Scanning electrochemical microscopy studies applied to single live cells.

摘要

Reactive oxygen species (ROS) have become an attractive topic because of their adverse and beneficial activities in live cells. Direct detection and quantification of ROS remains challenging due to their low concentrations and short half life time under physiological conditions. Scanning electrochemical microscopy (SECM) demonstrates the unique advantages of single live cell studies and permits the electrochemical measurements of ROS to be performed sensitively and selectively in discrete locations.;Subsequently, fundamental studies are explored to detect the extracellular H2O2 and O2 released from single COS-7 cells. The differential potential voltammetric results show O2 and H 2O2 can be deconvoluted by applying appropriate electrode potential. SECM images illustrated the variations of their concentrations above different cell organelles. A novel method which can generate numerous probe approach curves above different regions of cells in one depth image was developed.;In addition, SECM was applied to study the inflammatory response to infection by using H2O2 as the signalling messenger. The elevated release of H2O2 was observed from human bladder carcinoma cells stimulated with heat-killed Escherichia coli.;Furthermore, quantitative detection of H2O2 and O2 was demonstrated by simulation. Concentrations of H2O 2 and O2 at the surface of the membrane above the nuclear regions of COS-7, T24 and A498 cells were acquired. The extracellular amount of H2O2 under physiological conditions as well as H 2O2 and O2 release from B 16 cells upon stimulation with a tumor promoter, phorbol myristate acetate, were calculated.;This thesis specifically focuses on SECM studies of the major ROS, H 2O2, and the decomposition product of H2O 2 and O2-., O2. First, the comparison study of SECM with atomic force microscopy and confocal microscopy was conducted. The advantages of SECM as a chemical microscopy are clearly demonstrated in cell studies.;Finally, constant distance SECM technique was utilized to separate the topography from H2O2 and O2 release profile of RAW 264.7 cells using alternating current as the feedback signal. The disadvantage of mixing the surface information and chemical information in constant height SECM mode was minimized.;Keywords: reactive oxygen species, live cells, scanning electrochemical microscopy, electrochemistry, hydrogen peroxide, differential pulse voltammetry.