Herein, deoxyribonucleic acid (DNA)/multi-walled carbon nanotube (MWCNTs) with enhanced negative charged density was used as a novel electrochemical platform for oriented immobilization of bilirubin oxidase. The proposed support improved the direct electron transfer kinetics of BOD and its catalytic activity toward oxygen reduction reaction (ORR). In comparison to BOD enzyme which immobilized directly onto MWCNTs the current density increased three folds and reached to 270 μA cm−2 at 0.405 V with an onset potential of 0.57 V (vs. Ag/AgCl). The ability of this modified electrode as a biocathode is investigated after assembling with bioanode. The bioanode prepared with covalent attachment of glucose dehydrogenase enzyme (GDH) and nile blue (NB) as an efficient mediator for coenzyme regeneration onto glassy carbon electrode modified with amino-carbon nanotubes(MWCNTs-NH2) and carboxyl terminated polyamidoamin dendrimer (PAMAM-Den) as a multifunctional linker. Finally, the performance of one-compartment glucose/O2 biofuel cell without separators is also investigated. The open circuit voltage of the cell and maximum current density are obtained 660 mV and 172 μA cm−2, respectively, while the maximum power density of 45 μW cm−2 is achieved at 428 mV of the cell voltage in buffer solution saturated with O2 and containing 50 mM of glucose. The stability of the constructed EBFC is investigated under continuous operation at maximum power. It is observed that the biofuel cell can retain more than 85% of its power and potential performance after 24 h.
