Vol. 31, Supplement A (2015)

20.

We have developed the simple electrochemical immunochrimatography for albumin by detecting redox species generated by the enzyme reaction of glucose oxidase (GOx) captured by immunoreactions at the antibody immobilization area. The solution containing different concentrations of albumin is applied to the membrane. The added solution migrated toward the membrane ends by capillary force. When albumin arrived at the immobilized antibody, immunocomplexes were formed to capture albumin. GOx-labeled antibody was also captured in the area to form the sandwich structure. Finally, we added the substrates for enzyme, glucose and Fe(CN)₆^3- to produce Fe(CN)₆^4- by the enzyme reaction of captured GOx in the area and detected oxidation current of the generated Fe(CN)₆^4- by Pt electrode arranged downstream. The current response increased with increasing the concentration of albumin. Creatinine was also detected with the device from the oxidation current of hydrogen peroxide generated by successive reactions of three enzymes immobilized on the membrane. The total measurement time for both albumin and creatinine required with the present system is around 40 min.

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21.

Autonomous control of the transport of solutions in micro flow channels using a simple microvalve consisting of a self-assembled monolayer (SAM) formed on a platinum electrode is proposed. To realize autonomous control of microfluidic transport, we used switching of mixed potential. A microvalve in a main flow channel was opened when an electrolyte solution was injected into a controlling flow channel and the solution reached a zinc electrode. Controlled autonomous transport of solution in a network of flow channels could also be carried out using this microvalve.

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22.

Reagentless sensors are required for monitoring level of drugs which have fatal side effects. The author designed reagentless electrode sensors grafted with molecularly imprinted polymer containing a redox group grafted on electrode. Methylenebisacrylamide, methacrylic acid and acrylic acid was copolymerized in the presence of serotonin as a template by the photoinitiator immobilized on indium-tin oxide (ITO). The anodic current by ferrocenyl group detected by differential pulse voltammetry was sensitive to the template (serotonin) but insensitive to L-tryptophan. Heparin and vancomycin were also useful as the templates or the targets of sensing.

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23.

In this study, we investigated the insulin sensing method utilizing insulin binding DNA aptamer IGA3. It forms anti-parallel G-quadraplex folding single strands. It was reported that some anti-parallel G-quadruplexes bind hemin and show peroxidase activity by spectroscopic observation. In this study peroxidase activity of IGA3 with hemin was confirmed by spectroscopic measurement, and the activity was 2.75 times higher than hemin itself. Then IGA3 was immobilized onto a gold electrode to determine its activity electrochemically. Peroxidase activity of immobilized IGA3 with hemin was determined by Cyclic Voltammetry and cathodic peak current of the electrode showed the dependence on the concentration of H₂O₂. The cathodic peak current decreased by addition of insulin and the decrease depended on the concentration of insulin. As a result we demonstrated an aptamer-based detection method for insulin.

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24.

We report various states of mismatched methylcytosine in a DNA duplex by an immunochemical approach. There has been some discussion as to whether the conformation of a mismatched base is looped-out or stacked-in by physical techniques. Results obtained with our immunochemical approach imply that many bulged methylcytosines are looped-out because the affinity for a bulged methylcytosine is comparable to that for a freely accessible methylcytosine in a single strand DNA. In contrast, matched methylcytosine is in stacked state. This is because the antibody cannot bind with an inwardly turned methylcytosine in the duplex region owing to the large antibody size. By employing the difference between the affinity in the bulge and that in the duplex, we could determine selectively whether or not the target cytosine was methylated in an O6-methylguanine DNA methyltransferase (MGMT) promoter sequence.

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25.

We developed an electrochemical endotoxin (ET) detection system by using a combination of an ET-affinity microparticle (Cellufine^TM ETclean), a zinc complex-based probe for ET detection (ET probe), and an ultraflat nanocarbon film electrode. ET sample was captured on the microparticles modified with poly-epsilon-lysine with a high affinity to ET, and then the ET probe consisting of zinc complex parts and ET-affinity cetylpyridinium (CP) molecule was captured on the ET adsorbed microparticles via the ET-CP affinity interaction. The adsorbed ET probe was treated with acid solution, and finally we obtained zinc ion (Zn²⁺) containing solution. Anodic stripping voltammetry (ASV) was carried out using our nanocarbon film electrode to measure Zn²⁺ concentration. Our nanocarbon film electrode allowed us to detect Zn²⁺ with lower concentration compared with a conventionally available GC electrode. This was due to (1) the efficient capture of the ET and the ET probe on the microparticles, (2) the efficient accumulation of Zn²⁺ on the nanocarbon film, and (3) the very low noise made possible by the ultraflat surface. The current response was dependent on the amount of captured ET (LOD=0.2 ng/mL), which was superior result to our previous reports.

