论文标题
远程单分子förster共振能量转移零模式波导中的Alexa染料之间
Long-range Single Molecule Förster Resonance Energy Transfer Between Alexa Dyes in Zero-Mode Waveguides
论文作者
论文摘要
提出了在金属膜中铣削的零模式波导（ZMW）纳米蠕虫，以提高FRET效率，并在10 nm屏障之后实现单分子FRET检测，从而克服了均质培养基中衍射受限检测的限制。但是，较早的ZMW演示仅限于Atto 550 -Atto 647N荧光团对，询问了一个问题，即FRET增强观察是否是与这组特定的荧光染料相关的人工制品。在这里，我们使用Alexa Fluor 546和Alexa Fluor 647来研究ZMW内部超过10 nm的大供体 - 受体分离的单分子方格。与Atto对应物相比，这些Alexa荧光染料具有明显不同的化学结构，表面电荷和疏水性。我们在Alexa 546 -Alexa 647上的单分子数据表明，在超过10 nm的大分离下，有点效率提高，扩展了可用于FRET的空间范围，并确认了早期的结论。通过证明ZMW内部的FRET增强不取决于荧光染料的集合，该报告是建立ZMW的相关性以扩展FRET的灵敏度和检测范围的重要步骤，同时保留其在常规荧光染料对上工作的能力。
Zero-mode waveguides (ZMW) nanoapertures milled in metal films were proposed to improve the FRET efficiency and enable single molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550 - Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artefact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single molecule FRET at large donor-acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546 - Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.
