Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.