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研究生:林泱蔚
研究生(外文):Yang-Wei Lin
論文名稱:毛細管與晶片電泳於DNA之分離及量子點之製備與光電應用
論文名稱(外文):Capillary and microchip electrophoresis for DNA separation as well as synthesis and optoelectronic applications of quantum dots
指導教授:張煥宗張煥宗引用關係
指導教授(外文):Huan-Tsung Chang
學位類別:博士
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:2007
畢業學年度:94
語文別:英文
論文頁數:222
中文關鍵詞:DNA分離毛細管電泳晶片電泳量子點量子點薄膜
外文關鍵詞:DNA separationcapillary electrophoresismicrochip electrophoresisquantum dotsquantum dots films
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本論文可區分成兩大主題,第一部分主要是高效率及高再現性之毛細管與晶片電泳分離DNA之技術開發;第二部分著重於奈米粒子量子點之製備及其應用。首先,在毛細管電泳方面,利用線上濃縮技術及增加偵測光徑(氣泡容槽)方式來改善DNA片段靈敏度及分離效率。與傳統毛細管電泳操作模式比較,結果發現對89 bp DNA片段之偵測靈敏度可提升至170倍。此外,於氣泡容槽條件下,亦能提升大片段DNA(> 500 bps)之解析度。為了進一步降低分析時間及改善DNA於管壁吸附問題,於低濃度之篩分聚合物溶液(PEO)中添加少量十六烷基三甲基溴化銨(CTAB)進行DNA之分離。實驗結果發現,DNA吸附情形及與溴化乙錠(EtBr)間作用會隨著CTAB濃度增加而降低。在最佳化條件下,8分鐘內即可完成DNA Marker V及VI(18~2176 bp DNA片段)之分離,且對於18 bp DNA片段之偵測極限可降至2.0 ng/mL。在晶片電泳部分,首先利用聚合物溶液(PVP and PEO)和金奈米粒子(GNPs)來對微流體通道進行動態塗覆。以連續三層塗覆(PVP-PEO- GNPs)之微流體通道,在1.5% PEO(GNPs)聚合物溶液條件下,有效地改善18至2176 bp DNA片段之分離再現性(RSD 2.5%,n = 5)及解析度(R > 1.1)。另外,我們亦利用多層動態塗覆方式((PEO-PVP)2-PEO(GNPs))修飾微流體通道。在0.75% PEO(GNPs)溶液條件下,分離DNA Marker V及VI可得到快速(<3分鐘)、高效率(N > 1 700 000 plates/m)及高再現性(RSD 1.3%,n = 5)之結果。在奈米粒子量子點部分,本論文首先開發出利用雷射輔助-水相合成高量子效率量子點(CdSe)之技術。為了更進一步提升量子效率,除了形成核-殼形式量子點(CdSe@CdS)外,我們亦藉由紫外光照射降低其表面缺陷,成功地將量子點之量子效率提升至80%。此外,利用Stöber process形成核-殼-殼量子點(Silica-QDs-Silica)結構,更能有效地增加其光學、化學穩定性,未來將可應用於生物樣品之檢測及標識。最後,使用layer-by-layer assembly 技術,並且依據量子點間能量轉移和顏色混合之概念,我們可以製作出多色彩量子點薄膜。再結合微影技術,則可將多色彩量子點薄膜之尺度降低至微米範圍(10 um)。而此類薄膜在生物感測器、光子晶體及LED應用方面應極具發展潛力。
This thesis focuses on developing highly efficient and reproducible capillary electrophoresis (CE) and microchip CE (MCE) based techniques for DNA separation and preparing highly fluorescent quantum dots (QDs) for biosensing and optoelectronic devices. First, the sensitivity and resolution of DNA fragments have been optimized in CE, by applying on-line concentration and using a bubble cell (e.g. 300 um in diameter). When compared to that by conventional injection and use of a capillary without a bubble cell, up to 170-fold sensitivity improvements for the DNA fragments have been achieved. The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by CE in conjunction with laser-induced fluorescence (CE-LIF) detection using 0.75% poly(ethylene oxide) (PEO) solution is described. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. Under optimum condition, a mixture of DNA markers V and VI within 8 min at -375 V/cm was separated, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. In microchip electrophoresis for DNA separation section, we have demonstrated a simple method for dynamically coating the wall of the separation channels that were fabricated on poly(methyl methacrylate) (PMMA) plates using poly(vinyl pyrrolidone) (PVP), poly(ethylene oxide) (PEO), and gold nanoparticles (GNPs) in sequence. The three-layer (PVP-PEO-GNPs) coated PMMA chips provide improvements in resolution and reproducibility for DNA separation when using 1.5% PEO(GNPs), allowing the separation of DNA fragments ranging in the size of 18-2176 bp. Besides, multilayer coating of PMMA chips with PEO, PVP, and PEO containing gold nanoparticles [PEO(GNP)] is important for achieve high efficiency. Using a 2-(PEO-PVP)-PEO(GNP) PMMA chip, the separation of DNA markers V and VI by MCE in 0.75% PEO(GNP) was accomplished in 3 min. In QDs section, a simple synthetic route to the preparation of high-quality CdSe QDs in aqueous solution was present. The thermal synthesis, assisted by laser irradiation at 532 nm, allows the preparation of CdSe QDs that possess higher quantum yield. After UV irradiation, the as-prepared core–shell CdSe/CdS QDs become stable and fluoresce strongly in the visible range. For biocomplement, we also prepare highly water-soluble core-shell-shell (CSS) silica–QDs–silica NPs that exhibit greater QYs, photostability, and chemical stability. This feature, together with their narrow emission spectral profile, suggests that the CSS silica–QDs–silica NPs may have a great number of biological applications. QD films are fabricated through layer-by-layer (LBL) assembly using citrate-stabilized CdSe@CdS QDs, 3-mercaptopropionic acid (MPA)-stabilized CdTe QDs, and poly(diallyldimethylammonium chloride) (PDDA). The colors and emission intensities exhibited by the highly fluorescent QD films are readily tunable by controlling the deposition order and the number of bilayers of (PDDA-CdSe@CdS)n and (PDDA-CdTe)n.
Contents Page
中文摘要 I
Abstract III
Contents V
Table contents IX
Figure contents X

