Ultra-Bright Probes with Ultra-Narrow Emission for Molecular and Cellular Analysi

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DESCRIPTION (provided by applicant): Fluorescence based techniques, from cellular imaging to bioassays, have become an indispensable tool kit in both basic cellular studies of cancer and in clinical applications and in vitro diagnostics. Organic dyes appear to be the most versatile fluorophores used so far in these applications. However, the intrinsic limitations of conventional dyes, such as low absorptivity and poor photostability, have posed difficulties for the further development of high-sensitivity imaging techniques and high-throughput assays. As a result, there has been considerable interest in brighter and more stable fluorescent probes. For example, phycobiliproteins exhibit higher fluorescence brightness than small organic fluorophores, and there is also a great deal of interest in the development of brightly fluorescent nanoparticles, such as semiconductor quantum dots. This project aims to develop a new class of fluorescent probes, called Pdots, for use in the molecular analysis and study of cancer cells. To achieve this goal, we propose the following three aims: Aim 1: Develop multicolor ultra-bright Pdots with ultra-narrow emission bandwidth (Full Width at Half Maximum (FWHM) < 15 nm). Although Pdots are exceptionally bright, a severe drawback is the very broad emission spectra of currently available Pdot species, which limit their usefulness in multiplexed applications. There is an urgent need to develop new types of Pdots which can emit at different wavelengths with a narrow-band spectral width. Here, we propose to develop a series of Pdots with an ultra-narrow emission bandwidth of <15nm. Aim 2: Generation of Pdots with monodisperse size distributions. Although we can tune the size of Pdots from about 5nm to tens of nanometers in diameter, their size distribution is currently quite broad. Here, we proposed to develop an efficient and high-throughput method based on monodisperse filter pores to form Pdots of monodisperse size distributions. Aim 3: Generation of monovalent single-chain Pdots with controlled surface properties. It is often extremely difficult to control the number and geometrica distribution of chemical functional groups on a nanoparticle because of the presence of multiple reactive sites on its surface. In cell-based analyses, nanoparticle multivalence can cause cross-linking of cell-surface proteins involved in signal pathway activation, and thus dramatically reduce receptor binding capability. In molecular analysis, multivalence can also lead to cross linking of molecules and reduce the sensitivity of the assay. Here, we propose to take advantage of the ability to synthesize polymers with pre-defined number of functional groups to form monovalent Pdots.