零模波导中Alexa染料间的长程单分子Förster共振能量转移
Long-range Single Molecule Förster Resonance Energy Transfer Between Alexa Dyes in Zero-Mode Waveguides
摘要：
提出了在金属膜中研磨零模波导（ZMW）纳米孔以提高FRET效率并使单分子FRET检测超过10 nm势垒，克服了在均匀介质中衍射限制检测的限制。然而，早期的ZMW演示仅限于Atto 550 - Atto 647 N荧光团对，询问FRET增强观察是否是与这组特定荧光染料相关的人工产物。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-受体分离处的单分子FRET。这些Alexa荧光染料与Atto荧光染料相比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，FRET效率增强，扩展了FRET可用的空间范围，并证实了早期的结论。通过显示ZMW内部的FRET增强不依赖于荧光染料的集合，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一步，同时保留其在常规荧光染料对上工作的能力。
英文摘要：
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.
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