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We have characterized thiol-derivatized, single-stranded DNA (5‘-HS-(CH₂)₆-CAC GAC GTT GTA AAA CGA CGG CCA G-3‘, abbreviated HS-ssDNA) attached to gold via a sulfur−gold linkage using X-ray photoelectron spectroscopy (XPS), ellipsometry, and ³²P-radiolabeling experiments. We found that hybridization of surface-bound HS-ssDNA is dependent on surface coverage. The buffer concentration of the HS-ssDNA solution was found to have a profound effect on surface coverage, with adsorption greatly reduced at low salt concentration. More precise control over surface coverage was achieved by creating mixed monolayers of the thiol-derivatized probe and a spacer thiol, mercaptohexanol (MCH), by way of a two-step method, where first the gold substrate is exposed to a micromolar solution of HS-ssDNA, followed by exposure to a millimolar solution of MCH. A primary advantage of using this two-step …