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?