| DNA-based surface biosensors are being widely used for gene expression profiling, genotyping, and drug discovery. Biosensing in a surface format, particularly a microarray format, is advantageous as it can provide rapid parallel detection of multiple target biomolecules at very low concentrations. Nevertheless, there is a need for a better understanding of how the surface environment, which is very different from bulk solution, affects kinetic and thermodynamic biosensor measurements. The fundamental studies presented in this thesis are a step towards understanding such surface effects and predicting solution-phase or in vivo DNA or drug binding behavior from surface biosensor data. We use both surface plasmon resonance (SPR) angle-scanning spectroscopy and imaging to investigate the effects of DNA-based surface molecular architecture on DNA hybridization and duplex DNA-drug binding.; In designing a surface architecture for these studies, we found that there are strong sequence-dependent surface effects on the DNA self-assembly surface fabrication process. The kinetics and thermodynamics of immobilization for thiolated-DNA probe molecules on gold are controlled by non-specific nucleotide-surface interactions. We further optimized our DNA-based biosensing surfaces with an SPR imaging system developed in our laboratory. Angle-resolved imaging was employed to rapidly and directly quantify the DNA probe densities produced during surface fabrication.; Finally, we use our optimized surfaces and demonstrate for the first time, the application of SPR imaging to in-situ, label-free DNA-drug binding measurements. Using a well-studied anticancer drug compound, actinomycin-D, we compare the kinetics and thermodynamics of drug binding to surface-bound DNA with those obtained for DNA-drug binding in solution. We demonstrate the discrimination of drug binding to two different surface-immobilized DNA strands containing a binding site of specific affinity in parallel on a patterned surface. We are able to directly quantify the number of drug molecules bound to the surface and can detect less than one drug molecule per duplex (imaging limit of detection ∼5--6 x 1011 drug molecules/cm2). For all sequences, however, we find that the surface suppresses binding rates by ∼100 fold compared to bulk solution. |
