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

摘要

Macrophages produce reactive oxygen and nitrogen speciesn(ROS/RNS) in response to immunological challenges.nWe have previously reported the real-time detection andnquantification of released ROS/RNS by immunostimulatednmacrophages using constant potential amperometry,nat four different potentials, with platinized carbon microelectrodes.nAs a methodological extension to that work,nwe sought to develop an electroanalytical method thatnwould allow for the simultaneous monitoring of severalnROS/RNS. Triple potential-step chronoamperometry atnplatinized carbon microelectrodes was found to providensatisfactory sensitivity and signaloise ratio for thisnpurpose. The title method was applied to the detection ofnendogenously produced ROS/RNS by single IFN-γ/LPSPMA stimulated RAW 264.7 macrophages. Significantlynhigher fluxes of H2O2, ONOO-, and NO• responsesnwere detected over stimulated macrophages as comparednto unactivated macrophages, consistent with thenendogenous production of primary NO• and O2n•- bynboth the inducible isoform of nitric oxide synthasen(iNOS) and reduced nicotinamide adenine dinucleotidenphosphate (NADPH) oxidase enzymatic systemsnin stimulated cells. Crucially, significant temporalnvariations in the release of each of the aforementionednspecies was evidenced using this method, which wouldnnot have been achievable with the use of either constantnpotential amperometry or classical biochemicalnmethods such as the Griess assay.