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Title: | 以布朗動態法模擬由正向應力驅動DNA電泳分離 Brownian Dynamics Simulation of Electrophoretic DNA Separation by the Normal Stress Effect |
Authors: | Chao-Chen Kuo 郭朝琛 |
Advisor: | 謝之真(Chih-Chen Hsieh) |
Keyword: | 布朗動態法,DNA分離,電泳,微流道,正向應力, Brownian Dynamics,DNA Separation,Electrophoresis,Normal Stress, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | DNA分離於生物及醫學上的應用非常廣泛,相比傳統方法,透過微流道分離DNA具有便利、快速且成本低廉等特性,如今已成為研究趨勢。我們在過去的實驗裡發現DNA於漸縮-漸擴型通道中漸縮區前方加入圓柱陣列後,DNA產生向通道側壁偏移之現象,在無圓柱陣列之通道則觀察不到此現象。本研究以布朗動態法(Brownian Dynamics)模擬DNA於漸縮-漸擴型微流道的電泳行為,進一步了解DNA於通道中受正向應力遷移之現象,並提出以正向應力驅動DNA電泳分離之微流體通道設計。
我們首先模擬DNA於重覆漸縮-漸擴型通道的電泳行為,並探討圓柱陣列對偏移現象之影響。我們發現在通道中加入圓柱陣列後,掛勾於圓柱上的DNA會受到電場作用而伸展,再加上通過通道漸縮區時,DNA會更進一步受電場梯度作用而拉長,產生明顯的正向應力偏移效果。另外,為了比較電場與流場驅動對DNA遷移效果之影響,我們改以流場驅動DNA後,由於流體動力作用產生空乏層,導致DNA不容易停留於通道兩側牆壁區域,使偏移效果較電場為差。 接著為了詳細研究通道形狀對正向應力之影響,我們分別改變通道漸縮區與漸擴區形狀後,發現透過黛博拉數(De)可以預測不同分子量之DNA在不同形狀參數通道中是否會發生受正向應力偏移現象。若De>1,DNA會受正向應力偏移;若De<1則DNA不受影響,於通道中呈現平均分布。另外,我們利用此結果於重覆漸縮-漸擴型通道前方加入黛博拉數較高之通道使DNA預先產生偏移,能使DNA於重複通道入口處之兩側牆壁注入,更進一步提升分離效果。 此外,一般常用分岔型通道收集來通道中不同側向位置之DNA。而在我們的研究中,受到正向應力偏移之DNA會從靠近通道側壁之位置離開,因此我們透過三岔型通道於側邊通道收集受正向應力遷移之DNA。我們發現無論DNA分子量及電場大小,DNA皆會沿著電力線移動,故可以利用此現象估計分岔型通道收集DNA之效率。也可以透過調控側邊通道與中間通道電壓之比值來調控電力線之分布,能有效控制各分岔通道收集DNA之範圍。 雖然三岔型通道能有效於側邊通道收集受正向應力遷移之DNA,但不受正向應力遷移之DNA則因為布朗運動呈現平均分布,有部分同樣會從側邊通道離開,降低兩種DNA的分離效果。我們模擬利用負介電泳(nDEP)促使不受正向應力遷移之DNA向通道中央集中,並從三岔型通道之中間通道離開,透過控制介電泳大小能夠使受正向應力遷移之DNA仍然從側邊通道離開,進而達到幾乎完全分離之結果。 最後,我們嘗試單純利用正向應力分離多種DNA。為了達成此目的,我們設計連接兩段電場梯度不同之重覆漸縮-漸擴型通道以分離三種DNA。在第一段通道中分子量最大者De>1,受正向應力遷移從側邊通道離開;在第二段通道中,分子量次大者受正向應力遷移從側邊通道離開,分子量最小者則從中間通道離開。雖然側邊通道收集之DNA純度有限,仍可以透過側邊通道收集結果重複進入通道提高DNA純化效果。 由於實驗與模擬添加圓柱陣列後都能觀察到漸縮-漸擴型微流道中DNA受正向應力之偏移現象,希望能幫助預測此種通道實驗的結果趨勢,並提供分離方法設計之方向。 DNA separation and purification are widely used operations in biological and medical analyses. Compared with the traditional methods, DNA separation through microchannels by electrophoresis is more convenient and efficient. In the past experiments, we found that DNA migrated toward the convex wall as it passed through a converging-diverging microchannel. The migration was apparently driven by the normal stress, but it can only be observed when a post array was set at the inlet of the converging channel. In this study, Brownian Dynamics is used to simulate the behavior of DNA in microchannels to further understand the phenomenon. We also used the simulations to verify the design principles for improving the current device. We first investigated how the post array affects the normal stress induced DNA migration. We found that DNA hooked by a post will be stretched by the electric field. Since the normal stress is larger for more stretched DNA, the normal stress induced DNA migration is enhanced due to the presence of the post array. On the other hand, we have also replaced electric field with flow field. Although the normal stress induced migration is still observed, it is less prominent in flow field than in electric field due to the depletion of DNA at the channel walls caused by the hydrodynamic interactions. Second, we changed the shape of the converging and diverging channels to study the influence of the channel shape on the normal stress induced migration of DNA. We found that Deborah number (De) is a good indicator for the onset of the normal stress induced DNA migration. The migration only occurs for De>1 while DNA distributes evenly in the microchannel for De<1. We have applied this principle to design a pre-conditioning channel with higher De set in front of the separation channel. The separation efficiency of the device is clearly improved with the pre-conditioning channel. Third, we considered how to collect DNA samples at the end of the separation channel. In literatures, branched channel is usually employed for this purpose. However, the detailed design of the branched outlet channel could strongly affect the separation results. We found from our simulations that DNA usually moves along the electric field line, despite its molecular weight and the electric field strength. Therefore, we can use a fixed branched outlet channel with tunable applied electric potential in each outlet reservoir to control the electric field lines and therefore to optimize the collection of the separated samples. Although the normal stress induced DNA migration can be used to separate DNA with different sizes, the separation can never be complete. Thus, we simulated a special case that uses negative dielectophoresis (nDEP) to promote DNA moving away from the convex walls of the channel. Since the DEP force and the normal stress are competing, it is possible to find an optimal condition that could give a complete separation for a mixture of two different DNA samples. To conclude, we have successfully simulated the normal stress induced DNA migration in a converging-diverging channel by using Brownian dynamics. The simulations have helped us to understand the phenomenon more deeply. Moreover, we have developed several strategies to improve the separation efficiency of the device. We shall perform experiments to verify these strategies and hopefully invent a better DNA separation method in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7239 |
DOI: | 10.6342/NTU201902684 |
Fulltext Rights: | 同意授權(全球公開) |
metadata.dc.date.embargo-lift: | 2024-08-15 |
Appears in Collections: | 化學工程學系 |
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ntu-108-1.pdf | 9.94 MB | Adobe PDF | View/Open |
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