簡易檢索 / 詳目顯示

回結果列表
研究生： 曾彥勛
Zeng, Yan Syun
論文名稱： 環境敏感螢光探針標記SNAP-tag蛋白質： 具螢光增益並能抑制血漿中非專一性螢光訊號
Labeling Environment-Sensitive Fluorescence Probes to SNAP-tag Protein: Fluorescence Amplification and Nonspecific Signal Suppression Detection in Blood Plasma
指導教授： 陳貴通
Tan, Kui Thong
口試委員: 洪嘉呈
Horng, Jia Cherng
許馨云
Hsu, Hsin Yun
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： 2016
畢業學年度： 104
語文別： 中文
論文頁數： 128
中文關鍵詞： 螢光探針
外文關鍵詞： fluorescent probe
相關次數： 點閱：3  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，由於能快速、靈敏的偵測蛋白質和活性小分子，使螢光探針成為重要的醫療診斷工具。大多數螢光分子在緩衝液下偵測分析物有優異的表現，但是在生物樣品中偵測分析物，如血漿、血清等，螢光探針會與環境中的複雜蛋白產生鍵結，造成螢光訊號異常的改變。另外，環境敏感螢光分子在高極性水溶液中只會表現出微弱的螢光，而生物體內系統普遍上為高極性環境，這兩個問題為環境敏感螢光分子在生物環境中偵測分析物的一大限制。
    根據實驗室先前的研究，開發了環境敏感性螢光探針BGSBD，因為BGSBD有特殊的BG結構，在水溶液下，能與SNAP-tag蛋白產生專一性鍵結並產生螢光增益。因此本論文利用此概念，以環境敏感螢光分子SBD為主體，設計一種利用SNAP-tag蛋白保護探針的方法，能在血漿中偵測分析物並擁有螢光增益特性，且能阻隔探針與其他生物分子鍵結，抑制非專一性螢光訊號。為了驗證此方法，我們設計螢光探針BGSBD-NO¬2、BGSBD-N3和BGSBD-B(OH)2，分別檢測硝基還原酶(Nitroreductase)、硫化氫(Hydrogen Sulfide)和過氧化氫(Hydrogen Peroxide)，結果顯示我們成功的抑制非專一性螢光訊號，並能在血漿中達到螢光訊號放大效果，證明SNAP-tag蛋白保護概念在偵測真實臨床樣品具有相當大的潛力。

    Fluorescent probes are important tools in medical diagnosis, because they allow for rapid and sensitive monitoring target analytes. Although most fluorescent probes show excellent performance in clean buffers, they are easily foiled by nonspecific signals in complex real samples such as blood plasma. The problem is mainly caused by the nonspecific binding of fluorescent probes with other macromolecules. Furthermore, environment-sensitive dyes display weaker emission in aqueous buffers. Therefore, how to solve these two fundamental problems is an important issue.
    Previously, we’ve developed a BGSBD probe which exhibited dramatic fluorescence turn-on upon covalently labeled to SNAP-tag protein. Based on BGSBD, we design a novel protein-shield concept as demonstrated by SNAP-tag protein for fluorescence amplification and nonspecific signal suppression of fluorescence probes for detection of enzymes and reactive small molecules in blood plasma. Herein, we designed three different probes, BGSBD-NO2, BGSBD-N3 and BGSBD-(OH)2, to detect Nitroreductase (NTRase), Hydrogen Sulfide (H2S) and Hydrogen Peroxide (H2O2). We have successfully amplified the fluorescence signal and suppressed unspecific signal of the probes by our protein-shield strategy in human blood plasma.

