- Amperometric Enzyme Electrodes

Abstract

This thesis studies the conditions required to achieve direct electron transfer and the experimental tests needed to unequivocally demonstrate that it occurs. Many publications claim to observe direct electron transfer to redox enzymes (for example in the case of glucose oxidase) but the evidence presented is often incomplete and unconvincing. The first part of this thesis argues that the vast majority, if not all, of these claims of DET for GOx are incorrect. It presents results for glucose oxidase (GOx) adsorbed on multi‐walled carbon nanotubes (MWCNTs), a typical nanostructured GOx electrode, that clearly show that the surface redox peaks usually observed in these cases are due to free, adsorbed flavin and not due, as claimed, to DET to flavin within the enzyme. Also, the results can be explained by adsorption of enzymatically active GOx at the electrode surface and the detection of the decrease in the oxygen concentration at the electrode surface due to the enzyme catalysed oxidation of D‐glucose. The second part of this thesis establishes a flexible and structured method based on the use of site‐directed mutagenesis to introduce cysteine residues at specific locations on the enzyme surface followed by the reaction between the free thiol group and maleimide groups formed on the electrode surface to immobilise the mutated enzymes. It is preferable to immobilise redox proteins and enzymes in a specific orientation, but still with some flexibility to optimise reaction kinetics. Using cellobiose dehydrogenase (CDH) as a model system, multiwall carbon nanotube electrodes were first covalently modified with maleimide groups following a modular approach combining electrochemical surface attachment and solid phase synthesis methodology. Five CDH variants were used in this study, the CDH‐modified electrodes were tested for direct electron transfer (DET), showing high catalytic currents and excellent long‐term storage stability. A potential‐dependent Michaelis‐Menten model for the CDH modified GC/MWCNT has been constructed and a master equation employed to simulate the DET and MET experimental results. Several mechanisms were suggested to explain the DET and MET for CDH. The internal electron transfer (IET) has been shown to be the rate determining step in the proposed mechanism. This was confirmed by the simulated data along with the experimental results. The simulated data suggests the presence of two populations of immobilised enzymes, MET and DET enzyme. The validity of the aforementioned immobilisation method, was further examined. Three bilirubin oxidase (BOD) variants were used in this study, which were modified to bear a free cysteine residue in different positions at the surface of the enzyme, allowing fast and selective attachment to maleimide‐modified GC/MWCNT electrodes. The catalytic mechanism of O2 reduction by the Magnaporthe oryzae BOD covalently immobilized on multiwall carbon nanotube (MWCNT) electrodes, in the presence of Cl‾ and at different pH, was electrochemically investigated. The results highlight for the first time the influence of chloride ions on the direct oxygen reduction by MoBOD as a function of pH.