The structural and electrochemical characteristics of electrodeposited graphene oxide (GO) onto a single crystal gold bead electrode was studied using AFM and Cu electrodeposition. Optimal deposition uniformity was realized by rotation of the electrode, suggesting an initial electrophoretic deposition occurs followed by some reduction of the GO. Potential pulse treatments to reducing potentials for a short time significantly improve the electrochemical characteristics of the electrochemical reduced graphene oxide (ERGO). Both under-potential and over-potential Cu electrodeposition was used to characterize the quality of the ERGO deposit and showed that the gold surface was completely coated with ERGO only after potential pulsing treatments. AFM analysis of ERGO modified Au(111) facet on the gold bead electrode revealed the deposition was composed of sheets of GO/ERGO as well as much rougher deposits. AFM showed that the potential pulsing treatment resulted in the nucleation and growth of electrodeposited Cu nanoparticles over much of the ERGO surface, in contrast to the limited deposition of nanoparticles on the untreated GO deposit. The ERGO modified gold surface prepared using electrodeposition followed by potential pulse treatments results in conductive and electrochemically active ERGO surfaces.

Controlled the formation of monolayer or multilayers of RCs on a gold electrode Correlated electrochemical and photocurrent responses to the RC adsorbed conformations measured using AFM Adsorbed RC conformations affect photocurrent generation Photocurrent-potential response influenced by local redox mediator concentration and pH environment experienced by the adsorbed RCs

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 alkylthiolmodified 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.

Summary The formation of defined surfaces consisting of photosynthetic reaction centers (RCs) in biohybrid solar cells is challenging. Here, we start with the production of engineered RCs for oriented binding. RCs are deposited onto gold electrodes, and 6-mercapto-1-hexanol (MCH) is used to displace multilayers and non-specifically adsorbed RCs. The resulting electrode surfaces are analyzed for photocurrent generation using an intensity-modulated light and lock-in amplifier. Atomic force microscopy (AFM) is used to characterize the surface and the formation of RC structural assemblies. For complete details on the use and execution of this profile, please refer to Jun et al. (2021) .

Mixed DNA SAMs labeled with a fluorophore (either AlexaFluor488 or AlexaFluor647) were prepared on a single crystal gold bead electrode using potential-assisted thiol exchange and studied using Forster resonance energy transfer (FRET). A measure of the local environment of the DNA SAM (e.g., crowding) was possible using FRET imaging on these surfaces since electrodes prepared this way have a range of surface densities (Γ DNA ). The FRET signal was strongly dependent on Γ DNA and on the ratio of AlexaFluor488 to AlexaFluor647 used to make the DNA SAM, which were consistent with a model of FRET in 2D systems. FRET was shown to provide a direct measure of the local DNA SAM arrangement on each crystallographic region of interest providing a direct assessment of the probe environment and its influence on the rate of hybridization. The kinetics of duplex formation for these DNA SAMs was also studied using FRET imaging over a range of coverages and DNA SAM compositions. Hybridization of the surface-bound DNA increased the average distance between the fluorophore label and the gold electrode surface and decreased the distance between the donor (D) and acceptor (A), both of which result in an increase in FRET intensity. This increase in FRET was modeled using a second order Langmuir adsorption rate equation, reflecting the fact that both D and A labeled DNA are required to become hybridized to observe a FRET signal. The self-consistent analysis of the hybridization rates on low and high coverage regions on the same electrode showed that the low coverage regions achieved full hybridization 5× faster than the higher coverage regions, approaching rates typically found in solution. The relative increase in FRET intensity from each region of interest was controlled by manipulating the donor to acceptor composition of the DNA SAM without changing the rate of hybridization. The FRET response can be optimized by controlling the coverage and the composition of the DNA SAM sensor surface and could be further improved with the use of a FRET pair with a larger (e.g., > 5 nm) Forster radius.