Simultaneous Detection of Reactive Oxygen and Nitrogen Species Released by a Single Macrophage by Triple Potential-Step Chronoamperometry

Macrophages produce reactive oxygen and nitrogen species (ROS/RNS) in response to immunological challenges. We have previously reported the real-time detection and quantification of released ROS/RNS by immunostimulated macrophages using constant potential amperometry, at four different potentials, with platinized carbon micro-electrodes. As a methodological extension to that work, we sought to develop an electroanalytical method that would allow for the simultaneous monitoring of several ROS/RNS. Triple potential-step chronoamperometry at platinized carbon microelectrodes was found to provide satisfactory sensitivity and signal/noise ratio for this purpose. The title method was applied to the detection of endogenously produced ROS/RNS by single IFN-gamma/LPS/PMA stimulated RAW 264.7 macrophages. Significantly higher fluxes of H(2)O(2), ONOO(-), and NO(center dot) responses were detected over stimulated macrophages as compared to unactivated macrophages, consistent with the endogenous production of primary NO(center dot) and O(2)(center dot-) by both the inducible isoform of nitric oxide synthase (iNOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymatic systems in stimulated cells. Crucially, significant temporal variations in the release of each of the aforementioned species was evidenced using this method, which would not have been achievable with the use of either constant potential amperometry or classical biochemical methods such as the Griess assay.