活体肿瘤微环境复杂组分的实时在线及组学分析方法学研究

In vivo analysis, real-time on-line analysis and omics analysis are the emerging research areas of analytical chemistry. There is no doubt about the significance of developing methods which simultaneously achieve the analysis of all the three above requirements. This work carries out research on the important methodology issue, and plans to build a microdialysis combined with mass spectrometry technology, to realize in vivo, real-time on-line and omics analysis at the same time. In this work, we will investigate the various factors influencing the hyphenated method, including the factors affecting the recovery rate of microdialysis technology, the interface of the hyphenated method, etc. We propose to use ultra-low velocity microdialysis sampling technique to improve recovery, and build a stable and easy to use and durable nano-electrospray ionization (nano-ESI), nano-glow discharge ionization (nano-GDI) and MALDI ion source as interface of hyphenated method, and combine with high resolution and multistage mass spectrometry technology, to realize the real-time on-line and omics analysis with high sensitivity of the complex components from in vivo microdialysis sampling. The system will be used in the study of the metabolites of the tumor microenvironment. The implementation of this project is expected to provide methodology for in vivo study and to obtain a large number of real-time data in physiological and pathological conditions, which would lay a solid foundation for the understanding of biological events.

活体分析、实时在线分析、组学分析是分析化学新兴的研究领域，发展同时满足以上分析要求的方法，对于促进分析化学的发展，有着重大的意义。本课题针对这一重要的方法学问题开展研究，拟构建微透析结合质谱联用技术，同时实现活体复杂组分的实时在线及组学分析。工作中，我们将解决联用技术目前面临的诸多问题，包括微透析技术的回收率、联用技术的接口等。我们拟使用超低流速微透析取样技术提高微透析回收率。我们将研究并构建稳定、易用及耐用的纳升电喷雾电离、纳升辉光放电电离及MALDI离子源作为联用技术的接口，并结合高分辨及多级质谱技术，实现活体微透析取样中复杂组分的高灵敏度实时在线及组学分析。该体系将用于卵巢癌肿瘤微环境中复杂组分的研究。本课题的实施将为活体研究提供方法学，用以获取活体生理病理状态下大量的实时数据，为理解生物事件奠定基础。

微透析结合质谱联用技术，是有望同时实现活体、实时在线、组学分析要求的方法。但是，微透析结合质谱联用技术同时实现这些分析要求，尚需解决微透析技术回收率低的问题、联用技术的接口问题、多种组分的高灵敏度定性及定量分析问题。本课题针对这些方法学问题开展了一系列研究工作，包括构建了基于金属有机骨架化合物（Metal organic Frameworks，MOFs）、开放式活体微透析的新方法提高微透析对脂溶性物质的回收率；构建了文丘里环境喷雾、三层法电喷雾、Co-NC辅助的激光解吸电离源作为活体微透析及质谱联用的新接口，实现了活体微透析液中多组分的实时、在线、灵敏分析；并基于建立的方法，开展了大量的应用研究，取得了一系列成果，包括线监测鼠肝受到CCl4诱导的肝损伤活体透析液中的不同代谢物,发现葡萄糖被逐步上调，乳酸逐步下调。这些研究为活体研究提供方法学，用以获取活体生理病理状态下大量的实时数据，为理解生物事件奠定基础。活体微透析样本成分复杂，相应的质谱数据众多，对这些数据的深入挖掘，探讨其生物学意义，并进行验证，值得进一步研究。