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26.

An electrochemical enzyme biosensor with electronically type-sorted (metallic and semiconducting) single-walled carbon nanotubes (SWNTs) for use in aqueous media is presented. This research investigates how the electronic types of SWNTs influence the amperometric response of enzyme biosensors. To conduct a clear evaluation, a simple layer-by-layer process based on a plasma-polymerized nano thin film (PPF) was adopted because a PPF is an inactive matrix that can form a well-defined nanostructure composed of SWNTs and enzyme. For a biosensor with the glucose oxidase (GOx) enzyme in the presence of oxygen, the response of a metallic SWNT-GOx electrode was 2 times larger than that of a semiconducting SWNT-GOx electrode. In contrast, in the absence of oxygen, the response of the semiconducting SWNT-GOx electrode was retained, whereas that of the metallic SWNT-GOx electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting SWNT-GOx electrode, whereas the metallic SWNT-GOx electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. Electrochemical impedance spectroscopy was used to show that the semiconducting SWNT network has less resistance for electron transfer than the metallic SWNT network. Therefore, it was concluded that semiconducting SWNTs are more suitable than metallic SWNTs for electrochemical enzyme biosensors in terms of direct electron transfer as a detection mechanism. This study makes a valuable contribution toward the development of electrochemical biosensors that employ sorted SWNTs and various enzymes.

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27.

An electrochemical sensor for on-line monitoring of residual chlorine (R.C.) in tap water has been proposed. The potentiometric sensors using Pt or stainless steel as working electrodes ware fabricated. In this study, both Pt and stainless steel electrodes showed high performance. For example, the potential of SUS304 electrode responded to 1 mg/L free R.C. within about 4 minutes and response width was about 200 mV. Furthermore, interference of environmental factors such as pH ware evaluated. Various electrode materials represented almost same pH dependence of potential. It suggested that appropriate pairing of electrode materials could realize all-solid-type sensor device with compactness, low power consumption, inexpensiveness, robustness.

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28.

To develop on-site monitoring biosensors for salivary nitrate in stress science study, we have investigated sensing materials and also original biocompatible polymer membrane materials for high performance nitrate ion-selective field-effect transistors (ISFETs).
We prepared highly sensitive and selective prototype of FET NO_3- checkers based on NO_3--ISFETs using the suitable copper(I) complex as a nitrate-sensing material, 2-nitrophenyldodecylether (NPDDE) as a liquid membrane material (plasticizer) and a segmented poly(urethane-urea) (KP-13) as a bio-compatible polymer material using the commercialized FET pH checker. The FET NO_3- checker showed linear characteristics from 10_-5.5 M to 10_-0.5 M with a response time of less than a few seconds. The FET NO3- checkers showed specific NO_3- selectivity against coexisting major Cl- in human saliva. So, we challenged to apply human saliva without pretreatment using direct potentiometry for ship navigator's mental workload.

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29.

In this presentation, we report a reliable supramolecular sensor device utilizing an organic field effect transistor (OFET) to detect saccharides in water. The titration results showed that the OFET device can read out electrically a saccharide-recognition behavior of a phenylboronic acid-self-assembled monolayer. The OFET device can detect glucose concentrations higher than 5 mM, a sensitivity which covers the threshold for the diabetes diagnosis (the fasting plasma glucose level is 7 mM and the normal plasma glucose level is 11 mM). We believe that these results will widen the avenues for the potential development of future OFET-based supramolecular sensors used for saccharide detection in healthcare applications.

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30.

Field effect transistor (FET) biosensor is a promising device for medical application due to its potential for label-free and rapid sensing. Recently, an influenza pandemic is said to occur when non-human influenza viruses (IFV) gain ability for sustained and efficient human-to-human transmission. As a device preventing pandemic, FET biosensor having gate surface modified with glycan, which has sialic acid for specifically binding to hemagglutinin (HA) on the envelope of IFV, is expected to be useful. Glycans are believed to exceed other commonly-used probes such as antibodies, in discriminating whether the target IFV infects to human or avian, since both α2,6 and α2,3-sialylgalactose-terminated glycans can be immobilized on the FET gate surface. In this study, the capability of the glycan-immobilized FET to quantitatively detect and discriminate the host of IFV is evaluated by detecting HA dissociated from the envelope of IFV using non-ionic surfactant, Triton X-100.

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31.

