Glassy carbon and graphite electrodes modified with films of enzyme and osmium redox polymer, cross linked with poly (ethylene glycol) diglycidyl ether, were used for elaboration of a glucose/O-2 enzymatic fuel cell. The redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+) and [Os(4,4'-dichloro-2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+) were selected to facilitate transfer of electrons from the glucose oxidase (GOx) active site to the T1 Cu site of multicopper oxygenases of Trametes hirsuta laccase (ThLacc) and Myrothecium verrucaria bilirubin oxidase (MvBOD). Maximum power density at pH 5.5 of 3.5 mu W cm(-2) at a cell voltage of 0.35 V was obtained for an assembled membrane-less fuel cell based on ThLacc on glassy carbon as cathode, in the presence of 0.1 M glucose, 37 degrees C, with lower power observed at pH 7.4 and 4.5. Replacement of the ThLacc cathode with that of MyBOD produced 10 mu W cm(-2) at 0.25 V under pseudo-physiological conditions. Replacement of glassy carbon with graphite as base electrode material resulted in increased redox polymer loading, leading to an increase in power output to 43 mu W cm(-2) at 0.25 V under similar conditions. Improved stabilization of biofilms was achieved through covalent anchoring of enzyme and redox polymer on graphite electrodes, derivatized via electrochemical reduction of the diazonium cation generated in situ from p-phenylenediamine. Enzymatic fuel cells using this approach retained 70% power at 24 h, whereas fuel cells prepared without chemical anchoring to graphite retained only 10% of power over the same interval. (C) 2011 Elsevier B.V. All rights reserved.