Modification and characterization of fluorescent conjugated polymer nanoparticles for single molecule detection

Single molecule tracking using fluorescent dye or nanoparticle labels has emerged as a useful technique for probing biomolecular processes. Considerable interest arises in the development of nanoparticle labels with brighter fluorescence in order to improve the spatial and temporal resolution of single molecule detection and to facilitate the application of single molecule detection methods to a wider range of intracellular processes. The McNeill laboratory recently reported that conjugated polymer nanoparticles exhibit fluorescence cross-sections roughly 10--100 times higher than other luminescent nanoparticles of similar size, excellent photostability (2.2x108 photons emitted per nanoparticle prior to photobleaching), and saturated emission rates roughly 100 times higher than that of the molecular dyes and more than 1000 times higher than that of colloidal semiconductor quantum dots. One purpose of this graduate research is the development of highly fluorescent, bioconjugated nanoparticle labels based on conjugated polymers for demanding fluorescence applications such as single molecule tracking in live cells. Three surface modification methods (conjugated polymer nanoparticles encapsulated with lipid silica agents, conjugated polymer nanoparticles encapsulated with tetraethyl orthosilicate(TEOS) and hybrid nanoparticles with thiol pendant groups by the Stober Method (3-mercaptopropyl trimethoxysilane (MPS))) have been developed to protect the conjugated polymer, passivate the nanoparticle surface, and provide a chemical handle for bioconjugation such as nanoparticle encapsulation with alkoxysilanes and Stober method. After encapsulation, the fluorescence quantum yield of silica-encapsulated nanoparticles is improved by 20% as compared to bare conjugated polymer nanoparticles, while the photostability is improved by a factor of 2, indicating that some protection of the polymer is provided by the encapsulating layer. Another purpose of my research is the manipulation of the photophysics and photochemistry of conjugated polymer nanoparticles based on developing a more complete understanding of the various processes that control or limit nanoparticle brightness and photostability. The results indicate that a combination of photophysical processes including electron transfer to molecular oxygen, energy transfer, and exciton diffusion result in saturation and photobleaching phenomena that currently limit brightness. This study provides the potential methods and strategies aimed at manipulating such processes or limiting their effect on fluorescence brightness. Finally, efficient intra-particle energy transfer has been demonstrated in dye-doped CP nanoparticles, which provides a new strategy for improving nanoparticle fluorescence brightness and photostability, obtaining nanoparticles with red emission to avoid autofluorescence in mammalian tissue, and for designing novel sensitive biosensors based on energy transfer to sensor dyes.