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.Chemistry and BiochemistryChemistr

Oxygen plasma treatment of polystyrene and zeonor: substrates for adhesion of patterned cells

Plastic substrates made of polystyrene (PS) and Zeonor 1060R were treated with oxygen plasma to introduce polar groups (e.g., carbonyl and carboxylic acid) at the surface that render these materials hydrophilic and promote patterned adhesion of HeLa cells. Resultant surfaces were characterized using contact angle goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) to monitor changes in wettability, nanoscale topography, and chemical composition. Biocompatibility of the plastic surfaces was verified through fluorescence microscopy using three fluorophores, Alexa Fluor 488 conjugated to Annexin V, Hoechst 33258, and propidium iodide, indicating cells that undergo apoptosis and necrosis, respectively. The best cell growth was observed on PS treated at 5 W/sccm, for which the viability of adhering HeLa cells exceeded 90%. Patterning was accomplished using an elastomeric microcapillary system (\u3bcCS) made of poly(dimethylsiloxane) (PDMS) that consisted of a set of parallel channels to align cells in linear fashion. Densely populated bands were obtained on substrates of both plastic materials when the culture medium contained >2 7 105 cells/mL.Peer reviewed: YesNRC publication: Ye

Extending the lifetime of pH microelectrode with stabilized palladium hydride

We report a new fabrication method to produce palladium hydride pH microelectrode using a chemical approach to synthesize the palladium hydride. In contrast to electrochemically generated palladium hydride microelectrodes, chemically generated palladium hydride microelectrodes are longer lasting and importantly have a good analytical performance under aerobic conditions. Chemically generated palladium hydride microelectrodes perform best in acid to neutral electrolytes devoid of Cl−. They can readily be produced on 10 μm diameter disk platinum microelectrodes, which makes them attractive candidates for future localized electrochemical studies.</p

Probing Passivating Porous Films by Scanning Electrochemical Microscopy

Porous films are ubiquitous in electrochemistry. They frequently form on active electrodes due to the precipitation of insoluble reaction products. They can have beneficial effects, like the protection from electrochemical corrosion, or be of parasitic nature, as in the poisoning of fuel cell air cathodes. The effects of such layers on the electrochemical response of the substrate can be probed by Scanning Electrochemical Microscopy (SECM). Herein, we present modifications to the conventional analytical expressions for SECM microelectrode approach curves, to account for the effects of a porous layer. The modified expressions can be used to fit experimental approach curves and obtain film thickness and porosity parameters. Their performance is demonstrated through comparison to results obtained by finite element modeling, and by fitting experimental approach curves over well-defined filter membranes

Biological scanning electrochemical microscopy and its application to live cell studies

Peer reviewed: YesNRC publication: Ye

Altered Spatial Resolution of Scanning Electrochemical Microscopy Induced by Multifunctional Dual-Barrel Microelectrodes

The nonuniform diffusion profile to the edge of many multifunctional microelectrodes has the potential to give rise to distortions in its imaging capability, reducing the spatial accuracy of the techniques they are used in. In this work, numerical simulations are used to predict these distortions for dual-barrel electrodes used in the combined feedback/generation–collection mode of scanning electrochemical microscopy imaging a model substrate. The sensitivity of this distortion to tip–substrate distance, electrolyte composition, and size and shape of a reactive substrate feature are discussed