Real-time measurement of nucleic-acid hybridization using evanescent-wave sensors: steps towards the genosensor

Nucleic acids are used as receptors in biosensing for the determination of complementary nucleic-acid strands. This may be useful in clinical diagnostics, e.g., searching for DNA from viruses, or in other fields of hygiene or environmental monitoring. In this study we demonstrate the reversible binding of DNA oligonucleotides to immobilized DNA targets. Using both a grating coupler detector and surface plasmon resonance, the evanescent field is employed to distinguish between bound and unbound species. As a bridge for the immobilization of the DNA, streptavidin (or avidin) is coupled to the sensor surface. Three different targets have been investigated: (1) randomly biotinylated poly(dA); (2) 5'-biotinylated 24-mer consisting of balanced amounts of purines and pyrimidines; and (3) 5'-biotinylated 13-mer. The binding kinetics of poly(dT) have been investigated, demonstrating this fast process as multibinding complex formation, since no sequence specificity is involved. The binding of a variety of 13-mers to 24-mer and 13-mer templates has been observed and the binding kinetics analysed. In a 13-mer point mutations can be resolved by analysis of association and dissociation rate constants.