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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張煥宗 | |
| dc.contributor.author | Ming-Feng Huang | en |
| dc.contributor.author | 黃銘峰 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:17:13Z | - |
| dc.date.available | 2007-07-31 | |
| dc.date.copyright | 2006-07-31 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-28 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31672 | - |
| dc.description.abstract | 中文摘要
本論文主要是利用毛細管電泳的分離技術，結合雷射誘導螢光的偵測方式，分別針對胺類分子、馬兜鈴酸以及DNA 片段，開發出快速、高解析度且高靈敏度的分析技術。論文內容可分為四個部分：(1)利用毛細管電泳結合間接螢光偵測的方式，成功分離並偵測六種胺類分子。我們使用pH 3.5 的溶液，其中包括5.0% 甲醇, 0.1 mM 硫酸, 0.1mM cresyl violet, and 0.3 mM 鋰離子作為毛細管電泳的電解質溶液，來分離六種胺類分子，因為胺類分子並不具有螢光性質，因此利用與其具有相同官能機且淌度相近的螢光染料crysyl violet，在電泳過程中進行取代，因此可以得到胺間接螢光偵測的訊號。利用此方法其偵測極限可以達到µM 的程度，如果搭配pH junction 的濃縮方式，可降低其偵測極限達19 倍。(2) 馬兜鈴酸類化合物是常見於中藥中的一類成分，其具有腎毒性及致癌的危害，因為馬兜鈴酸為具有硝基苯的化合物，本身不具有螢光，因此在0.1 M 的鹽酸溶液中，利用鐵粉將硝基還原成胺基，被還原的馬兜鈴酸在390 nm 的光源激發下，會釋放出473 nm 的螢光。利用毛細管電泳暨雷射誘導螢光的技術，可在50.0 mM sodium tetraborate、10.0 mM SDS，pH 9.0 的條件下，在12 分鐘內分析出兩種自然界最常見的馬兜鈴酸類化合物－AA-I 與AA-II，其偵測極限可達到8.2 Nm (AA-I) and 5.4 nM (AA-II)，我們並II利用此技術分析61 種實際中藥材樣品中，並分析出其中24 種含有馬兜鈴酸成分。(3) 在第三部分中，我們利用加入金奈米粒子(GNPs)的低黏度聚環氧乙烷溶液來分離DNA 片段，我們發現當加入56 nm 的金奈米粒子後，分析片段大小為51 個鹼基對到2176 個鹼基對的marker DNA，可在5 分鐘內解析出其中28 個片段，另外針對較大片段的DNA，如片段大小為5 千到4 萬個鹼基對的5 kb ladder DNA，也可在30 分鐘內成功分離出其8 個片段，證明這是個高分離解析度與快速的DNA 分析技術。(4) 針對大小在數千個鹼基對以上的大DNA 片段，我們開發出新的快速且高解析度的毛細管電泳分離技術。我們在金奈米粒子的表面修飾聚合物分子，稱之為GNPPs。將GNPPs 填入到毛細管中作為DNA 的分離介質，DNA 藉由與金奈米 粒子間的作用力，以及與金奈米粒子上所修飾的聚合物分子所產生的摩擦力而分離，利用此技術我們可在5 分鐘內解析出大小為0.12 到23 kbp 的lambda DNA 的所有片段，另8.27 到48.5 kbp 的HMD DNA也可在6 分鐘之內分離出其所有片段。與傳統分析大片段DNA 的平板凝膠電泳或變換電壓式的毛細管電泳相較，其分析時間可從至少數小時縮短到數分鐘。 | zh_TW |
| dc.description.abstract | Abstract
This thesis focuses on developing efficient and sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) techniques for amines, aristolochic acid, and DNA. We developed a novel method for the analysis of amines under acidic conditions by CE in conjunction with indirect laser-induced fluorescence (CE-ILIF). The analysis of six amines by CE-ILIF using a solution, pH 3.5, containing 5.0% methanol, 0.1 mM sulfuric acid, 0.1 mM cresyl violet, and 0.3 mM lithium was complete in 5 min, with the limits of detection (LOD) on the level of µM.To further improve the sensitivity, on-line concentration based on pH junction has been demonstrated. When injecting the sample prepared in a solution of 0.2 mM sulfuric acid, pH 3.3, at 15 kV for 60 s to the above-mentioned solution, the LODs for the amines down to sub µM and the sensitivity improvements up to 19-fold when compared to that injecting at 15 kV for 5 s. Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Two predominant forms of AA are 8-methoxy-6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5- carboxylic acid (AA-I) and 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AA-II). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), the RAAs reduced from OAAs in 10.0 mM HCl containing iron powder is required prior to CE analysis. The RAAs exhibit fluorescence at 477 nm when excited at 390 nm. