CHARACTERIZATION OF DNA PROBES IMMOBILIZED ON GOLD SURFACES

We have characterized thiol-derivatized, single-stranded DNA (5'-HS-(C H2)(6)-CAC GAC GTT GTA AAA CGA CGG CCA G-3', abbreviated HS-ssDNA) att ached to gold via a sulfur-gold linkage using X-ray photoelectron spec troscopy (XPS), ellipsometry, and P-32-radiolabeling experiments. We f ound that hybridization of surface-bound HS-ssDNA is dependent on surf ace coverage. The buffer concentration of the HS-ssDNA solution was fo und to have a profound effect on surface coverage, with adsorption gre atly reduced at low salt concentration. More precise control over surf ace coverage was achieved by creating mixed monolayers of the thiol-de rivatized probe and a spacer thiol, mercaptohexanol (MCH), by way of a two-step method, where first the gold substrate is exposed to a micro molar solution of HS-ssDNA, followed by exposure to a millimolar solut ion of MCH. A primary advantage of using this two-step process to form HS-ssDNA/MCH mixed monolayers is that nonspecifically adsorbed DNA is largely removed from the surface. Thus, the majority of surface-bound probes are accessible for specific hybridization with complementary o ligonucleotides and are able to discriminate between complementary and noncomplementary target molecules. Moreover, the probe-modified surfa ces were found to be stable, and hybridization reactions were found to be completely reversible and specific in a series of experiments wher e duplex melting was examined.