Multilayered construction of glucose oxidase on gold electrodes based on layer-by-layer covalent attachment

A feasible approach to construct multilayered enzyme film on the gold electrode surface for use as biosensing interface is described. The film was fabricated by alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamine) (PAA). The covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy (EIS). X-ray diffraction (XRD) experiments revealed that the film was homogeneous and formed in an ordered manner with a thickness of 2.6 ± 0.1 nm per bilayer. The gold electrodes modified with the GOx/PAA multilayers showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayers. This suggests that the analytical performance such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayers. The six GOx/PAA bilayer electrode exhibited a sensitivity of 15.1 μA mM⁻¹ cm⁻² with a detection limit of 3.8 × 10⁻⁶ M. In addition, the sensor exhibited good reproducibility and stability.