Imaging horseradish peroxidase under electrochemical conditions

Ph. D. Thesis.In this work, potential dependent electrochemical characterisation has been performed on horseradish peroxidase (HRP) molecules immobilised on annealed highly oriented pyrolytic graphite (HOPG) surface immersed in phosphate buffer solution (PBS). Electrochemical impedance spectroscopy (EIS) was applied to investigate the HRP molecular capacitance. The observed HRP capacitance shows significant potential dependency, which is analogous to that of a typical metal oxide semiconductor (MOS) capacitor fabricated with p-type semiconductor materials. Accumulation region and depletion region has been observed from the capacitance (C)-potential (U) curve of the HRP molecules, and the “flat band potential”, at which there is no potential drop within the semiconductor layer, has been determined as the potential where dC/dU-U curve peaks. An MOS capacitor approximation is proposed to describe the potential dependent capacitance behaviour of HRP molecules. Scanning electrochemical potential microscopy (SECPM) and electrochemical scanning tunnelling microscopy (EC-STM) has been applied to monitor the potential drop in HRP molecules at single molecular level. SECPM maps the potential distribution within the electrochemical double layer (EDL) and can resolve HRP molecules with Angstrom resolution. Potential drop within HRP molecules is reflected as potential dependent morphological variation in SECPM images. The observed HRP apparent size peaks at the flat band potential. Similar potential dependency of HRP apparent size is observed using EC-STM, as the potential drop being an influence on the bias voltage between tip and substrate. SECPM was also used to directly measure the EDL profile with alternating electrode potential and electrolyte concentration. An exponential potential decay with reference to the tip-substrate separation has been observed as predicted by Gouy-ChapmanStern model. Delayed response of potential decay, with significant dependence on the local electrical field and ionic strength, was also observed at close tip-substrate distance. A quantitative approach based on a novel mechanism which suggests high resolution SECPM imaging could be attributed to direct electron exchange between tip and substrate is proposed to interpret the delayed response of potential decay