actively conducted. We focus on applying a mesoporous platinum electrode with a lot of high index surface as nonenzymatic glucose sensor. Nonenzymatic glucose sensors can perform sensing using various electrolytes. In our previous study, we reported that using NaOH as an electrolyte improves glucose sensing sensitivity compared with H₂SO₄¹⁾. This is considered that the ratio of optical isomer of glucose which is electrochemically more active at Pt electrode increases in high pH. In this study, by using LiOH and KOH solutions, which have the same pH as NaOH solution, the influence of coexisting ions in aqueous solution for glucose sensing was investigated. As a result, it was suggested that there is a difference in the complex formation depending on the coexisting ions, which affects the sensing sensitivity.

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20.

We have created a fluorescence detection aptasensor by modifying the surface of graphene or graphene oxide (GO) with an aptamer (single-stranded DNA) labeled with a fluorescent dye. We use this system to detect biologically important proteins such as cancer markers selectively and sensitively by molecular recognition of aptamer. We used two unique characteristics of graphene/GO, that it works as an excellent acceptor of fluorescence resonance energy transfer (FRET) in all wavelength regions of visible light, and that it adsorbs single stranded DNAs such as aptamers via π-π interaction on its sp2 domain. Therefore, in this aptasensor, the fluorescence intensity changes at protein detection depending on the FRET efficiency of the graphene/GO and the dye. So far, we have succeeded in increasing the sensitivity by the molecular design of the aptamer which increases the fluorescence intensity by increasing the distance between graphene/GO and a dye at detection of the protein. In this study, we studied the change in the fluorescence intensity at protein detection by changing on the number of layers of graphene/GO used to an aptasensor, and discuss the relationship between the number of layers of graphene/GO and the FRET efficiency.

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21.

In realizing miniaturized electrochemical devices, connection of solution usingliquid junctions poses a problem. In our previous report, we demonstrated that the compartments for the working, reference, and auxiliary electrodes could be separated and connected with metal wires with pH-sensitive ends. Based on the results, When the pH of the solutions in the working and reference electrode compartments were equal, the system behaved as if the reference electrode was connected with a liquid junction. The technique was also used for autonomous control of the transport of solutions in microfluidic systems. The metal junctions actually facilitate designing of integrated electrochemical systems.

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22.

We fabricated sputter-deposited carbon film with a nitrogen-containing group and estimated the efficacy of a surface nitrogen-containing group for detecting biomolecules. Two types of carbon films, one rich in graphite-like nitrogen-containing bonds and the other rich in pyridine-like bonds, were successfully fabricated without changing their nitrogen concentration, sp²/sp³ ratio or surface flatness. The pyridine-like bonds rich film shows a positive oxygen reduction peak of about 250 mV compared with pure carbon film and is also 200 mV more positive compared with film with graphite-like nitrogen-containing bonds. For detecting biomolecules, carbon film rich in pyridine-like bonds also exhibits more negative peak potentials for the oxidation of NADH and L-ascorbic acid, suggesting that carbon film rich in pyridine-like bonds will show improved performance for biomolecules detection. We also fabricated hydrophilic carbon film by NH₃ plasma treatment. Compared with water vapor plasma treated carbon film, the NH₃ plasma treated carbon film shows better biocompatibility and shows very little increase of peak separation when measuring 1mM Fe(CN)₆^3-/4- in the presence of 100 mg/mL bovine serum albumin (BSA).

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23.

Therapeutic drug monitoring (TDM) requires small sized and easily-operatable sensor is required. Sensor using molecularly imprinted polymer (MIP) as molecular recognition element may be potential analytic tool for TDM. In this study sensor chip including carbon paste electrode grafted with MIP of meropenem containing ferrocene is developed using cutting plotter and inkjet printer. The output current is sensitive to meropenem without addition of indicator. Thus, the sensor chip is feasible as a tool for TDM.

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24.