    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(protease and non-enzymatic protein) 1 §1-2 活性小分子(reactive small molecules) 2 §1-2-1 活性氧類(reactive oxygen species) 2 §1-2-2 活性氮類(reactive nitrogen species) 3 §1-2-3 活性硫類(reactive sulfur species) 4 §1-3 環境敏感螢光分子(environment-sensitive fluorophore) 5 §1-3-1 黏度敏感螢光分子(viscosity-sensitive fluorophore) 5 §1-3-2 極性敏感螢光分子(polarity-sensitive fluorophores) 6 第二章、文獻回顧與設計構想 8 §2-1 近代選擇性探針偵測方法 8 §2-1-1 小分子式螢光探針(small molecular fluorescent probes) 8 §2-1-2 量子點式螢光探針(quantum dot-based fluorescent probes) 10 §2-1-3 金奈米粒子探針(gold nanoparticle-based fluorescent probes, AuNPs) 11 §2-1-4 信標式螢光探針(beacon-based fluorescent probes) 13 §2-1-5 螢光蛋白式探針(fluorescent protein-based probes) 14 §2-1-6 自組裝式螢光探針(self-assembling fluorescent probes) 15 第三章、蛋白質探針之設計構想 17 §3-1 螢光分子選擇 17 §3-2 SNAP-tag 標記技術 19 §3-3 探針設計與策略 20 第四章、實驗結果與討論 22 §4-1 硝基還原酶之檢測 22 §4-2 硫化氫之檢測 32 §4-3 過氧化氫之檢測 37 §4-4 抗生物素蛋白之檢測 44 §4-5 Halo-tag蛋白之檢測 49 第五章、實驗結論 52 第六章、實驗部分 53 §6-1 一般實驗敘述 53 §6-2 螢光測試條件 55 §6-3 有機合成及光譜資料 58 參考文獻 85 附錄 91

    1. Admas, J. A. Chem. Rev. 2001, 101, 2271.
    2. Radzicka, A.; Wolfenden, R., Science 1995, 267, 90.
    3. Sorbi, D.; Boynton, J.; Lindor, K. Am. J. Gastroenterol. 1999, 94, 1018.
    4. Kang, J.; Lemaire, H.-G.; Unterbeck, A.; Salbaum, J. M.; Masters, C. L.; Grzeschik, K.-H.; Multhaup, G.; Beyreuther, K. & Muller-Hill, B. Nature 1987, 325, 733.
    5. Edwards, D. R.; Murphy, G. Nature 1998, 394, 527.
    6. Sohal, R. S.; Mockett R. J.; Orr W. C. Free Radic. Biol. Med. 2002, 33, 575.
    7. Gutteridge, J. M.; Halliwell, B. Ann. N. Y. Acad. Sci. 2000, 899, 136.
    8. Khodade, V. S.; Sharath Chandra, M.; Banerjee, A.; Lahiri, S.; Pulipeta, M.; Rangarajan, R.; Chakrapani, H. ACS Med. Chem. Lett. 2014, 5, 777.
    9. Kanvah, S.; Joseph, J.; Schuster, G. B.; Barnett, R. N.; Cleveland, C. L.; Landman, U. Acc. Chem. Res. 2010, 43, 280.
    10. Stadtman, E. R. Free Radical Res. 2006, 40, 1250.
    11. Harman, D. J. Gerontol. 1956, 11, 298.
    12. Mattson, M. P. Nature 2004, 430, 631.
    13. Houstis, N.; Rosen, E. D.; Lander, E. S. Nature 2006, 440, 944.
    14. Fruehauf, J. P.; Meysken, F. L., Jr. Clin. Cancer Res. 2007, 448, 789.
    15. Thomas, D. D.; Ridnour, L. A.; Isenberg, J. S.; Flores-Santana, W.; Switzer, C. H.; Donzelli, S.; Hussain, P.; Vecoli, C.; Paolocci, N.; Ambs, S.; Colton, C. A.; Harris, C. C.; Roberts, D. D.; and Wink, D. A. Free Radical Biol. Med. 2008, 45, 18.
