In this paper, we present a surface plasmon resonance (SPR)-based sensor for measuring DNA hybridization and DNA/small-molecule interactions. A mixed self-assembled monolayer (SAM) was used to optimize the biosensor sensitivity. It was observed that the mixed SAM formed by mixing 10 mM of 16-mercapto-1-hexadecanoic acid (16-MHA) and 6-mercapto-1-hexanol (6-MCH) at a 1: 10 molar ratio showed the best results. Subsequently, avidin was attached to the carboxyl groups on the SAM to serve as a binding element for biotinylated single-stranded (ss) DNA. The ssDNA-coated sensor was first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for synthetic 28-mer ssDNA. A linear calibration curve was observed in the range of 0.25-2.5 mu g/mL. Non-complementary DNA induced no significant SPR angle shift, which demonstrated the specificity of the assay. Secondly, the sensor was used to monitor the binding kinetics of DNA/small-molecule interactions in real time. The dissociation constant between immobilized DNA and sanguinarine was determined to be 8.0 x 10(-6) M. This complies with most data from the literature. In addition, the sensor could be regenerated with 0.01 M HCl and would be feasible for multiple testing. In conclusion, the experimental approach described in this study allows analysis of molecular interactions between DNA-binding drugs and selected targeted DNA sequences.
