Observation of Hybridization and Dehybridization of Thiol-Tethered DNA Using Two-Color Surface Plasmon Resonance Spectroscopy

In situ detection of hybridization can be through changes in mass changes in the optical properties, or changes in the electrochemical properties of the interface. Many of these methods require prior labeling of the DNA target or probe with special isotopes, fluorescence markers, or redox-active tags. Surface plasmon resonance (SPR) spectroscopy is known to be sensitive to the presence of unlabeled DNA at an interface. We report here the first quantitative in situ SPR study of the hybridization and dehybridization of a tethered unlabeled DNA film on a passivated gold surface. For the passivated gold surfaces used in these studies, no nonspecific adsorption of ss-DNA is observed, whereas a bare or partially covered gold surface will readily adsorb ss-DNA. For these studies, we use a novel two-color SPR method that allows us to quantify the number of ss-DNA molecules per unit area for tethered DNA films. These two-component films containing a thiol-derivatized ss-DNA molecule and a diluent thiol, mercaptohexanol (HS(CH sub(2)CH sub(2)) sub(3)OH), were prepared using molecular self-assembly techniques developed by Herne and Tarlov. In these films, the 25 base oligomer is tethered to the gold surface via an alkanethiol covalently linked at the 5' position of the ss-DNA. The mercaptohexanol serves to prevent nonspecific adsorption of ss-DNA. Using in situ SPR, we monitored the kinetics of hybridization for these films, determined the total number and percentage of active binding sites, and measured the hybridization activity of the film through five hybridization--dehybridization (melting) cycles.