結合流電場與基材表面結構於脂雙層上拉伸DNA之研究

Research of DNA Stretching on Lipid Bilayer by Combining Flow and Electric Field with Different Surface Structure

范子軒(Tzu-Hsien Fan)

指導教授 ： 謝之真

國立臺灣大學/工學院/化學工程學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803380

摘要

我們開發出操作簡易、成本低的新型DNA基因圖譜平台，其原理為在具有週期性溝槽的基材表面鋪上帶有正電荷的脂雙層後，帶負電的DNA會自發性的吸附在脂雙層上，又由於脂質本身的立體結構與彎曲曲面的幾何效應使得溝槽處有位能井之存在，使得DNA於此處能感應更多的正電荷，而自發性的吸附在溝槽處並沿溝槽夾角延展成一直線。因此我們能直接以螢光標定DNA的特定序列，以缺口標定法產生基因圖譜。
　　目前我們使用λ(48500 bp)與T4GT7 DNA(165647 bp)來研究脂雙層拉伸法，λ DNA自發性展開所需時間約為半小時，而T4則需6~8小時。為了進一步加速DNA自發性展開，我們嘗試以流場及電場幫助DNA更快的接觸位能井並幫助DNA展開。除了週期性溝槽的圖案玻片，我們還設計了具有類橢圓柱陣列之圖案玻片，希望利用DNA電泳時若被圓柱勾到會拉伸的現象與脂雙層能維持DNA拉伸型態的特性來幫助DNA拉伸。我們也嘗試以thiol-PEG在玻片表面產生無法吸附脂雙層的不沾黏層，進而產生帶狀脂雙層結構，以立體障礙配合流電場，以期能加速DNA伸展。為了能使平台進一步簡化，我們也嘗試以APTES與Ni2+對玻片表面改質使玻片帶正電，以取代對環境敏感度大的脂雙層。
　　實驗結果顯示，在以電場幫助DNA拉伸的過程中，理論上位能井應該足夠深，讓DNA不易掙脫出位能井，但實際操作電場會使DNA脫離位能井，還會造成水的電解，嚴重影響溶液pH值與離子強度，使DNA的螢光變暗，最終變質。流場雖能驅動溶液中的DNA，但吸附於脂雙層表面的DNA卻幾乎不受流場影響。以流電場幫助脂雙層上DNA拉伸的實驗結果不如預期，還需要進一步的改良。而以APTES與¬Ni2+改質之玻片表面雖能吸附DNA，卻讓DNA如靜止般固定在表面，反觀吸附於脂雙層之DNA則因脂雙層之流動性而有明顯布朗運動，因此作為吸附DNA的基材，脂雙層還是較適合的選擇。而在thiol-PEG改質之玻片上鋪上帶狀脂雙層後，我們發現DNA之形態與遷移率有異常，透過超音波洗淨光阻與改變氧電漿處理玻片之時機，DNA之狀態雖有改善，但仍然是有遷移率異常的現象。雖然實驗結果不如預期，但我們更深入了解了這個系統的潛在問題，要如何避免這些問題還必須進一步的研究。

關鍵字

流場 ； 電場 ； 脂雙層 ； DNA拉伸

並列摘要

We have recently developed a simple and low-cost DNA gene mapping platform that is merely a grooved glass slide covered with cationic lipid bilayers. Negatively charged DNA can adsorb and spontaneously extend along the grooves due to the existence of an electrostatic energy well originated from the geometric effect of the groove structure and steric effect of the lipid. After DNA extends, we can readily obtain the physical gene map by using nick-translating method.
　　We have used λ (48500 bp) and T4GT7 DNA (165647 bp) to test the platform, but found the time needed for DNA extension grows drastically with DNA size. For example, it takes about 0.5 hour for λ DNA to extend, but takes 6~8 hour for T4GT7. To facilitate the process, we first employed electric and flow field to help DNA to fall into the energy well and to extend faster. In addition to periodic groove pattern slides, we have also designed the ellipse-like column array. It is hoped that when the DNA is driven by electric or flow field, it will collide the posts and extend along the groove. We also used thiol-PEG to create a non-adhesive layer for lipids on the slide surface and expected the physical barrier can foster DNA extension in conjunction with applied electric or flow field. In order to simplify the platform, we also tried to modify the surface of the slide with APTES and Ni2+ to replace the environmentally sensitive lipid bilayers. We also tried to use thiol-PEG to treat the surface of the slide to form a non-stick layer for lipids.
The experimental results show that assisting DNA to extend by electric field will cause it leaving the energy well, against our theoretical prediction that the energy well should be deep enough to prevent this situation to happen. Moreover, applying electric field causes electrolysis of water and seriously affects the pH value and ionic strength of the solution, eventually quenching the fluorescence of DNA. Furthermore, DNA adsorbed on the surface of the lipid bilayer can hardly be driven by the flow field. To summary, accelerating DNA extension on the lipid bilayers with either flow or electric field are not working as expected. On the other hand, the surface of the slide modified with APTES and Ni2+ can adsorb DNA, but DNA looks immobile on the surface. In contrast, the DNA adsorbed on the lipid bilayer has obvious Brownian motion due to the fluidity of the lipid bilayers. Therefore, lipid bilayers is a more suitable choice for adsorbing DNA for analysis. After laying lipid bilayer on the thiol-PEG modified slide, we found that both the morphology and mobility of the DNA were abnormal. The state of DNA was improved by ultrasonic cleaning photoresist and changing the timing of oxygen plasma treatment. However, the DNA mobility is still much lower than usual. Although the experimental results are not as expected, we have a much deeper understanding of this system. Further research is still needed to improve the platform for practical use.

並列關鍵字

electric field ； flow field ； lipid bilayer ； DNA stretch

參考文獻

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