Abstract: | A rational strategy for the construction of a bioelectrocatalytic architecture by means of alternate electrostatic adsorption is described. Multilayer films containing glucose oxidase (GOx) and different polyelectrolytes were assembled onto a thiolated‐gold surface and the resulting bioelectrode was used for glucose biosensing. The supramolecular multistructure was prepared by assembling polyethylenimine and Nafion (as anti‐interference barrier), followed by the adsorption of polyethylenimine and DNA (as stabilizing film) and finally by the alternate deposition of polyethylenimine and glucose oxidase (as a biocatalytic layer). The influence of the deposition time and concentration of polyelectrolytes, organization and number of layers on the sensitivity and selectivity of the bioelectrode is discussed. The resulting enzymatic biorecognition layer exhibits very good analytical performance with a fast, sensitive (3.3±0.1)×104 nA M?1 and highly selective (0% interference for 6.0 mg % uric acid and 2.0×10?4 M ascorbic acid) response to glucose, demonstrating that the alternate electrostatic adsorption of conveniently selected polyelectrolytes allow a large improvement in the selectivity and sensitivity of a biosensor. |