Nanostructured direct electron transfer based biocathodes for applications in biofuel cells

Resumen

During the last years biofuel fuel cells (BFCs) have attracted great interest due to their possible applications, especially as electrical power sources for in vivo or ex vivo applications. In BFCs enzymes can be used as biocatalysts for fuel oxidation at the anode and oxidant reduction at the cathode. The majority of EFCs use oxygen-reducing enzymes at the cathode, and glucose-oxidizing enzymes at the anode, as they are very common substrates present in most human physiological fluids. Two multi-copper oxidases, laccase and bilirubin oxidase, and cellobiose dehydrogenase have been studied as possible biocatalysts for the oxygen reduction reaction and glucose oxidation, respectively. Laccases usually exhibit higher activity at acid pH and they are more inhibited in the presence of chloride ions than bilirubin oxidase. Therefore, native laccases have been engineered by directed evolution for obtaining mutants that show activity also under physiological conditions, and cysteine residues have been introduced by site-directed mutagenesis for oriented immobilization on gold electrodes. The major aim of the Thesis has been the development of biocathodes as they represent the rate-limiting part of the BFC due to the low O2 availability in human body. The development of the bioelectrodes was carried out paying special attention to the different electrode materials and immobilization strategies used to manufacture the biodevices. Indeed, a good immobilization strategy enhances the long-term stability of the biodevice while achieving efficient wiring of the enzyme. Additionally, a larger surface area of the support material allows higher enzyme loading, therefore increasing the current density developed. Gold nanorods, macroporous gold, indium tin oxide and carbonaceous materials have been used for this purpose, obtaining current densities up to 1.5 mA/cm2 for bioelectrocatalytic O2 reduction. Direct electron transfer (DET) based systems are preferred as some possible drawbacks of using mediators are overcome and allow making the miniaturization of the BFC easier. For these reason, all the immobilization strategies presented were developed in order to optimize DET between the enzyme and the electrode surface. Combination of a conventional BFC with electrochemical capacitors is also presented in order to overcome the limitations of both systems, achieving a maximum power output of 0.6 µW at an operating voltage of 0.15 V. This hybrid biodevice was also tested in ex vivo conditions by connecting it directly to the dorsal venous of a human volunteer.