甲烷氧化菌素耦合脂肪酶生物传感器差分脉冲伏安法对Cu2＋的检测

利用甲烷氧化菌素（methanobactin，Mb）可特异性捕获Cu2＋的特点，构建Mb耦合脂肪酶生物传感器，并利用荧光光谱、紫外光谱、傅里叶变换红外光谱以及透射电子扫描电镜对制备成功的固定化脂肪酶（Lipase@AuNPs）结构和形貌进行表征。借助生物传感器监测脂肪酶水解三油酸甘油酯时电信号的响应情况，Cu2＋与Mb特异性接合并在脂肪酶周围产生富集现象，抑制脂肪酶的催化活性，电流强度显著下降，从而实现对Cu2＋的快速定性、超痕量反定量检测。结果表明：采用差分脉冲伏安法测得最优检测体系为底物三油酸甘油酯溶于Tirs-HCl缓冲溶液的质量浓度2 g/100 mL、缓冲液pH 7.5，当Cu2＋浓度为1～100 nmol/L范围内时，传感器电流差值与其线性关系良好，回归方程为y＝0.228x＋0.774 8，R2＝0.995 0，检出限为0.03 nmol/L（RSN＝3）。本研究建立的新型脂肪酶生物传感器检测Cu2＋的方法，具有较高的灵敏度、专一性和稳定性，为实现食品中痕量、超痕量的重金属检测提供一定的参考。

Detection of Cu2+ Using Methanobactin-coupled Lipase Biosensor with Differential Pulse Voltammetry

In this study, a methanobactin (Mb)-coupled lipase biosensor was constructed based on the fact that Mb can specifically capture Cu2+. Lipase immobilized on gold nanoparticles (Lipase@AuNPs) was successfully prepared and its structure and morphology were characterized by fluorescence, ultraviolet-visible (UV-vis) and Fourier transform infrared spectroscopy (FTIR) and scanning transmission electron microscopy (STEM). The biosensor could monitor the response of electrical signals during the hydrolysis of triolein by lipase. Cu2+ specifically combined with Mb and was enriched around lipase, which inhibited the catalytic activity of lipase and significantly reduced the current intensity, thus allowing rapid qualitative and ultra-trace reverse quantitative detection of Cu2+. The results showed that the optimal detection conditions, as determined by differential pulse voltammetry (DPV), were as follows: substrate concentration 2 g/100 mL and buffer pH 7.5. The sensor current difference had a good linear relationship with Cu2+ concentration in the range of 1–100 nmol/L. The regression equation was as follows: y = 0.228x + 0.774 8, with a correlation coefficient (R2) of 0.995 0, and the detection limit of this method was 0.03 nmol/L (RSN = 3). Therefore, the new lipase biosensor has high sensitivity, specificity and stability, which lays a research foundation for the detection of trace and ultra-trace heavy metals in foods.