Electrochemical atomic force microscopy reveals potential stimulated height changes of redox responsive Cu-azurin on gold

The redox active metalloprotein Cu-azurin was directly chemisorbed on bare gold electrodes through disulfide forming groups (Cys3Cys26). Topological and electrochemical properties of the immobilized molecules were investigated by electrochemical atomic force microscopy (EC-AFM) in Peak Force Tapping mode and cyclic voltammetry (CV). Cu-azurin layers showed surface confined reversible electrochemical behavior. In-situ EC-AFM studies revealed a height difference of 0.32 nm between the oxidized (+0.45 V) and the reduced state (−0.10 V) for Cu-azurin by imaging single molecules independent of the ionic strength of the buffer solution. In contrast, no height change was detected for Zn-azurin which is non-redox active and served as control. The observed height changes of the Cu-azurin upon electrochemical redox switching are thought to originate from conformational changes of the protein and the variation in the orientation of immobilized proteins between the oxidized and reduced states. In this manner, the height of the Cu-azurin could be modulated reversibly by the applied potential.