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

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) mu mol/L and (0.069 +/- 0.001) s(-1), respectively, in 50 mmol/L PBS at pH 7.0. The catalytic efficiency of AP was evaluated to be (0.028 +/- 0.005) mu mol/L(-1)s(-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 (E degrees') 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 s(-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.