Immobilization of Redox Enzymes on Nanoporous Gold Electrodes: Applications in Biofuel Cells

Nanoporous gold (NPG) electrodes were prepared by dealloying sputtered gold:silver alloys. Electrodes of different thicknesses and pore sizes areas were prepared by varying the temperature and duration of the dealloying procedure; these were then used as supports for FAD-dependent glucose dehydrogenase (GDH) (Glomorella cingulata) and bilirubin oxidase (BOx) (Myrothecium verrucaria). Glucose dehydrogenase was immobilized by drop-casting a solution of the enzyme with an osmium redox polymer together with a crosslinked polymer, whereas bilirubin oxidase was attached covalently through carbodiimide coupling to a diazonium-modified NPG electrode. The stability of the bilirubin-oxidase-modified NPG electrode was significantly improved in comparison with that of a planar gold electrode. Enzyme fuel cells were also prepared; the optimal response was obtained with a BOx-modified NPG cathode (500 nm thickness) and a GDH-modified anode (300 nm), which generated power densities of 17.5 and 7.0 μW cm⁻² in phosphate-buffered saline and artificial serum, respectively.