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CE RAMAN SPECTROSCOPY OF ATP AND OTHER NEUROTRANSMITTERS

ATP 和其他神经递质的 CE 拉曼光谱

基本信息

批准号：
2771041
负责人：
MICHAEL DAVID MORRIS
金额：
$ 15.86万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-09-27 至 2000-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2771041
关键词：
PC12 cells Raman spectrometry adenosine triphosphate chemical kinetics chick embryo chromaffin cells cyclic AMP hydrolysis nucleotide metabolism

项目摘要

DESCRIPTION: Normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) will be used as on-line detection techniques for ribonucleotides and other neurochemicals preconcentrated by capillary electrophoresis. Raman spectroscopy will allow for identification and quantification of compounds from cell cultures and extracts. Isotachophoresis, with subsequent electrophoresis if needed will be used to preconcentrate compounds to 10-6 - 10-4 M for analysis by SERS and 10-2 - 10-1 M concentration for analysis by normal Raman spectroscopy. Conventional capillaries and microfabricated capillaries will be employed. The SERS system will employ lithographically fabricated silver micro-electrodes, which can be placed at the exit of a capillary in a field-free region. In microfabricated capillaries, the electrode system will be integrated into the separation channel. A locally designed confocal epi-illumination microprobe optical system will be used for Raman spectroscopy. A high throughput spectrograph, charge-coupled detector and a 2 Watt 532 nm solid-state laser will complete the Raman spectroscopy system. The Gran-Schmidt orthogonalization procedure and other statistical tests will be used to identify and quantify target compounds at initial concentrations as low as 10-9 M. Synthetic test mixtures of ribonucleotides will be used for development and testing of the normal Raman spectroscopy system. When initial development of the system is completed, performance in a complex biological matrix will be demonstrated by glutamate measurements in the giant dopamine neuron of the pond snail. The system will be used to study hydrolysis kinetics of ATP to ADP and AMP in cultured chromaffin cells and in chick brain embryo extracts. Presently, only the rapidly hydrolyzed ATP can be conveniently measured and the project is expected to yield more detailed and accurate release rates than are now available. Additionally, ATP/adenosine ratios will be measured in the same matrices. Finally, the system will be used to measure c-AMP release in PC12 cells.

描述：正常拉曼光谱和表面增强拉曼光谱（Sers）将被用作在线检测技术，核糖核苷酸和其他神经化学物质的毛细管预浓缩电泳拉曼光谱将允许识别和定量来自细胞培养物和提取物的化合物。等速电泳，如果需要，随后进行电泳，将化合物预浓缩至10-6 - 10-4 M用于Sers分析，并将10-2 - 10 -4 M用于SERS分析。 10-1 M浓度，用于通过常规拉曼光谱分析。将采用常规毛细管和微加工毛细管。 Sers系统将采用光刻制造的银微电极，其可以放置在毛细管的出口处，无场区在微加工毛细管中，电极系统将被整合到分离通道中。局部设计的共焦落射照明微探针光学系统将用于拉曼光谱。一种高通量光谱仪，电荷耦合探测器和2瓦532纳米固态激光器将完成拉曼光谱系统。格兰-施密特正交化法和其他统计测试将用于识别和量化目标化合物的初始浓度低至10-9 M。合成测试核糖核苷酸的混合物将用于开发和测试标准拉曼光谱系统。当系统的初始开发是完成后，将证明在复杂生物基质中的性能通过测量池螺巨大的多巴胺神经元中的谷氨酸盐。该系统将用于研究ATP水解为ADP和AMP的动力学在培养的嗜铬细胞和鸡脑胚胎提取物中。目前，只有快速水解的ATP才能方便地测量，该项目预计将产生比现在更详细和准确的释放率 available. 此外，ATP/腺苷比率将在相同的条件下测量。矩阵最后，将该系统用于测定PC 12中c-AMP的释放细胞