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研究生： 鍾紫宣
Chung, Tzu-Hsuan
論文名稱： 可控結合生物素探針於細胞成像及酵素催化螢光增益的應用
Enzyme-Catalyzed Fluorescence Signal Amplification and Cell-Imaging with Streptavidin-Biotin Controlled Binding Probe
指導教授： 陳貴通
Tan, Kui-Thong
口試委員: 黃郁棻
Huang, Yu-Fen
許馨云
Hsu, Hsin-Yun
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： 2018
畢業學年度： 106
語文別： 中文
論文頁數： 93
中文關鍵詞： 生物素探針 、鏈黴親合素 、螢光 、訊號增益 、酵素 、過氧化亞硝酸鹽
外文關鍵詞： caged-biotin-probe, streptavidin, fluorescence, signal amplification, enzyme, peroxynitrite
相關次數： 點閱：3  下載：0 
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    • 近年來發展出一種新型的籠閉生物素螢光探針模型(Caged-Biotin Probe, CBP)，利用鏈黴親和素蛋白與生物素間強勁的結合力(K⁓1015)，探針能夠在偵測到目標物時進行結構轉換，解除籠閉狀態的生物素即可與鏈黴親和素蛋白進行結合，具備快速專一且無背景訊號的特色，突破以往螢光探針易受背景干擾、單一訊號模式的限制，揭開新型螢光探針的序幕。
    銜接本實驗室設計的反應型籠閉生物素探針模型，我們合成出兩種新型的CBP (CBP-BA, CBP-N3)，兩者皆具備了簡易合成步驟、低背景、高螢光增益性等優點。我們將兩種探針進行不同的實驗研究。將CBP-BA進行細胞實驗的探討，不僅成功地偵測到內源性的過氧亞硝酸酶(peroxynitrite, ONOO)，並且擁有極佳的穩定性及偵測極限。我們也利用細胞影像觀察巨噬細胞遭受細胞激素 (cytokine)時的反應，更深入了解這些物質對疾病影響的程度。另一方面，CBP-N3實驗了兩種螢光放大機制模式：1. 藉著streptavidin上多個螢光分子放出訊號，運用於細胞實驗。2. 藉著streptavidin上的酵素及其對應的受質產生螢光，運用於孔盤實驗。孔盤實驗中，利用酵素催化可持續增益螢光訊號，提供了更可觀的螢光增益性質，具備了更優越的偵測極限。我們相信結合上述兩種探針的優點，可將CBP運用於細胞及活體實驗，且高增益性質的酵素催化優勢，能夠使我們更顯而易見地檢測目標物的濃度變化。

    Recently, many small molecule-based detection systems have been developed to sense enzyme activities and quantify small molecule concentration. In this thesis, a novel type of chemical probes—Target-Activated Streptavidin-Biotin Control Binding Probes (CBP) have been developed, which is based on the extremely high affinity between streptavidin and biotin. Incorporation of a functional group at the N’-1 urea nitrogen of biotin can dramatically reduce the streptavidin-binding affinity. This functional group can be designed by which target analytes will be detected. Streptavidin can be conjugated with multiple fluorophore units or enzymes to amplify fluorescence signals. Signal amplification can be achieved by the released fluorescence molecules after enzymatic reaction. Based on this approach, we applied the CBP concept not only on cell-imaging but also on enzyme-catalyzed fluorescence signal amplifications on maleimide-modified 96-well microplates. In the cell-imaging, the probe, CBP-BA, owns a very good sensitivity and selectivity toward peroxynitrite. We can utilize cell-imaging experiment of CBP-BA to study the biology about the inflammation of phagocyte. In the plate detection system, the detection limit of the probe, CBP-N3, in the concentration-dependent experiment has been improved by enzyme-catalyzed signal amplification. We believe that we have developed a versatile target detection method and have demonstrated the potential for diagnostic applications.

    摘要 I Abstract II 謝誌 III 目錄 IV 著作列表 VII 第一章 緒論 1 1-1 研究動機 1 1-2 自由基的形成與疾病的關係 1 1-2.1 活性氧物種 (Reactive Oxygen Species, ROS) 2 1-2.2 活性氮物種 (Reactive Nitrogen Species, RNS) 4 1-2.2.1 過氧化亞硝酸鹽 (Peroxynitrite) 8 第二章 參考文獻 10 2-1 籠閉螢光團 (caged-fluorophore) 12 2-2 籠閉螢光素 (caged-luciferin) 16 2-3 籠閉生物素 (caged-biotin) 20 第三章 探針構想與設計 22 3-1 設計方向 22 3-2 目標分析物及辨識端 23 3-3 探針如何標定於細胞 24 第四章 結果與討論 27 4-1 Biotin-NHS細胞測試 27 4-2 探針CBP-BA之外源性ONOO-細胞影像和其選擇性 28 4-3內源性ONOO-之釋放及其釋放量之比較 30 4-4 PMA, IFN-g釋放ONOO-之偵測極限 34 4-5 IFN-g刺激細胞時間與ONOO-釋放量之關係 37 4-6 抑制劑測試 39 4-7 探針穩定性與細胞毒性測試 41 第五章 酵素催化之訊號放大系統 45 5-1 酵素結合免疫吸附分析法ELISA 45 5-1.1直接捕獲vs間接捕獲(capture)及直接偵測vs間接偵測(detection) 45 5-1.2 ELISA不同類型之介紹 47 5-2 酵素催化訊號放大之優點 49 5-2.1 Target-Activated Plate Assay (TAPA) 50 5-3 探針CBP-N3之合成與設計 52 第六章 結果與討論 54 6-1 TAPA之條件改良 54 6-2 探針CBP-N3螢光測試 58 第七章 結論 62 第八章 實驗部分 64 8-1實驗藥品及器材 64 8-2有機合成與光譜資料 66 8-3 細胞培養及細胞影像實驗 73 8-3.1 培養液及試劑 73 8-3.2 細胞繼代培養 73 8-3.2.1 Hela細胞 73 8-3.2.2 RAW264.7細胞 73 8-3.2.3 RAW264.7及Hela細胞影像 74 8-3.3 細胞毒性測試 MTT Assay 74 8-4 TAPA 75 參考文獻 76 附錄 81

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