Nanoporous gold electrodes as matrices for enzyme immobilization for application in biosensors and biofuel cells

Robust nanoporous gold electrodes were fabricated by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloys were prepared with either a non-uniform or uniform distribution of silver alloy which showed clear differences in morphology on characterization with scanning electron microscopy. The surface area of these electrodes was up to 28 times that of the geometric surface area. The surface area accessible to modification by redox proteins was determined using cyt c as a model system. Covalent immobilization of cyt c at SAMs modified planar and nanoporous gold electrodes resulted in ca. 9 and 11 times higher surface coverages at uniform and non-uniform nanoporous gold, respectively, than at planar gold electrodes. Well defined mediatorless bioelecytrocatalytic reduction of oxygen was obtained on nanoporous gold electrodes prepared using a vacuum method and subsequently modified with Myrthecium verrucaria bilirubin oxidase (MvBOD). Diffusion limited faradaic response, with current densities of 0.8 mA/cm2, was observed when the enzyme modified electrode was stabilized with a layer the P017-epoxy polymer. The enzyme, Trametes hirsuta laccase (ThLc) also displayes direct electron transfer at unmodified nanoporous gold electrodes. The observed current densities of ca. 0.03 mA/cm2 were 10 times higher than the current densities at the ThLc modified electrode made by drop-casting and are in contrast to the absence of a response at unmodified polycrystalline gold electrodes. Nanoporous and planar gold electrodes modified with Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH) together with Os redox mediators and PEGDGE as a cross-linking agent resulted in glucose and lactose detecting biosensors. The sensors had (Imax,app, KM,app, sensitivity), limits of detection (LOD). GOx and CtCDH modified electrodes were utilized as anodes with MvBOD and Melanocarpus albomyces Lc (rMaLc) modified cathodes in biofuel cells. A maximal power density of 41 μA/cm2 for the CtCDH/MvBOD biofuel cell in 5 mM lactose and O2 saturated buffer was obtained. The power densities of the biofuel cells were also evaluated in artificial plasma, where decreased values were observed.