Nucleic acid films studied by surface plasmon resonance spectroscopy

The hybridization of complementary strands of DNA to form stable duplexes is important in biological and chemical research; for example, it is the underlying principle of all microarray-based techniques for analysis of DNA variation. Surface plasmon resonance (SPR) spectroscopy is one of the few in-situ methods that can be used to monitor the immobilization of thiol-modified DNA probes to form monolayer films and the subsequent capture of unlabeled single stranded DNA targets from solution to produce duplex DNA at the sensor interface.; In order to reproducibly fabricate DNA monolayer films for hybridization studies, we studied the covalent attachment via self-assembly of thiol-modified DNA probes onto gold and developed a model to fit the kinetic data. We found that the density of covalently attached DNA (probe density) can be controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential, and whether duplex or single stranded oligonucleotides are used. Most importantly, we found that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for hybridization independent of which probe immobilization strategy is used.; The hybridization of surface-immobilized DNA with solution-phase target DNA strands depends strongly on probe density. As the density decreases, the efficiency of duplex formation increases and the kinetics of target capture become more rapid. In addition to perfectly matched duplexes, we also studied the hybridization of targets with 1 or 2 base pair mismatches and shorter target strands that access different binding locations on the immobilized probe. Kinetic and equilibrium data were compared with various models for binding at interfaces in order to obtain equilibrium binding constants and to investigate the mechanism of binding.; For all studies, SPR optical data were analyzed to provide a quantitative measure of DNA coverage and DNA hybridization as a function of time, solution concentration, temperature or electrostatic potential. We describe methodologies developed for the interpretation of SPR data obtained under these conditions, such as temperature dependent probe-target melting, or upon application of interfacial electrostatic potential. Specific assumptions and calculations, such as the influence of refractive index anisotropy, are described and related to SPR data analysis.