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

描述(申请人提供)：基于荧光的技术，从细胞成像到生物测定，已经成为癌症基础细胞研究以及临床应用和体外诊断中不可或缺的工具包。有机染料似乎是迄今为止在这些应用中使用的最多用途的荧光团。然而，传统染料的固有局限性，如低吸光度和较差的光稳定性，给进一步发展高灵敏度成像技术和高通量分析带来了困难。因此，人们对更明亮、更稳定的荧光探针产生了相当大的兴趣。例如，藻胆蛋白表现出比小的有机荧光团更高的荧光亮度，而且对明亮荧光的开发也有很大的兴趣 纳米粒子，如半导体量子点。该项目旨在开发一种名为Pdots的新型荧光探针，用于癌细胞的分子分析和研究。为了实现这一目标，我们提出了以下三个目标：目标1：研制具有超窄发射带宽(半高全宽，15 nm)的多色超亮Pdots。尽管Pdots非常明亮，但一个严重的缺点是目前可用的Pdot物种的发射光谱非常宽，这限制了它们在多路应用中的用途。人们迫切需要开发新型的、能发射不同波长、具有窄带光谱宽度的光点。在这里，我们计划开发一系列具有15 nm超窄发射带宽的Pdots。目的2：生成具有单分散尺寸分布的Pdots。虽然我们可以调节Pdots的直径从大约5 nm到几十纳米，但它们的尺寸分布目前相当广泛。在这里，我们提出了一种基于单分散滤孔的高效、高通量的方法来形成单分散尺寸分布的Pdots。目的3：制备表面性质可控的单价单链Pdots。由于纳米粒子表面存在多个活性中心，控制其表面化学官能团的数量和几何分布往往是极其困难的。在基于细胞的分析中，纳米多价颗粒可以引起参与信号通路激活的细胞表面蛋白的交联性，从而显著降低受体结合能力。在分子分析中，多价态也会导致分子的交联，降低分析的灵敏度。在这里，我们建议利用合成具有预定义数量的官能团的聚合物来形成单价Pdots的能力。

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.