基于微流控液滴的表面增强拉曼光谱DNA检测研究

Nucleic acid analysis is of great significance in the life sciences, and the development of life sciences also continue to place higher demands on nucleic acid analysis. This project aims to develop novel methods for trace DNA analysis with no need to label, easy operation, good reproducibility, high sensitivity and integration, by combining microfluidic droplets and surface-enhanced Raman spectroscopy (SERS). Gold nanorods with small batch difference and uniform morphology will be prepared based on droplet micro-reactors, and then SERS strategies for DNA analysis will be developed by using gold nanorods as SERS substrate and droplets as micro-carriers. The methods will focus on the followings: 1) A microfluidic droplet-based SERS method for DNA analysis will be established with sample concentrated via droplet shrinking. 2) On-line SERS analysis for the products of droplet-based PCR will be developed, in which gold nanomaterials will assist droplet-based PCR amplification and serve as SERS substrates. 3) A single-cell analysis method will be built based on microfluidic droplets and SERS, which will be employed for measuring the structure and content of the DNA in the nucleus of nasopharyngeal carcinoma cells and for monitoring their apoptosis. These methods will lay foundation for the determination of HIV virus and the diagnosis of nasopharyngeal carcinoma.

核酸分析在生命科学中具有重要的意义，生命科学的发展也不断对核酸分析提出更高的要求。本项目拟将微流控液滴技术和表面增强拉曼光谱（SERS）相结合，建立无需标记、操作简便、重现性好、灵敏度高、集成化的痕量DNA分析新方法。基于液滴微反应器制备批次差异小、形貌均一的金纳米棒；以金纳米棒为SERS基底，液滴为微载体，发展SERS测定DNA的方法。这些方法包括：利用液滴收缩富集样品，建立基于微流控液滴的SERS测定DNA的分析方法；发展在线检测液滴PCR产物的SERS方法，将金纳米材料用于辅助液滴PCR扩增，又用作SERS基底；基于微流控液滴-SERS技术建立液滴内的单细胞分析方法，测定鼻咽癌细胞核内的DNA结构与含量及监测细胞凋亡。这些方法的建立将为HIV病毒检测和鼻咽癌肿瘤诊断奠定基础。

生物医学的发展对核酸等生命物质的检测提出了越来越高的要求。本项目利用微流控液滴和离心微流控技术，制备了均一性好、SERS活性高的Au-Ag2S纳米粒子和封装纳米粒子的凝胶微粒，为SERS检测提供了高质量的基底材料；将微流控液滴技术和SERS检测结合，发展了非标记、灵敏度高、快速的SERS方法，完成了鼻咽癌肿瘤标志物Epstein-Barr病毒DNA的定量检测；将PCR与SERS结合，进一步提高了方法的灵敏度和选择性，实现PCR扩增产物直接检测；采用微阵列芯片封装单细胞，开展细胞凋亡胞内组分的SERS分析，监测了鼻咽癌细胞凋亡过程中DNA等分子的变化。此外，还拓展性地发展了多种肿瘤细胞相关物质的SERS分析方法。本项目研究成果为开发高灵敏度和高重现性的SERS基底提供了新途径，为鼻咽癌等肿瘤疾病的诊断和治疗过程监测提供了方法学基础。