以奈米通道陣列結構探討分離不同長度DNA之物理機制

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研究生：	王琮玄 Wang, Chung-Hsuan
論文名稱：	以奈米通道陣列結構探討分離不同長度DNA之物理機制 Studies of the mechanism of sieving DNA by length with nano-channel matrices
指導教授：	周亞謙 Chou, Y.C.
口試委員:	周亞謙 Chou, Y.C. 連振炘 Lien, Chen-Hsin 林彩雲 Lin, T. Y. 黃國貞 Hwang, Gwo-Jen 周家復 Chou, Chia-fu
學位類別：	博士 Doctor
系所名稱：	理學院 - 物理學系 Department of Physics
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	136
中文關鍵詞：	去氧核醣核酸、電泳分析、生醫微積電、奈米通道陣列結構
外文關鍵詞：	DNA, electrophoresis alaysis, Bio-MEMS, nano-channel matrices
相關次數：	點閱：2 下載：0
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本論文研究主軸為利用先進奈米製程技術製作新穎的『人造奈米通道陣列結構』之元件，希望藉由此元件找到一種更為完整的解釋對於篩選不同長度之去氧核醣核酸(DNA)最有效率之方法與機制。
將傳統凝膠內錯綜複雜的結構視為多種大小不同的奈米和微米級通道互相交錯連接，利用去氧核醣核酸之持續長度(DNA-persistence length)及奈米通道孔徑大小之比例不同為出發點，製做三種不同奈米通道陣列結構：(1) 通道寬度和持續長度相當的50奈米通道陣列結構，(2) 通道寬度略大於持續長度的200奈米通道陣列結構，(3) 通道寬度約為持續長度10倍的450奈米通道陣列結構。藉由外加電壓的驅動及DNA在遇到不同寬度的奈米通道時，因為熵的變化及DNA內部彈性位能的改變會有所不同，故DNA在通過奈米通道時會有不同運動模式。
本實驗結果可分為三種：(1) 在50奈米通道陣列結構中，由於彎折DNA-persistence length需要克服其極大的內部彈性位能，此時DNA僅能以直線的型態通過通道，故較短的DNA具有較快的平均遷移速率，(2) 在200奈米通道陣列結構中，由於通道寬度大於DNA-persistence length，加上較長的DNA具有較大的表面積使得其接觸通道的機率增加，在外加電壓的驅動下，較長的DNA擁有較大的平均遷移速率，(3) 在通道寬度遠大於DNA-persistence length的450奈米通道陣列結構中，鬆持型態的DNA因為較短的DNA具有較小的截面積和通道接觸，而有較小的阻力使之具有較大的平均遷移速率。
本實驗不同於現有相關『利用人造結構篩選不同長度之DNA』的文獻在於不僅能觀察到DNA平均遷移率隨其長度增加而增加外亦觀察到相反的情形─DNA平均遷移率隨其長度增加而減少，後者是DNA在傳統凝膠電泳中的現象；在人造結構中能同時觀察到DNA平均遷移速率隨其長度增加而增加或減少是本論文和其它文獻僅能觀察到單一趨勢不同之處，故利用DNA-persistence length及奈米通道孔徑大小之比例不同為出發點，因為在不同比例大小情況下DNA在通過奈米通道時會有不同運動模式，除了藉此成功篩選不同長度之DNA外，本實驗在篩選不同長度DNA的分辨率比目前文獻記載內所使用的人造結構所得之分辨率來的高；本實驗的三種結果亦證明了不同長度DNA之平均遷移率和DNA-persistence length及奈米通道孔徑大小之比例不同而不同，以此觀點出發，提出在傳統凝膠電泳中，不同長度DNA之平均遷移率受通過凝膠中較小孔洞的難易度所影響，說明為何傳統凝膠電泳實驗中觀察到較短的DNA具有較高的平均遷移速率。本實驗以DNA-persistence length及奈米通道孔徑大小之比例不同為出發點，對於解釋在DNA分離技術中提出了一個新的觀點，使得在尋求篩選不同長度DNA最有效率之方法與機制的道路上更邁進一步。

The device with nano-channel matrices was fabricated and used to sieve DNA molecules. Channel matrices, which had been fabricated with the widths of individual nano-channel were 50, 200, and 450 nano-meters. The interval between two adjacent rows of nano-channels varies from 1 μm to 3 μm. Three different kinds of DNA molecules including Plasmid-2.8 kbps (~1 μm), λ-48.5 kbps (~16 μm) and T4-166 kbps (~55 μm) were used in these experiments. It was found that the mobility of shorter DNA molecules were not always greater than that of longer ones. Overall, we could divide the results into three parts. (1) In the width 50 nm channel matrices, the result of this is like the result of gel electrophoresis, which showed the mobility decreased monotonically with the length of DNA molecules. (2) In the width 200 nm channel matrices, the entropic trapping dominated, longer molecules have a higher probability to escape trapping regions due to high successful attacking frequency with the larger contact area, so the mobility increased monotonically with the length of DNA molecules and the result is reverse to the aforementioned result. (3) As different lengths of DNA molecules sieved in the width 450 nm channel matrices, the Ogston mechanism dominated, molecules could pass through the nanochannel without great deformation so the mobility decreased monotonically with the length of DNA molecules. Besides these results, compared with regular micro- and nano-trenches of previous authors' works, DNA molecules electrophoresis in these trenches was just one direction confined, but the motion of DNA molecules was confined in both directions perpendicular to the direction of drift of DNA molecules in our chip. Compared with time-consuming (1~24 hrs) and large sample consumption of conventional methods, gel DNA electrophoresis and Pulsed field gel electrophoresis, DNA molecules could be separated in 10 minutes and low sample consumption by this technique. Thus, this work not only provides a more helpful method for understanding separation processes in gel DNA electrophoresis, but also provides a more efficient method to sieve different lengths of DNA molecules.

中文摘要
Abstract                                                   
誌謝
Chapter 1 - Introduction(p1)                                       
1.1 Sieving mechanisms in gel electrophoresis(p2)                        
1.2 Sieving DNA molecules via artificial structures in previous literatures(p11)  
1.3 The concepts of this thesis(p24)                                    

Chapter 2 - Device fabrication processes and measurement instrument setup(p26) 
2.1 Design overview(p27)                                           
2.2 Accomplished nano-channel array(p36)                            
2.3 PSQ bonding method(p41)                                      
2.4 Sample preparation(p45)                                       
   2.4.1 DNA diluting and dyeing(p45)                               
   2.4.2 DNA sample protection and storing(p46)                      
2.5 Experimental setups(p48)                                      

Chapter 3 - Results and discussion(p56)                             
3.1 Sieving DNA molecules in 450 nm nano-channel array(p57)           
3.2 Sieving DNA molecules in 200 nm nano-channel array(p68)         
   3.2.1 The case of 200 nm nano-channel array with 1500 nm spacing(p68)   
   3.2.2 The case of 200 nm nano-channel array with 900 nm spacing(p81)    
3.3 Sieving DNA molecules in 50 nm nana-channel array(p86)        
3.4 Conclusion(p92)                                         

Chapter 4 - Summary(p95)                                   

Appendices                                                
Appendix A - Introduction of Debye length and electroosmosis(p98)      
Appendix B - Fabrication processes of the device(p102)                 
Appendix C - Optic proximity correction (OPC) effect(p128)              
Appendix D - Sample preparation                             
   D-I  Diluting DNA solution and dyeing DNA(p131)                  
   D-II  Steps of adding the protecting solution 
and PVP to stained DNA molecules(p133)                     

References(p134)                                                 


                                

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