A glucose biosensor based on glucose oxidase fused to a carbohydrate binding module family 2 tag that specifically...

The method of immobilization of glucose oxidase (GOD) on electrodes is especially important for the fabrication and performance of glucose biosensors. In this study, a carbohydrate binding module family 2 (CBM2) was successfully fused to the C terminal of GOD with a natural linker (NL) in endo-β-xylanase by genetic recombination, and a fusion GOD (GOD-NL-CBM2) was obtained. The CBM2 was used as an affinity adsorption tag for immobilization of the GOD-NL-CBM2 on a cellulose modified electrode. The specific activity of GOD-NL-CBM2 was comparable to that of the wild type GOD. In addition, the CBM2 tag of fusion GOD almost maintained its highest binding capacity under optimal catalytic conditions (pH 5.0, 50 °C). The morphology and composition analysis of the cellulose film reacted with and without GOD or GOD-NL-CBM2 confirmed the immobilization of GOD-NL-CBM2. The electrochemical properties of the GOD-NL-CBM2/cellulose film bioelectrode, with a characteristic peak of H₂O₂ at +0.6 V in the presence of glucose, revealed the capability of the immobilized GOD-NL-CBM2 to efficiently catalyze glucose and produce H₂O₂. Additionally, the current signal response of the biosensor to glucose was linear in the concentration range from 1.25 to 40 mM (r² ≥ 0.99). The sensitivity and detection limit of the GOD-NL-CBM2/cellulose film bioelectrode were 466.7 μA mol^-1 L cm^-2 and 0.475 mM (S/N = 3), respectively. Moreover, the glucose biosensor exhibited a rapid current change (< 5 s), high reproducibility (Relative standard deviation, RSD < 5%), substrate selectivity and stability, and retained about 80 % of the original current response after 2 months. The affinity adsorption-based immobilization strategy for GOD provides a promising approach to develop a high performance glucose biosensor.