Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting
DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand
DNA SAM was prepared using potential-assisted
thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a
methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal
gold bead
electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of
MB, (2) the surface density of
DNA, (3) the MB:AlexFluor488 ratio in the
DNA SAM, and (4) the local environment around the
DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of
DNA SAMs with its unlabeled
complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient
DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized
DNA SAM for surfaces with optimal
DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.