1. Introduction 1
1.1 Capillary electrophoresis 2
1.1.1 Electrophoretic mobility 4
1.1.2 Electroosmotic flow and Zeta potential 5
1.1.3 Efficiency and resolution 6
1.1.4 Sensitivity improvement 7
1.1.4.1 Design of detection cell 7
1.1.4.2 Detection system 7
1.1.4.3 Sample concentration 10
1.1.4.3.1 Field amplified sample stacking 10
1.1.4.3.2 pH mediated stacking 11
1.1.4.3.3 Isotachophoresis 12
1.1.4.3.4 Stacking using polymer solution in present of EOF 13
1.1.4.3.5 Sweeping 14
1.1.5 Capillary wall modification 15
1.1.5.1 Dynamic wall coating 15
1.1.5.2 Static coating 16
1.1.6 Applications 17
1.1.6.1 DNA sequencing 17
1.1.6.2 Protein analysis 19
1.2 Microchips based electrophoresis 20
1.2.1 Substrates and microfabrication 22
1.2.2 Surface modification 23
1.2.2.1 Chemical modification 23
1.2.2.2 Polymer coating 25
1.2.2.3 Protein adsorption 26
1.2.2.4 High energy treatment 27
1.2.2.5 Others method 28
1.2.3 Detection system 28
1.2.3.1 Confocal epifluorescence detection 29
1.2.3.2 Electrochemical detection 30
1.3 Semiconductor quantum dots 32
1.3.1 Optical properties of quantum dots 32
1.3.2 Synthesis of quantum dots 33
1.3.2.1 Core synthesis 33
1.3.2.2 Shell synthesis 33
1.3.3 Quantum dots modification 34
1.3.4 Applications 35
1.3.4.1 Photo-electronic materials 35
1.3.4.2 Biological sensors 36
1.4 Motive of research 37
1.5 References 39

2. Capillary electrophoretic separation of dsDNA under nonuniform electric fields
2.1 Abstract 67
2.2 Introduction 67
2.3 Experimental section 69
2.3.1 Apparatus 69
2.3.2 Materials 70
2.3.3 Bubble cell 70
2.3.4 Stacking and separation 71
2.4 Results and discussion 71
2.4.1 Sensitivity improvement 71
2.4.2 Separation in the absence of EOF 73
2.5 Conclusions 74
2.6 References 75

3. Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide
3.1 Abstract 82
3.2 Introduction 82
3.3 Experimental section 84
3.3.1 Apparatus 84
3.3.2 Materials 85
3.3.3 Preparation of PEO solution 86
3.3.4 Stacking and separation 86
3.4 Results and discussion 86
3.4.1 EOF in TB solution and PEO solution containing CTAB 86
3.4.2 DNA separation using PEO solution containing EtBr and CTAB 87
3.4.3 Separation of DNA markers V and VI 91
3.5 Conclusions 91
3.6 References 92

4. Analysis of dsDNA by microchip capillary electrophoresis using polymer solutions containing gold nanoparticles
4.1 Abstract 103
4.2 Introduction 103
4.3 Experimental section 105
4.3.1 Apparatus 105
4.3.2 Chemicals 106
4.3.3 DNA extraction and PCR products 107
4.3.4 Microfabrication 107
4.3.5 Coating 108
4.3.6 Separation 108
4.4 Results and discussion 109
4.4.1 Dynamic coating 109
4.4.2 Effect of background electrolytes 110
4.4.3 Stepwise changes in EtBr 112
4.4.4 Analysis of PCR products 113
4.5 Conclusions 113
4.6 References 114

5. Modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded DNA
5.1 Abstract 125
5.2 Introduction 125
5.3 Experimental section 128
5.3.1 Apparatus 128
5.3.2 Chemicals 129
5.3.3 Microfabrication 130
5.3.4 Dynamic coating 131
5.3.5 DNA extraction and PCR products 131
5.3.6 Electrophoretic procedure 132
5.4 Results and discussion 132
5.4.1 Dynamic coating and separation of FX 174 RF DNA-Hae III digest 132
5.4.2 Separation of DNA markers V and VI 135
5.4.3 Advantage and reproducibility 137
5.4.4 Separation of PCR products 138
5.5 Conclusions 139
5.6 References 139

6. Photoassisted Synthesis of CdSe and Core-Shell CdSe/CdS Quantum Dots
6.1 Abstract 149
6.2 Introduction 149
6.3 Experimental Section 152
6.3.1 Chemicals 152
6.3.2 Preparation of Citrate-stabilized CdSe QDs 152
6.3.3 Preparation of Core–Shell CdSe@CdS QDs 153
6.3.4 Photopassivation of core–shell CdSe@CdS QDs 153
6.3.5 Characterization of CdSe and CdSe@CdS QDs 153
6.4 Results and discussion 154
6.4.1 Laser-assisted Synthesis of CdSe QDs 154
6.4.2 Passivation of CdSe and Photoetching of Core–Shell CdSe@CdS 156
6.4.3 Effect of Laser Power 159
6.4.4 PL of Various Core–Shell CdSe@CdS QDs 160
6.5 Conclusions 160
6.6 References 161

7. Synthesis and Properties of Water-Soluble Core–Shell–Shell Silica–CdSe/CdS–Silica Nanoparticles
7.1 Abstract 172
7.2 Introduction 172
7.3 Experimental 175
7.3.1 Chemicals 175
7.3.2 Preparation of Citrate-stabilized Core–Shell CdSe@CdS QDs 176
7.3.3 Preparation of CSS Silica–QDs–Silica NPs 177
7.3.4 Characterization of QDs, MPS-QDs, and CSS Silica–QDs–Silica NPs 178
7.4 Results and discussion 178
7.4.1 Preparation of QDs 178
7.4.2 Modification of the Surfaces of QDs with Silica Precursors 179
7.4.3 Growth of CSS Silica–QDs–silica NPs 182
7.4.4 Stability of CSS Silica-QDs-silica NP 184
7.5 Conclusions 185
7.6 References 186

