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This thesis presents the development and redesign of a commercial electrochemical quartz crystal microbalance with dissipation (E–QCM–D). This was used to study factors affecting the efficiency of the four electron reduction catalysed by the fuel cell enzyme bilirubin oxidase from Myrothecium verrucaria immobilised on thiol modified gold surfaces. Within this thesis, the E–QCM–D was used to show that application of a constant potential to bilirubin oxidase adsorbed to thiol-modified gold surfaces causes activity loss that can be attributed to a change in structural arrangement. Varying the load by potential cycling distorts the enzyme by inducing rapid mass loss and denaturation. Attaching the enzyme covalently reduces the mass loss caused by potential cycling but does not mitigate activity loss. Covalent attachment also changes the orientation of the surface bound enzyme as verified by the position of the catalytic wave (related to the overpotential for catalysis) and reactive labelling followed by mass spectrometry analysis. The E–QCM–D was used to show how electrostatic interactions affect enzyme conformation where high pH causes a reduction in both mass loading at the electrode and a reduction in activity. At pH lower than the enzyme isoelectric point, there is a build up of multilayers in a clustered adsorption. When enzyme adsorbs to hydrophobic surfaces there is a rapid denaturation which completely inactivates the enzyme. Changing the surface chemistry from carboxyl groups to hydroxyl and acetamido groups shows that catalysis is shifted to more negative potentials as a result of an enzyme misorientation. Further to this, increasing the chain length of the thiol modifier indicates that an increased distance between surface and enzyme reduces activity, enzyme loading and results in a conformational rearrangement that permits electron transfer over longer distances.

Porous gold electrodes modified with the cyclic disulfide, 6,8-thioctic acid, are characterized and described for use in bioanalysis applications. The structure and composition of both the organic thin film resulting from exposure of the gold surface to thioctic acid as well as the immobilized bimolecular layer are investigated. The application of these porous electrodes to several flow-through and non flow-through applications is discussed. Thioctic acid is shown to polymerize on gold surfaces. The mechanism involves homolytic cleavage of the disulfide bond, yielding two thiyl radicals that form either a gold-thiolate bond, or attack a disulfide bond on a monomer nearby in solution. Such an attack results in free radical polymerization through repeating disulfide bonds. The carboxylic acid tails appear to dimerize via hydrogen bonding giving structural order to the surface in the form of large ring structures. These results provide direct chemical evidence for the cleavage of disulfide bonds upon chemisorption onto gold. Covalent immobilization of Immunoglobulin G on these surfaces was not successful as result of mass transport limitations involved in diffusion of the protein into the polymer, competing hydrolysis reactions or inactivation of the carboxylic acid tail due to dimerization. It is shown that antibody is immobilized by nonspecific binding. Porous gold electrodes prepared by sputter deposition of gold on porous nylon membranes are described for use in a competitive non-separation electrochemical enzyme immunoassay (NEEIA) for small molecules. Detection of the cardiac glycosides, digoxin and digitoxin was performed in buffer and undiluted serum. Detection limits for digoxin in serum were on the order of 0.1 pM, demonstrating the possible clinical utility of the immunoassay. The use of porous gold electrodes sputtered on both porous nylon and porous stainless steel are described for use as enzyme-based biosensors. Immobilized enzymes include alkaline phosphatase, $\beta$-d-galactosidase, glucose and tyrosinase. Tyrosinase modified porous gold electrodes are described for the detection of substituted phenols in both flow-injection analysis as well as following their separation by reversed phase liquid chromatography. The use of these electrodes for flow-injection immunoassay was not entirely successful due to low protein loading and hence inability to continuously regenerate the immunobinding surface.

An experiment has been completed which demonstrated quantum mechanical tunneling of electrons between two gold electrodes separated in vacuum. The tunneling current between the gold electrodes has been measured, for fixed voltages of 0.1 and 0.01 volts, as the electrode spacing was varied from a distance of approximately 2.0 nm down to a point where the electrodes touched. Current-voltage characteristics for fixed electrode spacing in the direct tunneling region have also been measured. Numerical calculations of the tunneling current based on the free-electron model of the electrodes and the barrier, an image-potential reduced barrier, and a WKB approximation for the tunneling probability have been performed and compared with Simmons' theory and with the experimental results. Within experimental error the results indicate that an image potential reduced barrier with the modifications suggested by Lang and Kohn gives a close approximation to the true barrier for metal-vacuum-metal tunneling. For the first time, the work function of the electrodes in a tunneling experiment has been deduced from experimental parameters independent of the tunneling device.