Studies on Controlled Protein Folding versus Direct Electron-Transfer Reaction of Cytochrome C on MWCNT/Nafion Modified Electrode Surface and Its Selective Bioelectrocatalytic H2O2 Reduction and Sensing Function

A systematic study on the relationship between direct electron-transfer (DET) function and the folding effect of the Cytochrome c (Cytc) protein has been performed. The aging effect of the Cytc protein-pH 7 phosphate buffer solution (PBS) at a fixed temperature of 5 °C was investigated using spectroscopic and electrochemical techniques. A strong correlation between the aging and electron-transfer (ET) function activity of the Cytc protein was observed. UV-Vis spectroscopic characterization of the aged Cytc protein showed a significant shift in the absorption signal from 405 nm to 413 nm due to the geometric transformation of the heme complex from rhombohedral to tetrahedral symmetry. Similarly, the FTIR and fluorescence spectroscopic results reveal cleavage of the amide-I and II bonds and opening of tryptophan amino-acid residues after aging up to 18 days. These results are similar to the literature reports on denaturants assisted by folding and unfolding behaviors of Cytc protein. A Cytc-pH 7 PBS aged over 18 days was chemically modified as a bioelectrode by successive drop casting of MWCNT-ethanol suspension (1st layer), Cytc-pH PBS (2nd layer), and 1% Nafion (Nf) solution (3rd layer) on GCE (GCE/MWCNT@Cytc-Nf), and the DET activity was studied. The protein electrode showed a highly stable and well-defined redox peak at E 0′ = -0.31±0.02 V vs Ag/AgCl and a Cytc active surface excess of 29.05 nmol cm-2 in N2-purged pH 7 PBS. The redox peak is found to be a surface-confined and proton-coupled ET reaction in characteristic. Furthermore, the GCE/MWCNT@Cytc-Nf system showed a highly stable and efficient response for the bioelectrocatalytic reduction of H2O2 demonstrated by cyclic voltammetry (CV) and rotating disc electrode (RDE) techniques in N2-purged pH 7 PBS. Bio-electrochemical sensing of H2O2 was tested by amperometric i-t and flow injection analysis (FIA) methods showing very stable sensing currents with a detection limit value of 1.6 μM. The following three factors are key for the successful development of an ET active Cytc-bioelectrode system: (i) Proper unfolding; (ii) a cooperative π-π, ionic, covalent, hydrophobic, hydrophilic, and hydrogen bondings with an underlying electrode; and (iii) proper orientation of the protein. © 2019 American Chemical Society. All rights reserved.