Aqueous CdSe quantum dot molecular beacon for RNA detection

The gold standard for nucleic acid detection uses PCR to amplify the nucleic acid followed by gel electrophoresis or fluorescent label for detection. As such, PCR is not suitable for in situ monitoring of genetic drug delivery in real time. Molecular beacons (MBs) are fluorescently labeled probes whose quenched fluorescence is unleashed when hybridized with a target gene that can be used for in-situ detection of a gene of interest in real time. Current fluorophore-based molecular beacons are specific for nucleic acid detection; however, the organic fluorescent dyes (fluorophores) are not very bright and are prone to photobleaching. Quantum dots (QDs) are fluorescent nanoparticles that do not photobleach and are much brighter than fluorophores. The goal of this thesis is to develop cadmium selenide (CdSe) aqueous quantum dots (AQDs) MBs with the CdSe AQD at the 5' end of the MB and a quencher, BHQ-2 on the 3' end to detect and target a 43-nt synthetic RNA sequence (from the orf1a gene of the SARS-CoV2 virus) as the model target RNA. In the absence of the target RNA, the AQD MB is in the hairpin conformation with the quencher near the AQD that reduces the fluorescence intensity. Upon binding to the target RNA sequence, the hairpin structure of the AQD MB opens. This moves the quencher away from the AQD and recovers the fluorescence of the AQD. The CdSe AQDs were first stabilized with dihydrolipoic acid (DHLA) prior to chemical conjugation to the quencher as a model, and eventually to the hairpin at various hairpin/AQD ratios. A 5:1 hairpin/AQD ratio yields the optimal efficiency, 99%, of hairpin conjugation due to the high density of carboxyl functionality on the AQD surface. The CdSe AQD MB successfully exhibits fluorescence recovery upon binding of target RNA. It was found that there was a correlation between the hairpin/AQD ratio, and the target RNA concentration needed for maximal PL recovery - less target RNA can achieve PL recovery with decreasing hairpin/AQD molar ratio. For the optimal hairpin/AQD ratio of 5:1, the recovery increases linearly with an increasing target RNA concentration up to 100 nM.