There are increasing demands to measure the respiratory activity of animal and microbial cells using samples and reagents of very small volumes. To this end, a micro analytical device with a miniaturized Clark-type oxygen electrode was fabricated. Water 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 dissolved oxygen concentration. In addition, evaporation of water from the electrolyte solution was avoided by adding sorbitol in the electrolyte solution. The device was used to measure the respiratory activity of E. coli. The current decrease depended on the density of E. coli. Also, various applications will be considered.

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25.

We demonstrated proof-of-concept experiment for a hydrogel-based touch sensor pad used for the non-invasive extraction and detection of sweat components. The sensor device was composed of an electrochemical L-lactate biosensor covered with an agarose gel in a phosphate buffer saline. When human skin contacts the agarose gel, L-lactate in sweat was continuously extracted into the gel, followed by in-situ potentiometric detection without controlled conditions. This novel type of sweat sensor is expected to enable the simple, non-invasive daily periodic monitoring of sweat biomarkers for advanced personal healthcare methods in the future.

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26.

Wearable ion sensors for monitoring biomarkers in sweat have been attached attention. Sodium ion concentration in sweat is a marker for electrolyte imbalance and is useful for the prevention of heatstroke and diagnosis of hyponatremia. Ammonium ion concentration in sweat is related to muscle fatigue. In this research, we newly fabricated a multi ion sensor to perform simultaneous quantitative monitoring of the two ions on a fabric substrate by heat transfer printing. The electrodes of the sensor were fabricated by screen printing. Responses of the sensor was performed by measuring the open circuit potential (EMF) measurements with changing in the concentration of ammonium chloride and sodium chloride (0.1-100 mM). The EMF of the sensor for sodium ion concentration was changed in 57.2 mV/log CNa⁺. The EMF change of the ammonium ion sensor was 56.7 mV/log CNH4+. These results indicated that the Nernst response was obtained. Since the concentration of ammonium ion in human perspiration is 0.1 to 1 mM and the sodium ion concentration is 30 to 100 mM in general, it was suggested that the sensor could be used for the determination of ammonium ion and sodium ion in perspiration.

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27.

The concentration of lactic acid in sweat is closely related to exercise intensity and physical fatigue. Thus, the concentration of lactate is considered to be extremely important as an index to improve athletes' training and health management. In order to detect changes in lactic acid concentration in sweat for a long time using a lactic acid biosensor as a wearable device, it is important to stably immobilize the enzyme and mediator that oxidize lactate on the electrode surface. In this study, the surface of the MgO template carbon, which is the electrode material, was modified with glycidyl methacrylate (GMA) by graft polymerization using electron beam irradiation treatment. The electrodes of the sensor were fabricated by screen printing. A lactate oxidase (LOx) was immobilized on the working electrode that bonds to the epoxy group of the GMA. The performance of the electrode of the sensor was evaluated by cyclic voltammetry. The current value at 0.4 V vs. Ag / AgCl was not significant change during 10 cycles. The current value remained at 95% after 10 cycle measurement, indicating that elution of lactate oxidase from the electrode surface was efficiently suppressed.

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28.

We have investigated human-stress measurement for salivary nitrate, which can be a candidate for stress markers derived from blood NO metabolites with ion-selective field-effect transistors (ISFETs). And we successfully demonstrated several acute stress tasks for healthy volunteers over 300 salivary samples using prototype of salivary nitrate checkers based on NO₃^--ISFETs using direct potentiometry. ISFETs have significant advantage for continuous on-site monitoring of salivary nitrate, however, ISFETs have problems on sensor drift characteristics. To realize accurate monitoring for nitrate in non-invasive biological sample, we investigated new polymers to prevent the drift of sensor characteristics compared with conventional matrix membranes, such as PVC as well as biocompatible polyurethane, such as KP-13. In this study, we investigated ISFETs using specific matrix polymer, for example KP-13, Pellethane^®, and P7281-PU. P7281-PU showed a prevention effect on the initial drift in various solutions including whole saliva. Moreover we studied the drift mechanism.

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29.

