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研究生: 洪媺媃
Hung, Mei-Jou
論文名稱: 利用牛血清蛋白包覆的金奈米團簇檢測兒茶酚類似物
Bovine Serum Albumin-Protected Gold Nanoclusters for the Detection of Catechol analogues
指導教授: 陳淑慧
Chen, Shu-Hui
學位類別: 碩士
Master
系所名稱: 理學院 - 化學系
Department of Chemistry
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 86
中文關鍵詞: 牛血清白蛋白金奈米團簇檢測應用兒茶酚
外文關鍵詞: Bovine serum albumin, Gold nanocluster, Sensor, Catechol
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  • 在過去十年以來,由於蛋白質包覆的金奈米團簇具有優異的光穩定性以及良好的生物相容性,因此在醫學和奈米生物技術領域中備受關注,且也被廣泛應用於物質的檢測,而在本研究中,我們以光穩定、無毒以及具有紅色螢光放射的牛血清蛋白包覆金奈米團簇 (BSA-AuNCs) 選擇性的檢測兒茶酚類似物質。
    首先我們利用螢光光譜儀、UV吸收光譜儀、MALDI-TOF質譜儀以及XPS對於合成後的BSA-AuNCs進行光學性質、質量及組成型態的鑑定,以確認BSA-AuNCs的形成,合成後的BSA-AuNCs整體質量大約為71 kDa,且具有635nm的螢光放射。
    實驗將兒茶酚類似物以鐵 (III) 離子氧化為鄰苯醌後與BSA-AuNCs反應,並觀察到了其螢光的淬滅,並藉由測量其螢光淬滅及UV吸收,可以選擇性的偵測兒茶酚類似物,再來我們更以蛋白質體學中由下而上的水解酵素法分析樣品,推測造成BSA-AuNCs螢光淬滅的原因主要來自氧化後的兒茶酚類似物與金團簇核心附近的BSA基板之間的親和作用力,進而發生金團簇螢光與兒茶酚類似物-鐵(III)錯合物及鄰苯醌之間的螢光共振能量轉移 ( FRET ) 和電子轉移 (Electron transfer)效應,造成螢光的淬滅。此檢測系統具有應用於真實樣品的可行性,在未來我們欲將其用於偵測茶葉中的兒茶素或添加於食用植物油中的沒食子酸丙脂。

    In the past decade, protein-protected gold nanoclusters have attracted much attention in the fields of medicine and nanobiotechnology due to their excellent photostability and good biocompatibility. It also was widely used in sensing applications. In this study, we utilized the fluorescent gold nanocluster synthesized by bovine serum albumin to detect catechol analogues.
    We found that the red fluorescence of BSA-AuNCs was quenched by catechol analogues after adding iron (III). This sensing system can selectively detect the catechol analogues by measuring the fluorescence quenching and UV absorbance.
    BSA-AuNCs reacted with oxidized catechol analogues were then analyzed by proteomics-based mass spectrometry techniques. It is speculated that the mechanism for the fluorescence quenching of BSA-AuNCs is mainly due to the fluorescence resonance energy transfer (FRET) between Au core and catechol analogues - iron(III) complex, and electron transfer effect between Au core and catechol quinone. We expect our developed sensing system could be applied to real samples, such as catechins in tea or propyl gallate in vegetable oil.

