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研究生: 黃詠泰
HUANG YANG-TAI
論文名稱: 發展功能性超順磁奈米粒子核酸純化試劑於自動化系統中
Development of the nucleic acid purification kits with functional superparamagnetic nanoparticles in automatic system
指導教授: 陳家俊
Chen, Chia-Chun
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 99
中文關鍵詞: 四氧化三鐵聚乙二醇核甘酸
英文關鍵詞: Fe3O4, PEG, nucleic acid
論文種類: 學術論文
相關次數: 點閱:86下載:9
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  • 本文實驗利用製備具有功能性磁性奈米粒子,以及分離純化所使用分解溶液、洗滌溶液以及析出溶液,利用磁性材料與溶液的搭配使用在純化核酸,並且發展出一套高效率自動化核酸純化試劑,應用於人類血液、病毒以及細胞中,從中萃取純化DNA與RNA。首先我們先合成出具有超順磁性的水相四氧化三鐵奈米粒子,並且加入聚乙二醇(polyethylene glycol, PEG)做混合,讓奈米粒子能夠均勻分散在溶液中,提升純化效率,並且在磁場下能從溶液中離開;接著再製備純化試劑,首先配製具有分解細胞效果的分解溶液(lysis buffer),讓核酸可以裸露出來,由於磁性奈米粒子的表面是帶有微負電荷,因此利用分解溶液中的Guanidine作為架橋,幫助DNA和RNA與磁珠結合,之後配置洗滌溶液(wash buffer),目的是為了將吸附在磁珠上的蛋白質及不純物質,藉由洗滌的步驟,將不純物質去除,最後利用析出溶液的配製,以低鹽調控的方式將吸附於磁珠上的核酸脫附下來。本實驗室的自動化的設備,在施以外加磁場的情況下,不需要離心的動作即可在數十分鐘內完成多組血液樣品、病毒以及細胞中核酸的純化過程,除了能夠在醫療的診斷上節省時間,也能夠節省人力資源,希望這套自動化純化核酸試劑,廣泛應用於疾病的檢測、親子鑑定上。

    In this work, we described a simple and convenient process for the purification of deoxyribonucleic acid(DNA) and ribonucleic acid(RNA) with functionalized polyethylene glycol(PEG) -mixed magnetic nanoparticles. First, the magnetic particles of iron oxide(Fe3O4) were coated with PEG to enhance the adsorptive capcity of nucleic acid. Second, The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, supercunducting quantum interference device. We also prepared solution Kits such as Blood DNA Auto Kit, Tissue DNA Auto Kit, Blood RNA Auto Kit and Cell RNA Auto Kit. The Kits include Lysis buffer, Wash buffer and Elution buffer. The Blood DNA Auto Kit and the Tissue DNA Auto Kit have been used in the separation of DNA from human blood and human tissue, respectively. The Blood RNA Auto Kit and the Cell RNA Auto Kit have been individually used in the separation of RNA from human blood and Mammalian cells. Furthermore, we analyzed the purified productions of DNA and RNA with gel electrophoresis, micro-volume UV-Vis spectrophotometer and polymerase chain reaction. Finally, the results indicate that the magnetic separation can be applied for clinical diagnoses.

