DNA-functionalized Gold Nanoparticles for Enhanced Molecular Sensing

Abstract

Since the advent of molecular beacon technology, much attention has been directed toward understanding RNA expression at a single-cell level. Our laboratory has designed an approach that harnesses the advantages of both molecular beacon and gold nanoparticle technology, termed hairpin DNA-functionalized gold nanoparticles (hAuNPs). hAuNPs improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNAse degradation, low cytotoxicity, and high specificity and sensitivity toward the target mRNA sequence. This dissertation highlights efforts to optimize the design of hAuNPs as well as new applications of these novel mRNA imaging probes. The optimization of the existing hAuNP construct resulted in a four-fold increase in signal-to-noise ratio. hAuNPs were used to image multiple biomarkers in three different types of living cancer cells. The fluorescence intensities for the probes in cells were measured by flow cytometry and confocal microscopy and showed good agreement with reported values for their target mRNA expression, thus confirming the ability to measure relative expression levels of mRNAs in living cells using hAuNPs. Specific imaging of RNA biomarkers was achieved in retinal tissue using in vivo retinal fluorescence imaging of laser-induced choroidal neovascularization in animal models. The use of gold nanorods (AuNRs) as the nanoparticle construct of hAuNPs was also investigated. Hairpin DNA functionalization was achieved on AuNRs after an overgrowth step in which a thin layer of gold atoms were reduced on a preformed AuNR, masking the trace silver ions that are thought to complicate other functionalization strategies. Finally, scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) mapping of AuNRs reveal new information on the structural and elemental characterization of AuNRs specifically the location of silver deposits on the surface of the rods, providing new insight into the mechanism by which AuNRs grow.