No abstract

The thermal stability of thiol based DNA SAMs prepared on gold surfaces is an important parameter that is correlated to sensor lifetime. The thermal stability of DNA SAMs was evaluated in aqueous buffer through the use of fluorophore labeled DNA, a single crystal gold bead electrode, and microscopy. The stability of different crystallographic regions on the electrode was studied for thermal treatments up to 95 °C for 90 min. Using a in situ combinatorial surface analytical measurement showed that the crystallography of the underlying gold surface played a significant role, with the square or rectangular lattices (e.g., 110, 100, 210) having the highest stability. Surfaces with hexagonal lattices (e.g., 111, 311, 211) were less stable toward thermal treatments. These crystallographic trends were observed for both high and low coverage DNA SAMs. High coverage DNA SAMs were the most stable, with stability decreasing with decreasing coverage on average. Increasing DNA SAM coverage appears to slow the kinetics of thermal desorption, but the coordination to the underlying surface determined their relative stability. Preparing the DNA SAMs under nominally similar conditions were found to create surfaces that were similar at room temperature, but had significantly different thermal stability. Optimal DNA sensing with these surfaces most often requires low coverage DNA SAMs which results in poor thermal stability, which is predictive of a poor shelf life, making optimization of both parameters challenging. Furthermore, the crystallographically specific results should be taken into account when studying the typically used polycrystalline substrates since the underlying surface crystallography maybe different for different samples. It appears that preparing DNA SAMs with low coverage and significant thermal stability will be challenging using the current SAM preparation procedures.

Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor development. The thermal stability of low-coverage DNA SAMs was studied for surfaces prepared using potential-assisted thiol exchange (E dep ) and compared to DNA SAMs prepared without control over the substrate potential (OCP dep ). Both surface preparation methods were studied using in situ fluorescence microscopy and electrochemistry with fluorophore or redox-modified DNA SAMs on a single-crystal gold bead electrode. Fluorescence microscopy showed that the influence of the underlying surface crystallography was important in both cases. The highest thermal stability was realized for square or rectangular surface atomic structure (e.g., surfaces from 110 to 100). The 111 and related surfaces were the least thermally stable. The low DNA coverage surfaces prepared by E dep had better thermal stability and higher DNA probe mobility as compared to OCP dep -prepared surfaces with the similar coverage. These results were correlated with methylene blue redox-tagged DNA probes, which directly measured the average DNA coverage. Both methods indicated that E dep DNA SAMs were more uniformly distributed across the electrode surface, while the surfaces prepared via OCP dep assembled into clusters with reduced mobility. The potentialassisted thiol-exchange approach to preparing low-coverage DNA SAMs was shown to quickly create modified surfaces that were consistent, had mobility characteristics which should yield superior DNA hybridization efficiencies, and having greater thermal stability which will translate into a longer shelf-life.

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.