國立臺灣師範大學博碩士論文全文系統

簡易檢索 / 詳目顯示

回結果列表

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：124 下載：5
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

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
                                

[1] Lemire, J. A.; Harrison, J. J.; Turner, R. J. Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nature Reviews Microbiology 2013, 11, 371-384.
[2] Percival, S. L.; Bowler, P. G.; Russell, D. Bacterial resistance to silver in wound care. Journal of Hospital Infection 2005, 60, 1-7.
[3] Chaloupka, K.; Malam, Y.; Seifalian, A. M. Nanosilver as a new generation of nanoproduct in biomedical applications. Trends in Biotechnology 2010, 28, 580-588.
[4] Monchy, S.; Benotmane, M. A.; Janssen, P.; Vallaeys, T.; Taghavi, S.; van der Lelie, D.; Mergeay, M. Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. Journal of Bacteriology 2007, 189, 7417-7425.
[5] Janssen, P. J.; Van Houdt, R.; Moors, H.; Monsieurs, P.; Morin, N.; Michaux, A.; Benotmane, M. A.; Leys, N.; Vallaeys, T.; Lapidus, A.; Monchy, S.; Médigue, C.; Taghavi, S.; McCorkle, S.; Dunn, J.; van der Lelie, D.; Mergeay, M. The complete genome sequence of Cupriavidus metallidurans strain CH34, a master survivalist in harsh and anthropogenic environments. PLOS ONE 2010, 5, e10433.
[6] Nies, D. H. The biological chemistry of the transition metal "transportome" of Cupriavidus metallidurans. Metallomics 2016, 8, 481-507.
[7] Zammit, C. M.; Weiland, F.; Brugger, J.; Wade, B.; Winderbaum, L. J.; Nies, D. H.; Southam, G.; Hoffmann, P.; Reith, F. Proteomic responses to gold(III)-toxicity in the bacterium Cupriavidus metallidurans CH34. Metallomics 2016, 8, 1204-1216.
[8] Reith, F.; Etschmann, B.; Grosse, C.; Moors, H.; Benotmane, M. A.; Monsieurs, P.; Grass, G.; Doonan, C.; Vogt, S.; Lai, B.; Martinez-Criado, G.; George, G. N.; Nies, D. H.; Mergeay, M.; Pring, A.; Southam, G.; Brugger, J. Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans. Proceedings of the National Academy of Sciences 2009, 106, 17757-17762.
[9] Maret, W.; Wedd, A. (2014). Binding, transport and storage of metal ions in biological cells
[10] Jian, X.; Wasinger, E. C.; Lockard, J. V.; Chen, L. X.; He, C. Highly sensitive and selective gold(I) recognition by a metalloregulator in Ralstonia metallidurans. Journal of the American Chemical Society 2009, 131, 10869-10871.
[11] Wiesemann, N.; Mohr, J.; Grosse, C.; Herzberg, M.; Hause, G.; Reith, F.; Nies, D. H. Influence of copper resistance determinants on gold transformation by Cupriavidus metallidurans Strain CH34. Journal of Bacteriology 2013, 195, 2298-2308.
[12] Giedroc, D. P.; Arunkumar, A. I. Metal sensor proteins: nature's metalloregulated allosteric switches. Dalton Transactions 2007, 3107-3120.
[13] Osman, D.; Cavet, J. S. Bacterial metal-sensing proteins exemplified by ArsR-SmtB family repressors. Natural Product Reports 2010, 27, 668-680.
[14] Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.; O'Halloran, T. V.; Mondragón, A. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301, 1383-1387.
[15] Brown, N. L.; Stoyanov, J. V.; Kidd, S. P.; Hobman, J. L. The MerR family of transcriptional regulators. FEMS Microbiology Reviews 2003, 27, 145-163.
[16] Chang, C.-C.; Lin, L.-Y.; Zou, X.-W.; Huang, C.-C.; Chan, N.-L. Structural basis of the mercury(II)-mediated conformational switching of the dual-function transcriptional regulator MerR. Nucleic Acids Research 2015, 43, 7612-7623.
[17] Philips, S. J.; Canalizo-Hernandez, M.; Yildirim, I.; Schatz, G. C.; Mondragón, A.; O’Halloran, T. V. Allosteric transcriptional regulation via changes in the overall topology of the core promoter. Science 2015, 349, 877-881.
[18] Ibáñez, M. M.; Checa, S. K.; Soncini, F. C. A single serine residue determines selectivity to monovalent metal ions in metalloregulators of the MerR family. Journal of Bacteriology 2015, 197, 1606-1613.
[19] Chen, P. R.; He, C. Selective recognition of metal ions by metalloregulatory proteins. Current Opinion in Chemical Biology 2008, 12, 214-221.
