微流控芯片与质谱联用进行单细胞核苷代谢轮廓分析研究

The study of single cell metabolomics is very useful for the eary diagnosis of disease and disease progress monitoring. It has very significant higher academic and application values. However, the conventional analytical methods is difficult for single cell manipulation, and the trace amounts of metabolites in single cells are easy to loss during the sample preparation process, which can lead the detection sensitivity largely reduced. Therefore, this research project is willing to establish the microfluidic and mass spectrometry combination system to conquer the above disadvantages, and takening the single cell metabolomic profiling of nucleosides as an example to research. In this project, a polydimethylsiloxane/glass microchip was fabricated using microfabrication technology, which contained single cells manipulation, immobilization and lysis unit and microsolid phase extraction unit. According to the structure of nanoESI source, a fused-silica capillary integrated with monolith was used to connect microfluidic device with mass spectrometry. It could realize the extraction and concentration, separation and online detection of metabolites of single cells, and the nucleoside metabolomic profiling spectra of single cells could be finally obtained. Besides, the metabolomics data analysis techniques were applied for the structure idenfication of nucleosides. Several parallel functional units could be integrated on a single platform for highthroughput single cell nucleoside metabolic profiling analysis, which could largely improve the analysis efficiency and reduce costs.

单细胞代谢组学研究有助于疾病的早期诊断和疾病进程的监测，具有十分重要的学术意义和应用价值。但是常规的分析方法对单细胞难于操纵，且样品预处理过程会导致单细胞内微量的代谢物部分丢失，极大地降低检测灵敏度。因此本项目建立微流控芯片与质谱联用的新技术来克服上述缺点，并以单细胞核苷代谢轮廓分析为例开展研究。本项目利用微加工技术在聚二甲基硅氧烷/玻璃复合芯片上制做用于单细胞操纵、固定、溶膜、微固相萃取柱等功能单元，并根据nanoESI源的结构特点直接用集成有整体柱的毛细管将微流控芯片与质谱连接，实现单细胞内代谢物的提取、富集、分离以及在线质谱检测，获得单细胞核苷代谢轮廓谱图。此外，还与代谢组学的数据分析技术相结合实现核苷的结构鉴定。该研究平台还可以通过集成多个平行的功能单元实现高通量的单细胞核苷代谢轮廓分析，极大地提高分析效率，并降低成本。

单细胞代谢组学研究有助于疾病的早期诊断和疾病进程的监测，具有十分重要的学术意义和应用价值。但是常规的分析方法对单细胞难于操纵，且样品预处理过程会导致单细胞内微量的代谢物部分丢失，极大地降低检测灵敏度。因此本项目拟通过选择微流控芯片制作材料；对其微通道结构、尺寸设计及加工制作；建立微流控芯片与MS联用体系；细胞的培养和药物对肿瘤细胞的刺激；微流控芯片技术对试剂和样品的操纵；样品预处理及条件优化等步骤，最终实现单细胞内核苷的代谢轮廓分析，并对潜在的核苷标记物进行生物验证。在本项目实时过程中我们设计了多种微通道结构以期实现单细胞的高效率捕获，主要包括具有鞘流微通道的微结构、微坑微阵列以及微柱微阵列，研究结果发现微坑微阵列和微柱微阵列的微流控芯片均能实现单细胞30%-40%的捕获。在单细胞微阵列的检测方面，我们将微流控芯片与nanoESI-MS和MALDI质谱分别进行了联用，结果发现nanoESI-MS由于有溶剂稀释效应，无法检测到单细胞含量极低的内源性小分子，且分析通量较低，而将微流控芯片与MALDI MS联用时，通过利用常见的MALDI基质或设计合成结构新颖的MALDI基质，并对这些基质或基质组合进行优化，同时对基质的喷涂方式进行优化，最终可以检测到细胞内12种脂类物质，且MALDI MS可以实现单细胞微阵列的自动化高通量检测。利用我们搭建的微流控芯片与MALD MS联用平台进行单细胞内源性代谢物分析，能极大提高分析效率，快速高效的发现单细胞间的显著差异代谢物，有助于疾病的早期诊断和预警，具有十分重要的科学意义和临床应用价值。