Consecutive Gated Injection-Based Microchip Electrophoresis for Simultaneous Quantitation of Superoxide Anion and Nitric Oxide in Single PC-12 Cells

As important reactive oxygen species (ROS) and reactive nitrogen species (ENS), cellular superoxide anion (O-2(center dot-)) and nitric oxide (NO) play significant roles in numerous physiological and pathological processes. Cellular O-2(center dot-) NO also have a close relationship and always interact with each other. Thus, the simultaneous detection of intracellular O-2(center dot-) and NO, especially at the single-cell level, is important. In this paper, we present a novel method to simultaneously detect and quantify O-2(center dot-) and NO in single cells using microchip electrophoresis based on a new consecutive gated injection method. This novel injection method achieved consecutive manipulation of single cells, guaranteeing an almost constant volumetric flow rate and thus good quantitative reproducibility. After cellular content separation by microchip electrophoresis and detection by laser-induced fluorescence (MCE-LIF), O-2(center dot-) and NO in single PC-12 cells were simultaneously quantified in an automated fashion. This is the first report of consecutive absolute quantitation at the single-cell level. The quantitative results obtained from single cells is beneficial for deep understanding of the biological roles of cellular O-2(center dot-) and NO. This new method constitutes a consecutive, accurate way to study the synergistic function of O-2(center dot-) and NO and other biomolecules in various biological events at the single-cell level.