8. Using a Layer-by-Layer Assembly Technique to Fabricate Multiply Colored Films of CdSe@CdS and CdTe Quantum Dots
8.1 Abstract 196
8.2 Introduction 196
8.3 Experimental 198
8.3.1 Chemicals 198
8.3.2 Synthesis of QDs. 198
8.3.3 Characterization of QDs 199
8.3.4 Layer-by-Layer Assembly 200
8.3.5 Fabrication of Microstructures 200
8.4 Results and Discussion 201
8.4.1 LBL assembly technique for QD films 201
8.4.2 Multicolor of the QD films 202
8.4.3 Patterned QD microstructure of different color 203
8.5 Conclusions 205
8.3 References 206

Conclusions and prospect 216

Publications and conferences 220

Table Contents
Table 1-1.LODs for different detection techniques in CE. 52
Table 1-2.Comparison of stacking efficiencies for different preconcentration methods. 53
Table 1-3.Polymeric additives and their applications for dynamic coating. 54
Table 1-4.Polymers for permanent coating. 55
Table 1-5.Material substrate and microfabrication for microchips. 56
Table 1-6.Four soft lithographic technique. 57
Table 2-1.Comparison of peak enhancement and resolution for the analysis of DNA at 10 kV. 77
Table 3-1.Repeatability of the migration times (tm) and fluorescence intensities (IF) for the DNA fragments at different CTAB concentrations. 95
Table 3-2.Repeatability of the migration times (tm) and fluorescence intensities (IF) for the DNA fragments by CE-LIF at different EtBr concentrations. 100
Table 4-1.Effect of the buffer used to prepare 1.5% PEO on the electrophorteic mobility of DNA. 118
Table 4-2.Migration time and peak height for the analysis of the digested PCR products of UGT1A7 gene. 119
Table 5-1.Comparison of the theoretical plates (N) and resolution (R) using different PMMA chips. 143
Table 6-1.Comparison of the PL QY for CdS and core–shell CdSe@CdS QDs prepared under different conditions. 164
Table 6-2.The effect that the intensity of light has on the preparation of the CdSe and core-shell CdSe/CdS QDs at 80 °C while irradiating with laser light for 2 h. 165
Table 7-1.Optical properties and sizes of QDs prepared using different methods. 189
Table 7-2.Size, optical properties and quantum yield of CSS silica–QDs–silica NPs, MPS-QDs, and QDs. 190

