Department of Biochemistry, Oxford University, South Parks Road, OX1 3QU, United Kingdom.
ACS Nano. 2013 Feb 26;7(2):1137-44. doi: 10.1021/nn3042122. Epub 2013 Jan 30.
Fluorescent nanoparticles have enabled many discoveries regarding how molecular machines function. Quantum dots have been the dominant class of fluorescent nanoparticles but suffer from blinking and from a substantial dark fraction--particles where the fluorescence is never seen--complicating any analysis of biological function. Nanoparticles composed of conjugated fluorescent polymers (Pdots) have recently been shown to have high brightness and no blinking. Here we develop a robust and efficient means to measure the dark fraction of Pdots, conjugating Atto dyes to the nanoparticles and testing fluorescence colocalization of dye and Pdot puncta. This established that the Pdots we generated had minimal dark fraction: ∼3%. The application of nanoparticles in biological environments is highly sensitive to surface functionalization. For Pdots we found that passivation with uncharged hydroxy-terminated polyethylene glycol caused a dramatic reduction in nonspecific cell binding and aggregation compared to a charged coating. Using carbonyl di-imidazole the hydroxy-Pdots were functionalized efficiently with streptavidin for high stability targeting, allowing specific labeling of mammalian cells. Type I insulin-like growth factor receptor (IGF1R) regulates cell survival and development, with roles in aging, heart disease, and cancer. We used hydroxy-Pdots to track the dynamics of IGF1R on a breast cancer cell-line, determining the diffusion characteristics and showing cholesterol-containing membrane nanodomains were important for receptor mobility at the plasma membrane. The near-unity bright fraction and low nonspecific binding of hydroxy-Pdots, combined with Pdot photostability and lack of blinking, provides many advantages for investigations at the single molecule level.
荧光纳米粒子使人们能够深入了解分子机器的工作原理。量子点是荧光纳米粒子的主要类别，但存在闪烁和显著的暗部分——荧光从未被观察到的粒子——这使得对生物功能的任何分析都变得复杂。最近，由共轭荧光聚合物（Pdots）组成的纳米粒子已经被证明具有高亮度和无闪烁的特性。在这里，我们开发了一种强大而有效的方法来测量 Pdots 的暗部分，将 Atto 染料与纳米粒子缀合，并测试染料和 Pdot 斑点的荧光共定位。这表明我们生成的 Pdots 具有最小的暗部分：约 3%。纳米粒子在生物环境中的应用对表面功能化非常敏感。对于 Pdots，我们发现与带电荷的涂层相比，用不带电荷的羟基封端的聚乙二醇进行钝化会显著降低非特异性细胞结合和聚集。使用羰基二咪唑，羟基-Pdots 可以有效地与链霉亲和素进行功能化，以实现高稳定性靶向，从而可以特异性标记哺乳动物细胞。I 型胰岛素样生长因子受体（IGF1R）调节细胞存活和发育，在衰老、心脏病和癌症中发挥作用。我们使用羟基-Pdots 来跟踪乳腺癌细胞系中 IGF1R 的动力学，确定扩散特征，并表明含有胆固醇的膜纳米区对于质膜上受体的流动性很重要。羟基-Pdots 的近全亮部分和低非特异性结合，加上 Pdot 的光稳定性和无闪烁特性，为单分子水平的研究提供了许多优势。
