Scanning electrochemical microscopy studies applied to biological systems

This dissertation specifically deals with scanning electrochemical microscopy (SECM) studies of cellular transport processes that involve ion channels or activated transport proteins. To study biological systems, the substrate generation-tip collection (SG-TC) mode of SECM is used. Because of the inherent difficulty in quantifying such measurements, a two electrode system is first used to understand the general behavior of SG-TC transients. Numerical simulations confirm that the transient currents measured in SG-TC mode of SECM agree with the experimental behavior of simple electroactive compounds. The influence of the ultramicroelectrode (UME) geometry on recorded and simulated transient response is discussed. Finally, significant experimental and theoretical differences in the feedback mode of SECM for hemispherical and disk UMEs are presented. This knowledge is subsequently applied to a biomimmetic system where an ion channel is inserted in a self-assembled monolayer. The transport of thallium ions across gramicidin ion channels is detected at a nearby hemispherical mercury UME. This transport system is a model system for real biological systems. Nevertheless, important kinetic information about differences in the transport energy barrier between the two ends of the gramicidin half-channel can be obtained. The small level current measured in the above work confirmed that the SG-TC mode of SECM had the sensitivity to measure the efflux of electroactive biological material from cells. As such, the uptake of menadione and subsequent release of an electroactive biological metabolite from yeast cells is also reported. The synthesis, electrochemical characterization and detection of the menadione glutathione conjugate export from yeast cells are discussed. Kinetic treatment of the collection currents from yeast aggregates revealed that the uptake of menadione is the slow dominant step in the experiment. Finally, single human liver cells are studied using SECM measurements while exposed to cytotoxic concentrations of menadione. A determined cytotoxic dose of menadione imposes a chemical stress on hepatocytes and leads to the export of the menadione-glutathione conjugate via an ATP-dependent pump. The process is observed and imaged for both isolated and differentiated cells and has some interesting biological relevance.