[20] Schultz, C. Fluorescent revelations. Chemistry & Biology 2009, 16, 107-111.
[21] Yang, F.; Moss, L. G.; Phillips, G. N. The molecular structure of green fluorescent protein. Nature Biotechnology 1996, 14, 1246-1251.
[22] Sample, V.; Newman, R. H.; Zhang, J. The structure and function of fluorescent proteins. Chemical Society Reviews 2009, 38, 2852-2864.
[23] Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W.; Prasher, D. Green fluorescent protein as a marker for gene expression. Science 1994, 263, 802-805.
[24] Heim, R.; Tsien, R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology 1996, 6, 178-182.
[25] Prevo, B.; Peterman, E. J. G. Forster resonance energy transfer and kinesin motor proteins. Chemical Society Reviews 2014, 43, 1144-1155.
[26] Broussard, J. A.; Rappaz, B.; Webb, D. J.; Brown, C. M. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nature Protocols 2013, 8, 265-281.
[27] Morise, H.; Shimomura, O.; Johnson, F. H.; Winant, J. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 1974, 13, 2656-2662.
[28] Xiong, T. C.; Ronzier, E.; Sanchez, F.; Corratgé-faillie, C.; Mazars, C.; Thibaud, J.-B. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter. Frontiers in Plant Science 2014, 5.
[29] Palmer, A. E.; Qin, Y.; Park, J. G.; McCombs, J. E. Design and application of genetically encoded biosensors. Trends in Biotechnology 2011, 29, 144-152.
[30] Sample, V.; Mehta, S.; Zhang, J. Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems. Journal of Cell Science 2014, 127, 1151-1160.
[31] Tseng, H. W.; Tsai, Y. J.; Yen, J. H.; Chen, P. H.; Yeh, Y. C. A fluorescence-based microbial sensor for the selective detection of gold. Chemical Communications 2014, 50, 1735-1737.
[32] Chen, P.; He, C. A general strategy to convert the MerR family proteins into highly sensitive and selective fluorescent biosensors for metal ions. Journal of the American Chemical Society 2004, 126, 728-729.
[33] Wegner, S. V.; Okesli, A.; Chen, P.; He, C. Design of an emission ratiometric biosensor from MerR family proteins: a sensitive and selective sensor for Hg²⁺. Journal of the American Chemical Society 2007, 129, 3474-3475.
[34] Chen, P.; Greenberg, B.; Taghavi, S.; Romano, C.; van der Lelie, D.; He, C. An exceptionally selective lead(II)-regulatory protein from Ralstonia Metallidurans: development of a fluorescent lead(II) probe. Angewandte Chemie International Edition 2005, 44, 2715-2719.
[35] Stoyanov, J. V.; Brown, N. L. The Escherichia coli copper-responsive CopA promoter is activated by gold. Journal of Biological Chemistry 2003, 278, 1407-1410.
[36] Liang, J.; Qin, M.; Xu, R.; Gao, X.; Shen, Y.; Xu, Q.; Cao, Y.; Wang, W. A genetically encoded copper(I) sensor based on engineered structural distortion of EGFP. Chemical Communications 2012, 48, 3890-3892.
[37] Liu, J.; Karpus, J.; Wegner, S. V.; Chen, P. R.; He, C. Genetically encoded copper(I) reporters with improved response for use in imaging. Journal of the American Chemical Society 2013, 135, 3144-3149.
[38] Wegner, S. V.; Arslan, H.; Sunbul, M.; Yin, J.; He, C. Dynamic copper(I) Imaging in mammalian cells with a genetically encoded fluorescent copper(I) sensor. Journal of the American Chemical Society 2010, 132, 2567-2569.
[39] Hessels, A. M.; Merkx, M. Genetically-encoded FRET-based sensors for monitoring Zn²⁺ in living cells. Metallomics 2015, 7, 258-266.
[40] Hessels, A. M.; Chabosseau, P.; Bakker, M. H.; Engelen, W.; Rutter, G. A.; Taylor, K. M.; Merkx, M. eZinCh-2: a versatile, genetically encoded FRET sensor for cytosolic and intraorganelle Zn²⁺ imaging. ACS Chemical Biology 2015, 10, 2126-2134.
[41] van Dongen, E. M. W. M.; Dekkers, L. M.; Spijker, K.; Meijer, E. W.; Klomp, L. W. J.; Merkx, M. Ratiometric fluorescent sensor proteins with subnanomolar affinity for Zn(II) based on copper chaperone domains. Journal of the American Chemical Society 2006, 128, 10754-10762.
