本文選題：液滴微流控芯片 + 軟光刻　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：真菌感染的日趨嚴(yán)重以及耐藥菌株的不斷出現(xiàn)對抗真菌新藥需求日趨增長。傳統(tǒng)孔板法已經(jīng)不能滿足抗真菌藥物活性分析的高通量和高內(nèi)涵。液滴微流控芯片作為微流控芯片的一個(gè)重要分支,具有分析速度快、靈敏度高、試劑消耗少、生物相容性高、無交叉污染、高通量、微型化、集成化、自動(dòng)化等特點(diǎn),可以在短時(shí)間內(nèi)分析大量的藥物參數(shù)和指標(biāo),加快篩選進(jìn)程。傳統(tǒng)孔板法需要將菌落孵育到一定濃度才能進(jìn)行檢測,常需數(shù)天時(shí)間。液滴作為微反應(yīng)器,包裹真菌細(xì)胞即可獲得較高的菌落濃度,大大減少真菌細(xì)胞孵育時(shí)間。液滴微流控芯片技術(shù)已逐漸應(yīng)用于藥物活性研究并凸顯優(yōu)勢,很有可能取代孔板,成為下一代主流的抗真菌藥物活性篩選平臺。本論文以具有良好研究基礎(chǔ)的抗真菌藥物為研究對象,以液滴微流控芯片平臺為手段,構(gòu)建抗白念珠菌藥物篩選的液滴微流控芯片系統(tǒng),初步實(shí)現(xiàn)細(xì)胞水平的抗真菌藥物活性分析。本文分為三個(gè)部分展開:首先,本文設(shè)計(jì)了多種通道結(jié)構(gòu)二維圖形,采用軟光刻法以聚二甲基硅氧烷(PDMS)為材料制作了大量的液滴微流控芯片,詳細(xì)討論了液滴微流控芯片的通道圖形設(shè)計(jì)和制作流程的優(yōu)化,為自行建立基于液滴微流控芯片的藥物篩選平臺打下基礎(chǔ),為實(shí)驗(yàn)室今后工作制訂較為通用的操作規(guī)程。其次,本文自主構(gòu)建了用于抗白念珠菌藥物篩選的液滴微流控芯片平臺,考察了自制液滴微流控芯片的性能,優(yōu)化了液滴生成和收集的條件,為下一步藥物篩選應(yīng)用做好準(zhǔn)備。以含1%EA-surfactant的HFE-7500為連續(xù)相、以RPMI 1640培養(yǎng)液為分散相生成了大小均一、單分散性良好的液滴。離心管收集液滴室溫靜置5天仍保持形狀穩(wěn)定。最后,進(jìn)行了基于液滴微流控芯片的抗白念珠菌藥物篩選初步探索。采用液滴生成芯片、液滴生成和直通道收集芯片分別進(jìn)行了抗真菌藥物篩選初步試驗(yàn),選擇了阿爾瑪藍(lán)作為終點(diǎn)指示劑,考察了芯片外孵育與芯片上孵育的可行性,根據(jù)試驗(yàn)結(jié)果選定了芯片外孵育的方法作為試驗(yàn)方法,最后進(jìn)行了4種已知陽性藥物和新藥(艾迪康唑)的活性篩選,并用孔板法進(jìn)行驗(yàn)證,初步證明自行設(shè)計(jì)構(gòu)建的液滴微流控芯片平臺可用于抗真菌藥物篩選,并且具有分析檢測速度快、試劑消耗量少的優(yōu)點(diǎn),為進(jìn)一步利用液滴微流控芯片技術(shù)開展高通量、高內(nèi)涵抗真菌藥物活性篩選提供技術(shù)支撐。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R91

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