Vol. 40, Supplement A (2024)

1.

In order to realize a sustainable society, the importance of ecological and environmental assessment is increasing. Recently, ecological research methods using environmental DNAs (eDNAs) have been attracting attention. Although this method can comprehensively evaluate fish species in environmental waters by analyzing their eDNAs contained in the waters using a next-generation sequencer, it requires a large amount of labor and money compared to the simplicity of water sampling and is overpowered when known eDNAs are going to be investigated. In this study, we developed DNA sensor array devices that enables an on-site eDNA evaluation on a single chip. For the study, we collected the water samples from fish tanks, including largemouth bass, an invasive species as an environmental indicator. At first, oligo DNAs were used to evaluate a basic performance of the prepared sensor array. It showed a large sensor response to a complementary DNA, but a small response to a mismatched DNA. Moreover, it showed a large response to the eDNA, indicating the eDNA from largemouth bass was successfully detected. Comparing with the results from the next-generation sequencing, it can be concluded that the sensor array revealed the presence of largemouth bass even among DNAs from other fishes.

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2.

In this study, we provide a method based on electrorotation (ROT) to monitor cellular responses to chemical stimulation without staining the cells. A novel ROT device was developed with two electrodes on the bottom of the microwells and one electrode arranged on the top of the microwells. This ROT device does not require 3D fabrication of microelectrodes and can be easily constructed by simply overlapping two electrode substrates. The application of an alternating (AC) voltage to the top electrode versus the two electrodes on the bottom of the microwells induces the formation of a non-uniform electric field in the microwells. Cells were captured within the well for a short period by positive dielectrophoresis, and then the induction of negative dielectrophoresis on the captured cells allows trapping only one cell in the central part of the micro-well while excluding others. The application of AC voltage with a phase shift of 120°to the three electrodes induces ROT to the captured cells. At this point, applying a higher voltage to the top electrode compared to the other two electrodes allows the rotation of cells with the center of rotation at the lower position of the microwells, even in the flow of the solution. We have demonstrated the ability to monitor an increase in cell membrane capacitance, induced by stimulation with 5 μM ionomycin, as an increase in rotation rate using this device. This method allows for the evaluation of the electrical properties of cells without the need for cell staining or contact with microelectrodes.

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3.

Electrochemiluminescence (ECL) is a light emission phenomenon intermediated by electron transfer through oxidation and reduction reactions at an electrode. Assuming that ECL and fluorescence (FL) differ only in the excitation energy source, we hypothesized that any luminophore having fluorescent properties and being able to undergo redox reaction on an electrode would exhibit ECL. In this study, we investigated the relationship between fluorescence properties and ECL properties of major fluorescent dye compounds by comparing the intensities of fluorescence and both anodic and cathodic ECL. The results suggest that compounds that exhibit fluorescence exhibit ECL, and that fluorescent dyes possible to be excited in a long wavelength range may exhibit ECL at low energies.

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4.

Inosine is one of the derivatives of adenosine triphosphate found in fish meat, and its concentration is an indicator of fish meat freshness. It is conventionally measured by high-performance liquid chromatography, but it is difficult to introduce the required substrate into the distribution field. The purpose of this study is to develop a real-time sensor for inosine. In this study, we synthesized an MIP using inosine as a template and developed a carbon paste electrode by mixing it with silicon oil containing silicon oil. The sensitivity of the developed electrode to inosine was examined and showed an increase in anodic current dependent on inosine concentration, but no sensitivity to inosinic acid and hypoxanthine. NIP also showed no sensitivity to inosine. Therefore, this MIP carbon paste electrode showed that the specific interaction with inosine was reflected in the anodic current. This result indicates that the fabricated electrode is promising as a highly selective and rapid sensor for inosine.

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5.

Adenosine triphosphate (ATP), which is present in both meat and fish meat, goes through various intermediate products over time, eventually changing to inosine (HxR) and hypoxanthine (Hx). This biochemical change is an important indicator of freshness of meat products, and we hypothesized that fabricating a sensor capable of detecting Hx would allow us to monitor fish freshness. The objective of this study is to fabricate an Hx sensor using MIP-CP. The response of the electrode to Hx and its selectivity to HxR were investigated by analyzing the oxidation and reduction currents; both the oxidation and reduction currents of MIP(Hx) were linearly correlated with the Hx concentration. No similar correlation was observed with NIP or inosine (HxR) concentrations. Thus, this MIP-CP showed that the specific interaction with the template Hx can be reflected in the reducing current. This result indicates that Hx-MIP-CP is a promising highly selective and rapid sensor for Hx.

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6.

