光谱和电化学方法研究自组装纳米簇模拟过氧化物酶

Investigation of Self-assembled Nano-cluster Artificial Peroxidase by Voltammetry and Spectroscopy

利用细胞色素c与Nafion,通过自组装方式,构建了纳米簇模拟过氧化物酶;利用电子透射显微镜、圆二色谱仪、紫外可见光分光光度仪及电化学工作站等设备测量模拟酶结构变化、酶促动力学参数以及电化学特性;在50 mmol/L Na2HPO4-NaH2PO4缓冲溶液(pH 10.0)中,测得模拟酶米氏常数Km、催化速率及催化效率分别为2.5μmol/L、0.069 s"1、0.028±0.005(μmol/L)"1s"1,其催化效率为天然辣根过氧化物酶的39%±5%,该模拟酶具有很好的稳定性和活性,能够替代辣根过氧化物酶。该模拟酶可以固定在功能化多壁纳米管与纳米金复合材料修饰玻碳电极上,在50 mmol/L Na2HPO4-NaH2PO4缓冲溶液(pH 7.0)中测得模拟酶与电极之间电子迁移速度常数ks为0.65 s"1。利用循环伏安法测量模拟酶催化浓度为0.02～10 nmol/L的H2O2,其阴极峰电流随H2O2浓度增加而增大,并且得出模拟酶催化H2O2检出限为0.05 nmol/L,电化学表观米氏常数Kmapp为2.3×10"10mol/L。

A nano-cluster with highly efficient peroxide activity was constructed self-assembly based on nafion（NF） and cytochrome c（Cyt c）,The UV-Vis spectrometry,Circular Dichroism（CD） and transmission electron microscopic（TEM） methods were utilized for additional characterization of the artificial peroxidase（AP）.The nano-cluster was composed of chain-ball structure,with an average ball size of approximately 40 nm detected by TEM method.The Michaelis–Menten（Km） and catalytic rate（kcat） constants of the AP were determined to be（2.5±0.4） μmol/L and（0.069±0.001） -1,respectively,in 50 mmol/L PBS at pH 7.0.The catalytic efficiency of AP was evaluated to be（0.028±0.005） μmol/Ls-1,which was 39%±5% as efficient as the native HRP.AP generated here can be used in place of HRP with high stability and activity.The AP was also immobilized on a functional MWNCTs-gold nano-complex modified glassy carbon electrode.The cyclic voltammetry of AP on the nano complex modified glassy carbon electrode showed a pair of well-defined quasi-reversible redox peaks with the formal potential of（Eo’） of（-45±2） mV（vs.Ag/AgCl） at scan rate of 0.05 V/s.The heterogeneous electron transfer rate constant was evaluated to be 0.65 -1 in 50 mmol/L Na2HPO4-NaH2PO4 buffer solution at pH 7.0.The cathodic peak current of the modified electrode increased with increasing concentration of hydrogen peroxide（from 0.02 to 10 nmol/L） with a detection limit of 0.05 nmol/L.The apparent Michaelis-Menten constant（Kmapp） was 0.23 nmol/L.