Electrochemical Quantification of Extracellular Local H2O2Kinetics Originating from Single Cells.
H2O2is 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 H2O2to 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 H2O2is 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 H2O2separately. Local extracellular [H2O2] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501-517.
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .
Monika
M
Bozem
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .
Phillip
P
Knapp
2 Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss Kiril i Metodij University , Skopje, Macedonia .
Valentin
V
Mirčeski
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .
Ewa J
EJ
Slowik
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .|3 Cardiovascular Physiology, University Medical Center, University of Göttingen , Göttingen, Germany .
Ivan
I
Bogeski
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .
Reinhard
R
Kappl
4 HEKA Elektronik Dr. Schulze GmbH , Lambrecht, Germany .
Christian
C
Heinemann
1 Department of Biophysics, Faculty of Medicine, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University , Homburg, Germany .
Markus
M
Hoth
D015374:Biosensing Techniques; D055664:Electrochemical Techniques; D004926:Escherichia coli / Q000276:immunology; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009000:Monocytes / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst; D059010:Single-Cell Analysis
D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide
ROS; SECM; electrochemistry; human monocytes; immunology; single-cell H2O2 quantification