Abstract:
This thesis describes the linking of enzymes to electrodes and their application in biofuel cells and as analytical devices.
Methanol dehydrogenase, an NAD independent enzyme was purified by two phase aqueous partition. The enzyme incorporated into a biofuel cell was capable of producing a current in the presence of either a soluble or insoluble mediator. Optimisation of the current was carried out and a variety of alternative membranes, mediators and electrodes were investigated for possible use in the biofuel cell. Although laboratory studies involved the use of platinum electrodes and the soluble mediator N,N,N',N' tetramethyl-p-phenylenediamine, other configurations were investigated, including modified membranes and electrodes. The ability to detect methanol coulometrically in solution led to the construction of a homogeneous poised potential sensor. The device was capable of detecting methanol at concentrations in solution as low as 0.02uM. the sensor was unaffected by many potential interfering compounds present in water supplies and was used in the analysis of water samples. The retention of the enzyme in the presence of the insoluble mediator 1,1'-dimethylferrocene allowed the construction of a probe. The probe was unaffected by fluctuations in the oxygen tension and was capable of detecting methanol in the range 0.9 uM to 0.1 mM.
Covalent immobilisation of glucose oxidase in the presence of 1,1'-dimethylferrocene enabled the amperometric determination of glucose in the range 0.01 - 30 mM. The device was independent of pH in the normal physiological range with a temperature coefficient of 4.0% degrees-1. The electrode was used to assay both buffered glucose samples and preliminary work was carried out on untreated samples from diabetic and non-diabetic patients.