We present here the size-selective capture of liposomes using dielectrophoresis (DEP) and its application to the detection of antigens through immuno-reactions. The device comprises an indium tin oxide (ITO) electrode substrate with a microwell pattern and another ITO substrate positioned above it. Application of an AC voltage (400 kHz, 3Vpp) induced positive DEP (p-DEP) in liposomes to trap them in the microwells size-selectively. The attractive force from p-DEP facilitated the formation of immuno-complexes between antibodies on both liposomes and microwells through antigens. Subsequently, negative DEP (n-DEP) was induced, to generate a repulsive force, ejecting liposomes that failed to form immuno-complexes from the microwells. The number of liposomes remaining in the microwells increased with increasing antigen concentrations. The injection of the solution with a high osmotic pressure to the device led to the rupturing of remaining liposomes, resulting in the absorption of phospholipid molecules to the electrode. This adsorption was detected as a decrease in the electrochemical oxidation current of ferrocyanide ions. In summary, our study successfully demonstrated the accelerated formation of immuno-complexes through DEP manipilation, and the quantitative detection of the number of trapped liposomes by inhibiting the diffusion of electrochemically active species to the electrode surfaces.

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7.

Wet-processable and highly conductive polymers are promising candidates for key materials in organic electronics. Poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS), commercially available as a water dispersion of colloidal particles, has potential applications to organic electronics because of its high electrical conductivity, transparency and thermal stability. Recently, we have developed a novel fully soluble self doped PEDOT (S-PEDOT) by chemical oxidative polymerization having a high conductivity without secondary dopants. In this study, the S-PEDOT was electrochemically synthesized at different current densities ranging from 0.5 to 5 mA/cm2. It was found that the electrical conductivity increased with increasing current density and attained as high as 427 S/cm at 5 mA/cm2 which was one order of magnitude higher than those so far reported in the literature. For the application to biosensors, flexible organic electronic circuits consisting of working, counter, and reference electrodes were fabricated by line-patterning the S-PEDOT water solution as a conductive ink on a Kapton film as a flexible substrate. The flexible glucose sensor was then fabricated by coating the bioink containing glucose oxidase, ferrocene, and chitosan on the working electrode. It was found that the flexible biosensor successfully worked where the electric current during the CV measurement increased with increasing the glucose concentration.

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8.

The various ions around us can cause health hazards and environmental damage. To prevent these problems, constant monitoring of wastewater is necessary, and compact, low-cost, high-performance environmental ion sensors are required. In this study, we focused on Sm-based perovskite-type oxides with high oxidation catalytic activity. Oxide fine powders were synthesized by the polymer precursor method, and thick-film type sensor elements were fabricated by the EPD method. The sensor was evaluated for its electrochemical response mainly to nitrite ions, and a linear response was observed in the range of 100 to 900 μM, confirming the concentration dependence.

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9.

Electrochemical measurements using a three-electrode system are widely used to study the properties of metallic materials. However, fabricating the measurement system requires many steps. To solve this problem, a sticker device for corrosion monitoring was fabricated by printing a reference electrode and a counter electrode on a polyurethane sheet by screen-printing and attaching the device to the target metal as the working electrode, which enables accurate and simple electrochemical measurement of a three-electrode system. In addition, an electrolyte gel was fabricated by gelatinizing a potassium chloride solution with PVA. The sticker device and gel were then combined to measure copper polarization, suggesting that the results are similar to those obtained using a typical three-electrode electrochemical cell.

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特別講演1

We developed the microarrayed immunosensors, which can be applied to single cell level measurement, based on two-dimensional surface plasmon resonance (2D-SPR) imaging. SPR sensitivity was drastically decreased in 10 μm wells because surface plasmon wave cannot propagate in such micro area. The use of shorter wavelength incident light might improve sensitivity of SPR sensing in micro wells. We have improved the sensitivity and resolution of 2D-SPR imaging in 10μm microwell array by using silver-based novel sensor chip and 505 nm incident light. This SPR technology using shorter wavelength incident light was also applied to the surface plasmon-enhanced fluorescence (SPEF) imaging, which is one of the near-fileld analytical method, based on SPR sensor principle. It was found that our SPR-SPEF imaging system could be captured SPEF images of FITC in FITC-BSA membrane, and Ru(phen)₃ in Nafion membrane for sensing of dissolved oxygen.

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10.

Rapid identification of contaminating microorganisms is important in food and beverage production control, and highly accurate discrimination of pathogenic microorganisms is required. Recently, bacterial identification methods using mass spectrometry such as MALDI-TOF-MS have attracted attention as a reliable and rapid testing method. However, the development of a rapid and simple bacterial identification method is required due to the several days required for incubation, the high cost of reagents and test equipment, and the need for trained operators. We have proposed a new bacterial identification technique called "colony fingerprinting", which uses machine learning to analyze image features (colony fingerprints: CFPs) extracted from the optical patterns of colonies. This technique has been shown to identify 15 bacterial species in 9 genera, including foodborne pathogens. However, because previous sensors were monochrome line sensors, the color information of the colonies was not available. The use of color information is expected to improve the discrimination accuracy. In this study, we developed a color line imaging sensor that can image the entire surface of a culture petri dish for highly accurate identification of pathogenic microorganisms.

