簡易檢索 / 詳目顯示

回結果列表
研究生： 李芳宏
Lee, Fang-Hong
論文名稱： 生物素化學探針結合抗生物素蛋白於螢光感測之應用
Chemical Probes based on Biotin and Avidin Conjugation: Applications in Fluorescence Sensing
指導教授： 陳貴通
Tan, Kui-Thong
口試委員: 王聖凱
Wang, Sheng-Kai
吳淑褓
Wu, Shu-Pao
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： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 78
中文關鍵詞： 螢光探針 、非專一性訊號 、抗生物素蛋白 、生物素
外文關鍵詞： fluorescence, nonspecific, avidin, biotin
相關次數： 點閱：2  下載：0 
分享至:
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

    • 近年來小分子螢光探針蓬勃發展，其具備快速、專一、高靈敏、操作簡便等優點，常用於偵測蛋白質及活性小分子，已成為醫療診斷和基礎研究不可或缺的工具。然而在目前，利用螢光探針於富含蛋白質的人體血清中對微量生物標記物進行定量偵測仍是一項具有挑戰性的工作。因多數探針所使用的螢光染料易與血清中的蛋白進行非專一且緊密的結合，造成螢光訊號異常的增益，以至於真實訊號被非專一性的訊號所干擾，結果令我們高估偵測對象之濃度產生錯誤判讀。本論文中，我們利用生物素與抗生物素蛋白之間強力專一的結合，將生物素化的探針封裝至抗生物素蛋白的疏水性孔洞，阻絕染料與血清中蛋白進行非專一性的結合，並將此蛋白質保護策略套用於極性敏感螢光染料SBD，經由修飾上不同的辨識端，設計出分別用以偵測酵素硝基還原酶及活性小分子過氧化氫之生物素化螢光增益探針，成功於富含蛋白質的血清樣品中得到專一性的螢光訊號。此外，在葡萄糖氧化酶的催化下，葡萄糖可與氧氣反應生成葡萄糖酸內酯並釋放出過氧化氫，因此過氧化氫可作為葡萄糖偵測時的訊號來源。在檢量線法與標準添加法的支持下，我們更證明BTSBD-B(OH)2可實際對人體血清中之葡萄糖進行定量。

    Recently, analyte-responsive small molecular fluorescent probes attract significant attention due to their specificity, high sensitivity, and ease of operations. They have become an indispensable tool in medical diagnosis and basic research. Although most fluorescent probes show excellent performance in aqueous buffers, their applications in protein rich human blood still are a great challenge as the real signal is usually obscured by nonspecific fluorescence. This problem occurs because most of the fluorescent dyes bind very tightly with blood proteins to produce a large fluorescence increase, resulting in overestimation of the biomarker concentrations and false positive diagnosis. In our research, we introduce a new concept to eliminate nonspecific fluorescence in protein rich blood samples, by utilizing the specific interaction between biotin and avidin, to encapsulate biotinylated fluorescent probes in avidin protein cavity and block out nonspecific binding of the dyes with other nontarget blood proteins. We combined this protein protective strategy with solvatochromic dye SBD to design two novel fluorescent probes, BTSBD-NO2 and BTSBD-B(OH)2, which successfully achieved quantitative detection of nitroreductase and hydrogen peroxide in human serum. Furthermore, since hydrogen peroxide produced stoichiometrically during the oxidation of glucose in the presence of glucose oxidase, hydrogen peroxide can be utilized as a signal transducer to detect glucose. By the support of standard addition and external calibration, we confirmed BTSBD-B(OH)2 can quantify the glucose level in human serum.

    摘要 i Abstract ii 謝誌 iii 著作列表 iv 目錄 v 第一章 緒論 1 §1-1 酶蛋白及非酶蛋白質(enzyme and non-enzymatic protein) 1 §1-2 環境敏感螢光分子(environment-sensitive fluorophore) 2 §1-2-1 黏度敏感螢光分子(viscosity-sensitive fluorophores) 2 §1-2-2 極性敏感螢光分子(polarity-sensitive fluorophores) 5 第二章 文獻回顧 7 §2-1 近代選擇性探針偵測方法 7 §2-1-1 偵測蛋白質之螢光探針 7 §2-1-2 偵測活性小分子之螢光探針 11 §2-2 非專一性訊號 16 第三章、探針設計 18 §3-1 生物素與抗生物素蛋白 18 §3-2 非專一性訊號抑制 19 §3-3 極性敏感螢光染料SBD 20 §3-4 生物素化螢光探針偵測方法 21 第四章 實驗結果與討論 22 §4-1 硝基還原酶之檢測 22 §4-2 過氧化氫之檢測 30 §4-3 葡萄糖之檢測 34 第五章 結論 37 第六章、實驗部分 38 §6-1 一般實驗敘述 38 §6-2 螢光測試條件 40 §6-3 有機合成及光譜資料 42 參考文獻 55 附錄 60

