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研究生：	陳依如 Chen, Yi-Ju
論文名稱：	以螢光共振能量轉移原理發展對銀離子具有選擇性的生物感測器 Fluorescence Resonance Energy Transfer-Based Biosensor for the Selective Detection of Silver Ions
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：	2017
畢業學年度：	105
語文別：	中文
論文頁數：	79
中文關鍵詞：	耐金屬貪銅菌、轉錄調控子CupR 、螢光共振能量轉移、藍綠色螢光蛋白、黃色螢光蛋白
英文關鍵詞：	Cupriavidus metallidurans CH34, transcriptional regulator CupR, FRET (fluorescence resonance energy transfer), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein)
DOI URL：	https://doi.org/10.6345/NTNU202202893
論文種類：	學術論文
相關次數：	點閱：123 下載：5
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銀離子獨特的抗菌能力被廣泛的被應用於醫療與生技產業，然而環境中過多的銀離子卻也會造成人類與生物體的危害。生物體為了保持體內金屬離子的平衡，發展一系列金屬調節機制，避免金屬離子可能引起細胞毒性。在實驗中，我們利用CupR建構以螢光共振能量轉移之生物感測器。CupR為MerR家族轉錄調控蛋白，來自耐金屬貪銅菌 (Cupriavidus metallidurans CH34) 。此生物感測器對銀離子具有良好專一性與選擇性，利用%FRET對銀離子濃度作圖，其結果在濃度0 - 10 µM具有良好的線性關係，最低偵測極限為0.45 µM，低於WHO所規範銀離子濃度之數值。我們嘗試比較感測器在不同溫度以及pH值時的蛋白質穩定性，並且測試不同的溶劑介質中，都有良好的檢測結果。此外，將蛋白質感測器製作於瓊脂糖凝膠中，增加感測的攜帶性及穩定性。

Silver ions are widely used for biomedical applications and antimicrobial agents. However, silver ions are highly poisonous and could be harmful and accumulate in human and living organisms. In order to maintain the balance of metal ions, organisms evolve series of metal regulation mechanisms to prevent the accumulation of metal ions to reduce cytotoxicity. In this work, we used CupR to construct a fluorescence resonance energy transfer (FRET) -based protein biosensor. CupR is a MerR family transcriptional regulator protein (from Cupriavidus metallidurans CH34). This biosensor exhibited high specificity and selectivity for silver ions. There was a linear relationship between %FRET and silver concentration response ranging from 0 to 10 µM. The limit of detection was 0.45 µM which was less than the maximal levels accepted by WHO. We examined the performance of biosensor at different temperature and pH to compare the stability of proteins. The samples in different aqueous media were successfully detected by our biosensor. Furthermore, agarose protein gel was used to increases the feasibility of portable and stable biosensors.