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS,the determination of aristolochic acid I and aristolochic acid II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The successful analysis of 61 medicinal samples and dietary supplements shows that the present CE-LIF is practical for the determination of AA-I and AA-II in real samples. Reproducible, rapid, and high-resolution DNA separations have been achieved using low-viscosity PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO(8 MDa) containing 56-nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO(2 MDa) containing 13-nm GNPs or 0.05% PEO(4 MDa) containing 32-nm GNPs. To separate long double strands of DNA by CE, with high efficiency, high speed, simplicity, and reproducibility, we describe a CE technique, which we call nanoparticle-filled CE (NFCE), using polymer-modified gold nanoparticles (GNPPs) in this topic. The gold nanoparticles were modified with poly(ethylene oxide) via noncovalent bonding to form GNPPs. The separations of high molecular weight (HMW) DNA with sizes ranging from 8.27 to 48.5 kbp and λ DNA (0.12–23.1 kbp) were accomplished within 6 and 5 min, respectively. The separation speed and resolution are greater than those by pulsed CE and slab gel electrophoresis. This is the first example for separating such high DNA molecules by CE-LIF using GNPPs. The results present in this thesis clearly demonstrate that CE-LIF based techniques are practical for analysis of amines, aristolochic acid, and DNA, with the advantages of rapidity, sensitivity, and reproducibility. The examples of separating DNA using PEO containing GNPs and GNPPs open the avenue of using nanoparticles for improved resolution and reproducibility for analysis of DNA. It is our strongly belief that the technique should be further applied to analysis of small solutes such as amines and acids and macromolecules like proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T03:17:13Z (GMT). No. of bitstreams: 1 ntu-95-D89223027-1.pdf: 1297110 bytes, checksum: 9bcd464258ea09f5805772b06e59aaed (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 中文摘要 I
Abstrate III Table contents X Fig. contents XI Chapter1 : Introductions of Capillary Electrophoresis and Gold Nanoparticles 1 1.1 History of Electrophoresis 1 1.2 Principle of Capillary Electrophoresis 3 1.2.1 Instrumental Design 4 1.2.2 Electroosmotic Flow 5 1.2.3 Electrophoretic Mobility 7 1.3 Types of Capillary Electrophoresis 7 1.3.1 Capillary Zone Electrophoresis (CZE) 7 1.3.1.1 On-line Sample Concentration Techniques in CZE 9 1.3.1.1.1 Field-Amplified Sample Stacking (FASS) 10 1.3.1.1.2 Large-Volume Sample Stacking (LVSS) 12 1.3.1.1.3 pH-Mediated Stacking 13 1.3.2 Capillary Gel Electrophoresis (CGE) 14 1.3.2.1 Property of Polymer Solution 16 1.3.3 Micellar Electrokinetic Chromatography (MEKC) 20 1.3.4 Capillary Isoelectric Focusing (CIEF) 23 1.3.5 Capillary Isotachophoresis (cITP) 25 1.3.6 Capillary Electrochromatography (CEC) 27 1.4 Applications of CE 27 1.4.1 DNA 27 1.4.1.1 Separation Models of DNA 28 1.4.1.2 Applications of DNA Analysis by CE 30 1.4.1.2.1 DNA Sequencing 30 1.4.1.2.2 Genotyping and Mutation Detection 31 1.4.1.2.3 RNA and Gene Expression 31 1.4.2 Biogenic Amines 32 1.4.3 Aristolochic Acid 33 1.5 Detection of Capillary Electrophoresis 34 1.5.1 UV-Visible Detection 35 1.5.2 Laser-Induced Fluorescence Detection 35 1.5.3 Indirect Laser-Induced Fluorescence Detection 36 1.6 Introduction of Gold Nanoparticles 38 1.7 Synthesis of GNPs 40 1.7.1 Citrate Reduction 40 1.7.2 The Brust-Schiffrin Method: Two-Phase Synthesis and Stabilization by Thiols 41 1.7.3 