突破衍射极限的针尖增强拉曼显微镜在细菌生物被膜无标记、高灵敏检测中的应用

Biofilm formation is governed by nanometer-sized structures with different chemical compositions. A detailed information of surface topography as well as chemical compositions of extracellular polymeric substance (EPS) play a crucial role for illustrating the mechanism of biofilm formation and decoding the antibiotic resistance. Highly sensitive, selective and high spatial resolution tip-enhanced Raman spectroscopic technique hopefully provides a novel approach for biofilm research without the interference of water. This project attempts to build up a near-field scanning optical microscopy based on a tuning fork shear-force feedback mechanism on basis of a commerical inverted confocal Raman microspectrometer. Finally, it will be a home-built multifunctional tip-enhanced Raman scattering microscopy capable of 3D scanning, which provides topograpgyic information at ca. 1 nm spatial resolution and optical information at a resolution down to ca. 10 nm. Sodium alginate fibers, a good model for EPS of biofilms at the nanoscale, will be studied by coventional Raman, surface-enhanced Raman and tip-enhanced Raman spectroscopy. Combining and comparing all the results based on these three techniques will result in a novel methodology for label-free characterization of nanostructures in biological system utilizing tip-enhanced Raman spectroscopy. On this basis, and gradually, label-free description of mono- and mutli-species (some model bacteria such as Pseudomonas Aeruginosa, Escherichi coli and etc) biofilms with high sensitivity could be accomplished on a scale of a few nanometers. More significantly, tip-enhanced Raman microscopy has seen the emergence of a powerful technique for characterizing other nanostructure of biological system with ultrahigh spatial resolution.

在纳米尺度上同时描述胞外多聚物的表面形貌和化学异质性对于揭示细菌生物被膜成膜机理，进而有效破解细菌抗药性至关重要。高灵敏、高选择性、高空间分辨和无水干扰的针尖增强拉曼光谱分析技术可望为其提供一种新的研究手段。本项目拟在商品化的"倒置型"共聚焦显微拉曼光谱仪上搭载基于音叉"剪切力反馈"机制的近场扫描探针显微镜，构建可实现三维高精度扫描成像的针尖增强拉曼散射显微镜（表面形貌与光学分辨率分别为1 nm和10 nm）。以海藻酸钠纤维作为胞外多聚物的模拟物，同时获取其普通拉曼、表面增强拉曼和针尖增强拉曼光谱数据，并进行充分的谱图比对、解析，以此建立起针尖增强拉曼光谱无标记检测生物体系中纳米结构的新方法。在此基础上，以铜绿假单胞菌等模式细菌为研究对象，运用上述方法逐步实现单株和多株细菌生物被膜的无标记、高灵敏成像检测，为细菌生物被膜乃至其他具有超精细结构的生物体系提供强有力的研究手段。

本项目开发出了包括普通拉曼光谱、表面增强拉曼光谱标记和无标记生物被膜成像技术，在此基础上，为实现细菌生物被膜表面精细结构与化学信息的可视化，研发出基于小分子介导的TERS成像新技术和相关装置。合成了一系列含叁键的拉曼信号分子，建立了基于叁键编码的传感分析原理和多靶向成像技术；更重要的是，首次提出了一种全新的光学信号输出技术，点击SERS；此外，通过目标物诱导来可控调节贵金属纳米粒子的聚集状态，从而改变生物（模拟）体系的光学性质的变化（包括SPR或SERS信号等），藉此来实现一系列生物分子及金属离子等目标物的高选择性和高灵敏检测。其四，设计制备了一系列不同组成、形状和尺寸的贵金属纳米粒子及其复合纳米材料，以其作为SERS增强基底，直接检测有机小分子、生物大分子及微生物细胞。.此外，在国际交流与合作上，项目主持人沈爱国教授先后出访澳大利亚、韩国等国家开展国际合作和交流和参加学术会议，并作重要学术报告。在研究生培养上，共计培养博士研究生和硕士研究生24名。受本项目资助，共发表SCI论文22篇，影响因子大于5的SCI论文8篇，其中Journal of the American Chemical Society 1篇，Analytical Chemistry 3篇，ACS Applied Materials & Interfaces 3篇，Chemical Communications 1篇。

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