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研究生： 陳慧瑜
Chen, Huei-Yu
論文名稱： 利用酵素連續催化顯色反應偵測酪胺酸及酪胺酸酶活性
The Enzyme Cascade-Triggered Colorimetric Reaction for the Detection of Tyrosine and Tyrosinase Activity
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： 75
中文關鍵詞： 酪胺酸 、酪胺酸酶 、4,5-多巴雙加氧酶 、甜菜醛胺酸 、甜菜色素 、酵素連續催化 、比色法生物感測器
英文關鍵詞： tyrosine, tyrosinase, 4,5-dopa dioxygenase, betalamic acid, betaxanthin, enzyme cascade, colorimetric biosensor
DOI URL： http://doi.org/10.6345/NTNU202000728
論文種類： 學術論文
相關次數： 點閱：82  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 ix 圖目錄 x Chapter 1 Introduction 1 1.1 生物感測器 (Biosensor) 1 1.1.1 前言 1 1.1.2 生物感測器的偵測機制介紹 1 1.1.3 生物感測器的種類 2 1.1.4 酵素生物感測器 (Enzyme-based Biosensor) 3 1.2 酪胺酸 (Tyrosine, Tyr) 5 1.2.1 酪胺酸介紹 5 1.2.2 酪胺酸代謝異常之相關疾病 6 1.3 酪胺酸酶 (Tyrosinase) 8 1.3.1 酪胺酸酶介紹 8 1.3.2 酪胺酸酶表達異常之相關疾病 9 1.4 多巴雙加氧酶 (DOPA-Dioxygenase) 11 1.5 甜菜醛胺酸及甜菜色素 (Betalamic Acid and Betalain) 13 1.5.1 甜菜醛胺酸的合成 13 1.5.2 甜菜色素的合成 14 1.6 文獻回顧 (Literature Review) 16 1.6.1 酪胺酸感測器 16 1.6.2 酪胺酸酶感測器 17 1.6.3 與先前研究文獻之感測器比較 18 1.7 實驗動機與目的 19 Chapter 2 Materials and Experimental Methods 20 2.1 實驗儀器 20 2.2 實驗藥品 21 2.3 酵素連續催化生物感測器之設計 23 2.4 蛋白質表達與純化 24 2.4.1 蛋白質表達與純化實驗流程 24 2.4.2 DOD蛋白表達之質體設計 24 2.4.3 勝任細胞的製作 25 2.4.4 轉化作用 26 2.4.5 DOD蛋白大量表達及純化 27 2.4.6 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 29 2.4.7 蛋白質濃度定量 33 2.5 酵素連續催化感測器的條件優化 34 2.5.1 環境pH值的影響 34 2.5.2 酪胺酸酶及4,5-多巴雙加氧酶的濃度影響 34 2.5.3 輔因子濃度的影響 34 2.5.4 酵素連續催化感測器用於酪胺酸之偵測 35 2.5.5 酵素連續催化感測器用於酪胺酸酶之偵測 35 2.5.6 甜菜色素之合成 36 2.5.7 胎牛血清與人工尿液樣品的配製 36 2.5.8 酪胺酸及酪胺酸酶之試紙型比色法感測器 36 2.5.9 酪胺酸酶抑制劑之篩選 37 2.5.10 偵測極限公式計算 37 Chapter 3 Results and Discussions 38 3.1 酵素連續催化感測器之實驗條件優化 38 3.1.1 環境pH值對酵素連續催化感測器之影響 38 3.1.2 酪胺酸酶及4,5-多巴雙加氧酶的濃度變化之影響 39 3.1.3 生成甜菜醛胺酸之時間追蹤 40 3.1.4 輔因子濃度對酵素連續催化感測器之影響 41 3.1.5 使用不同胺類縮合成甜菜色素 43 3.2 酪胺酸劑量反應 45 3.2.1 利用甜菜醛胺酸作為輸出訊號 45 3.2.2 利用甜菜色素作為輸出訊號 48 3.3 酪胺酸感測器之專一性測試 50 3.3.1 干擾物之吸收值比較 50 3.3.2 干擾物之混合干擾測試 52 3.4 酵素連續催化系統與酪胺酸酶系統之比較 54 3.5 酪胺酸感測器之應用 57 3.5.1 真實樣品之檢測 57 3.5.2 酪胺酸感測器應用於試紙之測定 61 3.6 酵素連續催化感測器偵測酪胺酸酶之實驗條件優化 63 3.7 酪胺酸酶劑量反應 64 3.7.1 利用甜菜醛胺酸作為輸出訊號 64 3.7.2 利用甜菜色素作為輸出訊號 65 3.8 酪胺酸酶感測器之專一性測試 66 3.9 酪胺酸酶感測器之應用 67 3.9.1 真實樣品之檢測 67 3.9.2 酪胺酸酶之抑制劑測試 69 Chapter 4 Conclusions 71 REFERENCE 72 附錄 本研究中所使用之菌種與質體 75

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