Microemulsion, Reversed Micelles, Surfactants, Membranes, and Polyelectrolytes 42 1.7.4 Seeding Growth 42 1.7.5 Polymers 43 1.7.6 Physical Methods: Photochemistry (UV, Near-IR), Sonochemistry, Radiolysis, and Thermolysis 43 1.8 Physical Properties of GNPs 44 1.8.1 The Surface Plasmon Resonance (SPR) 44 1.8.2 Fluorescence 45 1.9 DNA-GNPs Assemblies and Sensors 46 1.10 GNPs as Capillary Electrophoresis Separation Matrix 47 1.11 Motive of Research 49 1.12 References 51 Chapter 2 : Indirect Fluorescence of Amines in Capillary Electrophoresis Using Cresyl Violet 66 2.1 Abstract 66 2.2 Introduction 67 2.3 Experimental 68 2.3.1 Standard Chemical 68 2.3.2 Instrumentation 69 2.3.3 Separation and Calculation 69 2.4 Results and Discussions 70 2.4.1 Effect of the Probe and Light Source 70 2.4.2 Effect of Acids and Metal Ions 71 2.4.3 Separation of Amines 72 2.4.4 Stacking of Amines 73 2.5 Conclusion 75 2.6 References 76 Chapter 3 : Determination of Aristolochic Acid in Chinese Herbal Medicine by Capillary Electrophoresis with Laser-Induced Fluorescence Detection 84 3.1 Abstract 84 3.2 Introduction 85 3.3 Materials and Methods 87 3.3.1 Standard Chemicals 87 3.3.2 Medicinal Samples 87 3.3.3 Sample Preparation 88 3.3.4 Reduction of AAI and AAII 88 3.3.5 Characterization 89 3.3.6 CE-LIF Apparatus 89 3.3.7 CE Conditions and Quantitative Analysis 90 3.4 Results and Discussion 90 3.4.1 Reduction of OAAs and Characterization 90 3.4.2 Optimization of CE Separation and LIF Detection 92 3.4.3 Analysis of Medicinal Samples and Dietary Supplements 94 3.5 Conclusions 95 3.6 References 97 Chapter 4 : Improved Separation of dsDNA Fragments by Capillary Electrophoresis Using Poly(ethylene oxide) Solution Containing Colloids 107 4.1 Abstract 107 4.2 Introduction 108 4.3 Materials and Methods 109 4.3.1 Standard Chemicals 109 4.3.2 Apparatus 110 4.3.3 Synthesis of GNPs 110 4.3.4 Polymer Solutions Containing GNPs 111 4.3.5 DNA Separation by CE 111 4.4 Results and Discussion 112 4.4.1 GNPs in Polymer Solution 112 4.4.2 Separation of Small DNA Fragments 112 4.4.3 Separation of Large DNA Fragments 114 4.4.4 Size Dependence of DNA Mobility 115 4.5 Conclusion 117 4.6 References 118 Chapter 5 : Rapid and Highly Efficient Separation of Long Double -Stranded DNA by Nanoparticle-Filled Capillary Electrophoresis 129 5.1 Abstract 129 5.2 Introduction 130 5.3 Experimental Section 131 5.3.1 Standard Chemical 131 5.3.2 Apparatus 132 5.3.3 Synthesis of GNPs and GNPPs 133 5.3.4 NFCE 134 5.4 Results and Discussion 134 5.4.1 Property of GNPPs 134 5.4.2 Separation of λ-DNA 135 5.4.3 Salt Dependence 137 5.4.4 Separation of HMW DNA 137 5.4.5 Separation Mechanism 138 5.5 Conclusion 139 5.6 References 140 Chapter 6 : Conclusions and Perspectives 151 6.1 Conclusions 151 6.2 Prespectives 153 Table contents Table 2.1 Peak height and Bandwidth for methylamine obtained under different conditions of sulfuric acid lithium ions. 78 Table 2.2 Effect of sulfuric acid on the peak height, bandwidth, and peak area for10 μM benzylamine when injecting at 15 kV for 5 and 60 s 79 Table 3.1 Comparisons of OAAs and RAAs with respect to IR, Mass, 1H-NMR, UV-vis absorption, and fluorescence data 100 Table 3.2 Effect of SDS on speed, resolution, sensitivity, and reproducibility for RAA-I and RAA-II by CE-LIF 101 Table 3.3 Determinations of AA-I and AA-II in medicinal samples and dietary