Synthesis and Characterization of Fluorescent Silica Nanoparticles with Large Stokes Shifts for Multiplexed Assays and Imaging

Modern approaches to biological and biomedical analysis demand ever-increasing levels of sensitivity, selectivity, and throughput. This challenge has been addressed in the work described herein via the synthesis, characterization, and proofs of concept of a series of single and multidye copolymerized fluorescent silica nanoparticles with large Stokes shifts and near infrared fluorescence. The prepared fluorescent probes exhibit substantially enhanced fluorescence signals relative to their constituent dyes, good indicators of biocompatibility, and readily distinguishable fluorescence signals, promoting the simultaneous detection of multiple targets and reducing both the cost and time per assay. A novel NIR-fluorescent aminocyanine dye was designed for incorporation into silica nanoparticles, then synthesized and characterized as detailed in the second chapter. Spectroscopic characterization confirmed the intended dye structure and revealed a large Stokes shift, near infrared fluorescence, and a relatively high quantum yield, indicating the suitability of this compound for bioanalytical applications and incorporation into silica nanoparticles as either a standalone fluorophore or as a resonance energy transfer acceptor for other UV-visible dyes. In the third chapter, the incorporation of the novel dye and other commercial dyes into silica nanoparticles is discussed, along with characterization and proofs of concept for in vivo and in vitro applications of the resultant fluorescent labels. Nanoparticle synthetic approaches, dye concentrations, and surface coating densities were optimized for fluorescence intensities and biocompatibility. Nanoparticles containing single and multiple dye species were synthesized per the optimal parameters, producing a series of fluorescent tags with distinct fluorescence signatures and large Stokes shifts. Synthesized nanoparticles were characterized in terms of sizes, synthetic yields, quantum yields, limits of detection, stability, and synthetic reproducibility. The suitability of surface modified nanoparticles to in vitro and in vivo applications was demonstrated via biotin linkage to streptavidin microbeads and fluorescence microscopy imaging, hemocompatibility studies, and protein binding studies. Finally, in the fourth chapter, resonance energy transfer characteristics of multidye copolymerized nanoparticles were characterized. Energy transfer efficiencies between donor-acceptor pairs, numbers of dye molecules per nanoparticle, and average distances between dye molecules were calculated, then Förster radii for different donor-acceptor pairs were estimated using two approaches and compared