Quantitative and reproducible data can be obtained from surface-based DNA sensors if variations in the conformation and surface density of immobilized single-stranded DNA capture probes are minimized. Both the conformation and surface density can be independently and deterministically controlled by taking advantage of the preferential adsorption of adenine nucleotides (dA) on gold, as previously demonstrated using a model system in Opdahl, A.; Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9-14. Here, we describe the immobilization and subsequent hybridization properties of a 15-nucleotide DNA probe sequence that has additional m adenine nucleotides, (dA)(m), at the 5' end. Quantitative analysis of immobilization and hybridization for these probes indicates that the (dA)(m) block preferentially adsorbs on gold, forcing the probe portion of the strand to adopt an upright conformation suited for efficient hybridization. In addition, a wide range of probe-to-probe lateral spacing can be achieved by coimmobilizing the probe DNA with a lateral spacer, a strand of k adenine nucleotides, (dA)(k). Altering either the length or relative concentration of the (dA)(k) spacers added during probe immobilization controls the average surface density of probes; the density of probes, in turn, systematically modulates their hybridization with solution targets.

The structure and stability of single- and double-stranded DNA hybrids immobilized on gold are strongly affected by nucleotide-surface interactions. To systematically analyze the effects of these interactions, a set of model DNA hybrids was prepared in conformations that ranged from end-tethered double-stranded to directly adsorbed single-stranded (hairpins) and characterized by surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The stabilities of these hybrids were evaluated by exposure to a series of stringency rinses in solutions of successively lower ionic strength and by competitive hybridization experiments. In all cases, directly adsorbed DNA hybrids are found to be significantly less stable than either free or end-tethered hybrids. The surface-induced weakening and the associated asymmetry in hybridization responses of the two strands forming hairpin stems are most pronounced for single-stranded hairpins containing blocks of m adenine (A) nucleotides and n thymine (T) nucleotides, which have high and low affinity for gold surfaces, respectively. The results allow a qualitative scale of relative stabilities to be developed for DNA hybrids on surfaces. Additionally, the results suggest a route for selectively weakening portions of immobilized DNA hybrids and for introducing asymmetric hybridization responses by using sequence design to control nucleotide-surface interactions--a strategy that may be used in advanced biosensors and in switches or other active elements in DNA-based nanotechnology.