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Development of in situ detection methods for substances in brain by using bioelements and bilayer lipid membranes

利用生物元件和双层脂膜开发脑内物质原位检测方法

基本信息

批准号：
15350050
负责人：
SUGAWARA Masao
金额：
$ 8.51万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

For the design of in situ sensing systems for biologically important substances that participates in neuronal communication in acute mouse brain slices, the use of artificial and natural bilayer lipid membranes and biological elements has be investigated. A glass capillary-based enzyme electrode was designed to detect L-glutamate that is released under glucose-free and oxygen-deficient conditions (Ischemia) by using glutamate oxidase and a gall capillary having the ability of sampling a small volume of extracelluar solution. The sensor applied to acute mouse brain slices showed that release of L-glutamate is neuronal region-dependent and the amount of L-glutamate release increases in the order of CA1【approximately equal】CA3>DG. This order was in agreement with those obtained by differential imaging of L-glutamate fluxes using enzyme-immobilized membrane developed newly in this research project. A patch sensor containing NMDA receptors was developed for detecting GABA-induced release of glutamate in acute hippocampal slices. The results showed that in the CA1 region, glutamate release by GABA stimulation is induced via GABA_A receptor and in the CA3 region, release of glutamate was not observed due to inhibitory effect of GABA_B receptor. An excised patch membrane sensor for arachidonic acid was also developed and applied to detect L-glutamate-stimulated release of arachidonic acid. The results exhibited that release of arachidonic acid increases in the order of CA3>CA1【approximately equal】DG. Also, bilayer lipid membranes (BLMs) containing a single gramicidin channel was prepared by the tip-dip method. The BLMs exhibited current responses, corresponding to modulation of gramicidin monomer/dimer kinetics caused by the binding of analytes to membrane bound receptor sites in the BLMs. The BLMs were used for detecting antibodies.

为了设计参与急性小鼠脑切片神经元通讯的重要生物学物质的原位传感系统，研究了人工和天然双层脂膜和生物元件的使用。设计基于玻璃毛细管的酶电极，通过使用谷氨酸氧化酶和能够对少量细胞外溶液进行采样的胆毛细管来检测在无葡萄糖和缺氧条件（缺血）下释放的L-谷氨酸。应用于急性小鼠脑切片的传感器显示，L-谷氨酸的释放具有神经元区域依赖性，L-谷氨酸释放量按CA1【约等于】CA3>DG的顺序增加。该顺序与使用本研究项目新开发的酶固定膜对 L-谷氨酸通量进行差分成像所获得的结果一致。开发了一种含有 NMDA 受体的贴片传感器，用于检测急性海马切片中 GABA 诱导的谷氨酸释放。结果表明，在CA1区，GABA刺激引起的谷氨酸释放是通过GABA_A受体诱导的，而在CA3区，由于GABA_B受体的抑制作用，没有观察到谷氨酸的释放。还开发了花生四烯酸的切除贴片膜传感器并应用于检测L-谷氨酸刺激的花生四烯酸释放。结果表明，花生四烯酸释放量依次增加：CA3>CA1【约相等】DG。此外，还通过尖端浸入法制备了含有单短杆菌肽通道的双层脂质膜（BLM）。 BLM 表现出电流响应，对应于分析物与 BLM 中膜结合受体位点的结合引起的短杆菌肽单体/二聚体动力学的调节。 BLM 用于检测抗体。