Single-molecule studies on individual metal complexes
Single-molecule studies on individual metal complexes
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单个金属配合物的单分子研究
DOI：
10.1117/12.700430
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发表时间：
2007
影响因子：
1.8
通讯作者：
D. Herten
中科院分区：
文献类型：
--
作者：
A. Kiel;Anne Järve;J. Kovács;A. Mokhir;R. Krämer;D. Herten
一些荧光染料（例如 Atto635 或 Cy5）在存在各种金属离子和金属离子络合物时会表现出荧光发射的变化。我们利用这种效应设计了可以固定在玻璃表面上的染料-配体缀合物，并允许使用时间分辨单分子荧光光谱（SMFS）研究金属离子结合。双链DNA作为刚性支架，携带2,2'-联吡啶-4,4'-二羧酸作为螯合配体和荧光染料作为报告分子，放置在配体附近。在不存在金属离子的情况下，探针表现出高荧光量子产率，而在结合 Cu2+-离子时观察到强烈的荧光猝灭。时间分辨单分子测量揭示了高荧光（“开”）和低荧光（“关”）状态之间的随机切换。因此，金属离子与配体的配位通过染料的分子内荧光猝灭来指示。我们筛选了各种荧光染料对 Cu2+ 配位的敏感性，发现 Atto620 和 MR121 都非常适合此应用。使用 Atto620 对金属传感器的荧光寿命进行的整体研究表明，对金属离子浓度的依赖性很小，而单分子研究报告了荧光寿命的强烈变化，这与观察到的开启和关闭状态相关。我们的结果进一步表明，Atto620 的荧光在金属离子络合物缔合时并未完全猝灭；要么是因为形成了较少的荧光复合物，要么是因为用于荧光染料共价偶联的 C6 连接体的构象变化而导致分子内碰撞猝灭。
Some fluorescent dyes, like Atto635 or Cy5, exhibit alterations in fluorescence emission in the presence of various metal ions and metal ion complexes. We used this effect to design dye-ligand conjugates that can be immobilized on glass surfaces and allow studying metal-ion binding using time-resolved single-molecule fluorescence spectroscopy (SMFS). Double-stranded DNA served as a rigid scaffold carrying 2,2'-bipyridene-4,4'-dicarboxylic acid as chelating ligand and a fluorescent dye as reporter, placed in close vicinity to the ligand. In the absence of metal ions, the probes showed high fluorescence quantum yield, whereas strong fluorescence quenching upon binding of Cu2+-ions was observed. Time-resolved single-molecule measurements revealed stochastic switching between a highly fluorescent ("on") and a low fluorescent ("off") state. The coordination of the metal ion to the ligand is thus indicated by intramolecular fluorescence quenching of the dye. We screened various fluorescent dyes for their sensitivity to Cu2+-coordination, and found that both Atto620 and MR121 are well-suited for this application. Ensemble studies of the fluorescence lifetimes of metalsensors with Atto620 showed only small dependence on the metal-ion concentration, while single-molecule studies reported strong changes in the fluorescence lifetimes which were correlated with the observed on- and off-states. Our results further indicate that the fluorescence of Atto620 is not completely quenched upon association of the metal-ion complex; either because a less fluorescent complex is formed or because of intramolecular collisional quenching due to conformational changes of the C6-linker used for covalent coupling of the fluorescent dye.