Regmi Raju, Al Balushi Ahmed A, Rigneault Hervé, Gordon Reuven, Wenger Jérôme
CNRS, Aix Marseille Université, Centrale Marseille, Institut Fresnel, 13013 Marseille, France.
Department of Electrical Engineering, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Sci Rep. 2015 Oct 29;5:15852. doi: 10.1038/srep15852.
Diffraction ultimately limits the fluorescence collected from a single molecule, and sets an upper limit to the maximum concentration to isolate a single molecule in the detection volume. To overcome these limitations, we introduce here the use of a double nanohole structure with 25 nm gap, and report enhanced detection of single fluorescent molecules in concentrated solutions exceeding 20 micromolar. The nanometer gap concentrates the light into an apex volume down to 70 zeptoliter (10(-21) L), 7000-fold below the diffraction-limited confocal volume. Using fluorescence correlation spectroscopy and time-correlated photon counting, we measure fluorescence enhancement up to 100-fold, together with local density of optical states (LDOS) enhancement of 30-fold. The distinctive features of double nanoholes combining high local field enhancement, efficient background screening and relative nanofabrication simplicity offer new strategies for real time investigation of biochemical events with single molecule resolution at high concentrations.
衍射最终限制了从单个分子收集到的荧光，并为在检测体积中分离单个分子的最大浓度设定了上限。为了克服这些限制，我们在此介绍使用具有25纳米间隙的双纳米孔结构，并报告了在浓度超过20微摩尔的浓缩溶液中对单个荧光分子的增强检测。纳米间隙将光集中到低至70 zeptoliter（10^(-21)升）的顶点体积中，比衍射极限共焦体积低7000倍。使用荧光相关光谱和时间相关光子计数，我们测量到荧光增强高达100倍，同时光学态局部密度（LDOS）增强30倍。双纳米孔的独特特征结合了高局部场增强、高效背景筛选和相对简单的纳米制造，为在高浓度下以单分子分辨率实时研究生化事件提供了新策略。
