Sequence-specific analysis of oligodeoxynucleotides by precipitate-amplified surface plasmon resonance measurements

A flow injection SPR spectrometer is used to detect DNA hybridization via amplification by an enzyme-catalyzed precipitation reaction. Hybridization of an oligodeoxynucleotide (ODN) target with a surface-confined ODN probe, followed by hybridization between the overhang on the resultant duplex and a biotin-tagged ODN, yields a "sandwich" complex (a three-component double-stranded). A streptavidin-horseradish peroxidase conjugate (SA-HRP) can be attached to the sandwich complex-covered surface via the biotin/streptavidin complexation. In the presence of H2O2, the HRP catalyzes oxidation of 4-chloro-1-naphthol (CN) to form a precipitate on the sensor surface. The precipitated film dramatically changes the refractive index at the metal/dielectric interface and significantly lowers the detection level. The method is shown to be reproducible, to possess high ODN sequence specificity and a high sensitivity allowing detection of ODN target concentration as low as 10 fM.