    1. Bairoch, A. Nucleic Acids Res. 2000, 28, 304.
    2. Radzicka, A.; Wolfenden, R., Science 1995, 267, 90.
    3. Haidekker, M. A.; Theodorakis, E. A. J. Biol. Eng. 2010, 4, 1.
    4. Rumble, C.; Rich, K.; He, G.; Maroncelli, M. J. Phys. Chem. A 2012, 116.
    5. Haidekker, M. A.; Brady, T. P.; Lichlyter, D.; Theodorakis, E. A. J. Am. Chem. Soc. 2006, 128, 398.
    6. Ono, M.; Watanabe, H.; Kimura, H.; Saji, H. ACS Chem. Neurosci. 2012, 3, 319.
    7. Braun, D.; Rettig, W.; Delmond, S.; Letard, J. F.; Lapouyade, A. R. J. Phys. Chem. A 1997, 101, 6836.
    8. Choi, M. G.; Hwang, J.; Moon, J. O.; Sung, J.; Chang, S. K. Org. Lett. 2011, 13, 5260.
    9. Deliconstantinos, G.; Villiotou, V.; Stavrides, J. C. Biochem. Pharmacol. 1995, 49, 1589.
    10. Shinitzky, M. In: Physiology of membrane fluidity; Shinitzky, M., Ed.; CRC Press: Boca Raton, FL, 1984; pp 1−51.
    11. Nadiv, O.; Shinitzky, M.; Manu, H.; Hecht, D.; Roberts, C. T.; Leroith, D.; Zick, Y. Biochem. J. 1994, 298, 443.
    12. Rye, H. S.; Quesada, M. A.; Peck, K.; Mathies, R. A.; GIazer, A. N. Nucleic Acids Res. 1991, 19, 327.
    13. López-Duarte, I.; Vu, T. T.; Izquierdo, M. A.; Bull, J. A.; Kuimova, M. K. Chem. Commun. 2014, 50, 5282.
    14. Narayanaswamy, N.; Kumar, M.; Das, S.; Sharma, R.; Samanta, P. K.; Pati, S.K.; Govindaraju, T. Sci Rep. 2014, 4, 1.
    15. Goh, W. L.; Lee, M. Y.; Joseph, T. L.; Quah, S. T.; Brown, C. J.; Verma, C.; Teo, Y. N. J. Am. Chem. Soc. 2014, 136, 6159.
    16. Loving, G. S.; Sainlos, M.; Imperiali, B. Trends Biotechnol. 2010, 28, 73.
    17. Uchiyama, S.; Takehira, K.; Yoshihara, T.; Tobita, S.; Ohwade, T.; Org. Lett. 2006, 8, 5869.
    18. Kelkar, D. A.; Chattopadhyay, A. J. Phys. Chem. B. 2004, 108, 12151.
    19. Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K.; Biomed, Chromatogr. 2001, 15, 295.
    20. Kucherak, O. A.; Didier, P.; Mely, Y.; Klymchenko, A. S. J. Phys. Chem. l. 2010, 1, 616.
    21. Lee, M. H.; Kim, H. J.; Yoon, S.; Park, N.; Kim, J. S. Org. Lett. 2008, 10, 213.
    22. Huang, C.; Yin, Q.; Zhu, W.; Yang, Y.; Wang, X.; Qian, X.; Xu, Y. Angew. Chem. Int. Ed. 2011, 50, 7551.
    23. Urano, Y.; Sakabe, M.; Kosaka, N.; Ogawa, M.; Mitsunaga, M.; Asanuma, D.; Kamiya, M.; Young, M. R.; Nagano, T.; Choyke, P. L.; Kobayashi, H. Sci. Transl. Med. 2011, 3, 110.
    24. Xu, K.; Wang, F.; Pan, X.; Liu, R.; Ma, J.; Kong, F.; Tang, B. Chem. Commun. 2013, 49, 2554.
    25. Thurley, S.; Röglin, L.; Seitz, O. J. Am. Chem. Soc. 2007, 129, 12693.
    26. Zhang, R.; Zhao, J.; Han, G.; Liu, Z.; Liu, C.; Zhang, C.; Liu, B.; Jiang, C.; Liu, R.; Zhao, T.; Han, M. Y.; Zhang, Z. J. Am. Chem. Soc. 2016, 138, 3769.
    27. Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.; Shen, J. G. J. Am. Chem. Soc. 2006, 128, 6004.
    28. Montoya, L. A.; Pluth, M. D. Chem. Commun. 2012, 48, 4767.
    29. Adams, J. A. Chem. Rev. 2001, 101, 2271.
    30. Kamiya, M.; Asanuma, D.; Kuranaga, E.; Takeishi, A.; Sakabe, M.; Miura, M.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2011, 133, 12960.
    31. Rettig, W. Appl. Phys. B. 1988, 45, 145.
    32. Mizusawa, K.; Ishida, Y.; Takaoka, Y.; Miyagawa, M.; Tsukiji, S.; Hamachi, I. J. Am. Chem. Soc. 2010, 132, 7291.
