TECHNOLOGIES FOR CELLULAR NEUROMETABOLOMICS(RMI)

描述（由申请人提供）：了解健康和患病大脑的大脑功能需要了解神经元和构成大脑的支持细胞内发生的生物化学。众所周知，大脑中的单个细胞具有不同的信号分子补体和蛋白质补体，但对细胞代谢组的影响却知之甚少。哺乳动物中枢神经系统细胞代谢组的细胞间差异是什么？对于大多数代谢物，答案是未知的。使用无脊椎动物模型，测量了相邻神经元代谢组的巨大差异，例如氮能神经元中硝酸盐的毫摩尔水平，而非产生no的神经元中没有检测到硝酸盐。同样，氨基酸补体的巨大变化取决于神经元使用的信号分子。不幸的是，目前的技术限制不允许在单个哺乳动物神经元中测量主要代谢物。提出了一套技术开发和连接方法来创建仪器和方案来测量神经元簇、神经元组甚至单个神经元中的代谢物。这些开发工作包括独特的采样方案，基于微流体的样品调理单元，集成电泳分离，然后是天然荧光和质谱检测，必要时，将适当的代谢物捕获到纳米体积毛细管中进行纳米体积核磁共振光谱表征。这种独特的技术有望开启一种新的体积机制，以描述代谢物在细胞间的变化。这项技术将针对已知的最多样化的样本——哺乳动物的大脑——进行定制和验证。

DESCRIPTION (provided by applicant): Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.