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.