    目錄 致謝 I Abstract II 中文摘要 III 英文延伸摘要 IV 目錄 VIII 圖目錄 XII 表目錄 XV 簡稱用與對應表 XVI 第一章 研究內容 1 1.1 研究動機 1 1.2 研究方向與策略 2 第二章 文獻回顧 3 2.1 金奈米團簇 ( AuNCs ) 3 2.1.1 金奈米團簇簡介 3 2.1.2 金奈米團簇製備方法 4 2.1.3 金奈米團簇的應用與前景 5 2.2 牛血清蛋白金奈米團簇 ( BSA-AuNCs ) 5 2.2.1 牛血清白蛋白結構介紹 5 2.2.2 合成方法 7 2.2.3 螢光特性 7 2.2.4 結構分析 8 2.2.5 檢測方面應用 9 2.3 兒茶酚類似物 13 2.3.1 兒茶酚物質之化學特性及其毒性 13 2.3.2 兒茶酚轉變為鄰苯醌的方法 17 2.3.3 檢測兒茶酚類似物之方法回顧 18 第三章 實驗方法 20 3.1 實驗藥品與耗材 20 3.1.1 實驗藥品 20 3.1.2 實驗耗材與儀器 21 3.2 牛血清蛋白包覆金奈米團簇的合成 22 3.2.1 合成紅光牛血清蛋白包覆金奈米團簇 22 3.2.2 純化合成後的牛血清蛋白包覆金奈米團簇 22 3.3 基質輔助雷射脫附游離飛行式質譜儀(MALDI)的樣品製備 23 3.4 水解酵素法(In solution digestion) 24 3.4.1 變性酵素水解法 ( Denatured digestion) 24 3.4.2 原態酵素水解法 ( Native digestion ) 24 3.5 奈米電噴灑線性離子阱式軌道阱質譜儀 ( LTQ-Orbitrap ) 25 3.6 樣品性質鑑定之儀器 26 3.6.1 螢光光譜儀 ( Photoluminescence Spectrometer ) 26 3.6.2 紫外光可見光近紅外光分光光譜儀 ( UV / VIS / NIR Spectrometer ) 26 3.6.3 化學分析電子光譜儀 ( XPS) 26 3.7 以牛血清蛋白金奈米團簇檢測兒茶酚類似物 27 3.7.1 兒茶酚類似物標準液檢測 27 3.7.2 以變性水解酵素法分析與兒茶酚類似物反應完之BSA-AuNCs樣品溶液 27 3.7.3 以BSA-AuNCs檢測真實樣品 28 3.7.4 BSA-AuNCs於無氧環境下檢測兒茶酚-鐵(III) 28 3.8 數據分析軟體 29 第四章 結果與討論 31 4.1 牛血清包覆金奈米團簇的性質鑑定 31 4.1.1 紫外光可見光分光光譜儀 ( UV/VIS Spectrometer ) 分析 31 4.1.2 螢光光譜儀 ( Photoluminescence Spectrometer ) 分析 32 4.1.3 不同pH值BSA-AuNCs螢光強度及穩定性表現 34 4.1.4 基質輔助雷射脫附游離飛行質譜儀 ( MALDI-TOF ) 分析 35 4.1.5 化學分析電子光譜儀 ( XPS ) 分析 37 4.1.6 以由下而上方法搭配質譜分析BSA-AuNCs成核位點 39 4.2 兒茶酚轉變為鄰苯醌之鑑定 41 4.2.1兒茶酚類似物-鐵(III)離子比例之最佳化 41 4.2.2 兒茶酚類似物與鐵(III)離子反應之UV分析 45 4.3 以牛血清蛋白金奈米團簇檢測兒茶酚類似物 46 4.3.1 BSA-AuNCs與兒茶酚-鐵(III)離子反應後之UV/螢光鑑定 46 4.3.3 BSA-AuNCs與不同濃度兒茶酚類似物-鐵(III)離子反應之UV/螢光光譜圖 49 4.4 BSA-AuNCs偵測兒茶酚類似物之機制探討 50 4.4.1 BSA-AuNCs於無氧環境下檢測兒茶酚-鐵(III) 52 4.4.2 兒茶酚類似物-鐵(III)錯合物之UV吸收與BSA-AuNCs螢光淬滅之關聯性 53 4.4.3 以由下而上方法分析BSA-AuNCs與兒茶酚類似物-鐵(III)反應後之情形 56 4.5 BSA-AuNCs對於兒茶酚類似物之偵測專一性 60 4.6 BSA-AuNCs於真實樣品檢測 63 第五章 結論 67 第六章 參考文獻 69 附錄 77 BSA-AuNCs bottom-up peptide sequence coverage : 93% 77 BSA-AuNCs unmodified and modified ( CA、4OHE2、DA、PG) peptide 80 Annotated MS2 of modified peptide 84

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