Semiconducting Polymer Dots for Multiplexed Assays

描述（由申请人提供）：基于荧光的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，已经开发了各种荧光纳米颗粒。其中，共轭聚合物点（Pdot）表现出优异的上级特性，例如高亮度、快速发光速率和优异的光稳定性。然而，与Pdot相关的严重缺点是它们的宽发射光谱，这显著限制了它们在实际应用中的有用性。该提案描述了一类新的Pdot的改进，其发射不同波长的窄光谱带宽。为了实现这一目标，我们提出了以下目标：（1）开发具有与40 nm相当或小于40 nm的发射带宽FWHM的高亮度聚合物点。在这里，我们将进行各种光谱技术来表征Pdot的性质，如吸收截面，发射带宽，荧光量子产率，光稳定性和荧光寿命。还将进行单粒子荧光成像以提供Pdot与无机量子点（Qdot）的并排亮度比较（2）优化纳米粒子表面性质以减少非特异性标记。在这里，检查Pdot的非特异性标记，我们将使用一系列的技术，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)演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Pdot对细胞环境中的生物分子进行多重检测。具体而言，将进行流式细胞术和荧光成像，以评价标记特异性并比较标记靶标的荧光亮度。将建立可靠的协议，同时标记三个或更多个细胞的目标与DPdot。这将为这些明亮的窄带Pdot应用于多重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.