Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Anal Chem. 2015 Nov 3;87(21):11048-57. doi: 10.1021/acs.analchem.5b03059. Epub 2015 Oct 20.
Fluorescence-based imaging techniques critically rely on bright and photostable probes for precise detection of biological molecules. Recently, a new class of multichromophoric probes based on fluorescent dendrimer nanoconjugates (FDNs) was developed for single molecule fluorescence microscopy (SMFM). FDNs are generated by covalent conjugation of multiple fluorescent dyes onto macromolecular polymeric scaffolds and show marked increases in brightness and long-term photostability relative to their single organic dye constituents. Multichromophoric probes, however, are generally known to suffer from transient fluorescence emission intensities and long excursions into dark states. To overcome these issues, photostabilizers can be added to bulk solution, though some small molecule additives may exhibit poor aqueous solubility or biological toxicity. In this work, we develop enhanced FDN derivatives by covalently linking a redox-active photostabilizer (Trolox) directly onto FDN molecular scaffolds. In one approach, multiple organic dyes (Cy5) and Trolox molecules are randomly distributed on dendritic scaffolds in tunable stoichiometric amounts, and in a second approach, Cy5 dyes are covalently linked to Trolox in a precise 1:1 stoichiometry followed by covalent attachment of Cy5-Trolox conjugates onto dendrimers. In all cases, FDN-Trolox conjugates show increases in photostability, brightness, and reduced fluctuations in transient fluorescent intensity relative to FDN probes. Bulk and single molecule photophysical data for FDN probes are compared to single self-healing dye systems such as Cy5-Trolox, and as a proof-of-principle demonstration, we use FDN-Trolox derivatives for bulk immunofluorescence imaging. Overall, our work suggests that self-healed multichromophoric systems such as FDN-Trolox probes present a useful strategy for increasing fluorescent probe photostability.
基于荧光的成像技术严重依赖于明亮和光稳定的探针，以实现对生物分子的精确检测。最近，一类新的多色荧光树枝状纳米复合物（FDN）基于单分子荧光显微镜（SMFM）被开发出来。FDN 通过将多个荧光染料共价连接到大分子聚合物支架上而产生，与它们的单个有机染料成分相比，显示出明显的亮度和长期光稳定性增加。然而，多色探针通常被认为会经历瞬态荧光发射强度和长时间进入暗态。为了克服这些问题，可以在体相溶液中添加光稳定剂，尽管一些小分子添加剂可能表现出较差的水溶性或生物毒性。在这项工作中，我们通过将氧化还原活性光稳定剂（Trolox）共价连接到 FDN 分子支架上来开发增强的 FDN 衍生物。在一种方法中，多个有机染料（Cy5）和 Trolox 分子以可调节的化学计量随机分布在树枝状支架上，在另一种方法中，Cy5 染料通过共价键连接到 Trolox 中以精确的 1:1 化学计量比，然后将 Cy5-Trolox 缀合物共价连接到树枝状聚合物上。在所有情况下，与 FDN 探针相比，FDN-Trolox 缀合物显示出光稳定性、亮度的提高和瞬态荧光强度波动的减少。与 FDN 探针的体相和单分子光物理数据进行了比较，对于单自修复染料系统，如 Cy5-Trolox，并作为原理证明，我们使用 FDN-Trolox 衍生物进行体相免疫荧光成像。总的来说，我们的工作表明，自修复多色系统，如 FDN-Trolox 探针，为提高荧光探针的光稳定性提供了一种有用的策略。
