Microanalytical methods for the detection of reactive nitrogen species

描述（由申请人提供）：氧化应激被认为是许多神经退行性疾病的主要原因，包括阿尔茨海默病、帕金森病和肌萎缩侧索硬化症（ALS）。在这些和其他情况下，由内皮细胞、星形胶质细胞和胶质细胞与附近神经元反应产生的活性氧和活性氮可引起神经元损伤或死亡。星形胶质细胞在感染或炎症期间释放的细胞因子和其他肽引起诱导型一氧化氮合酶（iNOS）的诱导，导致大量一氧化氮释放到神经血管间隙。这种一氧化氮可以与神经元或其他细胞内产生的超氧化物反应，产生过氧亚硝酸盐以及其他活性氧和氮。本提案的总体目标是开发基于mems的分析方法，使研究人员能够获得有关构成神经血管空间的细胞产生的活性氮浓度的定量信息。基于微芯片的设备为这些研究提供了一些独特的优势。微芯片上通道的小尺寸使得测量体积非常小的样品（包括单个细胞中包含的皮升体积）成为可能。芯片的集成格式使得检测短寿命物种成为可能，例如过氧亚硝酸盐，因为它们是由化学，酶促或生物反应形成的。通过微芯片电泳，可以在不到一分钟的时间内快速有效地分离这些瞬态物种。此外，检测器可以直接集成到芯片格式的选择性和敏感检测氧化还原活性或荧光分析物。我们的计划是开发基于微芯片的系统来检测过氧亚硝酸盐和其他活性氧和氮。该方法将用于监测化学反应和生物系统中过氧亚硝酸盐及其反应产物的生产。最初的研究将集中在巨噬细胞产生的过氧亚硝酸盐的检测上，因为已知巨噬细胞在激活后会产生大量的这种化合物。然而，最终的目标将是测量星形胶质细胞和内皮细胞产生的过氧亚硝酸盐，这些细胞存在于神经血管空间和血脑屏障中。分析将在单细胞水平和活细胞培养中进行。这种方法可以用来更好地理解氧化应激在神经退行性疾病的发生、进展和调节中的作用。公共卫生相关性：本提案涉及一种用于测量生命系统中过氧亚硝酸盐的分析方法的发展。过氧亚硝酸盐是一种自由基，在氧化应激条件下在大脑中产生，被认为在包括阿尔茨海默病和帕金森病在内的神经退行性疾病中起主要作用。提出的方法将有可能更好地阐明这种极短的物种在神经退行性疾病中的作用。

DESCRIPTION (provided by applicant): Oxidative stress has been proposed to be a major cause of many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these and other conditions, neuronal damage or death can be caused by reactive oxygen and nitrogen species, which are generated by endothelial cells as well as astrocytes and glia, reacting with nearby neurons. Cytokines and other peptides released by astrocytes during infection or inflammation cause the induction of inducible nitric oxide synthase (iNOS), which results in the release of large amounts of nitric oxide into the neurovascular space. This nitric oxide can react with superoxide generated inside neurons or other cells to produce peroxynitrite as well as other reactive oxygen and nitrogen species. The overall goal of this proposal is to develop MEMs-based analytical methodology that will enable researchers to obtain quantitative information regarding the concentration of reactive nitrogen species generated by the cells that make up the neurovascular space. Microchip-based devices provide some unique advantages for these studies. The small dimensions of the channels on a microchip make it possible to measure samples with very small volumes (including the picoliter volumes contained in a single cell). The integrated format of the chip makes it possible to detect short-lived species, such as peroxynitrite, as they are formed by chemical, enzymatic, or biological reactions. Fast and efficient separations of these transient species can be accomplished in less than a minute by microchip electrophoresis. In addition, detectors can be directly integrated into the chip format for the selective and sensitive detection of redox-active or fluorescent analytes. Our plan for this proposal is to develop microchip-based systems for the detection of peroxynitrite and other reactive oxygen and nitrogen species. This methodology will be used to monitor the production of peroxynitrite and its reaction products both in chemical reactions and in biological systems. Initial studies will concentrate on the detection of peroxynitrite generated by macrophages because they are known to produce large quantities of this compound upon activation. However, the ultimate goal will be to measure the peroxynitrite generated by astrocytes and endothelial cells present in the neurovascular space and at the blood-brain barrier. Analyses will be performed both at the single cell level and with live cells in culture. This methodology can then be used to obtain a better understanding of the role of oxidative stress in the development, progression, and regulation of neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: This proposal is concerned with the development of an analytical method for the measurement of peroxynitrite in living systems. Peroxynitrite is a free radical species that is generated in the brain during conditions of oxidative stress and is believed to play a major role in neurodegenerative diseases including Alzheimer's and Parkinson's disease. The proposed methodology will make it possible to better elucidate the role of this extremely short lived species in neurodegenerative disease.