Aqueous CdSe Quantum Dot Molecular Beacon for RNA Detection

Thursday, May 30, 2024

11:00 AM-1:00 PM

BIOMED Master's Thesis Defense

Title:
Aqueous CdSe Quantum Dot Molecular Beacon for RNA Detection

Speaker:
Hannah Slagle, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
 
Advisors:
Wan Y. Shih, PhD
Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Wei-Heng Shih, PhD
Professor
Department of Materials Science and Engineering
College of Engineering
Drexel University

Details:
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, which are ideal for in-situ detection of a gene of interest in real time. While current fluorophore-based molecular beacons are specific for nucleic acid detection 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 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 in close proximity to the AQD that greatly reduces its fluorescence intensity. Upon binding to a target RNA, the hairpin structure of the AQD MB opens. This greatly moves the quencher away from the AQD and recovers the fluorescence of the AQD. The CdSe AQDs are first synthesized in water and further stabilized with dihydrolipoic acid (DHLA) prior to chemical conjugation. For conjugation, they are first conjugated to the quencher as a model to evaluate the conjugation efficacy, and eventually to the hairpin at various hairpin/AQD ratios for optimal target RNA detection. It is found a 5:1 hairpin/AQD ratio yields the optimal conjugation efficiency of the hairpin at 99% due to the high density of carboxyl functionality on the AQD surface. The CdSe AQD MB successfully exhibit fluorescence recovery upon binding of target RNA. There is a correlation between the hairpin/AQD ratio and the maximum target RNA concentrations up to which the AQD MBs exhibit fluorescent recovery. For the optimal hairpin/AQD ratio of 5:1, the fluorescent recovery increases linearly with an increasing target RNA concentration up to 100 nM, relevant concentrations in gene delivery applications.

Contact Information

Natalia Broz
njb33@drexel.edu