Fluorescent Nanoconjugate Derivatives with Enhanced Photostability for Single Molecule Imaging

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• 作者：Daniel T. Reilly ; Sung Hoon Kim ; John A. Katzenellenbogen ; Charles M. Schroeder
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 3, 2015
• 年：2015
• 卷：87
• 期：21
• 页码：11048-11057
• 全文大小：595K
• ISSN：1520-6882

文摘

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鈥揟rolox conjugates onto dendrimers. In all cases, FDN鈥揟rolox 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鈥揟rolox, and as a proof-of-principle demonstration, we use FDN鈥揟rolox derivatives for bulk immunofluorescence imaging. Overall, our work suggests that self-healed multichromophoric systems such as FDN鈥揟rolox probes present a useful strategy for increasing fluorescent probe photostability.