Molecular beacons are lengths of DNA, fluorescently labeled at one end and linked to a quencher at the other. Unbound, they adopt a hairpin loop conformation: the fluorophore is positioned next to the quencher and fluorescence is quenched. Upon binding to a complementary sequence, the beacon opens, the fluorescent label and quencher move apart and fluorescence appears. Sensitivity is dependent upon the ratio of fluorescence intensities in the open and closed conformations. In preliminary experiments, the 1.4 nm Nanogold gold cluster has demonstrated equal quenching ability to the fluorophores usually used, and since it absorbs at a wide range of wavelengths, it quenches a variety of fluorophores. Two formulations of the Nanogold label, and larger covalently linked gold clusters, will be investigated as quenchers. Synthetic modification of the gold cluster surface, cross-linking protocols and fluorophores will be used to incorporate weak affinities between the fluorophore and gold cluster to bring them closer together and increase quenching efficiency in the closed conformation. The ratio of fluorescence intensities for the open and closed conformations, and hence sensitivity, will be increased beyond that currently available. Optimized gold cluster-quenched beacons will be cross-linked to fiber optic cables to prepare highly sensitive and specific biosensors.
The proposed technology enables the real-time detection of specific DNA sequences in cells and other highly localized systems. Biosensors using the new probes may be used for rapid genetic screening of tissues and organs for transplantation, and for a variety of other applications such as rapid cancer screening and fetal testing which would benefit from their high specificity, portability and reduced invasiveness.
