(392b) Dual-Electrode Electrochemical Detection for Microchip Electrophoresis: Voltammetric Identification of Chemically Labile Species

Reactive nitrogen species such as nitric oxide (NO^.) and peroxynitrite (ONOO^-) are chemically labile species that participate in oxidative stress and nitration/nitrosylation in vivo. These species have been implicated in several cardiovascular and neurodegenerative diseases. The short life time of these molecules makes them difficult to detect, often requiring indirect methods of analysis. Microchip electrophoresis coupled to amperometric detection (ME-EC) offers fast separations and sensitive detection— allowing these species to be characterized before significant degradation. Amperometric detection normally utilizes migration time to identify analytes in a sample. For complex samples such as cell lysates, analyte identification solely utilizing migration time becomes problematic when contamination protrudes. Therefore, a ME-EC method with dual electrodes was developed for identification of analytes by voltammetric characterization. Voltammetric information for analytes was obtained through a current ratio generated by employing two working electrodes in a series configuration. The current ratio can be unique to analytes with different half-wave potentials and deviations from such can imply impurities.  The electrodes were integrated into a 5 cm simple “T” microchip. In this setup, the first electrode is in in-channel configuration while the second electrode is in end-channel configuration. Nitrite, tyrosine and hydrogen peroxide standards were used to optimize the system. Current ratios for these standards were generated by correcting sensitivity differences between two working electrodes. Applying this to commercially available peroxynitrite samples, it was found that test samples were contaminated with hydrogen peroxide, which is used in peroxynitrite synthesis. This method will be employed to identify RNS production in bulk cell lysates. The ultimate goal of this project is to identify the heterogeneity of reactive nitrogen species production in single cells.