Sensing the Concentration of Biological Reductants through Single-Molecule Fluorescence Blinking

Fluorescence blinking is a photophysical phenomenon where a fluorophore undergoes transitions between the dark state and the fluorescent state. The addition of reductants and oxidants can induce the fluctuation of emitted photons via nonfluorescent radical ion species of fluorophores, which are formed by redox reactions. Fluorescence blinking based on redox reactions has been harnessed to sense the microenvironments surrounding the fluorophores. Here, we report a fluorescence blinking system based on redox reactions to sense the concentrations of the reductants. The excess of coexisting oxidant p-nitrophenylphosphate can generate nonfluorescent radical cations of fluorophores, and an intermolecular electron transfer reaction with reductants (ascorbic acid and glutathione) regenerates fluorophores in a reductant concentration-dependent manner. The developed fluorescence blinking system using red-emitting fluorescent dyes ATTO 647N and JF₆₄₆, allowed us to detect the concentration of glutathione through single-molecule fluorescence measurements, demonstrating that fluorophore molecules can serve as sensors for the local concentration of reductants.