Enzyme immobilized electrode was fabricated using transparent cellulose aqueous solution containing glucose oxidase (GOx). Transparent cellulose aqueous solution was prepared by using the clear layer of diluted cellulose dispersion solution produced by wet jet milling. GOx cellulose solution was applied on Pt-Ir alloy wire and was dried for at least two days in room temperature. The obtained GOx-immobilized electrodes provided good response current up to 22.4 mM. Good linear relationship between glucose concentration and response current was observed. Correlation coefficient of 0.994 was obtained ranging from 0 to 11 mM on the electrode prepared using 500 mg/L cellulose and 250 mg/L GOx mixture solution. The stability of GOx-immobilized electrode response was measured for 30 days. After initial decrease of response current for the first few days, relatively constant responses were obtained for twenty days.

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32.

Immobilized conditions of enzymes onto carbon fibers were investigated.Each of a disk type of the immobilized enzymes was applied to a flowsystem and then,the performance characteristics were performed.

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特別講演

We here describe the development of immunosensing systems with chemically-amplified responses and those based on the dielectrophoretic manipulation of microparticles. Chemical amplification that involves a reaction sequence to generate a relatively large amount of product near the transducer surface is a useful way to enhance the sensor response effectively. The dielectric manipulation of microparticles provides simple and rapid immunoassay protocols. Highly-sensitive, rapid and simple imunosensing methods are suitable for the purpose of point-of-care testing of biomarkers.

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33.

Aiming to develop the practical biosensor, the miniaturized Surface Plasmon Resonance (SPR) sensor has been investigated. The miniaturized sensing device requires a high-sensitivity, a high-selectivity, and a rapid detection. High sensitivity and high-speed detection are not produced by only transducer ability but also kinetic factors. In this study, the biosensing based on indirect competitive inhibition method is investigated. According to the Langmuir isotherm, the enhancement mechanism was analyzed. It was found that the sensitivity for detection of clenbuterol was enhanced from 2 ppt to 0.05 ppt, although the affinity constant (K₃) for immunoreaction of Au nanoparticle-labeled secondary antibody was smaller than that of unlabeled secondary antibody.

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34.

Conventional localized surface plasmon resonance (LSPR) sensors have advantages over propagating surface plasmon resonance (SPR) sensors in terms of sensing volume, optical setup, and possibility of miniaturization. However, an LSPR sensor requires space for light dispersion and mechanical scanning of a grating, which limit the cost and size reduction of LSPR sensors. In this study, we propose and demonstrate potential-scanning LSPR sensors as a new class of LSPR sensors that can detect refractive index changes by potential scan instead of wavelength scan. Since the sensors only need a small laser diode or light emitting diode combined with a band-pass filter and an electronic circuit for potential scanning, the sensors are cost-effective and suitable for miniaturization. Here we used an ITO electrode loaded with gold nanoparticles for a potential-scanning LSPR sensing. Potential scanning at a certain wavelength in an aqueous electrolyte solution containing sucrose provided a potential-scanning extinction spectrum with a peak. The peak potential shifted negatively with increasing refractive index of the solution, as expected from the principle. The results indicate that the potential-scanning LSPR sensor can be applied to chemical sensing and biosensing based on refractometry.

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35.

Previously, we have developed enzyme-sensors based on a 2D-SPR imager that used oxidases such as lysine oxidase(LysOx) and galactose oxidase(GalOx) and Os polymer, which causes refractive index change according to the redox state. And we demonstrated that these sensors are useful for detecting these bio-markers for metabolic disorders more easily and faster than the conventional methods. Furthermore, we have also succeeded to detect the two substrates simultaneously by means of co-immobilizing LysOx/Os-HRP polymer and GalOx/Os-HRP polymer on the adjacent two part of the gold chip which was divided into two regions. In this study, we aimed at developing the 2D-SPR enzyme-sensor using another combination of dehydrogenase and Os polymer. Lactate dehydrogenase(LDH) was chosen in this time. We have successfully determined the lactate from 50 µM to 200 µM in the sample solutions.These results indicated that our sensing strategy might be useful for fabricating dehydrogenase based 2D-SPR sensors.

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36.

An interferometric analytical device was fabricated using a microfabricated optical directional-coupler (DC). With the DCs of different lengths, optical intensities measured at the output ports fit well to the corresponding fitting curve and the curve calculated based on the coupled mode theory. The sensor could differentiate different liquids. Also, the sensor could detect single stranded DNAs conjugated with quantum-dots (QDs) adsorbed at sensor surface. Measured relative optical intensity changes induced by changes in cladding refractive index (nclad) agreed well with values calculated based on the coupled mode theory.