    16. Ferguson, L. R. Mutat. Res. 2010, 690, 3.
    17. Kalyanaraman, B. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11527.
    18. Lancaster, J. R. Nitric Oxide. 2008, 19, 68.
    19. Sugiura, H.; Ichinose, M. Nitric Oxide. 2011, 25, 138.
    20. Brannan, R. G. J. Agric. Food Chem. 2010, 58, 3767.
    21. Ito, K.; Kawanishi, S. Biochem. Biophys. Res. Commun. 1991, 176, 1306.
    22. Chatgilialoglu, C.; Ferreri, C.; Ballestri, M.; Mulazzani, Q. G.; Landi, L. J. Am. Chem. Soc. 2000, 122, 4593.
    23. Laggner, H.; Hermann, M.; Sturm, B.; Gmeiner, B. M. K.; Kapiotis, S. FEBS Lett. 2005, 579, 6486.
    24. Kimura, H. Antioxid. Redox Signal. 2014, 20, 783.
    25. Köhn, C.; Dubrovska, G.; Huang, Y.; Gollasch, M. Int. J. Biomed. Sci. 2012, 8, 81.
    26. Yang, G.; Wu, L.; Wang, R. FASEB J. 2006, 20, 553.
    27. Zanardo, R. C. O.; Brancaleone, V.; Distrutti, E.; Fiorucci, S.; Cirino, G.; Wallace, J. L. FASEB J. 2006, 20, 2118.
    28. Kimura, Y.; Goto, Y.-I.; Kimura, H. Antioxid. Redox Signal. 2010, 12, 1.
    29. Perna, A. F.; Ingrosso, D. Nephrol. Dial. Transplant. 2012, 27, 486.
    30. Fiorucci, S.; Antonelli, E.; Mencarelli, A.; Orlandi, S.; Renga, B.; Rizzo, G.; Distrutti, E.; Shah, V.; Morelli, A. Hepatology. 2005, 42, 539.
    31. Han, Y.; Qin, J.; Chang, X.; Yang, Z.; Du, J. Cell. Mol. Neurobiol. 2006, 26, 101.
    32. Rettig, W. Appl. Phys. B. 1988, 45, 145.
    33. Rumble, C.; Rich, K.; He, G.; Maroncelli, M. J. Phys. Chem. A 2012, 116.
    34. Haidekker, M. A.; Brady, T. P.; Lichlyter, D.; Theodorakis, E. A. J. Am. Chem. Soc. 2006, 128, 398.
    35. Ono, M.; Watanabe, H.; Kimura, H.; Saji, H. ACS Chem. Neurosci. 2012, 3, 319.
    36. Braun, D.; Rettig, W.; Delmond, S.; Letard, J. F.; Lapouyade, A. R. J. Phys. Chem. A 1997, 101, 6836.
    37. Choi, M. G.; Hwang, J.; Moon, J. O.; Sung, J.; Chang, S. K. Org. Lett. 2011, 13, 5260.
    38. Yang, Z.; He, Y.; Lee, J.-H.; Park, N.; Suh, M.; Chae, W. S.; Cao, J.; Peng, X.; Jung, H.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2013, 135, 9181.
    39. Loving, G. S.; Sainlos, M.; Imperiali, B. Trends Biotechnol. 2010, 28, 73.
    40. Uchiyama, S.; Takehira, K.; Yoshihara, T.; Tobita, S.; Ohwade, T.; Org. Lett. 2006, 8, 5869.
    41. Kelkar, D. A.; Chattopadhyay, A. J. Phys. Chem. B. 2004, 108, 12151.
    42. (a) Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K.; Biomed, Chromatogr. 2001, 15, 295.
    (b) Santa, T.; Fukushima, T.; Ichibangase, T.; Imai, K. Biomed. Chromatogr. 2008, 22, 343.
    43. Kucherak, O. A.; Didier, P.; Mely, Y.; Klymchenko, A. S. J. Phys. Chem. l. 2010, 1, 616.
    44. Lee, M. H.; Kim, H. J.; Yoon, S.; Park, N.; Kim, J. S. Org. Lett. 2008, 10, 213.
    45. Huang, C.; Yin, Q.; Zhu, W.; Yang, Y.; Wang, X.; Qian, X.; Xu, Y. Angew. Chem. Int. Ed. 2011, 50, 7551.
    46. Alam, J.; Cook, J. L. Anal. Biochem. 1990, 188, 245.
    47. Kamiya, M.; Asanuma, D.; Kuranaga, E.; Takeishi, A.; Sakabe, M.; Miura, M.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2011, 133, 12960.
    48. Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 10629.
    49. Leutwyler, W. K.; Bürgi, S. L.; Burgl, H. Science 1996, 271, 933.
    50. Banerjee, S.; Kar, S.; Perez, J. M.; Santra, S. J. Phys. Chem. C 2009, 113, 9659.
    51. Pompella, A.; Visvikis, A.; Paolicchi, A.; De Tata, V.; Casini, A. F. Biochem. Pharmacol. 2003, 66, 1499.
    52. Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.-T. Anal. Chem. 2009, 81, 875.