A simple electrochemical sensor for in-line monitoring of residual chlorine in tap water has been proposed. The sensor device which consists of pair of Pt and stainless steel electrodes exhibited high performance. However, interferences of conventional factors such as conductivity, pH, dissolved oxygen have to be evaluated for practical application. In this study, we have investigated the effect of pH on Pt and stainless steels electrode potential in synthetic tap water. It is found that both electrodes didn’t show Nernst slope (59 mV/pH) in dilute solution like tap water and initial potentials level were unstable. It indicated that the self-compensation ability of this method was not perfect. For example, Pt electrode show about 40 mV/pH and stainless steels show about 20 mV/pH. On the other hand, this kind mismatch would be relatively small against fluctuations of conductivity and dissolved oxygen concentration.

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30.

Residual chlorine is disinfectants made by adding sodium hypochlorite etc. to tap water. It acts to kill and denature harmful substances such as microbe and viruses by strong oxidizing power.　It is necessary components but then it is also cause for the chlorine smell if it is present too much. Therefore, it is necessary to control the amount appropriately. Generally, to measure the residual chlorine concentration, test solutions colored using a reagent, and measured by absorbance. Alternatively, it applies a voltage to the metal electrode and the reduction current is measured. However, the former requires many operations for each measurement. The latter is a large and expensive apparatus, moreover the risk of elution of the electrode and often request maintenance. Therefore, either of them is unconvenient and is not applied to the terminal water tap. This paper introduces a potentiometric residual chlorine sensor as a solution to their problems. In addition, we investigated the water quality factors that may affect the performances of the sensor.

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31.

The FET （Field effect transistor : FET） biosensor is a promising cost-effective and rapid, label-free detection tool for biomolecules. To detect uncharged molecules by the FET biosensor, negatively charged aptamers have been used as receptors. In this case, the FET biosensor detects the change of charge-density within the Debye length due to structural transformation of aptamers caused by the capture of the uncharged molecule. However, the inhibition of structural transformation due to the steric hindrance of adjacent aptamers may reduce the sensitivity of the aptamer-immobilized FET biosensor. In this study, we propose immobilization method using structurally altered aptamers (target-aptamer complex). The aptamer-immobilized gate surface which fabricated by removing targets from target-aptamer complex is expected to reduce the steric hindrance. In this presentation, we investigated the formation conditions of the target-aptamer complex in the solution. We also report the detection results using the method.

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32.

Chemical imaging sensor, which is one of the semiconductor-based chemical sensors, has been developed as a powerful tool in the field of analytical chemistry. There have been many reports which utilized advantages of the sensor such as label-free measurement, flat sensor surface or multi-point measurement. In this study, we focus on another advantage of large-area measurement. A novel measurement system which is able to scan an area of 130 mm x 130 mm was designed to observe the pH distribution of plant rhizosphere. In addition to a large-area scanning stage, the measurement system also included an LED light source to simulate sunlight, a camera module to observe the extension of root, and a thermometer to record the temperature variation. It was demonstrated that the rhizopheric pH distribution could be visualized by the system.

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33.

Although many types of analytical methods for cells and their metabolites have been developed, it is difficult to monitor bioactivity (such as carbohydrate metabolism) continuously and easily. In this regard, we have decided to develop a novel microfluidic device with an extended-gate type organic field-effect transistor (OFET) for real-time glucose monitoring. The drive part (i.e. OFET) is connected to the detection part composed of the microfluidic channel (height: 50 µm, width: 100 µm) and the extended-gate electrode. The extended-gate electrode functionalized with phenylboronic acid derivative (PBA) as a glucose receptor showed the real-time electrical detection of glucose concentrations in an aqueous solution. Alternately injection of the phosphate buffer solution (100 mM, pH 7.4) with or without glucose into the microfluidic channel exhibited a real-time reversible response in the drain current. The result obtained indicates that the fabricated device would be used in monitoring of the bioactivity in liver cells.

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34.

