Ultra Sensitive Single Molecule Spectroscopy With Plasmonic Antennas

描述（由申请人提供）： 这项研究提出在等离子体天线上开发单分子光谱（SMS），以实现1 nm的空间分辨率，1 μ s的时间分辨率和1 mM的工作浓度，比最先进的SMS提高了3个数量级。SMS的发明使得有可能在真实的时间内跟踪单个酶分子的生化反应，并对具有长读段（10-15 kb）的单个DNA分子进行测序。然而，有两个关键障碍阻碍了SMS的广泛使用：1）。浓度屏障。为了检测单个分子，平均而言，要求检测体积内一次不超过一个分子。因此，难以在高于1微摩尔的浓度下进行单分子检测，该浓度超出高微摩尔至毫摩尔范围的生理相关浓度范围。2）。决议 屏障目前单分子弗斯特共振能量转移光谱（SM-FRET）的时空分辨率分别约为4 nm和1 ms，而大多数蛋白酶的动力学是在亚纳米长度尺度和纳秒到微秒的时间尺度上，太小太快以至于SMS无法捕获。该项目提出通过利用最近在等离子体天线方面的突破所带来的极端光操纵能力来打破浓度和分辨率障碍。等离子体激元学是一个蓬勃发展的科学和技术领域，它利用金属纳米结构的表面等离子体来限制、路由和操纵纳米长度尺度的光。在过去的十年中，由于更好的电磁波模拟算法，大量的计算资源和越来越精确的纳米纤维技术，等离子体天线已被证明可以1）相干放大荧光信号，2）抑制单个荧光分子的光漂白和闪烁，以及3）减少背景噪声。因此，天线已被证明可以将荧光信噪比提高多达3个数量级，从而提供了一种打破浓度和分辨率障碍的方法。为了开发这个“天线实验室”平台，本项目将：1）利用计算机模拟、单分子超分辨显微镜和原子力显微镜（AFM）辅助制造技术，合理设计和制造用于高分辨率SMS的等离子体天线; 2）利用新型聚合物辅助方法大规模制造等离子体天线; 3）以DNA分子尺、多肽和腺苷酸激酶为模型，建立单酶瞬态动力学捕获方案。通过利用等离子体的进步，“天线实验室”将成为一种通用而强大的工具，以空间和时间分辨率可视化在单分子水平上工作的酶，这是以前无法实现的。

DESCRIPTION (provided by applicant): This research proposes to develop single-molecule spectroscopy (SMS) on plasmonic antennas to achieve 1nm spatial resolution, 1¿s temporal resolution, and 1mM working concentration, representing improvement by 3 orders of magnitude over state-of-the-art SMS. The invention of SMS has made it possible to follow the biochemical reactions of an individual enzyme molecule in real time, and sequence a single DNA molecule with long reads (10-15 kb). However, two key barriers have hampered the expanded use of SMS: 1). Concentration barrier. To detect a single molecule requires that, on average, no more than one molecule be within the detection volume at a time. Therefore, it is difficult to carry out single-molecule detection at concentrations above 1micromolar, out of the range of physiologically relevant concentrations of high micro- to millimolar range. 2). Resolution barrier. The spatiotemporal resolution of current single-molecule F¿rster resonance energy transfer spectroscopy (SM-FRET) is about 4nm and 1ms, respectively, whereas the dynamics of most protein enzymes is on a sub-nanometer length scale and nano- to microsecond time scale, too small and too fast for SMS to capture. This project proposes to break the concentration and the resolution barriers by harnessing the extreme light manipulation power from recent breakthroughs in plasmonic antennas. Plasmonics is a flourishing field of science and technology that exploits the surface plasmon of metallic nanostructures to confine, route, and manipulate light at the nanometer length scale. In the past decade, thanks to better electromagnetic wave simulation algorithms, massive computational resources, and increasingly precise nanofabrication techniques, plasmonic antennas have been demonstrated to 1) coherently magnify fluorescent signals, 2) suppress photo-bleaching and blinking of single fluorescent molecules, and 3) reduce background noise. As a result, the antennas have been shown to enhance the fluorescence signal-to-noise ratio by up to 3 orders of magnitude, offering a means to break the concentration and resolution barriers. To develop this "lab-on-antennas" platform, this project will: 1) use computer simulation, single-molecule super-resolution microscopy, and atomic-force microscopy (AFM) assisted fabrication to rationally design and fabricate plasmonic antennas for high-resolution SMS; 2) fabricate plasmonic antennas on a large scale with novel polymer-assisted methods; and 3) use DNA molecular ruler, polypeptides, and adenylate kinase as models to establish the protocols of capturing transient dynamics of a single enzyme. By leveraging the advance of plasmonics, "lab-on-antennas" will be a general and powerful tool to visualize enzymes working at the single-molecule level with spatial and temporal resolution that has been previously unattainable.