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研究生： 張璧名
Be-Ming Chang
論文名稱： 利用螢光奈米鑽石做生物標記的開發與應用
Development and Use of Albumin-Conjugated Fluorescent Nanodiamond for Biolabeling
指導教授： 張煥正
Chang, Huan-Cheng
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2011
畢業學年度： 99
語文別： 中文
論文頁數： 48
中文關鍵詞： 螢光奈米鑽石 、白蛋白 、抗體
英文關鍵詞： fluorescencet nanodiamond, Albumin protein, antibody
論文種類： 學術論文
相關次數： 點閱：187  下載：2 
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    • 生物研究上,螢光標記是常用的研究方法之一,但常用的螢光標記物 質,如有機螢光染劑、量子點等有光漂白的問題或是具生物毒性而影響生物活性, 這些都會降低研究結果的準確性。相較而言,螢光奈米鑽石能永久維持螢光性質 並具有非常好的生物相容性,沒有量子點會光閃爍而不利於觀察的缺點,在生物 標記上也能做長期及連續的追蹤,因此做為生物標記的工具極具潛力。然而螢光 奈米鑽石在生物研究常用的緩衝溶液中容易聚集 (agglomeration) 的問題,讓 生物標記無法成功達到專一性及良好的影像解析。因此本篇利用白蛋白(albumin protein) 以簡易吸附的方式修飾到螢光奈米鑽石表面上,避免螢光奈米鑽石聚 集的問題,且證明能長時間在生物緩衝溶液 (Phosphate buffered saline, PBS) 中維持良好的懸浮性。
    再者,本實驗利用懸浮性良好的螢光奈米鑽石 (albumin-FND)表面的白 蛋白之胺基 (amine group) 位置,將生物素 (biotin) 分子共價鍵結在白蛋白 上成為我們的標記探針 (biotin-albumin conjugated FND)。而我們將具有生物 素分子的 CD44 抗體或霍亂毒素次單元 B 在辨認細胞表面專一性的位置後,利用 卵白素 (streptavidin protein) 與生物素之間極高的親和性來與標記探針 (biotin-albumin conjugated FND) 做連接,並成功得到專一性的螢光標記及良 好的螢光解析。

    Immunofluorescence labeling is a common method offering visualization and detection of key cellular processes. Fluorescent probes, such as, organic dye and quantum dots suffer from some limitations, like photo bleaching and toxicity. Recently, fluorescent nanodiamond (FND) has emerged as a promising tool for fluorescence analysis owing to its non-photobleaching and high biocompatibility characteristics. Here we show that albumin-conjugated FNDs disperse well in high ionic strength bio-medium without agglomeration and can keep good stability for at least 9 days just using simple non-covalent conjugation methods. Also, we demonstrate that this albumin-conjugated FNDs coupled with biotins, through covalent cross-linkage with the albumin amine groups, can bind selectively with streptavidin and subsequently with biotinylated CD44 antibodies or biotinylated Cholera toxin subunit B. With this technique, we achieve high-specificity targeting of membrane proteins or glycolipid, and obtain high-quality fluorescence images of fixed and live cells.

    中文摘要..........................................................................................I Abstract .......................................................................................II 目錄 .......................................................................................... III 表目錄.......................................................................................... VII 圖目錄 ..........................................................................................IX 第一章 諸論 ........................................................1 1.1 前言 ....................................................1 1.2 螢光奈米粒子 ............................................1 1.3 奈米鑽石之結構與特性 ....................................3 1.4 螢光奈米鑽石 ............................................6 1.5 螢光標記方式 ............................................9 1.6 標記抗體 ...............................................11 1.6.1 CD44 GLYCOPROTEIN....................................13 1.6.2 GLYCOLIPID RECEPTOR GANGLIOSIDE GM1......................14 1.7 研究動機 ...............................................15 第二章 藥品與儀器..................................................17 2.1 藥品 ...................................................17 2.2 儀器 ...................................................18 第三章 實驗方法....................................................20 3.1 螢光奈米鑽石(FND)的製備 ................................20 3.2 製備 BSA 修飾之奈米鑽石粒子 .............................22 3.2.1 30NM 螢光奈米鑽石吸附 BOVINE SERUM ALBUMIN PROTEIN (BSA) .22 3.2.2 30NM 螢光奈米鑽石吸附Α-LACTALBUMIN PROTEIN (Α-LA).....22 3.2.3 15NM 螢光奈米鑽石吸附Α-LACTALBUMIN PROTEIN ............23 3.3 利用 BSA 修飾螢光奈米鑽石做細胞膜上標記 .................23 3.3.1 CELL CULTURE ........................................23 3.3.2 利用 BSA-30 NM FND 非專一性吸附於細胞膜表面 ........24 III 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 製備 BIOTIN-BSA-FND 鑽石粒子 ........................24 利用有機螢光染劑標記 HELA CELL 之 CD44 抗原 ...........25 CD44 LABELING USING BIOTIN-BSA-FND PARTICLES .............26 CD44 LABELING CONTROL USING BIOTIN-BSA-FND ..............27 封片膠 MOWIOL 的製備 ................................27 GM1 LABELING ........................................28 第四章 結果與討論..................................................29 4.1 奈米鑽石吸附 ALBUMIN PROTEIN ...............................29 4.1.1 4.1.2 4.1.3 奈米鑽石吸附 BOVINE SERUM ALBUMIN .....................29 奈米鑽石吸附 Α- LACTALBUMIN PROTEIN ...................30 BSA 修飾的螢光奈米鑽石非專一性標記在 HELA CELL 膜上 ..34 4.2 CD44 LABELING .............................................38 4.2.1 利用 BIOTIN-FLUORESCEIN 標記 HELA CELL 之 CD44 抗原.......38 4.2.2 利用螢光奈米鑽石標記細胞膜上 CD44 抗原 .............39 4.3 GM1 GLYCOLIPID LABELING .....................................42 4.3.1 利用 30 NM 螢光奈米鑽石標記未聚集及已聚集之 GM1 ......42 第五章 結論........................................................44 第六章 參考文獻....................................................45

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