Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O2−) and hydrogen peroxide (H2O2), respectively. The method permits direct, real-time in vitro determination of both extra- and intracellular O2− and H2O2 produced by human neutrophil granulocytes. The rate of O2− production by stimulated neutrophils was calculated to about 10−17 mol s−1 per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H2O2 instead of O2− was obtained from stimulated neutrophils with the rate of about 3 · 10−18 mol s−1 per single cell. When the H2O2 release was discontinued, fast H2O2 utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O2− sensitive amperometric response. By contrast, the H2O2 sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H2O2, produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.