Use of combined scanning electrochemical and fluorescence microscopy for detection of reactive oxygen species in prostate cancer cells.

Release of ROS from prostate cancer (PC3) cells was studied using scanning electrochemical microscopy (SECM) and fluorescence microscopy. One-directional lateral scan SECM was used as a rapid and reproducible tool for simultaneous mapping of cell topography and reactive oxygen species (ROS) release. Fluorescence microscopy was used in tandem to monitor the tip position, in addition to providing information on intracellular ROS content via the use of ROS-reactive fluorescent dyes. A unique tip current (iT) vs lateral distance profile was observed when the tip potential (ET) was set at -0.65 V. This profile reflects the combined effects of topographical change and ROS release at the PC3 cell surfaces. Differentiation between topographical-related and ROS-induced current change was achieved by comparing the scans collected at -0.65 and -0.85 V. The effects of other parameters such as tip to cell distance, solvent oxygen content, and scan direction on the profile of the scan were systematically evaluated. Cells treated with tert-butyl hydroperoxide, a known ROS stimulus, were also evaluated using the lateral scanning approach. Overall, the SECM results correlate well with the fluorescence results. The extracellular ROS level detected at the SECM tip was found to be similar to the intracellular ROS level monitored using fluorescence microscopy. While the concentration of each contributing ROS species has not been determined and is thus part of the future study, here we have successfully demonstrated the use of a simple two-potential lateral scan approach for analysis of ROS released by living cells under real physiological conditions.