基于微流控芯片结合多维液质联用技术的抗真菌药物活性分析的方法学研究

There is an urgent need for anti-fungal new drugs due to the more serious mycotic infection and the increasing drug-resistant strains. The anti-fungal activity screening using 96-well plates cannot meet the requirement of the high-throughput and high content assays. Recently, the miniaturized and integrated microfluidics has shown great potential in drug activity analysis that it might be a promising substitute for 96-well plates. This project aims to develop the micro-droplet microfluidic chip system in view of Candida albicans and C.elegans. The microdroplet generation,merge and online fluorescent detection were integrated in the device,which would be used for high-throughput activity assay of anti-fungal candidate compounds. Morever, the multiple gradient generator followed by an array of microscale cell or worms culture chambers and on-chip solid-phase extraction columns for sample pretreatment prior to mass analysis was integrated in the device which was fabricated in one single step. The quantitative characterization of the anti-fungal activity and high content screening are to be obtained using an integrated microfluidic device coupled to a mass spectrometer. All work attempts to develop a feasible new methodology for high-throughput activity analysis of anti-fungal candidate compounds, which would be better than the screening with 96-well plates and quantify the activity. This project will provide new measure and technical support for the research and development of anti-fungal new drugs.

真菌感染的日趋严重以及耐药菌株的不断出现对抗真菌新药需求日趋增长。传统孔板法已经不能满足抗真菌药物活性分析的高通量和高内涵。微型化和集成化的微流控芯片技术已逐渐应用于药物活性研究并凸显优势，很有可能取代孔板，成为下一代主流的药物活性筛选平台。本课题拟以具有良好研究基础的抗真菌药物为研究对象，以微流控芯片为载体，从细胞（白念珠菌）和模式生物（线虫模型）两个层面，构建白念珠菌和感染白念珠菌线虫模型的液滴微流控芯片系统，将液滴生成、融合和在线荧光检测等功能集成，实现高通量的抗真菌药物活性在线分析；进而结合多维液质联用技术，将培养、浓度梯度生成、在线荧光检测、生理指标检测、前处理等功能集成，对以上高通量分析获得的新化合物进行活性的定量表征，实现高内涵分析。本项目旨在探索抗真菌药物活性分析的创新方法，建立优于传统孔板法并能实现活性定量表征的微流控芯片系统，为抗真菌新药研究提供新的思路和技术借鉴。

抗真菌新药筛选的紧迫性由于不断出现的耐药菌株和严重的真菌感染日益凸显，传统孔板法已经不能满足抗真菌药物活性分析的高通量和高内涵。液滴微流控芯片技术逐渐应用于药物活性研究，很有可能取代孔板成为未来主流的抗真菌药物活性筛选平台。.本项目成功构建了抗白念珠菌药物筛选的液滴微流控芯片系统，实现细胞水平的抗真菌药物活性分析。考察了自制液滴微流控芯片的性能，优化了液滴生成和收集的条件，为下一步药物筛选应用做好准备。以含1% EA-surfactant的HFE-7500为连续相、以RPMI 1640培养液为分散相生成了大小均一、单分散性良好的液滴。离心管收集液滴室温静置5天仍保持形状稳定。采用液滴生成芯片、液滴生成和直通道收集芯片分别进行了抗真菌药物筛选初步试验，选择了阿尔玛蓝作为终点指示剂，考察了芯片外孵育与芯片上孵育的可行性，最后进行了已知阳性药物和新药（艾迪康唑）的活性筛选，并用孔板法进行验证；传统孔板法需要将菌落孵育到一定浓度才能进行检测，常需数天时间。液滴作为微反应器，包裹真菌细胞即可获得较高的菌落浓度，大大减少真菌细胞孵育时间。证明自行设计构建的液滴微流控芯片平台用于抗真菌药物筛选的可行性，并且具有分析检测速度快、试剂消耗量少的优点。成功构建了白念珠菌感染秀丽隐杆线虫模型，进行线虫液滴微流控芯片的设计与制作，选择0.25%的琼脂糖为悬浮剂，包裹单线虫达30%；设计分选芯片并尝试微注射加样，用活死荧光染料对线虫进行染色，用于荧光检测。我们还进行了新型数字液滴芯片的设计，实现对线虫液滴在平板上的驱动操控；以及采用通道修饰微流控芯片进行肿瘤细胞的捕获与固定，建立在芯片上结合荧光染料进行抗肿瘤药物活性研究的分析方法。本项目研究成果具有较高的学术价值，在抗真菌药物的活性筛选方面有很高的应用价值，有望应用于抗菌药物研发，产生良好的社会影响和经济效益。