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11.

A simple and rapid microRNA detection technology is useful for early diagnosis of cancer. The signaling probe-based DNA microarray which we have developed for microbiological testing was applied to detect human miRNAs. This microarray does not require fluorescent labeling of target nucleic acids and washing operations, thus enabling simple and rapid nucleic acid detection. In this study, we designed probes for miR-21 and miR-155 detection, which are diagnostic markers for breast cancer, and evaluated their specificity. The designed probes bound specifically to their targets. In addition, the target miRNAs could be detected in serum without washing. This study demonstrated the specificity evaluation of signaling DNA arrays for miRNA detection and simultaneous detection of two type of miRNAs.

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12.

Field-effect transistors made of polymer semiconductive materials (PFETs) show switching profiles by applying gate voltages. Therefore, PFETs can be applied to chemical sensor platforms by introducing appropriate molecular recognition materials. In this study, we herein propose an extended-gate type flexible PFET for chemical sensing applications. The polymer semiconductive material (i.e., poly{2,5-bis(3-tetradecylthiophene-2-yl)thieno[3,2-b]thiophene}) was employed to achieve a solution-processable device fabrication. In addition, the surface of the flexible PFET was fully covered with a hydrophobic layer made of a fluorinated polymer material, which allowed stable device operation under ambient conditions. The PFET fabricated on a flexible plastic substrate showed reproducible device characteristics with negligible hysteresis. Furthermore, the potential of the flexible PFET was revealed by a cycle test under a certain bending stress. The presentation will show the strategy of device design, details of device fabrication, basic transistor characteristics under mechanical stresses, and applications for chemical sensing.

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13.

Histamine (Hm), a toxic compound, is produced due to an enzymatic reaction when histidine (His)-rich redfish such as tuna and mackerel or their processed products are improperly stored. The rise in demand for Hm analysis. However, the conventional immunoassay method is costly, time-consuming, and technically demanding, burdening wholesalers heavily. Molecularly Imprinted Polymer grafted Carbon Paste (MIP-CP) is a feasible sensor tool due to its high selectivity, stability, and ease of production. In this study, we developed a disposable sensor using MIP-CP for Hm sensing. The cathodic current at the MIP-CP electrodes was sensitive to Hm with high linearity but was insensitive His. This sensitivity was specific to the MIP-CP electrodes, whereas the NIP-CP electrodes did not exhibit sensitivity to Hm. Those results suggest that the changes observed in the MIP-CP electrodes are due to the specific interaction between Hm and the Hm-imprinted cavities in the MIP.

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14.

Vancomycin (VCM) is the first-line treatment for the most common nosocomial mesitylene-resistant Staphylococcal Aureus (MRSA) infection. Overdosage of this drug causes kidney damage, so strict blood concentration monitoring is required. This study aimed to develop a drug sensor for rapid monitoring of blood drug levels of VCM. Molecularly imprinted polymers (MIPs) with VCM as a template were grafted onto the surface of graphite particles and kneaded with oil to produce an MIP-immobilized carbon paste (MIP-CP). The MIP-CP was applied to a substrate screen-printed with carbon ink to fabricate a VCM sensor. The current obtained by differential pulse voltammetry of this sensor in human serum containing VCM was linearly correlated with the VCM concentration. The serum individual differences in sensitivity were small, showing promise for the VCM sensor's clinical use.

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15.

Ion-selective electrodes (NH₄⁺-ISE and K⁺-ISE) are commonly used for the determination of ammonium (NH₄⁺) and potassium (K⁺) ions. Selectivity for the target ion (NH₄⁺ or K⁺) is expressed by using nonactin for NH₄⁺-ISE or valinomycin for K⁺-ISE as an ionophore within the sensing membrane. However, since the ionic radii of NH₄⁺ and K⁺ are close, they act as interfering ions for each other's ISE. Therefore, it is difficult for accurate measurement is when both ions coexist in the aqueous solution. We established a method for simultaneous determination of NH₄⁺ and K⁺ using the theoretical equation of ISE expressed based on the distribution equilibrium by ion transfer between the aqueous and membrane phases, and investigated the construction of a simultaneous determination method for multiple ions.

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16.

We report on a multichannel surface plasmon resonance sensor for cell evaluation based on pattern recognition of components of culture medium. The sensor consists of five cysteine derivatives immobilized on five Au thin films as probes, and can simultaneously acquire 10-dimensional kinetic information on cellular secretions in the culture medium. Our sensor succeeded in identifying changes in cellular states induced by anticancer drugs with a single introduction of culture medium, which had been obtained by combining several biochemical assays.

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