Antimicrobial resistance (AMR) is a global problem in medicine. Therapeutic drug monitoring (TDM) of antibacterial drugs are necessary for overcoming the AMR problem. Vancomycin (VCM) is a typical antibacterial agent which requires a strict TDM. In this study, we developed a VCM sensor using a graphite paste electrode on which a molecularly imprinted polymer (MIP) is immobilized. The sensitivity of the electrode with a phosphate buffered sample solution or bovine whole blood sample solution containing vancomycin was evaluated by differential pulse voltammetry (DPV) method. As a result, the current value increased with vancomycin concentration in PBS and bovine whole blood, but the sensitivity was lower in bovine whole blood. Therefore, whole blood was measured separately for each component in order to identify the factor of sensitivity reduction, and the sensitivity in blood excluding low molecular weight formation by membrane filtration was almost equal to that in PBS, and it was found that the low molecular weight component in bovine whole blood is the cause of the sensitivity decrease. From this fact, the MIP carbon paste electrode can accurately monitor the concentration of vancomycin in blood by correcting the concentration of low molecular components in blood.

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35.

Therapeutic drug monitoring (TDM) is strongly recommended for vancomycin (VCM), which is the first-line antibacterial agent against hospital-acquired infection. However, the conventional vancomycin analysis method is cumbersome in operation and forced to outsource inspection to external organizations, so the time lag of TDM is too long to for preventing resistant-bacteria creation. Therefore, we developed a vancomycin sensor for TDM which can measure rapidly and conveniently using molecularly imprinted polymer (MIP) which has a specific binding ability and can be produced easily and economically. At present, there is a problem that current values differ in buffer solution and in blood. In this study, we compared the VCM-sensitivity of the MIP electrode among media: whole blood, plasma, and washed blood cell suspension, and protein-removed serum. As the result, current in the protein removed-serum. The result indicates that the low molecular weight component in blood is the most important factor which raises the out put current at the MIP-electrode. Plasma

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36.

Discrimination of the contaminant microorganisms is an important technique for manufacturers of foods and medicines. Conventional methods based on biochemical properties and genetics information of microorganisms require long assay time and skillful operators. To solve this problem, we have proposed a rapid and simple method for discrimination of microorganisms called “colony fingerprinting”. In colony fingerprinting, discrimination was conducted based on analyses of the colony images, called “colony fingerprints”, which were obtained using a two-dimensional imaging sensor. However, the two-dimensional imaging sensor only allowed imaging of a part of culture plates. On the other hand, a scan system using a line imaging sensor can visualize a number of culture plates in one shot, leading to high throughput analysis. Toward this goal, in this study, we confirmed whether it is possible to discriminate Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis based on the colony fingerprinting using line imaging sensor. Using support vector machine (SVM) and random forest (RF), discrimination was successfully demonstrated with 100% of accuracy. This result suggests that drastic high-throughput discrimination of microorganism is possible by expanding scan distance in the future study.

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37.

Circulating tumor cells (CTCs) were defined as the tumor cells circulating in the peripheral blood of patients with metastatic cancer. Genetic analysis of CTCs at single cell level is believed to provide detailed information of cancer patients and to reveal mechanism of cancer metastasis. However single-cell genetic analysis of CTC is still difficult challenge because they are extremely rare (<100 CTCs per 50 billions of normal blood cells). Recently we have developed simple and rapid method for single-CTC isolation based on microcavity array (MCA) and gel-based cell manipulation (GCM). This method could visualize single-CTCs by hydrogel-encapsulation and enable us to isolate them by tweezers. In this study, we developed the protocol for single-cell gene expression analysis based on MCA/GCM system and evaluated the performance. Furthermore, we also carried out single-cell transcriptome analysis of CTCs isolated from metastatic gastric- and pancreatic-cancer patients to evaluate metastatic potential of CTCs at single-cell level.

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38.

In this presentation, a non-invasive method for measuring cellular membrane capacitance based on an electrorotation is proposed by using a device with three-dimensional interdigitated array (IDA) electrodes in a high-throughput manner. Electrorotation of cells has been usually performed using four square electrodes arranged symmetrically around a point to measure a membrane capacitance. However, the important of throughput in a main issue to apply electrorotation to estimate cellular properties statically. We fabricated a novel device consisted of two IDA electrodes that were placed orthogonally via a fluidic channel to form grids. The array of grids allows to form rotational electric fields in grids. The electrorotation of cells trapped in grids was simultaneously conducted by applying an AC electric field to determine membrane capacitances of four different types of cells.

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