Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

中文翻译：

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DNA折纸纳米天线热点中的荧光团光稳定性和饱和度。

在光漂白之前，用于单分子光谱的荧光染料可能经历数百万次激发-发射循环。由于等离子体热点中光的高度集中，因此在DNA折纸纳米天线中对荧光染料的条件要求更高。在这里，我们简要地回顾了单分子成像的荧光团稳定的当前状态，并揭示了与等离子体荧光增强相关的其他因素。我们显示，尽管DNA折纸纳米天线提高了单分子荧光团的光稳定性，但它们在等离子热点中的强电场中的性能仍然受到潜在的光物理过程（如形成暗态和光异构化）的限制。这些光物理过程限制了光子计数率，增加异质性并加剧荧光增强因子的定量。这些因素还降低了在生物物理单分子实验中可以达到的时间分辨率。最后，我们展示了如何使用等离激元DNA折纸纳米天线来影响带有荧光团-猝灭剂对的DNA发夹分析的光物理性质，从而导致其在基于荧光的诊断分析中的应用。尤其是，我们表明，此类检测可通过黑暗淬灭剂的过早光漂白产生假阳性结果。我们展示了如何使用等离激元DNA折纸纳米天线来影响具有荧光团-猝灭剂对的DNA发夹分析的光物理性质，从而导致其在基于荧光的诊断分析中的应用。尤其是，我们表明，此类检测可通过黑暗淬灭剂的过早光漂白产生假阳性结果。我们展示了如何使用等离激元DNA折纸纳米天线来影响具有荧光团-猝灭剂对的DNA发夹分析的光物理性质，从而导致其在基于荧光的诊断分析中的应用。尤其是，我们表明，此类检测可通过黑暗淬灭剂的过早光漂白产生假阳性结果。