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研究生： 夏啟揚
Hsia, Chi-yang
論文名稱： 對磺胺類藥物偵測與蛋白質偵測之螢光探針合成
Sulfonamide Drugs and Protein Detection with Fluorescent Probes
指導教授： 陳貴通
Tan, Kui-thong
口試委員: 王聖凱
Sheng-kai, Wang
陳貴通
Kui-thong, Tan
許馨云
Hsin-yun, Hsu
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： 2014
畢業學年度： 103
語文別： 英文
論文頁數： 215
中文關鍵詞： 探針 、半合成螢光生物感測器
外文關鍵詞： probe, semisynthetic fluorescent sensor
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    • 代謝物與蛋白質在人體內扮演重要角色，參與生長、發育、繁殖等功能，當兩者濃度失常時常造成各種疾病。傳統偵測方法須經過一系列步驟，較不便且費時。
    本論文中以螢光方式對磺胺類代謝物與 hCAII、avidin 蛋白進行偵測，在代謝物偵測上使用半合成生物感測器，蛋白質偵測則以螢光探針的形式進行檢測。半合成生物感測器是由 SNAP-hCA 融合蛋白與螢光探針所組成，以生物轉殖方法表現SNAP-hCA融合蛋白；螢光探針則是以 BG 基團搭配環境敏感螢光分子 SBD 並連接磺胺類基團組成。SNAP-hCA 融合蛋白中的SNAP 可辨識BG 基團，將螢光探針以共價鍵方式連接於 SNAP 蛋白上，磺胺類基團則可與hCAII 結合，使感測器形成環狀構型。環狀構型造成螢光分子周圍環境較為疏水，SBD 分子產生明顯螢光訊號。當目標磺胺類藥物加入後與探針競爭hCAII 活性位置，使構型呈開環狀態，降低環境疏水性，造成螢光訊號下降。蛋白質螢光探針利用環境敏感螢光分子GFP 與 Bodipy，連接苯磺醯胺（Benzene-sulfonamide）或生物素（biotin），可分別與hCAII、avidin形成專一性結合。探針與目標蛋白結合與否，會造成螢光分子周圍環境改變，使螢光訊號產生變化。

    In recent years, scientists discover that proteins and metabolites are involved in many diseases. The concentration of proteins and metabolites often influence patient’s conditions. Therefore, methods which can detect concentration of proteins and metabolites are important in biology and medicine. Here we introduce two approach for the detection of proteins and metabolites, fluorescence sensor protein and fluorescence probe.
    Fluorescent sensor proteins offer the possibility to study the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as these sensors rely on a conformational change of a protein upon ligand binding to generate sinal. Here we devalope a strategy to overcome this limitation. We use a self-labeling protein tag to link with hCA to form SNAP-hCA. This recombinant protein was labeled with defferent synthetic moleculer containing BG group and fluorophore.
    In our fluorescent sensor protein, the metabolite of interest competes with labeling ligand. The fluorescence decrease, as the metabolite displaces the intramolecular ligand and makes the cyclic system open. The defference of readout can also be fitted with dose-response equation to obtain IC50. Furthermore, the modular design of our sensors provides a cyclic system that can change ligand binding protein or self-labeling protein. This adventage can let us detect defferent metabolite by aimply changing ligand binding protein in further approaches.
    In the second part, fluorescence probe containing a fluorophore and a ligand is designed to detact protein. Since the ligand part of fluorescence probe can specificly inhibit target protein, our probe can show a high specificity for target protein identification. Our fluorophore in this part not only has polarity sensitivity but also viscosity sensitivity. When the ligand part of our probe recognizes the binding site of target protein, the fluorophore will be closer to the binding pocket. Subsequently the fluorescent turn on, because of the restricted environment.

    謝誌 i 摘要 ii Abstract iii 縮寫對照表 vi 第一章 緒論 1 §1-1蛋白酶與非蛋白酶 1 §1-2代謝物 3 §1-2.1一般代謝物 3 §1-2.2磺胺類藥物 5 §1-3環境敏感螢光分子 10 §1-3.1極性敏感螢光分子 10 §1-3.2黏度敏感螢光分子 12 第二章 文獻回顧 14 §2-1細胞內小分子代謝物檢測 14 §2-1.1週質結合蛋白質變構式生物感測器 14 §2-1.2適體型感測器 16 §2-1.3螢光受質型偵測方法 18 §2-1.4量子點型偵測方法 20 §2-1.5金奈米粒子型偵測方法 22 §2-1.6半合成生物感測器偵測方法 24 §2-2近代選擇性蛋白質檢測 26 §2-2.1螢光探針 26 §2-2.2抗體型偵測方法 28 §2-2.3螢光蛋白型偵測方法 30 §2-2.4適體信標式偵測方法 32 §2-2.5高分子型螢光偵測方法 33 §2-2.6量子點型偵測方法 34 §2-2.7金奈米粒子型偵測方法 35 第三章 磺胺類藥物檢測之設計與方法 37 §3-1 設計構想 37 §3-1.1文獻參考 37 §3-1.1設計探討 38 §3-2 方法 41 §3-2.1環境敏感螢光探針選擇 41 §3-2.2 SNAP-tag 技術 42 §3-2.3 合成策略 43 第四章 磺胺類藥物檢測之實驗結果與討論 45 §4-1探針開發 45 §4-2磺胺類藥物IC50值檢測 53 §4-3結論 65 第五章 蛋白檢測之設計與方法 66 §5-1 設計構想 66 §5-1.2文獻參考 66 §5-1.1設計探討 69 §5-2方法 70 §5-2.1 GFP 螢光分子合成策略 70 §5-2.2 Bodipy 螢光分子合成策略 70 第六章 實驗結果與討論 72 §6-1 GFP 螢光分子 72 §6-2 Bodipy 螢光分子 79 第七章 實驗總結論 96 第八章 實驗部分 97 §8-1一般實驗敘述 97 §8-2有機合成部分與光譜資料 99 §8-3蛋白質表現與純化 145 §8-3.1蛋白質之蛋白表現 145 §8-3.2蛋白質純化 147 §8-3.3 SDS-膠體電泳（SDS-page） 150 參考文獻 152

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