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

本研究利用固定在机械手上的微渗析探头制成的铂黑小体积在线传感器，在活体条件下测定脑神经元细胞外L-谷氨酸的释放浓度。首先，在体外实验中，利用BAS100B/W工作站和小电流模块获得传感器输出的L-谷氨酸的线性校正曲线，以提高灵敏度和降低噪声水平。产生的过氧化氢在保持在600 mV的铂电极上被电化学氧化，氧化电流被监测为传感器输出与谷氨酸浓度之间的线性关系。至于体内实验，我们试图消除细胞外抗坏血酸对测定系统的干扰。我们还使用碳体香炉进行了实验，使用的是Os聚冥想器而不是铂黑。该方法将电极电位控制在0 mV，不受儿茶酚胺和抗坏血酸的干扰。体内在线检测的另一个问题是不可避免地会受到谷氨酰胺的污染，这就需要一种对谷氨酸具有高度专一性的谷氨酸氧化酶。这种酶的纯化目前正在进行中。随着该酶的问世，完成了整个检测体系的建立。

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.