ESTABLISHMENT OF AN ONLINE ASSAY SYSTEM OF INTRACEREBRALLY RELEASED GLUTAMATE BY MEANS OF MICROBIOCENSOR

微生物传感器脑内释放谷氨酸在线测定系统的建立

基本信息

批准号：
06557038
负责人：
KITO Shozo
金额：
$ 10.75万
依托单位：
University of the Air
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557038/
关键词：
glutamate platinum black sensor hydrogen peroxide sensor output ascorbic acid platinum tube glutamate oxidase glutamine glutamine oxidase ptチューブカイニン酸 glucocorticoid nicotine dexamethasone in situ hybridization in vivo microd

项目摘要

This study was undertaken to assay concentrations of extracellular L-glutamate release from imtracerebral neurons in vivo with a small-volume online sensor made of platinum black consisted of microdialysis probe fixed at the manipulator.As the first step, in vitro studies to get a linear calibration curve of L-glutamate determined with sensor output with use of the BAS100B/W workstation and low current module to increase the sensitivity and lower the noise level.The generated hydrogen peroxide is oxidized electrochemically at a Pt electrode held at 600mV and the oxidized current was monitored as a linear relationship between sensor output and glutamate concentration.As for in vivo experiments, we tried to remove extracellular ascorbic acid which interfere the assay system. We also performed experiments using carbon bulk censer with Os poly meditator instead of platinum black. In this method, the potential of the electrode can be held at 0 mV so that there is no interference by either catecholamine or ascorbic acid.An another problem in vivo online measurement is that we can not avoid, contamination with glutamine from this assay system, we need a glutamate oxidase which is extremely specific to glutamate. Purification of this kind of enzyme is currently in progress. With this enzyme coming up, establishment of the whole assay system is completed.

本研究采用由固定在机械手上的微透析探针组成的铂黑制成的小体积在线传感器测定体内脑神经元释放的细胞外 L-谷氨酸浓度。使用 BAS100B/W 工作站和低电流模块，通过传感器输出确定 L-谷氨酸的校准曲线，以提高灵敏度并降低噪音水平。产生的过氧化氢在 Pt 下被电化学氧化电极保持在600mV，氧化电流被监测为传感器输出和谷氨酸浓度之间的线性关系。对于体内实验，我们试图去除干扰测定系统的细胞外抗坏血酸。我们还使用带有 Os 聚冥想剂的碳散装香炉代替铂黑进行了实验。在这种方法中，电极的电位可以保持在0 mV，这样就不会受到儿茶酚胺或抗坏血酸的干扰。体内在线测量的另一个问题是我们无法避免该检测系统受到谷氨酰胺的污染，我们需要一种对谷氨酸具有极高特异性的谷氨酸氧化酶。这种酶的纯化目前正在进行中。有了这个酶，整个检测体系的建立就完成了。