Semiconducting Polymer Dots for Multiplexed Assays

描述（申请人提供）：荧光技术在现代细胞生物学和生物医学研究中发挥着重要作用。由于缺乏合适的荧光探针，这些技术的进一步发展受到阻碍。为了克服这一挑战，各种荧光纳米粒子已经被开发出来。其中，共轭聚合物点（Pdots）具有亮度高、发射速率快、光稳定性好等优点。然而，与Pdots相关的一个严重缺点是它们的发射光谱宽，这大大限制了它们在实际应用中的有用性。这一提议描述了一种新型Pdots的改进，该Pdots在不同的波长发射，具有窄的光谱带宽。为了实现这一目标，我们提出了以下目标：(1)开发发射带宽FWHM等于或小于40 nm的多色明亮聚合物点。在这里，我们将采用各种光谱技术来表征Pdot的特性，如吸收截面、发射带宽、荧光量子产率、光稳定性和荧光寿命。单粒子荧光成像也将进行Pdots与无机量子点（Qdots）的并排亮度比较。(2)优化纳米颗粒表面特性，减少非特异性标记。在这里，为了检查Pdots的非特异性标记，我们将使用一系列技术，包括凝胶电泳，动态激光散射，亲和层析，流式细胞术和荧光光谱/成像。(3)展示细胞环境中生物分子的多重检测。在这里，我们将使用明亮的窄带Pdots对细胞环境中的生物分子进行多重检测。具体来说，流式细胞术和荧光成像将评估标记特异性和比较标记目标的荧光亮度。将建立可靠的协议，以同时标记三个或更多的细胞目标与多色Pdots。这将为将这些明亮的窄带Pdots应用于多路检测和生物成像奠定基础。

DESCRIPTION (provided by applicant): Fluorescence based techniques play an essential role in modern cell biology and biomedical research. Further development of these techniques has been hindered by the lack of suitable fluorescent probes. To overcome this challenge, various fluorescent nanoparticles have been developed. Among those, conjugated polymer dots (Pdots) exhibited superior properties such as high brightness, fast emission rate, and excellent photostability. However, a severe drawback associated with Pdots is their broad emission spectra, which significantly limit their usefulness in practical applications. This proposal describes the refinement of a new class of Pdots that emit at different wavelengths with narrow spectral bandwidth. To achieve this goal, we propose the following aims: (1) Develop multicolor bright polymer dots with emission bandwidth FWHM that is comparable to or less than 40 nm. Here, we will carry out various spectroscopic techniques to characterize the Pdot properties such as absorption cross section, emission bandwidth, fluorescence quantum yield, photostability, and fluorescence lifetime. Single-particle fluorescence imaging will also be performed to provide side-by-side brightness comparisons on Pdots versus inorganic quantum dots (Qdots) (2) Optimize nanoparticle surface properties to reduce nonspecific labeling. Here, to examine nonspecific labeling of Pdots, we will use a range of techniques, including gel electrophoresis, dynamic laser scattering, affinity chromatography, flow cytometry, and fluorescence spectroscopy/imaging. (3) Demonstrate multiplex detection of biomolecules in cellular environments. Here, we will use the bright, narrow-band Pdots for multiplex detection of biomolecules in cellular environments. Specifically, flow cytometry and fluorescence imaging will be performed to evaluate the labeling specificity and compare fluorescence brightness of the labeled targets. Reliable protocols will be established for simultaneous labeling of three or more cellular targets with multicolor Pdots. This will lay the foundation of applying these bright, narrow-band Pdots for multiplex detection and biological imaging.