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

描述（由申请人提供）：从细胞成像到生物测定，基于荧光的技术已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通用的荧光团。然而，传统染料固有的局限性，如低吸收率和光稳定性差，为进一步发展高灵敏度成像技术和高通量检测带来了困难。因此，人们对更亮、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，并且在开发亮荧光的藻胆蛋白方面也存在很大的兴趣。 纳米颗粒，例如半导体量子点。该项目旨在开发一类新的荧光探针，称为Pdot，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：开发具有超窄发射带宽（半峰全宽（FWHM）< 15 nm）的超高亮度Pdot。虽然Pdot特别亮，但严重的缺点是目前可用的Pdot种类的非常宽的发射光谱，这限制了它们在多路复用应用中的有用性。迫切需要开发能够以窄带光谱宽度发射不同波长的新型Pdot。在这里，我们建议开发一系列具有<15nm的超窄发射带宽的Pdot。目的2：产生具有单分散尺寸分布的Pdot。虽然我们可以将Pdot的直径从约5nm调整到数十纳米，但它们的尺寸分布目前相当广泛。在这里，我们提出开发一种基于单分散过滤孔的高效和高通量的方法，以形成单分散尺寸分布的Pdot。目的3：产生具有受控表面性质的单价单链Pdot。由于纳米颗粒表面存在多个反应位点，因此控制纳米颗粒上化学官能团的数量和几何分布通常是极其困难的。在基于细胞的分析中，纳米颗粒多价性可引起参与信号通路激活的细胞表面蛋白的交联，从而显著降低受体结合能力。在分子分析中，多价也可导致分子交联并降低测定的灵敏度。在这里，我们提出利用合成具有预定义数量的官能团的聚合物以形成单价Pdot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.