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37.

Rapid and label-free bacteria sensor has urgently been required for preventing foodborne illness and hospital infection. However, conventional methods, such as culturing, require a substantial amount of time. We have thus demonstrated a novel approach to a highly selective and rapid bacteria detection method in a label-free format using bacterial surface structure transferred to a conducting polypyrrole (PPy) film. A thin black film was formed on the electrode after electrochemical polymerization of pyrrole in a solution containing bacteria. Scanning electron microscope (SEM) images of the film revealed dense doping of the target bacteria. The bacteria observed on the PPy film were almost completely removed after the overoxidation process, indicating the formation of cavities that were shape-complementary to the target bacteria. This film enabled us to detect target bacteria at concentration as low as 10³ CFU/mL within 3 min, without any bacterial pretreatment.

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38.

Cell viability assay is important in drug screening and diagnosis prior to implantation. Cell respiratory activity measurement is one of the evaluation methods for cell viability. We have developed Bio-LSI for a highly sensitive, amperometric bio-imaging platform with 250 µm-pitched 400 sensor electrodes. In this study, we used Bio-LSI with Pt electrode for the cell respiratory measurement of human liver carcinoma cell (HepG2) spheroid. The averaged results from 4 measurement point about the decrease of reduction current measured by Bio-LSI at −0.5 V vs Ag/AgCl were correlated with the respiratory activity measurement by scanning electrochemical microscopy (SECM) (R²=0.749). To obtain oxygen consumption rate of the spheroid, we designed and fabricated a Bio-LSI with higher-density electrode configuration (100 µm in averaged electrode distances). By using this higher-density Bio-LSI, the oxygen consumption rate of a HepG2 cell spheroid was obtained 2.97×10^-13 (mol/s), which is appropriate value from the data obtained with SECM.

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39.

Cytokines are produced by immune cells in response to viral or bacterial pathogens and therefore have high diagnostic value. Here, we developed a novel device, consisting of large number of glass capillaries filled by monolithic silica (monolithic silica-CP), that is capable of single-cell arraying and of the detection of interferon gamma (IFN-γ) released from single cells. The fabrication of this device started with the monolithic silica synthesis with different concentration of PEG for the control of silica porous structure. Based on the capillary action at the individual capillary, dropped solution containing human PBMC (peripheral blood mononuclear cell) was simultaneously aspirated from top to bottom. As a consequence, the cells were arrayed on this device at single cell level. Furthermore, using anti-IFN-γ antibody immobilized on monolithic silica-CP, IFN-γ was successfully detected from arrayed single cells. The rapid, easy and efficient process using the developed new device for cytokine detection from single-cells will be expanded in the future to enable of application for diagnosis/monitoring of various infectious diseases and of detail functional analysis of immune cells in response to antigens.

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40.

Mental stress is closely related to the function of the immune system. To estimate the degree of the mental stress, the phagocytotic activity of neutrophils was measured by measuring the change in the oxygen consumption under the existence of E. coli using a microfabricated Clark-type oxygen electrode coupled with a microfluidic channel. Clear reproducible responses were observed with the oxygen electrode. The current changed linearly up to 8.0×10⁸ cells/mL E. coli concentration. The respiratory activity of E. coli changed accompanying the decrease in the number of the bacteria by the phagocytosis. Neutrophils were separated from whole blood obtained from a normal mouse and a stressful mouse that was restricted in warm water for 2 h. A clear difference was observed in the oxygen consumption between the two cases. The results demonstrate that our device and method can be used to estimate the degree of the mental stress.

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41.

We have developed electrochemical apoptosis sensing methods using Asp-Glu-Val-Asp-p-methoxyaniline (DEVD-pMA) as a novel substrate for caspase-3. First we tried the amperometric detection of pMA released from DEVD-pMA by cell apopsosis. From the cyclic voltammogram obtained from 500 μM DEVD-pMA and 500 μM pMA in PBS solution, we employed 0.55 V vs Ag/AgCl for the applied potential of amperometry. Then, we fabricate interdigitated array electrode chip device with 3×3 measurement points for simultaneous multiple sensing of cell apoptosis. The detection is based on the redox cycling of quinoneimine (QI; produced by oxidation of pMA) and p-aminophenol (pAP; produced by reduction of QI). In both experiments, we successfully detected the apoptosis of human liver carcinoma cell line (HepG2) exposed to 5 μg/mL doxorubicin for 24 h. These electrochemical sensing methods can be applied to easy and on-site monitoring of the cell viability in wide fields of bioscience and medicine including transplantation therapy and drag discovery.

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