Detection of Intracellular Oxygen Species

描述(申请人提供)：细胞内氧物种的检测在疾病的生物分子基础研究中的基本问题是，活性氧物种(ROS)，如超氧化物(O2-)和过氧化氢，是否由细胞持续地从线粒体或其他部位产生，以及这些ROS的水平是在生理和病理条件下产生的。解决这一问题的一个基本要求是开发能够在细胞和亚细胞水平上分析O2-和H2O2的非侵入性技术。本研究的长期目标是开发定量技术来检测和定量单细胞中的ROS，重点是线粒体ROS的产生，并将这些技术的结果与常用的ROS检测方法(如使用氧化敏感探针的荧光显微镜)相关联。为了实现这些目标，我们将利用电化学纳米传感器和自旋捕获电子顺磁共振(EPR)光谱领域的发展。将使用以下方法在细胞培养和线粒体上进行研究：1)合成并应用新型亲脂的自旋捕获DEPMPO衍生物来捕获O2-，2)将EPR光谱和EPR血氧仪的数据关联起来以获得有关O2-释放环境的信息，3)表征基于官能化纳米光纤上的共价连接酶的纳米传感器用于细胞内O2-和/或H2O2的检测，4)将这些技术的结果与更容易获得的方法，例如荧光显微镜的结果进行比较，以及5)研究在损伤条件下O2-和H2O2水平的变化。希望这些研究能解决以下问题：i)生理和病理刺激后O2-和H2O2的实际浓度是多少？Ii)特定疾病是否与ROS水平升高或降低有关？以及iii)在不损害ROS信号功能的情况下改善或逆转ROS变化的最佳治疗策略是什么？

DESCRIPTION (provided by applicant): Detection of Intracellular Oxygen Species Among the fundamental questions in research on the biomolecular basis of disease is whether reactive oxygen species (ROS), e.g. superoxide (O2-) and H2O2, are continuously produced by cells -from mitochondria or other sites, and what levels of these ROS are generated under physiological and pathological conditions. An essential requirement to address this is development of noninvasive techniques capable of assaying O2- and H2O2 at the cellular and subcellular level. The long-term objective of the present research is to develop quantitiative techniques to detect and quantify ROS in single cells, with a focus on mitochondriaL ROS production, and to correlate results from these techniques with commonly-used assays for ROS such as fluorescence microscopy using oxidation-sensitive probes. To achieve these goals, we will exploit developments in the fields of electrochemical nanosensors and spin-trapping electron paramagnetic resonance (EPR) spectroscopy. Studies will be carried out in cell cultures and mitochondria using the following approach: 1) synthesize and apply new lipophilic derivatives of the spin trap DEPMPO to trap O2-, 2) correlate EPR spectra and data from EPR oximetry to obtain information about the environment in which O2- is released, 3) characterize nanosensors based on covalently attached enzymes on functionalized optical nanofibers for intracellular detection of O2- and/or H2O2 , 4) compare results from these techniques with those from more accessible methods, e.g. fluorescence microscopy, and 5) study changes in O2- and H2O2 levels under injury conditions. It is hoped that these studies will address questions such as: i) What are actual concentrations of O2- and H2O2 after physiologic and pathologic stimuli? ii) Are specific diseases associated with elevated vs. decreased ROS levels? and iii) What are the best therapeutic strategies to ameliorate or reverse changes in ROS without compromising their signaling functions?