Characterization of DNA Probes Immobilized on Gold Surfaces

TLDR

The authors immobilized thiol‑derivatized single‑stranded DNA on gold and characterized the resulting monolayers with XPS, ellipsometry, and 32P labeling, then refined surface coverage by a two‑step deposition of HS‑ssDNA followed by mercaptohexanol to form mixed monolayers. Hybridization of the surface‑bound probes was shown to depend on surface coverage and buffer salt concentration, with the two‑step HS‑ssDNA/MCH method yielding largely nonspecific‑DNA‑free, stable, and reversible monolayers that discriminate complementary from noncomplementary targets.

Abstract

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 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 oligonucleotides and are able to discriminate between complementary and noncomplementary target molecules. Moreover, the probe-modified surfaces were found to be stable, and hybridization reactions were found to be completely reversible and specific in a series of experiments where duplex melting was examined.