Cyclic voltammetry, square wave voltammetry or electrochemical impedance spectroscopy? Interrogating electrochemical approaches for the determination of electron transfer rates of immobilized redox proteins.

In this article, we cross-examine three well-established electrochemical approaches, namely cyclic voltammetry (CV), cyclic square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) to dissect the electron transfer (ET) rate of electrostatically immobilized cytochrome c on Ag electrodes. A detailed analysis supported by simulations of redox transition provided three distinct values for the heterogeneous electron transfer (HET) rate constant of cyt c interfaced on COOH-terminated C-long alkanethiol, i.e., k= 47.8 (±2,91) in CV, k= 64.8 (±1,27) in SWV, and k= 26.5 in EIS. We discuss the obtained discrepancies obtained from electrochemical methods and compare them with the data from spectro-electrochemical experiments. A comprehensive selection list is created from which the most applicable approach can be chosen for studying proteins of interest. CV is most applicable to study the interfaced proteins exhibiting k of ca. 0.5 - 70 , SWV is suitable for a broader range of k of 5 - 120 and EIS for k of 0.5 to 5 if alkanethiols are used as immobilization strategy.