臺灣博碩士論文加值系統

複雜基質往往是在分析鑑定生物樣品時最大的阻礙，所以擁有有利且實用的純化工具是非常重要的。具有選擇性、快速且簡單合成特性的分子生物模板，顯然能成為現代生物化學純化的最佳利器之一。其中抗原決定基 (表位) 印跡法 (Epitope imprinting) 為現代研究大分子蛋白質常見的方法，其運用抗原-抗體通過一小段活化位點進行專一性辨識的概念，結合分子生物模板純化欲分析之目標蛋白質。
本研究合成分子生物模板於Fe3O4奈米粒子上，藉以純化人類血清白蛋白，經由十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 和MALDI-TOF MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry) 進行純化鑑定。本實驗Fe3O4奈米粒子是藉由共沉澱法合成，在奈米粒子表面修飾TEOS (Tetraethoxysilane) 避免Fe3O4氧化，再修飾具有-NH2官能基的APTES ((3-Aminopropyl)triethoxysilane)，讓Lomant's Reagent能將磁性奈米粒子與模板分子Human Serum Albumin胜肽鍵結在一起，並以高分子包覆聚合，再使用DTT (Dithiothreitol) 水溶液將模板分子移除後，形成最終的分子生物模板。磁性模板分子能快速地吸附蛋白質標準品及真實樣品中的目標蛋白質，並能藉由其磁力迅速的分離模板和溶液，藉由SDS-PAGE和MALDI-TOF MS證實此技術之可行性。

Biological samples are usually accompanied with complex matrices, which cause a huge obstacle in the identification of biological analytes. For the reason, a convenience and useful tool for the selective purification of targeted analytes is necessary. Molecularly imprinting polymers possess several advantages, including selectivity, simple for preparation and rapid for purification, which become one of appropriate implements for purification and extraction of targeted analytes. Epitope imprinting has been used for the purification of large macromolecule protein. The principle is similar with the interaction between antibodies and antigens, which need a small portion of antigen to bind with antibody. In this study, a small exposed peptide of protein was used as the template to formation of Fe3O4@MIP, which could be able to recognize the targeted protein.
Fe3O4 magnetic nanoparticles were synthesized by the use of co-precipitation method and subsequently coated with TEOS and APTES. The primary amines on magnetic nanoparticles and targeted peptide were reacted with Lomant's Reagent to form stable amide bonds. Targeted specific recognition shells, which the size, shape and functional groups corresponding to peptide template, were formed by polymerization with functional monomers. The peptide template could be removed by utilizing dithiothreitol and the resulting MIPs were employed to purify the targeted proteins. The resulting magnetic molecularly imprinting polymers absorbed targeted protein rapidly, which could be separated easily from complex matrix sample with a magnet. The absorption of targeted protein could be analyzed and confirmed by SDS-PAGE and MALDI-TOF MS.

摘要 I
ABSTRACT II
第一章、 緒論 1
1.1. 蛋白質體學 1
1.2. 磁性奈米粒子 2
1.2.1. 磁性奈米簡介 2
1.2.2. 磁性奈米粒子穩定化反應 7
1.3. 分子生物模板之簡介 9
1.3.1. 分子印跡合成技術 10
1.3.1.1. 共價型分子印跡 10
1.3.1.2. 非共價型分子印跡 10
1.3.2. 分子印跡聚合物的製備方法 11
1.3.2.1. 整體印跡法 (Bulking imprinting) 11
1.3.2.1.1.本體聚合法 12
1.3.2.1.2.沉澱聚合法 12
1.3.2.1.3.懸浮聚合法 12
1.3.2.2. 表面印跡法 (Surface imprinting) 13
1.3.2.3. 抗原決定基法 (Epitope approach) 13
1.3.3. 分子印跡技術基本原理 14
1.3.3.1. 功能性單體 (Functional monomer) 14
1.3.3.2. 起始劑 (Initiator) 17
1.3.3.3. 目標分子 (Targeted template) 17
1.3.3.4. 交聯劑 (Cross-linker) 17
1.4. 質譜儀 20
1.4.1. 質譜儀簡介及其原理 20
1.4.2. 基質輔助雷射脫附游離飛行時間串聯式質譜 (Matrix-Assisted Laser Desorption Ionization) 20
1.4.3. 飛行時間質量分析器 (Time-of-Flight, TOF) 21
1.4.4. 基質輔助雷射脫附游離化機制[73] 22
1.4.5. 基質 24
1.4.6. MALDI樣品盤 (MALDI plate) 25
1.4.7. 儀器構造 27
1.5. 研究動機 29
第二章、 實驗藥品、儀器及步驟 32
2.1. 實驗藥品 32
2.2. 實驗器材 34
2.3. 藥品配置 35
2.4. 實驗步驟 36
2.4.1. Fe3O4磁性奈米粒子製備 36
2.4.2. Fe3O4@SiO2磁性奈米粒子製備 36
2.4.3. Fe3O4@SiO2@NH2 磁性奈米粒子製備 37
2.4.4. Fe3O4@SiO2@NH-DTSP-HSA製備 37
2.4.5. Fe3O4@SiO2@NH-DTSP-HSA@Silane製備 38
2.4.6. Fe3O4@MIP 製備 38
2.4.7. HSA peptide 吸附測試 38
2.4.8. HSA peptide 脫附測試 39
2.4.9. Standard HSA protein 吸附測試 39
2.4.10. Standard HSA protein 脫附測試 40
2.4.11. Real sample 部分 40
2.4.11.1.人類血清之取得方法 40
2.4.11.2.Real sample 吸附測試 41
2.4.11.3.Real sample 脫附測試 41
2.4.12. 逆相高效能液相層析儀 (RP-HPLC) 參數 42
2.4.13. MALDI參數 42
第三章、 結果與討論 46
3.1. 磁性奈米粒子鑑定 46
3.1.1. 運用TEM觀測磁性奈米粒子粒徑大小 46
3.1.2. 運用FT-IR觀測磁性奈米粒子特定官能基 46
3.1.3. 運用DLS觀測磁性分子生物模板粒徑大小 51
3.1.4. 運用VSM鑑定Fe3O4磁性奈米粒子之磁力 51
3.2. 條件探討 54
3.2.1. 溶劑是否除水之選擇 54
3.2.2. Fe3O4是否要包覆SiO2 56
3.3. 反應量優化 56
3.3.1. 優化藥品DTSP及HSA的反應量 56
3.3.2. 優化藥品APTES的反應量 59
3.4. 反應時間 59
3.5. 功能性膠體厚度 62
3.6. 吸附效率之測試 62
3.6.1. 胜肽測試 62
3.6.2. 標準品測試 65
3.6.2.1. 標準品 65
3.6.2.2. 標準品之還原雙硫鍵 65
3.6.2.3. 標準品之還原雙硫鍵與烷化硫基 68
3.6.2.4. 標準品之還原雙硫鍵與烷化硫基與過濾 68
第四章、 結論 73
第五章、 參考資料 74

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