| Project/Area Number |
11640612
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
分離・精製・検出法
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
YAO Toshio Graduate School of Engineering, Osaka Prefecture University Associate Professor, 工学研究科, 助教授 (50081310)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | microprobe biosensor / enzyme sensor / in vivo sensor / highly sensitive sensor / enzyme reactor / L-glutamin acid / neurotransmitter / 基質リサイクリング |
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| Research Abstract |
1. A microdialysis enzyme electrode involving amplification for L-glutamate was developed in this work. This enzyme electrode was prepared by cross-linking L-glutamate oxidase and glutamate dehydrogenase with glutaraldehyde on the poly (1,2-diaminobenzene) film-coated platinum fiber as a working electrode and a silver / silver chloride fiber as a reference electrode within its cavity. The L-glutamate was recycled enzymatically in the presence of an excess of NADH in the measuring buffer solution, . As a result, L-glutamate was detected with a 300-fold increase in sensitivity compared with the unamplified responses. The detection limit was 0.1 μM.
2. A bioelectrochemical in vivo flow-injection system with an on-line microdialysis sampling was developed for the assay of the trace amounts of L-glutamate released from rat brain cells. In the first stage of the operation, the dialysate from the probe was delivered to the sample loop of the six-way autoinjector by perfusing Ringer's solution. The calibration of the detection system including enzyme reactors was performed simultaneously. In the second stage, the diaiysate in sample loop was automatically injected into the flow-injection line and assayed. An L-glutamate oxidase / glutamate dehydrogenase coimmobilized reactor was used as an on-line amplifier based on the substrate recycling. The detection limit was less than 0.5 μM.
By the proposed methods, the variation of L-glutamate level released from rat brain cells were assayed in vivo and also after continuous stimulation of KC1, to demonstrate the reliability of the system.
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