Figure Contents
Figure 1-1.Illustration of double layer and zeta potential. 58
Figure 1-2.Flow profiles of electrically driven systems and pressure driven systems. 59
Figure 1-3.Scheme diagram of a capillary electrophoresis separation. 60
Figure 1-4.Approaches to path length extension that are reported in CE. (a) Bubble cell, (b) Z-type flow cell, (c) Multireflection cell. 61
Figure 1-5.Scheme of the sheath flow post column reactor is shown. 62
Figure 1-6.Commercial microfluidic system by Caliper, Agilent and Shimadzu. 63
Figure 1-7.Scheme of confocal epifluorescence detection is shown. 64
Figure 1-8.Energy level diagram showing promotion of an electron from the valence band to the conduction band, leaving a hole behind. 65
Figure 1-9.Scheme of the two general methods used to disperse hydrophobic QDs in aqueous solution. 66
Figure 2-1.The capillary with a 300-um bubble cell. 78
Figure 2-2.Analyses of 2.0 mg/mL DNA markers V and VI in the presence of EOF at 10 kV. 79
Figure 2-3.Effects of bubble cell and electric field strength on separations of DNA in the absence of EOF. 80
Figure 2-4.Separation of a sample containing 0.2 ug/mL DNA markers V and VI in the absence of EOF. 81
Figure 3-1.The separations of 0.5 ug/mL fx 174 RF DNA-HaeIII digest at different concentrations of CTAB. 97
Figure 3-2.The fluorescence intensities of the mixtures of 0.5 ug/mL fx 174 RF DNA-HaeIII digest, 100 mM TB (pH 8.0), 25.0 μg/mL EtBr and different concentrations of CTAB. 98
Figure 3-3.The separations of 0.25 ug/mL fx 174 RF DNA-HaeIII digest at different concentration of EtBr. 99
Figure 3-4.The separations of 0.25 ug/mL fx 174 RF DNA-HaeIII digest at different concentration of PEO solution. 100
Figure 3-5.Electropherogram of 0.1 ug/mL DNA markers V and VI by CE-LIF at -375 V/cm. 101
Figure 3-6.Linearity between the peak heights for some of the DNA fragments in a mixture of DNA markers V and VI and injection volumes. 102
Figure 4-1.Comparison of the separation of fx 174 RF DNA-HaeIII digest using four different dynamic coating approaches. 120
Figure 4-2.Separations of a mixture of equal volume of DNA markers V and VI under different condition. 121
Figure 4-3.TEM images of 1.5% PEO(GNPs) prepared in three different buffers shown in Fig. 4-2. 122
Figure 4-4.Electropherogram of the DNA separation by a stepwise CE technique. 123
Figure 4-5.Separation of the digested PCR products. 124
Figure 5-1.Schematic diagrams of a MCE separation and detection system (A) and a representative PMMA microchip (B). 144
Figure 5-2.Comparison of the separation of fx 174 RF DNA-HaeIII digest using three differently coated PMMA chips. 145
Figure 5-3.Separations of a mixture containing DNA markers V and VI using X-(PEO-PVP)-PEO(GNP) PMMA Chips. 146
Figure 5-4.UV-vis spectra for X-(PEO-PVP)-PEO(GNP) PMMA plates. 147
Figure 5-5.Separation of hsp65 gene fragments of mycobacterium HaeIII digests. 148
Figure 6-1.Schematic depiction of the setup used for the laser-assisted synthesis of CdSe QDs in aqueous solution. 166
Figure 6-2.Effect of temperature and laser irradiation on the optical properties of the CdSe QDs. 167
Figure 6-3.Comparison of the optical properties of the core–shell CdSe@CdS QDs before and after UV irradiation for 24 h. 168
Figure 6-4.Characteristic of CdSe QDs and core-shell CdSe/CdS QDs. 169
Figure 6-5.The absorption spectra of the core–shell CdSe(80)@CdS QDs and CdSe(80). 170
Figure 6-6.Absorption and PL spectra of the core–shell CdSe@CdS QDs prepared under different experimental conditions. 171
Figure 7-1.TEM images of (A) QDs and (B) MPS-QDs (24 h). (C) PL of QDs and MPS-QDs (D) PL ratios (I0/I) plotted against the number of MPS molecules per QD nanoparticle. 191
Figure 7-2.(A–D) TEM images of CSS silica–QDs–silica NPs that were prepared using MPS-QDs that had been incubated for 10 min, 12, 24, and 48 h, respectively. (E) PL spectra and (F) emission wavelengths and sizes of the CSS silica–QDs–silica NPs. 192
Figure 7-3.(A–D) TEM images of CSS silica–QDs–silica NPs that were prepared using 12, 25, 50, and 100 nM MPS-QDs, respectively. (E) PL spectra and (F) emission wavelengths and sizes of the CSS silica–QDs–silica NPs. 193
Figure 7-4.(A) HR-TEM images and EDX spectra of the CSS silica–QDs–silica NPs. (B) EDX spectra recorded in the areas suggested by the arrows. (C) Silica NPs with holes were produced by etching out the QDs shell of CSS silica–QDs–silica NPs. 194
Figure 7-5.(A) Photobleaching behavior under CW Ar+ laser radiation. (B) Effect of pH on photoluminescence. (C) Effect of salt concentration on stability. 195
Scheme 8-1.Cartoon representation of the LBL fabrication of multilayered QD. 209
Figure 8-1.Plots of the fluorescence intensities of the different QD films as a function of the number of preparation cycles. 210
Figure 8-2.(A) Optical images of the five films. (B) The corresponding fluorescence spectra of the films, presented as black, red, green, blue, and light-blue curves, respectively. 211
Figure 8-3.(A) Optical images of (PDDA-CdTe)20-(PDDA-CdSe@CdS)n QD films. For n = 0, 2, 4, 6, and 8, the colors are red, orange, olive-green, chartreuse, and green, respectively. (B) The corresponding fluorescence spectra. 212
Figure 8-4.Optical images and the corresponding fluorescence spectra of (PDDA-CdTe)20-(PDDA-PSS)n-(PDDA-CdSe@CdS)2 QD films for n = 0, 2, and 5. 213
Figure 8-5.(A) Optical image and (B) fluorescence intensity profile across the dashed line of a QD microstructure. 214
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