(382c) Determining Reactive Oxygen and Nitrogen Species in Cells By Microchip Electrophoresis

Microchip electrophoresis is a powerful tool for the analysis of biological samples. In particular, the ability to perform fast efficient separations makes it possible to monitor multiple compounds simultaneously with high temporal resolution. The small sample volume requirements of the chip make it ideal for the analysis of microdialysis samples and single cells.  Reactive oxygen and nitrogen species (RNOS), such as nitric oxide, peroxynitrite and superoxide, have been implicated in a number of cardiovascular and neurodegenerative diseases.  RNOS are generated as part of the immune response and can react with DNA, proteins and lipids leading to cell death or dysfunction. Approaches for the detection and quantitation of multiple RNOS simultaneously are lacking, especially at the single cell level. In this presentation, methods for the detection of RNOS using microchip electrophoresis with electrochemical and laser induced fluorescence will be described. Many RNOS, including as nitrite, peroxynitrite, hydrogen peroxide and nitric oxide, are electroactive and can be separated and detected using microchip electrophoresis with electrochemical detection (MEEC).  An advantage of MEEC is that intracellular oxidants, which play a protective role against oxidative stress (glutathione and ascorbate), can be measured under the same conditions. Alternatively, laser induced fluorescence detection (LIF) can be used for the indirect detection of RNOS including nitric oxide, superoxide and peroxynitrite. ME-LIF requires derivatization of the analytes with specific fluorophores prior to analysis.  In this case, the electrophoretic separation is used to isolate the desired product from reaction side products, allowing for more precise quantitation. Examples of the detection of RNOS in macrophage cells using both ME-EC and ME-LIF will be presented.

Funding from the National Science Foundation is gratefully acknowledged.