Monitoring of oligonucleotide hybridization using light-addressable potentiometric and evanescent wave fluorescence sensing

Hybridized pairs of oligonucleotides, one biotinylated and the other FITC-labeled, were captured on streptavidincoated surfaces via biotin-streptavidin binding. Detection of the fluoresceinated probe was achieved (1) potentiometrically by light-addressable potentiometric sensing (LAPS) using urease-conjugated anti-fluorescein antibodies and (2) by evanescent wave spectroscopy (EWS) using light internally reflected along a quartz fiber on which the hybrid duplexes were captured facilitating excitation of the FITC-labeled probe in the evanescent wave zone. As little as 20 pg fluoresceinated oligonucleotide could be detected using the LAPS, whereas, the limit of detection using EWS was 50 ng. A dose-dependent reduction of signal was observed in the presence of a nonfluoresceinated oligonucleotide of identical sequence as that of the fluoresceinated target using both methods. Specificity of hybridization was confirmed by the ineffectiveness of added genomic DNA from several species in suppressing the signal. These data indicate that the LAPS and EWS methodologies may be useful for rapid and sensitive detection of oligonucleotides of defined sequence.