基于液滴微流控芯片的抗白念珠菌药物筛选平台初步构建及应用研究

发布时间：2018-05-03 01:30

本文选题：液滴微流控芯片 + 软光刻　；参考：《第二军医大学》2017年硕士论文

【摘要】：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。液滴微流控芯片作为微流控芯片的一个重要分支,具有分析速度快、灵敏度高、试剂消耗少、生物相容性高、无交叉污染、高通量、微型化、集成化、自动化等特点,可以在短时间内分析大量的药物参数和指标,加快筛选进程。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。液滴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,成为下一代主流的抗真菌药物活性筛选平台。本论文以具有良好研究基础的抗真菌药物为研究对象,以液滴微流控芯片平台为手段,构建抗白念珠菌药物筛选的液滴微流控芯片系统,初步实现细胞水平的抗真菌药物活性分析。本文分为三个部分展开:首先,本文设计了多种通道结构二维图形,采用软光刻法以聚二甲基硅氧烷(PDMS)为材料制作了大量的液滴微流控芯片,详细讨论了液滴微流控芯片的通道图形设计和制作流程的优化,为自行建立基于液滴微流控芯片的药物筛选平台打下基础,为实验室今后工作制订较为通用的操作规程。其次,本文自主构建了用于抗白念珠菌药物筛选的液滴微流控芯片平台,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。最后,进行了基于液滴微流控芯片的抗白念珠菌药物筛选初步探索。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,根据试验结果选定了芯片外孵育的方法作为试验方法,最后进行了4种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,初步证明自行设计构建的液滴微流控芯片平台可用于抗真菌药物筛选,并且具有分析检测速度快、试剂消耗量少的优点,为进一步利用液滴微流控芯片技术开展高通量、高内涵抗真菌药物活性筛选提供技术支撑。
[Abstract]:The demand for new antifungal drugs is increasing with the increasing severity of fungal infection and the emergence of drug-resistant strains. The traditional pore plate method can not meet the high throughput and high content of antifungal activity analysis. As an important branch of microfluidic chips, droplet microfluidic chips are characterized by high analytical speed, high sensitivity, low reagent consumption, high biocompatibility, no cross contamination, high throughput, miniaturization, integration, automation, etc. A large number of drug parameters and indicators can be analyzed in a short time to speed up the screening process. The traditional orifice plate method needs to incubate the colony to a certain concentration to detect, which often takes several days. Liquid droplets can be used as microreactors to encapsulate fungal cells and obtain higher colony concentration, which greatly reduces the incubation time of fungal cells. Droplet microfluidic chip technology has been gradually applied to the study of drug activity and highlights its advantages, which is likely to replace the pore plate and become the next mainstream screening platform for antifungal activity. In this paper, a droplet microfluidic chip system for the screening of anti-Candida albicans drugs was constructed by using droplet microfluidic chip platform and antifungal drugs as the research object. Preliminary analysis of antifungal activity at cell level was carried out. This paper is divided into three parts: firstly, this paper designs two dimensional patterns of various channel structures, and makes a large number of droplet microfluidic chips by soft lithography using polydimethylsiloxane (PDMS) as the material. The channel graphic design and fabrication process optimization of droplet microfluidic chip are discussed in detail, which lays the foundation for the establishment of a drug screening platform based on droplet microfluidic chip, and provides a general operating procedure for laboratory work in the future. Secondly, the platform of droplet microfluidic chip for drug screening of Candida albicans was constructed, the performance of self-made droplet microfluidic chip was investigated, the conditions of droplet generation and collection were optimized, and the preparation for drug screening and application in the next step was made. HFE-7500 containing 1-surfactant was used as continuous phase and RPMI 1640 as dispersed phase to form droplets with uniform size and good monodispersity. Centrifuge tube collection droplets at room temperature remain stable for 5 days. Finally, the screening of anti-Candida albicans drugs based on droplet microfluidic chip was preliminarily explored. Using droplet generation chip, droplet generation chip and direct channel collection chip, the screening experiments of antifungal drugs were carried out respectively. Almaran was selected as the terminal indicator, and the feasibility of incubation outside the chip and on the chip was investigated. According to the experimental results, the method of extracellular incubation was selected as the test method. Finally, the activity of four known positive drugs and new drugs (Aidiconazole) were screened and verified by the pore plate method. It is preliminarily proved that the self-designed droplet microfluidic chip platform can be used for the screening of antifungal drugs, and has the advantages of fast analysis and detection, less reagent consumption, and high throughput for the further use of droplet microfluidic chip technology. Screening of high-content anti-fungal drugs provides technical support.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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