Single-molecule imaging is a powerful technique to study functions of biological molecules. As an application of this method, dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction and chaperonin-asisited protein folding were visualized by using total internal reflection fluorescence microscopy. Single molecule analysis revealed for the first time that the release of GroES from GroEL was governed by the two successive timers; a lag period (-3s) preceded the net release process (-5s). The protein folding in the immobilized cis complex, monitored using green fluorescent protein and triggered by photolysis of caged-ATP, also started after a lag period (-3s). Thus, the first timer defines the 3s lifetime of a novel intermediate state in which both non-native protein and GroES attach to GroEL. Although increasing number of reports on the observation and analysis of a single protein molecule have been published recently, the study presented here is the first “live-scene” visualization of functional association-dissociation between proteins and of protein folding. The power of this approach will not be restricted in the studies on chaperonin but be extended to the studies on other protein-protein interaction.

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A method based on microsystems for the physical dissection of DNA and the recovery of the dissected fragment is developed for the acquisition of an aimed position on a DNA strand. The device consists of a glass substrate, onto which a sacrificial layer, DNA carrier layer, and a pair of electrodes are deposited. Fluorescence labeled DNA is electrostatically stretched and immobilized onto the carrier layer with one of its molecular end aligned on the electrode edge. Using an AFM probe as a knife, an aimed portion of the DNA together with the carrier layer is dissected under the fluorescence microscope. By dissolving the sacrificial layer, the DNA fragment on the carrier piece is recovered onto a membrane filter. The carrier layer is then melted to obtain DNA fragments in solution. The yield of DNA recovery in such a device is investigated by PCR assay, and fluorescence labeling is identified as a major factor that may deteriorate the yield, in particular when DNA is dried and irradiated by the excitation light. A new DNA labeling is developed, where the fluorescence dye is incorporated only to the molecular ends, and the yield close to unity is demonstrated.

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The mesoporous materials SBA-15 and SBA-16 from the self-assembled template organic framework inorganic compound materials are successfully prepared with powder and film states. The powder state of mesoporous material exhibit a variety of properties and possibilities of these kinds of materials. The film state of these SBA-15 and SBA-16 are successfully fabricated into the surface photo voltage (SPV) type gas sensor device as a gas adsorption insulator layer. These kinds of gas sensors device exhibit NO and NO₂ gas sensing properties dependent on their mesoporous film structure. We are succeeded in indication about a possibility of mesoporous silicate film for the SPV type gas sensor application.

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A new fluid flow inside the microchannel was successfully developed. The flow created here involves segmented flow injection of plural organic phases into a microchannel followed by contact with a single aqueous phase to form stable organic-aqueous two-layer flow inside the microchannel. Fundamental study on the developed flow inside the microchannel was performed by monitoring the dye-doped segmented organic phases by thermal lens microscopy (TLM). Excellent repeatability and very small injection volume in developing segmented flow were realized. The new fluid flow created here is expected to allow us multi-ion sensing, which is not easily demonstrated by conventional ion sensor technology using a solvent polymeric membrane, by combining with neutral ionophore-based ion pair extraction using plural numbers of organic phases containing different ionophore molecules.

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An organophosphorus pesticide, ethylthiometon (0.01-0.2 ppm) was determined by using a surface-electrochemical sensor system: the monolayer formation (chemisorption)-reductive desorption of thiocholine was applied to monitor the activity change of cholinesterase caused by the pesticide.

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An optical biosensor that detects substrates of oxidases was developed as an element of an integrated chemical sensor. The sensor consisted of a spot of layers of enzyme and electron mediator on a thin gold film. The substrate concentration was converted into the refractive index change of electron mediator, and the refractive index was measured by Surface Plasmon Resonance (SPR). SPR was also used to choose the sensing area to give maximum sensitivity within the enzyme spot. SPR detection has advantages in biological binding reaction measurements because it does not need chemical modification for the target molecules, but it had not been suitable for the detection of enzymatic reactions. Our method will enable detection of many types of biological reactions in a uniform SPR measurement format.

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Airborne benzene, toluene, and xylene (BTX) are aromatic volatile organic compounds (VOCs) of great social and environmental significance. Since they are toxic even at ppb concentrations and their levels of toxicity are different, quantitative identification of VOCs in the air is required. We developed small (37cm x 28 cm x 10cm) VOC detection system (fig.1) comprising microfluidic device (concentration and detection cells, 1 cm x 3 cm, respectively), a pump, a deuterium (D₂) lamp, an UV spectrometer, and a SCSI interface. When BTX mixture gas was introduced into this detector, we successfully obtained the component ratios.

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