(376e) Surface Diffusion of DNA Oligonucleotides on Patterned Silane Surfaces

In detection modalities that require waveguides, the study of lipid bilayers, and binding events on the surfaces of living cells, detection typically occurs on flat surfaces. Here, dilute analyte concentrations can give rise to large capture times owing to the long distances analytes must travel to reach probes, and the fairly low probability of capture once they reach the probe. To speed the detection of analytes in planar biosensors, we are pursuing the use of patterned surfaces that direct their analytes to their complementary probes by surface diffusion. This reduction of dimensionality (RD) enhancement reduces the distance a molecule must traverse from bulk solution to reach a fixed detection site. We have designed surfaces that possess probe domains surrounded by regions that encourage surface diffusion along the detection surface while allowing the analyte to sample many conformations resulting in higher capture probabilities. Surfaces were constructed from patterned self-assembled monolayers (SAMs) of the positively charged silane 6-aminohexyl-3-aminopropyl trimethoxy silane (AHTMS). Following adsorption of AHTMS, a photomask with rectangular dark patches was placed over the AHTMS surface and UV irradiated, leaving behind regularly patterned rectangles of AHTMS surrounded by glass. A crosslinking agent was then used to attach DNA probes to the amine terminus of the remaining regions of AHTMS to serve as probe sites. The remaining areas of bare glass are then backfilled with either AHTMS or another silane that will serve as the pathway for surface diffusion. The roughness and thickness of the SAMs were confirmed by X-ray reflectivity studies and AFM yielding expected values. For surface diffusion to occur over reasonable time scales, the adsorbed DNA must be reversibly adsorbed to the pathways. This was assessed using a total internal reflection-fluorescence (TIRF) / fluorescence recovery after patterned photobleaching (FRAPP) apparatus on an inverted microscope stage. Using the TIRF/FRAPP instrument, we have studied adsorption, desorption, and surface diffusion of fluorescently labeled DNA oligonucleotides on patterned AHTMS surfaces under high and low ionic strength conditions. The use of TIRF ensures that only molecular processes occurring very close to the surface are detected. Total adsorbed amounts are measured by the TIRF intensity, while the FRAPP yields surface diffusion coefficients. At all ionic strengths, an initial, fast adsorption was observed followed by a second, slower adsorption proceeding after 100 seconds. This can be explained by two sequential molecular processes, the first driven by electrostatic interactions, and the second driven by hydrophobic interactions or base-stacking. Desorption experiments showed little desorption after a surface pre-adsorbed with DNA in pure buffer was rinsed with DNA-free buffer (50 mM Tris HCl, pH 8.0). However, nearly all of the material on the surface desorbed in low and high ionic strength buffers. This non-monotonic behavior again indicates that two distinct processes are controlling the adsorption. Further, the total adsorption at low and high ionic strengths was higher than in 50 mM Tris HCl. The good agreement of adsorption and desorption data demonstrate that the adsorbed DNA is in equilibrium with the DNA in solution as required for appropriately fast surface diffusion. Accordingly, surface diffusion coefficients measured by TIRF/FRAPP agree well with literature values (Dsurf °Ö 1 x 10-8 cm2/s). In order to identify the conditions necessary for fast analyte capture, we have systematically varied the size of capture regions, the concentration of probes within that region, and the concentration of probes in solution. These data were described within a mass-transfer model accounting for both 2D and 3D diffusion. Implications for the improved design of bioanalytical devices will be discussed.