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研究生: 王登甲
TENG-CHIA WANG
論文名稱: 快速純化高密度脂蛋白結合電化學品質分析於臨床研究
Rapid Purification of High Density Lipoprotein and Quality Analysis by Electrochemistry
指導教授: 陳建光
Jem-Kun Chen
口試委員: 鄭智嘉
Chih-Chia Cheng
黃群耀
Chun-Yao Huang
林豐彥
Feng-Yen Lin
陳建光
Jem-Kun Chen
學位類別: 碩士
Master
系所名稱: 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 140
中文關鍵詞: 高密度脂蛋白鐵奈米粒子磁離純化抗體DHR螢光檢測法電化學抗氧化能力心血管疾病蛋白質G
外文關鍵詞: High Density Lipopotein, iron nanoparticle, magnetic separation purification, Protein G, antibody, DHR fluorescence detection, electrochemistry, antioxidant capacity, cardiovascular disease
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  • 摘要 I Abstract III 致謝 VI 目錄 VIII 圖目錄 XII 第1章 前言 1 1.1 研究背景 1 1.2 研究動機與目的 4 1.3 臨床和體外研究道德和患者人口統計學 6 第2章 實驗理論與文獻回顧 7 2.1 HDL 分離法 7 2.2 人類高密度脂蛋白之螢光氧化試劑法檢驗 10 2.3血管內皮前驅細胞(endothelial progenitor cell/EPC) 15 2.4 電化學偵測 15 2.4.1 電化學反應系統 15 2.4.2 電化學感測器 17 2.4.3 循環伏安分析法 18 2.4.4 電化學阻抗頻譜 21 2.4.5 生物感測器於電化學之應用 23 2.4.6 網版印刷電極 24 2.5超順磁性四氧化三鐵奈米粒子 26 2.5.1磁性材料特性 26 2.5.2 共沉澱法(Co-precipitation) 32 2.5.3 微乳化法(Micro-emulsion) 32 2.5.4 水熱法(Solvothermal reaction) 32 2.6表面分子固定法 33 2.7 抗體(Antibody) 35 2.8 蛋白質檢測法(Bio-Red Protein Assay) 38 第3章 儀器原理 40 3.1 高解析度場發射掃描式電子顯微鏡(Field-emission scanning electron microscope,FE-SEM) 40 3.2 場發射穿透式電子顯微鏡(Field-emission transmission electron microscope,FE-TEM) 41 3.3 X光繞射分析儀(X-ray diffractometer,XRD) 45 3.4 傅立葉轉換紅外線光譜儀(Fourier transform infrared spectrometer,FT-IR) 48 3.6 動態光散射粒徑分析儀(Dynamic light scattering,DLS) 53 3.7 表面電位分析儀(Zeta-potential) 54 3.8 超導量子干涉磁量儀(Superconducting quantum interference device magnetometer,SQUID) 56 3.9 雷射掃描式共軛焦顯微鏡 (Laser scanning confocal microscope,LSCM) 59 3.10恆電位分析儀 (Potential Stat) 61 3.11 全波長多功能微盤分析儀 62 第4章 實驗流程與方法 65 4.1 實驗流程圖 65 4.2 實驗藥品 66 4.3 實驗儀器 68 4.4 實驗步驟 70 4.4.1 表面羧基化四氧化三鐵奈米粒子(FeNPs@L-Cystein)的製備 70 4.4.2 FeNP@L-Cystein@Protein G 核殼奈米粒子的製備 71 4.4.3 PBS緩衝溶液製備 73 4.4.4 HBS溶液製備 73 4.4.5 電化學之電解液製備 74 4.4.6 Protein G濃度定量 74 4.4.7 FeNP@L-Cystein@ProteinG 核殼奈米粒子之抗體修飾 75 4.4.8 FeNP@ L-Cystein @Protein G 抗體修飾反應 75 4.4.9 FeNP@L.C.@Protein G@IgG 抓取HDL並以DHR 123螢光試劑檢測其抗氧化能力 76 4.4.10 HDL以電化學檢測其抗氧化能力並與DHR 123螢光變化率比較分析 77 第5章 結果與討論 78 5.1 SEM表面型態分析 78 5.1.1 FeNP SEM影像分析 78 5.1.2 FeNP@ L-Cystein@Protein G影像分析 80 5.2 FeNP@L-Cystein奈米粒子綜合定性分析 82 5.2.1 FeNP@ L-Cystein定性分析 82 5.2.2 FT-IR 定性分析 82 5.2.3 XRD結晶分析 84 5.2.4 XPS 定性分析 85 5.2.5 DLS 粒徑分析 87 5.2.6 Zeta 表面電位分析 89 5.2.7 SQUID 磁性分析 90 5.3 FeNP@L-Cystein@Protein核-殼奈米粒子綜合定性分析 92 5.3.1 FeNP@ L-Cystein@Protein G FT-IR 定性分析 92 5.3.2 FeNP@ L-Cystein@Protein G DLS 粒徑分析 94 5.3.3 FeNP@ L-Cystein@Protein G Zeta 表面電位分析 96 5.3.4 FeNP@ L-Cystein@Protein G@Anti-HDL IgG SQUID 磁性分析 97 5.4 UV-vis光譜 98 5.4.1 FeNP@ L-Cystein@Protein G 包覆量分析 98 5.4.2 FeNP@L-Cystein@Protein G @Anti-HDL IgG 接枝量分析 102 5.5 FeNP@L-Cystein@Protein G @Anti-HDL IgG @HDL 抗體專一性檢驗 105 5.5.1 CLSM螢光檢驗 105 5.5.2 FeNP@ L-Cystein@Protein G -Ig@HDL 抗氧化螢光能力檢驗 106 5.5.3 FeNP@L.C.@Protein G-Ig 抓取率穩定測試 108 5.6 FeNP@L-Cystein@Protein G-Ig-HDL臨床小樣本之DHR抗氧化能力檢驗 110 5.7 北醫檢體HDL電化學訊號與DHR 123螢光變化率之結果與相關性 113 5.8 HDL DHR123螢光變化率和電化學訊號與血管內皮前驅細胞(endothelial progenitor cell/EPC)相關係數分析 117 第6章 結論 118 參考文獻 120

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