口試委員會審定書    #
誌謝       i
中文摘要   ii
Abstract  iii
目錄      iv
圖目錄    ix
表目錄    xii
第一章    緒論    1
1    微生物與金屬離子    1
1-1    微生物調控金屬離子之方法    1
1-2    銀離子    2
2    Cupriavidus metallidurans CH34    2
2-1    Cupriavidus metallidurans CH34之金誘導調節組cup regulon    2
2-2    轉錄調控蛋白    4
2-3    MerR家族轉錄調控因子    4
3    螢光蛋白介紹與應用    7
3-1    螢光蛋白    7
3-2    螢光共振能量轉移 (fluorescence resonance energy transfer, Förster resonance energy transfer, FRET)    9
3-3    生物冷光共振能量轉移 (bioluminescence resonance energy transfer, BRET)    10
3-4    螢光蛋白應用    10
4    以轉錄調控蛋白設計生物感測器之文獻回顧    11
4-1    全細胞感測器    11
4-2    蛋白質感測器    12
4-2-1    螢光蛋白質感測器    12
4-2-2    單一螢光基團之蛋白質感測器    13
4-2-3    FRET-based蛋白質感測器    14
5    銀離子感測器文獻回顧    15
6    動機與目標    18
第二章    實驗材料與方法    19
1    實驗儀器    19
2    實驗藥品    20
3    引子    22
4    質體    23
5    菌株    25
6    實驗方法    27
6-1    質體設計    27
6-2    質體基因工程    29
6-2-1    設計引子    29
6-2-2    複製基因片段    29
6-2-3    抽取質體    30
6-2-4    限制內切酶裁切    30
6-2-5    接合作用    31
6-2-6    轉形作用    31
6-2-7    篩選    32
6-2-8    定序    32
6-2-9    儲存    32
6-3    蛋白質表現、純化與定量    33
6-3-1    勝任細胞    33
6-3-2    重組蛋白質大量表現、純透析    34
6-3-3    鎳樹脂再生    35
6-3-4    十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)    36
6-3-5    蛋白質濃度定量    37
6-4    篩選以螢光共振能量轉移所建立的CupR(s)螢光蛋白感測器    38
6-4-1    篩選CupR(s)螢光蛋白感測器之受體螢光蛋白    38
6-4-2    篩選螢光蛋白CFP與調控蛋白CupR(s)之間不同鏈長的螢光蛋白感測器    38
6-4-3    篩選不同CupR片段的螢光蛋白感測器    38
6-5    螢光蛋白感測器之專一性    39
6-6    銅離子對藍綠色螢光蛋白的影響    39
6-7    螢光蛋白感測器對銀離子的劑量反應    39
6-8    螢光蛋白感測器之金屬干擾性    40
6-9    不同溫度對蛋白質感測器的影響    41
6-10    不同pH值對蛋白質感測器的影響    41
6-11    不同介質之金屬離子對蛋白質感測器的影響    41
6-12    蛋白質感測器之穩定性    42
6-13    蛋白質瓊脂糖凝膠感測器    42
6-14    不同濃度之瓊脂糖凝膠對蛋白質感測器的影響    43
6-15    蛋白質瓊脂糖凝膠感測器對銀離子的劑量反應    43
6-16    不同介質之銀離子對蛋白質瓊脂糖凝膠感測器的影響    44
6-17    蛋白質瓊脂糖凝膠感測器之穩定性    44
6-18    蛋白質瓊脂糖凝膠感測器之螢光顯微鏡圖    45
6-19    CupR(s)蛋白質感測器結晶    45
第三章    實驗結果與討論    46
1    建構pET-29b-cfp-L6cupR(s)-ypet質體    46
2    螢光蛋白感測器純化    49
3    螢光蛋白感測器機制    50
4    篩選以螢光共振能量轉移所建立的螢光蛋白感測器    51
4-1    篩選螢光蛋白感測器之受體螢光蛋白    51
4-2    篩選藍綠螢光蛋白與調控蛋白CupR(s)之間不同鏈長的螢光蛋白感測器    52
4-3    篩選不同CupR(s)片段的螢光蛋白感測器    53
5    螢光蛋白感測器之金屬專一性    55
6    銅離子對藍綠色螢光蛋白的影響    57
7    CupR(s)螢光蛋白感測器對銀離子的劑量反應    59
8    CupR(s)螢光蛋白感測器之金屬干擾性    60
9    不同溫度對CupR(s)蛋白質感測器的影響    61
10    不同pH值對CupR(s)蛋白質感測器的影響    62
11    不同介質之銀離子對蛋白質感測器的影響    64
12    CupR(s)蛋白質瓊脂糖凝膠感測器    65
13    不同濃度之瓊脂糖凝膠對蛋白質感測器的影響    66
14    CupR(s)蛋白質瓊脂糖凝膠感測器對銀離子的劑量反應    67
15    不同介質之銀離子對CupR(s)蛋白質瓊脂糖凝膠感測器的影響    68
16    CupR(s)蛋白質感測器之穩定性    69
17    CupR(s)蛋白質瓊脂糖凝膠感測器之螢光顯微鏡圖    69
18    CupR(s)蛋白質感測器結晶    70
第四章    結論    73
參考資料    74
                                

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