Electrochemical sensor based on rGO/Au nanoparticles for monitoring H2O2 released by human macrophages

•Amperometric sensor for detection of H2O2 released by living cells (human THP-1 macrophages) was developed.•H2O2 diffuses through cell membrane and can be checked in the extracellular space.•Macrophages highly provide Reactive Oxygen Species generation in inflamed tissues.•Macrophages were exposed to pro- and anti-oxidant treatments and H2O2 release was measured in conditioned medium.•Results of the electrochemical sensor agree with those obtained by flow cytometry using the same cells. Increased oxidative burden contributes to the pathogenesis of most inflammatory diseases and is associated with aging and chronic inflammation. Macrophages contribute to the generation of reactive oxygen species (ROS) within inflamed tissues. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric biochemical assays. Hydrogen peroxide (H2O2) diffuses through the cell membrane and can be monitored in the extracellular space. Herein, we present a sensor for H2O2 detection released by cells in culture supernatants. H2O2 sensing performance was evaluated using chronoamperometric detection. A sensitivity of 0.0641 μA μM−1 cm−2 with a limit of detection of 6.55 μM and excellent selectivity against many interferents was found. H2O2 release was also measured in conditioned medium from human THP-1 macrophages exposed to pro-oxidant and anti-oxidant treatments. The results were compared with those obtained by flow cytometry using the same cells stained with carboxy-H2DCFDA and MitoSOX Red, which detect intracellular ROS and mitochondrial superoxide, respectively. The addition of pro-oxidants lipopolysaccharide (LPS) and nigericin resulted in a significant increase in the cathodic current due to the H2O2 reduction, indicating an increased release of H2O2. The addition of 17-oxo-DHA, which inhibits LPS- and nigericin-dependent responses, decreased the LPS- and nigericin-induced release of H2O2. All the results obtained with the sensor were consistent with those obtained using flow cytometry. The operation of the sensor directly in the cell culture growth medium had no impact on cell viability. The sensor is highly sensitive, fast, and cost effective, and it can potentially be used for real time monitoring of oxidative stress.