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Using surface plasmon resonance (SPR) spectroscopy in the Kretschmann configuration, we followed the self-assembly of organic ultrathin films which resulted from exposure of gold surfaces to solutions of CH3(CH2) n - 1SH (n = 8, 12, 16, and 18) in ethanol and heptane. We monitored film growth in situ and continuously for up to 72 h with an overall thickness resolution of <1 Å. Film dielectric constants, necessary for accurately calculating average film thicknesses from SPR spectra, were determined unambiguously for fully formed films by comparing spectra from organic films in different solvents. In addition, we introduce a novel two-color SPR experiment with which we can obtain both film thickness and film dielectric constant without changing solvents. We studied the chain length dependent and concentration dependent kinetics of film formation in ethanol and found that there are at least three distinct kinetics steps. The kinetics of the first, most rapid, step and the third, slowest, step can be described well with Langmuir adsorption models. The kinetics of the second step are zeroth order and depend on alkanethiol chain length, concentration, and partial film thickness. The formation kinetics in heptane can be described with a single step Langmuir adsorption model. We find that films formed by continuous self-assembly in solution are always thicker than films for which the process of self-assembly has been interrupted by rinsing with neat solvents. We also present evidence that, by rinsing the partially formed film before reimmersion into thiol solution, we can change the final film thickness and structure of a hexadecanethiolate film. Our results are most consistent with a formation mechanism involving adsorption of both chemisorbed and physisorbed molecules during film formation in ethanol, with the overall formation kinetics determined by the relative solubility of the alkanethiol in the solvent.

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We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.