    53. Huang, C.-C.; Chiang, C.-K; Lin, Z.-H.; Lee, K.-H.; Chang, H.-T. Anal. Chem. 2008, 80, 1497.
    54. Wang, C. I.; Chen, W. T.; Chang, H. T. Anal. Chem. 2012, 84, 9706.
    55. De Bundel, D.; Sarre, S.; Van Eeckhaut, A.; Smolders, I.; Michotte, Y. Sensors 2008, 8, 5171.
    56. Oh, K. J.; Cash, K. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128, 14018.
    57. Oh, K. J.; Cash, K. J.; Plaxco, K. W. Chem. Eur. J. 2009, 15, 2244.
    58. Okochi, M.; Sugita, T.; Tanaka, M.; Honda, H. RSC Adv. 2015, 5, 91988.
    59. Sapsford, K. E.; Berti, L.; Medintz, I. L. Angew. Chem. Int. Ed. 2006, 45, 4562.
    60. Chung, C.-I.; Makino, R.; Dong, J.; Ueda, H. Anal. Chem. 2015, 87, 3513.
    61. Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem. Biol. 2009, 4, 535.
    62. Mizusawa, K.; Ishida, Y.; Takaoka, Y.; Miyagawa, M.; Tsukiji, S.; Hamachi, I. J. Am. Chem. Soc. 2010, 132, 7291.
    63. Johnson, I. The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition; Life Technologies Corporation, 2010.
    64. Zanetti-Domingues, L. C.; Tynan, C. J.; Rolfe, D. J.; Clarke, D. T.; Martin-Fernandez, M. PloS one 2013, 8, e74200.
    65. Hughes, L. D.; Rawle, R. J.; Boxer, S. G. PloS one 2014, 9, e87649.
    66. Ogawa, T.; Aoyagi, S.; Miyasaka, T.; Sakai, K. Sensors 2009, 9, 8271.
    67. Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B. Chem. Rev. 2000, 100, 1973.
    68. Hawe, A.; Sutter, M.; Jiskoot, W. Pharm. Res. 2008, 25, 1487.
    69. Uchiyama, S.; Kimura, K.; Gota, C.; Okabe, K.; Kawamoto, K.; Inada, N.; Yoshihara, T.; Tobita, S. Chemistry (Easton) 2012, 18, 9552.
    70. (a) Duguid, E. M.; Rice, P. A.; He, C., J. Mol. Biol. 2005, 350, 657. (b) Kufer, S.; Dietz, H.; Albrecht, C.; Blank, K.; Kardinal, A.; Rief, M.; Gaub, H., Eur. Biophys. J. 2005, 35, 72.
    71. Gautier, A.; Juillerat, A.; Heinis, C.; Corrêa Jr, I. R.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. biol. 2008, 15, 128.
    72. Liu, T. K.; Hsieh, P. Y.; Zhuang, Y. D.; Hsia, C. Y.; Huang, C. L.; Lai, H. P.; Lin, H. S.; Chen, I. C.; Hsu, H. Y.; Tan, K. T. ACS Chem. Biol. 2014, 9, 2359.
    73. 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.
    74. Mattila, T. K.; de Boer, A. Drugs 2010, 70, 2229.
    75. Liu, Y.; Zhu, J.; Xu, Y.; Qin, Y.; Jiang, D. ACS Appl. Mater. Interfaces 2015, 7, 11141.
    76. Los, G.; Encell, L.; McDougall, M.; Hartzell, D.; Karassina, N.; Zimprich, C.; Wood, M.; Learish, R.; Ohana, R.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D.; Bulleit, R.; Wood, K. ACS Chem. Biol. 2008, 3, 373.
    77. Chen, B.; Wang, P.; Jin Q.; Tang, X. Org. Biomol. Chem. 2014, 12, 5629.
    78. Meyer, Y.; Richard, J.-A.; Delest, B.; Noack, P.; Renard, P.-Y.; Romieu, A. Org. Biomol. Chem. 2010, 8, 1777.
    79. Sayre, L. M.; Perry, G.; Smith, M. A.; Chem. Res. Toxicol. 2008, 21, 172.
    80. d'Ischia, M.; Napolitano, A.; Manini, P.; Panzella, L. Chem. Res. Toxicol. 2011, 24, 2071.
    81. Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86.
    82. Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153.

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)

    QR CODE