葡萄糖氧化酶在枯草芽孢杆菌芽孢表面的展示及其酶电极制备

Display of glucose oxidase on Bacillus subtilis spore surface and preparation for enzyme electrode

摘要:

葡萄糖生物传感器是目前最为常见的电化学生物传感器，绝大多数葡萄糖生物传感器采用在电极表面修饰葡萄糖氧化酶的方法来制备，但是，在电极的固定化过程中需要酶的纯化，使得成本增加，已成为固定化酶电极开发领域的瓶颈。文中主要以芽孢衣壳蛋白CotX为锚定蛋白将葡萄糖氧化酶 (Glucose oxidase，GOD) 展示到枯草芽孢杆菌芽孢表面，通过Western blotting分析、免疫荧光分析以及酶活检测均证明GOD在芽孢表面有效表达，发酵获得重组芽孢 (Spore-GOD)。再采用滴涂法和电沉积法制备了氧化石墨烯/普鲁士蓝沉积膜修饰玻碳电极，将Spore-GOD固定在修饰电极表面，最后滴加一层Nafion溶液，制成了电化学生物传感器，用于葡萄糖的灵敏测定。葡萄糖在该酶电极传感器上的循环伏安图表明，该反应在0.42 V处出现明显的氧化峰，并且氧化还原峰电流与葡萄糖浓度在0.1–7.0 mmol/L之间具有良好的线性关系，校正曲线方程为：I=1.304 7Cglucose+3.639 (R2=0.992 9)，其检测限为7.5 μmol/L (S/N=3)。此修饰电极具有良好的导电性能、稳定性和重现性，可用于葡萄糖的分析测定。

关键词:枯草芽孢杆菌，表面展示，葡萄糖氧化酶，氧化石墨烯，普鲁士蓝沉积，生物传感器

Abstract:

Glucose biosensor is currently the most common electrochemical biosensor. Most glucose biosensors are prepared by modifying glucose oxidase on the electrode surface. However, in the process of electrode immobilization, enzyme purification is required, which increases the cost and has become a bottleneck in the field of development of immobilized enzyme electrodes. In this study, glucose oxidase (GOD) was displayed on the surface of Bacillus subtilis using the spore capsid protein CotX as an anchor protein. By Western blotting analysis, immunofluorescence analysis and enzyme activity detection, GOD was effectively expressed on the surface of spores, and recombinant spores (Spore-GOD) were obtained by fermentation. The graphene oxide/prussian blue deposition film modified glassy carbon electrode was prepared by the drop coating method and the electrodeposition method. The surface of the modified electrode was fixed with Spore-GOD, and finally covered with a layer of Nafion solution to make an electrochemical biosensor for sensitive determination of glucose. The cyclic voltammogram of glucose on the enzyme electrode sensor showed a well-defined oxidation peak at 0.42 V, and the redox peak current has a good linear relationship with the glucose concentration in the range of 0.1–7.0 mmol/L. The calibration curve equation is: I=1.305Cglucose+3.639 (R2=0.992 9), and its detection limit is 7.5 μmol/L (S/N=3). This modified electrode has good conductivity, stability and reproducibility, and can be used for the analysis and determination of glucose.

Key words:Bacillus subtilis, surface display, glucose oxidase, graphene oxide, Prussian blue deposition, biosensor