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研究生: 石家宜
Shih, Chia-I
論文名稱: 開發以4,5-多巴雙加氧酶為基礎之全細胞生物感測器用於檢測二價銅離子及多巴胺
Development of DOPA 4,5-Dioxygenase-Based Whole-Cell Biosensors for the Detection of Copper(II) and Dopamine
指導教授: 葉怡均
Yeh, Yi-Chun
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 74
中文關鍵詞: 二價銅離子多巴胺螢光感測器4,5-多巴雙加氧酶綠色合成甜菜色素全細胞生物感測器
英文關鍵詞: Cu2+, dopamine, fluorogenic sensors, DOPA 4,5-dioxygenase, green synthesis, betaxanthin, whole-cell biosensors
DOI URL: http://doi.org/10.6345/NTNU202000805
論文種類: 學術論文
相關次數: 點閱:112下載:0
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  • 誌謝 i 中文摘要 ii ABSTRACT iii Abbreviations iv 目錄 v 表目錄 vii 圖目錄 viii 第 1 章 緒論 1 1.1 生物感測器 1 1.1.1 原理及發展 1 1.1.2 全細胞生物感測器 3 1.2 兒茶酚胺類神經傳導物質 5 1.3 4,5-多巴雙加氧酶 7 1.4 甜菜醛胺酸與甜菜色素 9 1.4.1 甜菜醛胺酸 9 1.4.2 甜菜色素 10 1.5 文獻回顧 12 1.5.1 銅離子感測器 12 1.5.2 多巴胺感測器 14 1.6 實驗動機與目的 16 第 2 章 材料與實驗方法 18 2.1 實驗儀器 18 2.2 實驗藥品 19 2.3 實驗設計 21 2.3.1 銅離子感測器實驗設計 21 2.3.2 多巴胺生物感測器實驗設計 22 2.4 實驗方法 23 2.4.1 勝任細胞的製備 23 2.4.2 轉化作用 23 2.4.3 甜菜色素的合成 24 2.4.4 甜菜色素偵測二價銅離子 25 2.4.5 甜菜色素試紙應用 25 2.4.6 定點突變實驗 26 2.4.7 突變蛋白質活性篩選 30 2.4.8 蛋白質的表現及粗萃取 32 2.4.9 蛋白質膠體電泳 34 2.4.10 突變蛋白質用於多巴胺的檢測 37 2.4.11 數據處理 37 第 3 章 實驗結果與討論 38 3.1 甜菜色素的合成 38 3.1.1 輔因子濃度之影響 38 3.1.2 甜菜色素的合成條件 39 3.1.3 不同胺類合成之甜菜色素 40 3.2 甜菜色素對二價銅離子的偵測 42 3.2.1 不同相位羧基合成甜菜色素加銅離子後的螢光影響 42 3.2.2 優化2-AIPA-BX生成條件 44 3.2.3 金屬離子選擇性 45 3.2.4 螢光淬滅之時間追蹤 46 3.2.5 銅離子的劑量反應 47 3.2.6 銅離子與2-AIPA-BX之作用關係 48 3.2.7 應用於人工尿液樣品及試紙 50 3.2.8 與先前文獻比較 52 3.3 突變蛋白質的活性篩選 53 3.3.1 突變胺基酸位點的選擇 53 3.3.2 單點突變 54 3.3.3 雙點突變 57 3.4 突變蛋白質專一性測試 59 3.4.1 選擇性比較 59 3.4.2 左旋多巴與多巴胺混合干擾 60 3.4.3 檢測線性比較 61 第 4 章 結論 63 REFERENCE 64 附錄 本研究使用之引子、質體及菌種 67

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