CNRS, Aix-Marseille Université, Ecole Centrale Marseille, Institut Fresnel, Campus de St Jérôme, 13397 Marseille, France.
Nat Nanotechnol. 2013 Jul;8(7):512-6. doi: 10.1038/nnano.2013.98. Epub 2013 Jun 9.
Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel 'antenna-in-box' platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.
单分子荧光技术在许多应用中是关键的，包括 DNA 测序、分子和细胞生物学以及早期诊断。不幸的是，通过衍射受限的光学观察单分子受到限制，只能检测飞升级别的检测体积，并且需要皮升级或纳升级别的浓度，远远低于大多数生物反应发生的微升级别。这种限制可以通过等离子体纳米结构来克服，等离子体纳米结构能够将光限制到纳米级别的体积。尽管这些纳米天线增强了荧光亮度，但大的背景信号和/或对金属表面的非特异性结合阻碍了在高浓度溶液中对单个荧光分子的检测。在这里，我们引入了一种新型的“天线盒”平台，该平台基于纳米孔内的间隙天线。这种设计结合了荧光信号增强和背景筛选，在微升级别样品浓度和 zeptolitre 级别的检测体积下提供了高的单分子灵敏度（荧光增强高达 1100 倍和微秒传输时间）。如我们使用各种生物分子所证明的那样，天线盒装置可以针对生理相关浓度的单分子荧光研究进行优化。
