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研究生： 卓見
Cho, Chien
論文名稱： 化學探針應用於金屬離子及氟離子成像之發展
Development of Chemical Probes for the Imaging of Metal and Fluoride Ions
指導教授： 陳貴通
Tan, Kui-Thong
口試委員: 許馨云
Hsu, Hsin-Yun
黃郁棻
Huang, Yu-Fen
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： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 121
中文關鍵詞： 化學探針 、細胞表面 、籠閉式生物素 、金屬離子 、氟離子
外文關鍵詞： chemical probes, cell membrane, cage-biotin, metal ions, fluoride ions
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    • 儘管螢光探針在偵測小分子的應用上不斷地突破，仍有個基本的問題有待解決：在高濃度蛋白質的樣品內受到的非專一性結合所產生的感擾訊號。在本篇論文當中，我們將介紹一種封裝在抗生物素蛋白質腔中的螢光離子探針的新穎概念，來阻隔非專一性結合。我們在環境敏感性分子－1,8-naphthalimide螢光基團修飾上生物素(biotin)基團與鋅離子辨識端，透過將探針和抗生物素蛋白結合，以阻隔複雜蛋白質環境非專一性之干擾。我們結合SNAP-tag標記技術與avidin-biotin系統，成功地在Hela細胞表面標記上螢光探針，並且快速偵測到鋅離子產生之螢光影像。
    此外，我們也開發一種非螢光化學探針，透過生物素(biotin)與含有Cy3螢光基團之鏈黴親合素蛋白(streptavidin-Cy3)專一性結合的特點，成功開發出可偵測氟離子之反應型籠閉生物素探針，並且應用於小分子微陣列(Small-Molecule Microarrays, SMMs)之技術。透過小分子微陣列小型化、高通量篩選的特性，將探針固定於微陣列載體上觀測探針與streptavidin-Cy3結合後所產生之螢光影像。

    Despite the promising improvements made recently in fluorescence probes for the small molecules detection, there is one problem remain: strong nonspecific signals in samples containing high protein levels. In this thesis, we introduce a novel fluorescent probe encapsulated in protein cavity concept as demonstrated by avidin protein. The probes were constructed by following the current probe design for zinc ions with a short biotin linker for the conjugation to avidin protein to overcome the nonspecific fluorescence in blood samples by blocking out nonspecific dye−protein interactions. Furthermore, The AGT (O6-alkylguanine-DNA alkyltransferase) labeling methodology provides a way to direct localization of the Zn(II) probe on the surface of living cells and to visualize qualitatively a turn-on emission response to addition of zinc.
    In addition, we introduce a novel sensing mechanism based on caged biotin for fluoride ion detection in small-molecule microarrays (SMMs). The reactable caged biotin probes were modified with fluoride-cleavable groups at the urea nitrogen. Upon uncaging with fluoride ions, the caged biotin probes are transferred to the streptavidin-Cy3 protein to emit stronger fluorescence. We then designed and synthesized novel caged biotin probes that could immobilized on the surface of microarrays.

    摘要 i Abstract ii 謝誌 iii 著作列表 iv 目錄 v 第一章 緒論 1 1-1前言 1 1-2感測器的組成 2 1-3感測器的特性 5 1-4化學感測器的種類與應用 6 1-5螢光化學感測器(Fluorescent Chemosensor) 8 1-5-1光誘導電子轉移(PET, Photoinduced Electron Transfer) 9 1-5-2鎖住基團的螢光分子(Block group fluorophore) 13 1-5-3螢光共振能量轉移(Förster resonance energy transfer, FRET) 14 Part 1. 17 第二章 文獻回顧 18 2-1 環境敏感螢光分子(environment-sensitive fluorophore) 18 2-1-1 極性敏感螢光分子(polarity-sensitive fluorophores) 18 2-2鋅離子之螢光偵測 20 2-2-1鋅離子之亞細胞影像偵測 21 2-2-1.1粒線體中偵測鋅離子之影像 21 2-2-1.2高基氏體中偵測鋅離子之影像 22 2-2-1.3細胞表面中偵測鋅離子之影像 24 第三章 探針設計與構想 26 3-1探針設計 26 3-1-1 探針構想 26 3-1-2 探針設計 30 第四章 結果與討論 33 4-1 鋅離子螢光探針性質測試 33 4-2 細胞表面實驗 36 4-3 實驗結論 39 Part 2. 40 第五章 文獻回顧 41 5-1籠閉探針(caged probe) 41 5-1-1反應型籠閉螢光探針(reactive caged-fluorescence probe) 42 5-1-1.1籠閉型羅丹明(caged-rhodamine) 42 5-1-1.2籠閉型東京綠(caged-Tokyo Green) 43 5-1-1.3籠閉型香豆素(caged-cumarin) 45 5-1-2籠閉型非螢光探針 45 5-1-2.1籠閉生物素(caged-biotin) 46 5-2小分子微陣列(Small-Molecule Microarrays) 47 5-2-1小分子微陣列製備方法(Array fabrication strategies) 48 5-2-2共價鍵結固定(covalent immobilization) 49 5-2-3光活化( Photoactivation) 49 5-2-4原位合成(in situ synthesis) 50 第六章 探針設計與構想 52 6-1探針設計 52 6-1-1探針構想 52 6-1-2 探針設計 53 第七章 結果與討論 56 7-1 小分子微陣列影像與分析 56 7-2 實驗結論 59 第八章 實驗部分 60 8-1 一般實驗敘述 60 8-2 螢光測試條件 62 8-3轉染後Hela細胞影像 62 8-4 有機合成及光譜資料 63 文獻參考 83 附錄 88

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