利用微流體晶片系統即時分析DNA與其轉譯蛋白質之相互關係

Real-time Analysis of DNA Incorporation and Protein translation by Microfluidic Chip System

吳宗鴻(Tsung-Hung Wu)

指導教授 ： 吳立真

國立暨南國際大學/科技學院/應用化學系/碩士(2013年)

摘要

基因表現分析對於早期癌症診斷、治療和藥物傳遞、開發等生物相關研究有很大助益。目前對於定量細胞和組織中的DNA及蛋白質含量其方法有：測量吸光值、二維膠電泳法(2D-gel electrophoresis)、原位聚合脢連鎖反應(In situ PCR)和同位素親和標示(isotope-coded affinity tag，ICAT)等來做定量，但無法同時偵測DNA與其表達之蛋白質含量。因此，本研究發展了一套微流體晶片電泳分析系統來觀測：(1) 細胞轉染的效率。(2) 即時偵測基因與其轉譯蛋白質含量之間的關係。(3) 轉染基因與其轉譯蛋白質兩者之比率。本實驗先利用化學修飾的方式將帶有藍色螢光蛋白(pTagBFP-N)的質體修飾上化學冷光(luminol)，再將其轉染至細胞內(293T cell line)於微流體晶片系統中測得其螢光強度，並可以獲得轉染至細胞內的質體濃度對於其轉譯蛋白質之間的濃度關係。
在微流體晶片的部份採用SU-8光阻液作為母模結構，PDMS作為微流體晶片材料，在完成翻模後會以掃描式電子顯微鏡(SEM)確定其結構是否完整，而PDMS本身的光穿透性和氣體通透性對於螢光偵測和細胞進樣是較佳的環境。本實驗也會探討利用氧電漿系統接合晶片時其灌注氧氣時間、施打氧電漿時間及施加功率所造成之影響，並在接合後，利用微注射幫浦將試劑注入到微流道中，檢查晶片接合後是否有液漏。
本系統利用微流體晶片電泳結合化學冷光修飾，測定基因與蛋白質之間的螢光強度關係之比較，不僅對基因表現分析上有相當大的助益，對生醫領域的發展更有重大的貢獻。

關鍵字

微晶片電泳 ； 聚二甲基矽氧烷 ； 化學冷光 ； 質體 ； 蛋白質表現

並列摘要

Gene expression analysis for the early cancer diagnosis, treatment, drug delivery and drug development play a critical role. Several ways to quantitative the DNA and protein content of cell and tissue have been developed, including absorption measurement, two-dimensional gel electrophoresis, in situ PCR and isotope-coded affinity tag (ICAT) analysis. These methods determinate the changes of protein contents, but can’t detect the level of incorporated DNA contents and the translated protein contents simultaneously. Therefore, we developed a microfluidic chip analysis system to determine the (1) transfection efficiency; (2) real-time determination of the contents of incorporated plasmid DNA and translated protein contents; (3) the ratio of transfected DNA and translated protein.
In this study, we used plasmids encoding blue fluorescent protein (pTagBFP-N) to be modified with chemiluminescence (CL) substance (luminol), and transfected into 293T cell line to determine the fluorescence intensity by microfluidic chip system. The concentrations of incorporated DNA and translated protein were then measured. The microfluidic device using SU-8 photoresist as template structure, polydimethylsiloxane (PDMS) as microfluidic chip material, was subjected to scanning electron microscope (SEM) to examine the structural integrity of microfluidic channel. PDMS holds better transmittance and gas permeability, so that it can be exerted to carry on fluorescence detection and cell cultivation, respectively. The parameters of oxygen plasma system for chip binding, including oxygen filling times, oxygen plasma treatment times, and oxygen plasma treatment power were investigated. Leakage test of the microfluidic channel was also preformed.
This device successfully integrated microfluidic chip system with chemiluminescence modified plasmids to determine the relationship between incorporated DNA and translated protein contents. This research may provide the insights to gene expression analysis, and benefit the field of biomedicine.

並列關鍵字

microfluidic chip ； polydimethylsiloxane ； chemiluminescence ； plasmid ； protein expression

參考文獻

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