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研究生:余婉琪
研究生(外文):Wan-Chi Yu
論文名稱:奈米粒子對毛細管電泳分離胺及酸之影響
論文名稱(外文):The impact of nanoparticles on the separation of DNA and amines by capillary electrophoresis
指導教授:張煥宗張煥宗引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:92
中文關鍵詞:奈米粒子毛細管電泳DNA分離生物胺分離
外文關鍵詞:nanoparticlescapillary electrophoresisseparation of DNA and biological amines
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本論文是在研究毛細管電泳對生物分子分離之應用,主要分成二個部分,第一個部分是探討毛細管電泳中,於聚合物溶液中加入添加物對DNA分離之影響。實驗所用的聚合物為乙基纖維素(2-hydroxyethyl cellulose,HEC),分子量為1,300 000,加入添加物thiourea之後,相較於單純以0.35% HEC分離DNA marker Ⅴ、Ⅵ,可大幅提高394-653 bp範圍的9個DNA片段之解析度,另外,我們也探討金奈米粒子對DNA分離之影響,金奈米粒子可以使DNA的分離在較短的時間內完成(整個分離時間可在五分鐘內完成),且能維持住相當的解析度,加入這些添加物可提高DNA之分離解析度,可能的原因為:添加物與聚合物作用因而改變聚合物的構型,此構型的改變有助於DNA分離解析度的提高。
第二部分則是研究毛細管表面修飾對生物胺及其代謝有機酸分離的影響,我們探討毛細管壁修飾聚離胺酸(poly-L-lysine,PLL)及二氧化矽奈米粒子(SiONP)對分析物分離的影響。毛細管壁修飾聚離胺酸(以(PLL-SiO2)n-PLL表示)時,最佳的條件是修飾(PLL-SiO2)2-PLL,並以添加3% acetonitrile (ACN) 之10 mM甲酸溶液(pH 3.7)進行分離。在此條件下,對酸的靈敏度較高,像是對5-HIAA及5-HT的偵測極限分別可達1 nM及35 nM;另一方面,毛細管壁修飾二氧化矽奈米粒子時(以(PLL-SiONP)n表示),是以修飾(PLL-SiONP)3的毛細管,並在10 mM甲酸溶液(pH 3.7)進行分離可得最佳的分離結果,在此條件下,對胺的靈敏度較高。像是對TA及5-HIAA的偵測極限為0.09 nM及19 nM。因此可依據欲分析的生物分子是胺或酸而選擇毛細管壁以聚離胺酸或二氧化矽奈米粒子修飾來進行分離。
In this thesis, we focus on the impact of nanoparticles on the separation of biological molecules by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) detection. In the first part of this thesis, the impact of additives in 0.35% 2-hydroxyethyl cellulose (HEC, Mw 1,300 000 g/mole) on DNA separation by CE-LIF is studied. By using 0.35% HEC prepared in 40 mM glycine buffer (pH 9.9) containing 1% thiourea, 32-nm gold nanoparticles, and 1.27μM ethidium bromide, 9 DNA fragments with their sizes ranging from 394 to 653 bp were well resolved within 5 min. The separation is fast, highly efficient, and reproducible, mainly because of the change in HEC morphologies as a result of the interaction among thiourea, HEC, and gold nanoparticles. In the second part of this thesis, we have investigated the separation of biological amines and acids by CE-LIF using capillaries coated with poly-L-lysine (PLL) and silica nanoparticles (SiONPs). The capillaries coated with (PLL-SiONP)3 and (PLL-SiONPs)2-PLL are suitable for the separations of amines and acids, respectively. By using a (PLL-SiONP)3 capillary filled with a solution consisted of 10 mM formic acid (pH 3.7) and 3% acetonitrile (ACN), the limits of detection (LOD) at signal-to-noise ratio (S/N) = 3 are 1.2 nM and 35.0 nM for 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptamine (5-HT), respectively. The LODs are 0.09 nM and 19.0 nM for TA and 5-HIAA, respectively, when using a (PLL-SiONPs)2-PLL capillary filled with 10 mM formic acid ( pH 3.7). The practical application of the method has been validated by the analysis of urine samples, with great sensitivity, high speed, efficiency, and good reproducibility.
目 錄

摘要 Ⅰ
目錄 Ⅳ
表目錄 Ⅷ
圖目錄 Ⅹ

第一章 毛細管電泳與奈米粒子之應用概論…………………………1
1.1毛細管電泳發展歷史 …………….1
1.2毛細管電泳的原理 4
1.2.1 電泳 4
1.2.2電滲流與Zeta電位 5
1.2.3分離解析度 8
1.2.4 分離效率 8
1.2.5影響分離效率的因素 9
1.2.5.1 焦耳熱 9
1.2.5.2樣品吸附 10
1.2.5.3進樣長度 10
1.3 DNA的電泳分離機制 11
1.3.1 Ogston模型 12
1.3.2 Reptation模型 14
1.3.3聚合物溶液 15
1.4毛細管電泳之偵測系統 16
1.4.1雷射激發誘導螢光(laser-induced fluorescence,LIF).. 17
1.4.1.1 DNA的偵測…………………………………………...18
1.4.1.2具生物活性胺類分子及有機酸的偵測...…………......19
1.5奈米粒子的簡介 19
1.5.1 金奈米粒子的應用 21
1.5.2 二氧化矽奈米粒子的應用………………………………..22
1.6研究動機 24
1.7 參考文獻 26
1.8 本章圖表 ….31
第二章 毛細管電泳之DNA分離:聚合物溶液中添加物的影響..38
2.1 導論 38
2.2 實驗 41
2.2.1實驗試藥 41
2.2.2實驗試劑配製 42
2.2.3毛細管之前處理 44
2.2.4毛細管電泳系統 44
2.2.5雷射誘導螢光偵測系統 45
2.2.6 DNA分離實驗 45
2.3實驗結果與討論 46
2.3.1添加物對DNA分離之影響 46
2.3.2不同聚合物溶液配製方法對DNA分離之影響 47
2.3.3添加物thiourea作用之探討 47
2.4 結論 48
2.5 參考文獻 50
2.6 本章圖表 53
第三章 毛細管壁修飾聚離胺酸和二氧化矽奈米粒子對生物胺及代謝有機酸分離之影響……………………………………………………60
3.1 導論 60
3.2 實驗 62
3.2.1實驗儀器 62
3.2.2實驗試藥 63
3.2.3毛細管前處理 63
3.2.4 緩衝溶液的製備…………………………………...……...64
3.2.5 尿液成份分析……………………………………………..64
3.3 實驗結果與討論 65
3.3.1 緩衝溶液pH值的影響 65
3.3.2 以(PLL-SiONP)n-PLL修飾之毛細管分離胺及酸…….....66
3.3.3 以(PLL-SiONP)n修飾之毛細管分離胺及酸 67
3.3.4 有機溶劑acetonitrile (ACN)的影響………………………67
3.3.5 尿液成份分析與定量……………………………………..68
3.4 結論 69
3.5 參考文獻 71
3.6 本章圖表 73
第四章 總結..………………………………………………………...91
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