Optimization and characterization of covalent immobilization of glucose oxidase for bioelectronic devices

•EDC/NHS-mediated immobilization of glucose oxidase was optimized.•Inclusion of Tween-20 in washing buffer was effective in removing physically adsorbed GOx.•Covalently bound glucose oxidase amounted to 3.3±0.3mg-GOx/mg-MWCNTs.•Electrode with maximum amount of enzyme resulted in massive enhancement of current density. Enzyme electrodes are widely applied to miniature implantable bioelectronic devices such as biofuel cells and biosensors. The main obstacle associated with miniaturization is the reduced surface area of electrodes for the accommodation of enzymes, leading to poor power output or detection signals. This study aimed to maximize the loading of glucose oxidase (GOx) on the surface of multi-walled carbon nanotubes (MWCNTs), thereby enhancing the generation of electric power or sensing signals. Because the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and glucose oxidase significantly affected the immobilization efficiency, these factors were optimized by the Box–Behnken design. The physically adsorbed enzyme was almost completely removed by washing the GOx-bound MWCNTs with buffer solution containing 5g/L of Tween-20. Enzyme loading was found to be ∼3.3±0.3 mg-GOx/mg-MWCNTs under the optimal conditions (430mM NHS, 52mM EDC and 8.7mg/mL GOx). The formation of carboxyl group on the surface of MWCNTs and the covalent bonding between GOx and MWCNTs, and immobilized GOx were observed by FTIR and AFM, respectively. The biochemical analysis showed that the immobilized GOx possesses high activity for the conversion of glucose into gluconic acid. The cyclic voltammetry data showed that the anodic current density of electrodes loaded with the highest amount of GOx was much higher than those of electrodes loaded with smaller amounts of GOx.