دانلود رایگان مقاله بهینه سازی بیحرکتی کووالانسی اکسیداز گلوکز

Abstract

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 5 g/L of Tween-20. Enzyme loading was found to be ∼3.3 ± 0.3 mg-GOx/mg-MWCNTs under the optimal conditions (430 mM NHS, 52 mM EDC and 8.7 mg/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.

4. Conclusions

This study demonstrates that GOx can be covalently attached to MWCNTs, without physical adsorption, by EDC/NHS-mediated immobilization. The covalently bound GOx amounted to 3.3 mgGOx/mg-MWCNTs, which is one of the highest achieved values to our knowledge. This was accomplished by optimizing the concentrations of EDC, NHS, and GOx. The Box–Behnken design method was a useful approach to determine the optimal conditions. Washing the immobilized GOx with buffer solution containing Tween-20 was a key step to minimize the physical adsorption of GOx during the immobilization process. The TGA revealed the successful carboxylation of MWCNTs. The FTIR and AFM analyses clearly showed the covalent bonding between GOx and MWCNTs, and GOx covering the entire surface ofMWCNTs. The biochemical analysis showed that the immobilized GOx possesses high activity for the conversion of glucose into gluconic acid. The electrode covered with GOx-MWCNTs obtained under the optimal conditions generated a current density of 148 A/cm2, which is much higher than that generated by the electrode covered with a smaller amount of GOx. This study will contribute to solving problems associated with miniaturization of bio-electronic devices such as biofuel cells or biosensors.