Single-molecule electrochemical imaging resolves the midpoint potentials of individual fluorophores on nanoporous antimony-doped tin oxide.

We report reversible switching of oxazine, cyanine, and rhodamine dyes by a nanoporous antimony-doped tin oxide electrode that enables single-molecule (SM) imaging of electrochemical activity. Since the emissive state of each fluorophore is modulated by electrochemical potential, the number of emitting single molecules follows a sigmoid function during a potential scan, and we thus optically determine the formal redox potential of each dye. We find that the presence of redox mediators (phenazine methosulfate and riboflavin) functions as an electrochemical switch on each dye's emissive state and observe significantly altered electrochemical potential and kinetics. We are therefore able to measure optically how redox mediators and the solid-state electrode modulate the redox state of fluorescent molecules, which follows an electrocatalytic (EC') mechanism, with SM sensitivity over a 900 μm field of view. Our observations indicate that redox mediator-assisted SM electrochemical imaging (SMEC) could be potentially used to sense any electroactive species. Combined with SM blinking and localization microscopy, SMEC imaging promises to resolve the nanoscale spatial distributions of redox species and their redox states, as well as the electron transfer kinetics of electroactive species in various bioelectrochemical processes.