    總目錄.........................................................I 圖目錄......................................................VIII 表目錄......................................................XIII 中文摘要..................................................XIV 英文摘要...................................................XV 謝誌.......................................................XVI 第一章 緒論.........................................................1 1-1奈米科學.........................................................1 1-2磁性奈米粒子.........................................................5 1-2-1奈米效應下的磁性物質.........................................................5 1-2-2磁性介紹.........................................................5 1-2-3磁性種類.........................................................6 1-2-4磁滯曲線.........................................................7 1-3磁性奈米粒子在生物醫學的應用........................................................11 1-4 去氧核糖核酸與核糖核酸簡介........................................................16 1-4-1去氧核糖核酸........................................................16 1-4-2核糖核酸........................................................19 1-5傳統方式純化DNA與RNA.......................................................21 1-6 聚合酵素鏈鎖反應........................................................23 1-7反轉錄聚合酶連鎖反應........................................................26 1-8磁性奈米材料於DNA純化的應用........................................................27 1-8超順磁氧化鐵奈米粒子的介紹與製備方式........................................................32 第二章 實驗........................................................35 2-1研究動機與目的........................................................36 2-2製備功能性四氧化三鐵奈米粒子(Fe3O4@PEG) ..............................................38 2-2-1實驗藥品........................................................38 2-2-2 實驗儀器........................................................39 2-2-3 實驗步驟........................................................40 2-3 DNA分離純化之緩衝溶液........................................................41 2-3-1配製流程........................................................41 2-4 功能性十八烯酸包覆四氧化三鐵奈米粒子分離純化血液中DNA ......................................................42 2-4-1 實驗藥品........................................................42 2-4-2 實驗儀器........................................................42 2-4-3 實驗步驟........................................................42 2-5 RNA與功能性PEG包覆四氧化三鐵奈米粒子(Fe3O4@PEG)...............................................45 2-5-1 實驗藥品........................................................45 2-5-2 實驗儀器........................................................45 2-5-3 實驗步驟........................................................45 2-6功能性PEG包覆四氧化三鐵奈米粒子(Fe3O4@PEG)分離純化 B型肝炎病毒中的DNA.......................................................50 2-6-1 實驗藥品........................................................50 2-6-2 實驗儀器........................................................50 2-6-3 實驗步驟........................................................51 2-7聚合酵素鏈鎖反應(PCR) ....................................................53 2-7-1血液純化DNA之PCR.......................................................53 2-7-1-1 實驗藥品........................................................53 2-7-1-2 實驗儀器........................................................53 2-7-1-3實驗步驟........................................................54 2-7-2血液中純化之RNA 的cDNA製備與PCR分析.....................55 2-7-2-1實驗藥品........................................................55 2-7-2-2實驗儀器........................................................56 2-7-2-3實驗步驟........................................................56 2-8 DNA與RNA之電泳分析........................................................59 2-8-1 實驗藥品........................................................59 2-8-2 實驗儀器........................................................59 2-8-3 實驗步驟........................................................60 第三章 結果與討論........................................................61 3-1 Fe3O4@PEG磁性奈米粒子TEM分析........................................................61 3-2 Fe3O4@PEG 磁性奈米粒子之XRD與SEM index分析.............64 3-3 Fe3O4@PEG奈米粒子的 FT-IR光譜分析........................................................67 3-4 Fe3O4@PEG 奈米複合材料的熱重分析........................................................68 3-5 Fe3O4@PEG 磁性奈米粒子的SQUID分析........................................................69 3-6 Fe3O4@PEG 磁性奈米粒子的 Zeta Potential 與DLS分析......70 3-7 Fe3O4 @PEG 磁性奈米粒子的細胞毒性測試........................................................73 3-8 Fe3O4 @PEG 磁性奈米粒子的細胞吞噬測試........................................................75 3-9 利用磁性奈米複合材料自動化分離血液中DNA ......................................................77 3-9-1純化過程中分解溶液Guanidine thiocyante濃度對純化DNA的影響RNA.......................................................77 3-9-2純化過程中分解溶液EDTA濃度與pH值對純化DNA的影 響........................................................80 3-9-3 Fe3O4@PEG 磁性奈米粒子與市售純化分離血液中DNA ..........................................................82 3-10 利用磁性奈米複合材料自動化分離乳癌細胞與血液中RNA.........85 3-10-2 Fe3O4@PEG 磁性奈米粒子純化分離人類血液中RNA.......................................................87 3-10-3 Fe3O4@PEG 純化血液中 RNA進行PCR與定序分析........................................................88 3-11 Fe3O4@PEG 純化人類B型肝炎病毒之DNA與PCR................92 3-12 Fe3O4@PEG 材料存放時間與穩定度測試........................................................93 第四章 結論........................................................94 未來展望........................................................95 參考文獻........................................................96

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