Surface Plasmon Resonance Imaging Measurements of DNA Hybridization Adsorption and Streptavidin/DNA Multilayer Formation at Chemically Modified Gold Surfaces

A combination of scanning and imaging surface plasmon resonance (SPR) experiments is used to characterize DNA hybridization adsorption at gold surfaces and the subsequent immobilization of streptavidin. Single-stranded oligonucleotides are immobilized at gold surfaces, and the hybridization of biotinylated complements from solution is monitored with SPR. The subsequent attachment of streptavidin to the biotinylated complements provides a method of enhancing the SPR imaging signal produced as a result of the hybridization and leads to a 4-fold improvement in the hybridization detection limit of the SPR imaging apparatus. In situ scanning SPR experiments are used to measure a 60 ± 20% hybridization efficiency between immobilized single-stranded DNA and biotinylated complements. From the information provided by both the in situ imaging and scanning SPR experiments, an absolute surface coverage of immobilized single-stranded DNA is estimated to be ∼3 × 1012 molecules/cm2. The SPR signal resulting from hybridization onto immobilized probes is further amplified by the formation of streptavidin/DNA multilayers which grow by a combination of DNA hybridization and biotin−streptavidin binding. DNA/DNA multilayers without streptavidin are used as an additional method of amplifying the SPR signal.