Electrochemical scanning tunneling microscopy as a tool for probing protein function: from electron transfer to molecular recognition and chemosensation

Electron transfer (ET) is involved in many fundamental biological processes. While spectroscopic and electrochemical techniques have established the theoretical framework governing protein ET, they typically capture average responses across large populations or characterize ET in fixed molecular arrangements. Electrochemical scanning tunneling microscopy (EC-STM) has emerged as a unique single-molecule technique that combines atomic-scale spatial resolution with electrochemical control, enabling direct investigation of charge transport in proteins within a dynamic framework. Initially focused on redox proteins, EC-STM has evolved from imaging to spectroscopic measurements, including current-distance, current-voltage, and break-junction approaches, revealing electrochemically regulated transport pathways and dynamic fluctuations. More recently, EC-STM has been extended to non-redox proteins and to the study of charge transport in increasingly complex biological systems, such as inter-protein ET and molecular recognition. These studies have demonstrated that ligand binding can modulate the electrical response of proteins, providing a direct readout of the conformational and electronic changes associated with biological recognition events. These observations reinforce EC-STM as a powerful approach for studying protein function beyond classical ET systems, bridging the gap between nanoscale biophysics and molecular recognition. This mini review traces the evolution of EC-STM applied to protein ET from its origins in intraprotein ET of redox proteins to its development into a versatile tool for studying protein function through molecular recognition. We analyze how EC-STM has provided new insights into ET mechanisms, protein-protein interactions, and ligand-induced electrical responses, and we conclude by considering its potential for the development of protein-based biosensors and bioelectronic technologies.