Improving biomolecular sensing at surfaces and investigating oligomeric DNA hybridization kinetics in solution

This thesis focuses on understanding the kinetics of two model systems: protein-DNA and DNA-DNA interactions as measured using a biosensor capable of quantitative, high throughput, real time, label free detection that is readily extended to other biomolecular interactions. To this end, we built an automated, angle scanning surface plasmon resonance imaging instrument along with customized patterning and fluidic delivery devices; we use this biosensor to report, for the first time, rate constants of human alpha-thrombin binding to its single stranded DNA aptamer. However, the formation of intermolecular aptamer G-quartet formation, not considered at the time, may have interfered with these measurements. Another interference, non-specific binding or fouling was observed despite the use of mixed component oligo(ethylene glycol) self-assembled monolayers (OEG SAMs). OEG SAMs, which contain reactive probe attachment sites while minimizing non-specific binding, or fouling, are thought to provide reliable biosensor measurements. We show that the fabrication method alters OEG SAM stability, as measured by reductive desorption, and protein dependent fouling in ways not appreciated in earlier work. Our results suggest that different packing densities are created by each method, which cause OEG to take on different conformations and could explain the differences in fouling.;DNA-DNA hybridization kinetics were measured in solution by monitoring the hysteresis in heating and cooling of an oligonucleotide duplex. Though DNA hybridization has long been investigated, oligonucleotide kinetics are not understood. We focus on hybridization rate constants and activation energies, showing that both are significantly influenced by DNA sequence length but not GC content The data shows that rate constants for longer DNA sequences display a stronger ionic strength dependence compared with shorter strands, suggesting that longer strands hybridize via a more extended nucleus. Furthermore, the length dependence of the hybridization rate constant and hysteresis separation do not follow known polynucleotide trends. Although limited to only ten sequences, this study shows that length dependent kinetics of oligonucleotides merit further investigation. Further measurements may aid in the development appropriate kinetic models which are not currently available. Extended to surfaces, such models may find use in understanding oligonucleotide kinetics in DNA microarrays and other biosensors.