单粒子拉曼读出的编码微球对宫颈癌高危病毒基因组的同步检测

High-risk human papillomavirus (HPV) infection is the direct index of the early diagnosis of cervical cancer, it is urgent to develop a rapid and simple method for the simultaneous identification and detection of different types of HPV in clinical practice. In this program, we'll explore single-particle Raman readout and the encoded method to achieve the simultaneous detection of different types of HPV: (1) designing Au and Ag multilayered shell nanostructures with hollow interiors and the method for assembling the Raman tag molecules at the interlayers, explaining the relationship of electromagnetic coupling and the hot-spot enhancement by controlling the thickness and the gap of the multilayered shells, preparing the nanotags with highly dense hot spots, strong and uniform single-particle Raman readout; (2) exploring the method for the encoding of multilayered shell nanoparticles with Raman-active molecules and the modification of oligonucleotide sequences, and verifying the selectivity and sensitivity to the target HPV and the uniform readout of the nanotags; (3) designing the sandwiched structures of magnetic immobilization capture-HPV nucleic acid-labeled nanoprobe based on the selective separation of magnetic nanoparticles, achieving the simultaneous detection of different types of HPV by the fingerprint recognition of encoding molecules, and establishing the relationship and its recognition mode between the Raman-encoded signals and the qualitative and quantitative analysis of HPV. The results of the program will be expected to provide a new way for the early diagnosis of cervical cancer.

高危型人乳头瘤病毒（HPV）感染是宫颈癌早期诊断的直接指标，临床上急需对不同基因型别HPV进行同步鉴定和检测的快速简便方法。本项目拟探索单粒子拉曼读出和编码的方法实现不同基因型别HPV的同步检测：（1）设计Au和Ag复合多层壳空心纳米粒子和拉曼标签分子在壳层间的组装方法，通过多层壳厚度和间隙的调控，阐明电磁耦合和热点增强之间的关系，合成出具有高密度热点、强信号输出且强度一致的单粒子拉曼读出纳米标签；（2）探索拉曼活性物质对复合多层壳空心纳米粒子进行编码及寡核苷酸修饰方法，验证拉曼纳米标签对目标HPV的选择性和敏感性及标签信号输出的一致性；（3）设计基于磁性粒子选择性分离的"磁性固相载体-HPV核酸-标记探针"三明治检测结构，通过编码分子的指纹识别实现对不同基因型别HPV的同步检测，建立拉曼编码信号与HPV的定性定量对应关系及其识别模式。本项目的预期成果将为宫颈癌的早期诊断找到一条新的途径。

本项目针对表面增强拉曼散射（SERS）技术在生物检测和成像中的应用需求，重点克服其重复性差和无法定量的难题，设计和制备了高活性的SERS纳米探针，实现了对痕量生物分子及肿瘤细胞的检测和成像分析。主要发现点和成果如下：（1）制备了单粒子拉曼读出的纳米探针（包括金纳米空心球、多层壳编码微球和银包金纳米粒子），实现了对细胞内抗癌药物以及自由基的超敏感检测和成像；（2）构建了银包金/氮化碳（Au@Ag/ g-C3N4）复合结构的SERS纳米探针，实现了对痕量肿瘤细胞的富集和诊断；（3）发展了SERS芯片组装和试纸印刷的方法，为快速、现场和廉价的生化分析提供了新手段。上述研究工作取得了多项具有创新性和系统性的研究成果，部分研究工作已经在国际期刊Anal. Chem.，Chem. Commun.和RSC Adv.等发表论文7篇。此外，还获得授权国家发明专利2项。