权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study on microdialysis probe biosensors for real-time assay of neurotransmitters

实时检测神经递质的微透析探针生物传感器的研究

基本信息

批准号：
11640612
负责人：
YAO Toshio
金额：
$ 1.86万
依托单位：
Osaka Prefecture University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640612/
关键词：
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

项目摘要

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.

1.研制了一种用于谷氨酸放大的微透析酶电极。该酶电极是在聚（1，2-二氨基苯）膜涂层的铂纤维上用戊二醛交联L-谷氨酸氧化酶和谷氨酸脱氢酶而制成的，铂纤维作为工作电极，银/银氯化物纤维作为参比电极。在测量缓冲液中存在过量NADH的情况下，酶促回收L-谷氨酸。结果，与未扩增的响应相比，检测到灵敏度增加300倍的L-谷氨酸。检测限为0.1 μ M。建立了一种在体流动注射生物电化学在线微透析进样系统，用于测定大鼠脑细胞释放的微量谷氨酸。在操作的第一阶段，通过灌注林格氏溶液将来自探头的透析液输送至六通自动注射器的样品环。同时进行了包括酶反应器在内的检测系统的校准。在第二阶段，样品环中的透析液自动注入流动注射管线并进行测定。采用L-谷氨酸氧化酶/谷氨酸脱氢酶共固定化反应器作为基于底物循环的在线放大器。检测限小于0.5 μM。利用所建立的方法，在活体及连续KCl刺激后，测定了大鼠脑细胞释放L-谷氨酸的变化，验证了该系统的可靠性。