Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy

The redox metalloprotein azurin self-chemisorbed onto Au(111) substrates has been investigated by electrochemically controlled scanning tunneling (STM) and scanning force/lateral force microscopy (SFM/LFM) and cyclic voltammetry (CV) in aqueous solution. The combined use of STM and SFM/LFM under electrochemical control in the negative side of the azurin redox midpoint (+116 mV vs. SCE) has delivered unique information on the nature of the STM images. While in STM the bright spots, believed to be associated with azurin molecules, are visible only for potential values higher than -125 mV, the concurrent electrochemical SFM results show adsorbed proteins over the whole potential range investigated (from -225 to +75 mV). Stepping the potential back and forth (between -25 and -125 mV) in STM imaging, it has been possible to make bright spots appearing and disappearing repeatedly, indicating that STM image formation arises possibly through resonant tunneling via the redox levels of azurin. These results represent the first clear evidence of potential-dependent tunneling in proteins adsorbed onto a conductive substrate.