| Budget Amount *help |
¥34,500,000 (Direct Cost: ¥34,500,000)
Fiscal Year 1988: ¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1987: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1986: ¥19,500,000 (Direct Cost: ¥19,500,000)
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| Research Abstract |
The present study is concerned with solid-state biosensors with protein monolayers (Langmuir-Blodgett films), which is thought to be one of the most promising molecular devices. The first stage of the study was focused to establish the growth method of proteinimmobilized LB films. Enzyme molecules were adsorbed onto the lipid monolayers over the water subphase in the LB trough, and the adsorbed film was deposited onto a solid substrate. The kind of lipidmaterials, enzyme specific activity, surface pressure during the adsorption, and sequence in the adsorption were varied to study their influences on the activity of enzyme LB films, numbers of immobilized enzyme molecules, film morphology (observed by electron microscope), biosensor sensitivity, etc. GOD (glucose oxidase) was used for typical enzyme in the present study. The selection of lipid material and the subphase pH was effective for keeping the charging polarity of the lipid inverse frfom that of GOD, increasing the electrostatic
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force in the adsorption, and enhancing the properties of GOD LB films for biosensors. Furthermore, the sequence of expansion and recompression ot the monolayer after the adsorption showed similar good effects. When glucose sensors were fabficated by using the GOD LB films, they demonstrated the wide measurement range from 5 to 5000 mg/dl, and the reperition life time more than one hundred times. The present glucose sensor is the first reported one with the enzyme-immobilized LB filem.
The next stage was devoted to the study of the relation between the film property and the GOD conformation. It was found that the denatured GOD is suitable for the stable immobilization in the lipid monolayer and the conformation is recovered by the surface compression over the subphase. Consequently, the deposited GOD film shows activity high enough for a biosensor. That conclusion is understood reasonable if the nature of protein molecules spread over the water surface is taken into account.
In the final stage of the present study, biosensors with further developed functions were studies. The following two sensors were successfully prepard; i.e., sensor with a mediator function by immobilizing GOD in a ferrocene derivative lipid monolaer, and an insullin sensor by using an insulin-antibody immobilized LB film and alcohol-dehydrogenase, which give rise to chemical amplification in this immunosensor. Those results suggest the promising future of protein-immobilized LB films. Less
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