[42] van Dongen, E. M. W. M.; Evers, T. H.; Dekkers, L. M.; Meijer, E. W.; Klomp, L. W. J.; Merkx, M. Variation of linker length in ratiometric fluorescent sensor proteins allows rational tuning of Zn(II) affinity in the picomolar to femtomolar range. Journal of the American Chemical Society 2007, 129, 3494-3495.
[43] Vinkenborg, J. L.; Nicolson, T. J.; Bellomo, E. A.; Koay, M. S.; Rutter, G. A.; Merkx, M. Genetically encoded FRET sensors to monitor intracellular Zn²⁺ homeostasis. Nature Methods 2009, 6, 737-740.
[44] Anand, T.; Sivaraman, G.; Anandh, P.; Chellappa, D.; Govindarajan, S. Colorimetric and turn-on fluorescence detection of Ag(I) ion. Tetrahedron Letters 2014, 55, 671-675.
[45] Hung, Y.-L.; Hsiung, T.-M.; Chen, Y.-Y.; Huang, Y.-F.; Huang, C.-C. Colorimetric detection of heavy metal ions using label-free gold nanoparticles and alkanethiols. The Journal of Physical Chemistry C 2010, 114, 16329-16334.
[46] Saran, R.; Liu, J. A silver DNAzyme. Analytical Chemistry 2016, 88, 4014-4020.
[47] Chang, C.-C.; Lin, S.; Wei, S.-C.; Chu-Su, Y.; Lin, C.-W. Surface plasmon resonance detection of silver ions and cysteine using DNA intercalator-based amplification. Analytical and Bioanalytical Chemistry 2012, 402, 2827-2835.
[48] Chang, Y.; Zhang, Z.; Hao, J.; Yang, W.; Tang, J. BSA-stabilized Au clusters as peroxidase mimetic for colorimetric detection of Ag⁺. Sensors and Actuators B: Chemical 2016, 232, 692-697.
[49] Vilela, D.; González, M. C.; Escarpa, A. Sensing colorimetric approaches based on gold and silver nanoparticles aggregation: Chemical creativity behind the assay. A review. Analytica Chimica Acta 2012, 751, 24-43.
[50] Bülbül, G.; Hayat, A.; Andreescu, S. Portable nanoparticle-based sensors for food safety assessment. Sensors 2015, 15, 30736-30758.
[51] Kumar, B.; Asha, K.; Chauhan, S. DNAzyme mediated post-transcriptional gene silencing: A novel therapeutic approach. WebmedCentral 2013, 1-12.
[52] Kleinke, K.; Saran, R.; Liu, J. Label-free Ag⁺ detection by enhancing DNA sensitized Tb³⁺ luminescence. Sensors 2016, 16, 1370.
[53] Bian, L.; Ji, X.; Hu, W. A novel single-labeled fluorescent oligonucleotide probe for silver(i) ion detection in water, drugs, and food. Journal of Agricultural and Food Chemistry 2014, 62, 4870-4877.
[54] Krizkova, S.; Huska, D.; Beklova, M.; Hubalek, J.; Adam, V.; Trnkova, L.; Kizek, R. Protein-based electrochemical biosensor for detection of silver(I) ions. Environmental Toxicology and Chemistry 2010, 29, 492-496.
[55] Zhou, W.; Ding, J.; Liu, J. 2-Aminopurine-modified DNA homopolymers for robust and sensitive detection of mercury and silver. Biosensors and Bioelectronics 2017, 87, 171-177.
[56] Lee, J.; Park, J.; Hee Lee, H.; Park, H.; Kim, H. I.; Kim, W. J. Fluorescence switch for silver ion detection utilizing dimerization of DNA-Ag nanoclusters. Biosensors and Bioelectronics 2015, 68, 642-647.
[57] WHO (2011). Guidelines for drinking-water quality, fourth edition. Geneva: World Health Organisation. P. 415.
[58] Verma, N.; Singh, M. Biosensors for heavy metals. Biometals 2005, 18, 121-129.
[59] Chiu, T.-Y.; Yang, D.-M. Intracellular Pb²⁺ content monitoring using a protein-based Pb²⁺ indicator. Toxicological Sciences 2012, 126, 436-445.
[60] Ji, Q.; Zhao, B. S.; He, C. A highly sensitive and genetically encoded fluorescent reporter for ratiometric monitoring of quinones in living cells. Chemical Communications 2013, 49, 8027-8029.
[61] Péterffy, J.; Szabó, M.; Szilágyi, L.; Lányi, S.; Ábrahám, B. Fluorescence of a histidine-modified enhanced green fluorescent protein (EGFP) effectively quenched by copper(II) ions. part II. molecular determinants. Journal of Fluorescence 2015, 25, 871-883.
[62] Kim, I. J.; Kim, S.; Park, J.; Eom, I.; Kim, S.; Kim, J.-H.; Ha, S. C.; Kim, Y. G.; Hwang, K. Y.; Nam, K. H. Crystal structures of Dronpa complexed with quenchable metal ions provide insight into metal biosensor development. FEBS Letters 2016, 590, 2982-2990.
[63] Rahimi, Y.; Goulding, A.; Shrestha, S.; Mirpuri, S.; Deo, S. K. Mechanism of copper induced fluorescence quenching of red fluorescent protein, DsRed. Biochemical and Biophysical Research Communications 2008, 370, 57-61.

簡易檢索 / 詳目顯示

相關論文