Electrochemical Quantification of Extracellular Local H₂O₂ Kinetics Originating from Single Cells

H₂O₂ is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local hydrogen peroxide concentrations ([H₂O₂]) originating from single cells is required.

Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H₂O₂] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H₂O₂] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H₂O₂] 5-8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H₂O₂ to an unstimulated MC, the local [H₂O₂] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H₂O₂ is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex^® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H₂O₂ separately. Local extracellular [H₂O₂] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501-517.