supplements 102 Table 4.1 Effect of the GNPs on the separation efficiency and migration time for the DNA Markers V and VI 121 Table 4.2 Effect of the GNPs on the migration time and separation efficiency for the KiloBase DNA 122 Table 4.3 Comparison of migration time and resolution for the 5 kb DNA ladder fragments in the presence and absence of the GNPs 123 Table 5.1 Comparison of migration time, reproducibility, and resolution using GNPPs under different conditions 143 Fig. contents Fig. 1.1 Movement of charged particles in electric field 1 Fig. 1.2 Instrumental design of capillary electrophoresis 5 Fig. 1.3 Electroosmotic flow 6 Fig. 1.4 Model of field-amplified sample stacking 11 Fig. 1.5 Model of large-volume sample stacking 13 Fig. 1.6 Model of pH-mediated stacking 14 Fig. 1.7 The intrinsic structure of a polymer solution depends on the polymer concentration 18 Fig. 1.8 Mechanism of indirect laser-induced fluorescence detection. 37 Fig. 2.1 Structure of cresyl violet in acidic solution. 80 Fig. 2.2 Electropherogram of the separation of six model amines 81 Fig. 2.3 Separation of a beer sample 82 Fig. 2.4 On-line concentration and separation of six amines 83 Fig. 3.1 Reduction of OAAs to RAAs 103 Fig. 3.2 UV-vis spectra of OAAs and RAAs and fluorescence spectra of RAAs 104 Fig. 3.3 Electropherogram of the separation of RAA-I and RAA-II 105 Fig. 3.4 Electropherograms of the separations of real samples 106 Fig. 4.1 Separations of DNA Markers V and VI using PEO(8 MDa) and PEO(8 MDa) containing GNPs 124 Fig. 4.2 Separations of KiloBase DNA marker using PEO(2 MDa) and PEO(2 MDa) containing GNPs 125 Fig. 4.3 Separations of 5 kb DNA Ladder using PEO(2 MDa) containing GNPs and PEO(4 MDa) containing GNPs 126 Fig. 4.4 Plots of the electrophoretic mobilities for the DNA Markers V and VI as a function of the fragment size 127 Fig. 4.5 Plots of the electrophoretic mobilities for the kiloBase DNA fragments as a function of the fragment size 128 Fig. 5.1 TEM of (A) 32nm - PEO GNPPs and (B) 32nm - PEO GNPPs + EtBr 144 Fig. 5.2 Separation ofλ-DNA fragments using different concentration of GNPPs 145 Fig. 5.3 Separation ofλ-DNA fragments using 5X GNPPs containing different concentration of EtBr 146 Fig. 5.4 UV-Vis absorption and emission spectra of 5X GNPPs containing different concentration of EtBr 147 Fig. 5.5 Separation ofλ-DNA fragments using 5X GNPPs containing NaCl 148 Fig. 5.6 Separation of HMW DNA fragments 149 Fig. 5.7 The scheme shows long dsDNA separated through GNPPs 150 | |
| dc.language.iso | en | |
| dc.subject | 馬兜鈴酸 | zh_TW |
| dc.subject | 胺 | zh_TW |
| dc.subject | 毛細管電泳 | zh_TW |
| dc.subject | DNA | zh_TW |
| dc.subject | Capillary | en |
| dc.subject | elecrophoresis | en |
| dc.subject | amines | en |
| dc.subject | aristolochic acids | en |
| dc.subject | DNA | en |
| dc.title | 胺、馬兜鈴酸及DNA之毛細管電泳分析 | zh_TW |
| dc.title | Capillary electrophoresis for the analyses of amines, aristolochic acids and DNA | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 劉春櫻,林萬寅,吳信隆,魏國佐,王書蘋 | |
| dc.subject.keyword | 毛細管電泳,胺,馬兜鈴酸,DNA, | zh_TW |
| dc.subject.keyword | Capillary,elecrophoresis,amines,aristolochic acids,DNA, | en |
| dc.relation.page | 155 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-30 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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