Microanalytical methods for the detection of reactive nitrogen species

检测活性氮的微量分析方法

• 批准号: 7828006
• 负责人: Susan M Lunte
• 金额: $ 17.99万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7828006
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Astrocytes; Biological; Blood - brain barrier anatomy; Brain; Cells; Cessation of life; Chemicals; Detection; Development; Devices; Dimensions; Endothelial Cells; Free Radicals; Goals; Infection; Inflammation; Life; Measurement; Measures; Methodology; Methods; Microchip Electrophoresis; Monitor; Neurodegenerative Disorders; Neuroglia; Neurons; Nitric Oxide; Nitrogen; Organism; Oxidation-Reduction; Oxidative Stress; Oxygen; Parkinson Disease; Peptides; Peroxonitrite; Play; Production; Reaction; Reactive Nitrogen Species; Regulation; Research Personnel; Role; Sampling; Sulfhydryl Compounds; Superoxides; System; analytical method; base; biological systems; chemical reaction; cytokine; detector; human NOS2A protein; macrophage; method development; microchip; public health relevance

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.

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）。在这些和其他条件下，神经元损伤或死亡可由活性氧和氮物质引起，其由内皮细胞以及星形胶质细胞和神经胶质细胞产生，与附近的神经元反应。在感染或炎症期间由星形胶质细胞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，这导致大量一氧化氮释放到神经血管空间中。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物质。该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物质浓度的定量信息。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。该芯片的集成格式使其能够检测短寿命的物种，如过氧亚硝酸盐，因为它们是通过化学，酶或生物反应形成的。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时物种。此外，检测器可以直接集成到芯片格式中，用于选择性和灵敏地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，用于检测过氧亚硝酸盐和其他活性氧和氮物种。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产生。最初的研究将集中在检测由巨噬细胞产生的过氧亚硝酸盐，因为已知它们在活化时会产生大量这种化合物。然而，最终目标将是测量神经血管空间和血脑屏障中星形胶质细胞和内皮细胞产生的过氧亚硝酸盐。将在单细胞水平和培养物中的活细胞水平进行分析。这种方法可用于更好地了解氧化应激在神经退行性疾病的发展，进展和调节中的作用。公共卫生关系：该建议涉及的分析方法的发展，在生活系统中的过氧亚硝酸盐的测量。过氧亚硝酸盐是在氧化应激条件下在大脑中产生的自由基物质，并且被认为在神经退行性疾病（包括阿尔茨海默病和帕金森病）中起主要作用。所提出的方法将有可能更好地阐明这种寿命极短的物种在神经退行性疾病中的作用。

项目成果

Determination of Methylarginines in Infant Plasma by CE-LIF.

通过 CE-LIF 测定婴儿血浆中的甲基精氨酸。

• DOI: 10.1039/c4ay00340c
• 发表时间: 2014
• 期刊: Analytical methods : advancing methods and applications
• 作者: Linz,ThomasH;Lunte,SusanM
• 通讯作者: Lunte,SusanM

Optimization of the separation of NDA-derivatized methylarginines by capillary and microchip electrophoresis.

• DOI: 10.1177/2211068211424551
• 发表时间: 2012-02
• 期刊: Journal of laboratory automation
• 作者: Linz TH;Snyder CM;Lunte SM
• 通讯作者: Lunte SM

Micro total analysis systems: fundamental advances and biological applications.

• DOI: 10.1021/ac403688g
• 发表时间: 2014-01-07
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Culbertson, Christopher T.;Mickleburgh, Tom G.;Stewart-James, Samantha A.;Sellens, Kathleen A.;Pressnall, Melissa
• 通讯作者: Pressnall, Melissa

An integrated microfluidic device for monitoring changes in nitric oxide production in single T-lymphocyte (Jurkat) cells.

• DOI: 10.1021/ac401665u
• 发表时间: 2013-11-05
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Metto, Eve C.;Evans, Karsten;Barney, Patrick;Culbertson, Anne H.;Gunasekara, Dulan B.;Caruso, Giuseppe;Huvey, Matthew K.;Fracassi da Silva, Jose Alberto;Lunte, Susan M.;Culbertson, Christopher T.
• 通讯作者: Culbertson, Christopher T.