Electrochemical Quantification of Extracellular Local HO Kinetics Originating from Single Cells.

AIMS

HO 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 ([HO]) originating from single cells is required.

RESULTS

Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [HO] 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 [HO] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [HO] 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 HO to an unstimulated MC, the local [HO] 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 HO 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 HO separately. Local extracellular [HO] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501-517.