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

用于分子和细胞分析的超窄发射超亮探针

• 批准号: 8737597
• 负责人: Daniel T Chiu
• 金额: $ 20.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737597
• 关键词: Address; Affect; Binding; Binding Sites; Biological; Biological Assay; Boron; Caliber; Cell Surface Proteins; Cells; Chemicals; Communities; Complex; Development; Diagnostic; Dyes; Electrostatics; Energy Transfer; Exhibits; Fluorescence; Fluorescent Probes; Generations; Goals; Imaging Techniques; In Vitro; Label; Lanthanoid Series Elements; Lead; Malignant Neoplasms; Methods; Molecular; Molecular Analysis; Particle Size; Performance; Phycobiliproteins; Polymers; Property; Quantum Dots; Reagent; Reporting; Semiconductors; Series; Signal Pathway; Surface; Surface Properties; Swelling; Techniques; Technology; Time; Water; Width; absorption; base; cancer cell; cellular imaging; clinical application; crosslink; density; design; fluorophore; functional group; high throughput screening; interest; nanometer; nanoparticle; particle; public health relevance; receptor binding; tool

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.

描述（由申请人提供）：基于荧光的技术，从细胞成像到生物测定，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，传统染料的固有局限性，例如吸收率低和光稳定性差，给高灵敏度成像技术和高通量检测的进一步发展带来了困难。因此，人们对更亮、更稳定的荧光探针产生了极大的兴趣。例如，藻胆蛋白比小的有机荧光团表现出更高的荧光亮度，并且人们对开发明亮的荧光材料也产生了很大的兴趣。 纳米颗粒，例如半导体量子点。该项目旨在开发一类新型荧光探针，称为 Pdots，用于癌细胞的分子分析和研究。为了实现这一目标，我们提出以下三个目标： 目标 1：开发具有超窄发射带宽（半高全宽（FWHM）< 15 nm）的多色超亮 Pdot。尽管 Pdot 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，这限制了它们在多重应用中的用途。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超窄发射带宽<15nm的Pdot。目标 2：生成具有单分散尺寸分布的 Pdot。尽管我们可以将 Pdot 的尺寸从直径约 5 纳米到数十纳米进行调节，但它们的尺寸分布目前相当广泛。在这里，我们建议开发一种基于单分散过滤孔的高效、高通量方法，以形成单分散尺寸分布的 Pdot。目标 3：生成具有受控表面特性的单价单链 Pdot。由于纳米颗粒表面存在多个反应位点，控制纳米颗粒上化学官能团的数量和几何分布通常极其困难。在基于细胞的分析中，纳米颗粒多价性会导致参与信号通路激活的细胞表面蛋白发生交联，从而显着降低受体结合能力。在分子分析中，多价还会导致分子交联并降低测定的灵敏度。在这里，我们建议利用合成具有预定数量官能团的聚合物的能力来形成单价 Pdot。