Profiling H2O2 from single COS-7 cells by means of scanning electrochemical microscopy

Abstract

We report quantitative determination of extracellular H₂O₂ released from single COS-7 cells with high spatial resolution, using scanning electrochemical microscopy (SECM). Our strategy of depth scan imaging in vertical x-z plane was conveniently utilized to a single cell for obtaining probe approach curves (PACs) to any positions on the membrane of a live cell by simply drawing a vertical line on one depth SECM image. This SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The H₂O₂ concentration at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O₂, and determined to be 0.020 mM by overlapping the experimental PAC with the simulated one having a known H₂O₂ release value. The H₂O₂ profile determined in this way gives insight into physiological activity of single live cells. In addition, intracellular H₂O₂ profile was demonstrated using confocal microscopy by labelling the cells with a luminomphore, 2',7'-dichlorodihydrofluorescein diacetate. The two methodologies have illustrated complementary experimental results of H₂O₂ detection, indicating that H₂O₂ generation is centered at endoplasmic reticula.

Keywords: COS-7 live cells; Confocal microscopy; Depth scan imaging strategy; Extracellular H(2)O(2) imaging; Reactive oxygen species; Scanning electrochemical microscopy (SECM).