    33. Lushchak, V. I. J. Amino Acids 2012, 736837.
    34. Lushchak,V.I. Aquat. Toxicol. 2011, 101, 13.
    35. Singh, N.; Dhalla, A.K.; Seneviratne, C.; Singal, P.K. Mol Cell Biochem. 1995, 147, 77.
    36. Halliwell, B. Biochem. J. 2007, 401, 1.
    37. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. Int. J. Biochem. Cell Biol. 2007, 39, 44.
    38. Turrens, J. F. J Physiol. 2003, 552, 335.
    39. Simon, H. U.; Haj-Yehia, A.; Levi-Schaffer, F. Apoptosis 2000, 5, 415.
    40. Zhang, R.; Zhao, J.; Han, G.; Liu, Z.; Liu, C.; Zhang, C.; Liu, B.; Jiang, C.; Liu, R.; Zhao, T.; Han, M. Y.; Zhang, Z. J. Am. Chem. Soc. 2016, 138, 3769.
    41. Patel, R. P.; McAndrew, J.; Sellak, H.; White, C. R.; Jo, H.; Freeman, B. A.; Darley-Usmar, V. M. Biochimica et Biophysica Acta. 1999, 1411, 385.
    42. Alayash, A.I.; Ryan, B.A.; Cashon, R.E. Arch. Biochem. Biophys. 1998, 349, 65.
    43. Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.; Shen, J. G. J. Am. Chem. Soc. 2006, 128, 6004.
    44. Kimura, H. Antioxid. Redox Signal. 2014, 20, 783.
    45. Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chem. Soc. Rev. 2015, 44, 4596.
    46. Montoya, L. A.; Pluth, M. D. Chem. Commun. 2012, 48, 4767.
    47. Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics 2002, 1, 845−867.
    48. Das, D. K.; Mondal, T.; Mandal, A. K.; Bhattacharyya, K. Chem. Asian J. 2011, 6, 3097−3103.
    49. Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B. Chem. Rev. 2000, 100, 1973−2012.
    50. Kim, Y. K.; Lee, J. S.; Bi, X.; Ha, H. H.; Ng, S. H.; Ahn, Y. H.; Lee, J. J.; Wagner, B. K.; Clemons, P. A.; Chang, Y. T. Angew. Chem., Int. Ed. 2011, 50, 2761−2763.
    51. Wu, Y. Y.; Yu, W. T.; Hou, T. C.; Liu, T. K.; Huang, C. L.; Chen, I. C.; Tan, K. T. Chem. Commun. 2014, 50, 11507.
    52. Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.; Ascenzi, P. Mol. Aspects. Med. 2012, 33, 209.
    53. Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N. Nat. Mater. 2007, 6, 765.
    54. Reisch, A.; Klymchenko, A. S. Small 2016, 12, 1968.
    55. Søndergaard, R. V.; Christensen, N. M.; Henriksen, J. R.; Kumar, E. K. P.; Almdal, K.; Andresen, T. L. Chem. Rev. 2015, 115, 8344.
    56. Johnson, I. The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition; Life Technologies Corporation, 2010.
    57. Terai, T.; Maki, E.; Sugiyama, S.; Takahashi, Y.; Matsumura, H.; Mori, Y.; Nagano, T. Chem. Biol. 2011, 18, 1261.
    58. Wilchek, M.; Bayer, E. A. Methods Enzymol. 1990, 184, 5−13.
    59. McMahon, R. J. Avidin-Biotin Interactions: Methods and Applications; Humana Press: Totowa, NJ, 2008.
    60. Dundas, C. M.; Demonte, D.; Park, S. Appl. Microbiol. Biotechnol. 2013, 97, 9343−9353.
    61. Uchiyama, S.; Kimura, K.; Gota, C.; Okabe, K.; Kawamoto, K.; Inada, N.; Yoshihara, T.; Tobita, S. Chemistry (Easton) 2012, 18, 9552.
    62. Li, Y.; Sun, Y.; Li, J.; Su, Q.; Yuan, W.; Dai, Y.; Han, C.; Wang, Q.; Feng, W.; Li, F. J. Am. Chem. Soc. 2015, 137, 6407.
    63. Cui, L.; Zhong, Y.; Zhu, W. P.; Xu, Y. F.; Du, Q. S.; Wang, X.; Qian, X. H.; Xiao, Y. Org. Lett. 2011, 13, 928.
    64. Mattila, T. K.; de Boer, A. Drugs 2010, 70, 2229.

    QR CODE