自從1962年由日本科學家下村脩從維多利亞發光水母分離出綠色螢光蛋白後，螢光蛋白相關的研究就如同雨後春筍般冒出，並時常用於紀錄蛋白質交互作用、基因標誌、化學感測器等。近年來在環境與生物體上偵測陰離子成為重要的目標，因此陰離子感測器開始成為重大的研究方向。我們合成出含尿素基團之螢光蛋白衍生物o-BCUBDI，可以以氫鍵為作用力與F－進行配位進而加以辨識。從螢光圖譜可見o-BCUBDI逐漸加入後，本身在439 nm產生的螢光，其強度逐漸下降，並逐漸長出在較長波長502 nm的螢光，此是為一消一長之比例式螢光。相較於螢光增強、螢光淬熄等螢光感測器，比例式螢光具有兩種放射螢光因此具有內標準法的效果，可以增加訊雜比，並且提高目標物偵測之準確性。以此又繼續探討o-BCUBDI對F－之偵測極限、鍵結計量以及結合常數，並且也以逐漸加入水將F－抓回去的方式，證明o-BCUBDI與F－之螢光的改變並非受到催化反應的影響，最後又以NMR滴定的方式推測o-BCUBDI與F－配位方式。而除了陰離子之外，加入Cu2+、Fe3+等金屬離子亦會使o-BCUBDI有比例式螢光。然後我們也進行酸鹼滴定，輔以了解o-BCUBDI 與待測物結合後產生比例式螢光之機制。

Since 1962 , Green Fluorescence Protein (GFP) was purified by Osamu Shimomura, from the Aequorea jellyfish. Later, the research on the GFP sprung out , and then the was applied to protein interaction, gene expression and chemosensor. Nowadays，the anion chemosensor has been paid in substantial effort. We then synthesized GFP derivative with urea moiety, o-BCUBDI, which could bind with F－ through hydrogen bonding to recognize it. We could see the fluorescent spectroscopy that the original fluorescence at 439 nm decreased, and the fluorescence at 502 nm gradually grew, this kind of fluorescence was called ratiometric fluorescence. In comparison with fluorescence enhance or fluorescence quench, ratiometric fluorescence can act as an internal reference to enhance the signal-to-noise ratio and improve the accuracy of the detection. We also discussed the detection limit, binding stoichiometry, binding constant between o-BCUBDI and F－. The back titration of F－ through gradually adding water was also performed, which means that the interaction of F－ and o-BCUBDI is not a catalytic reaction. Then the F- NMR titration waa also performed to discuss the binding site of F- and o-BCUBDI. Metal ions such as Cu2+ and Fe3+ could cause ratiometric fluorescence to o-BCUBDI, too. The acid and base titration were also performed to assist the discussion on the ratiometric fluorescence mechanism of o-BCUBDI and its analyte.

[1] Shimomura, O.; Johnson, F.; Saiga, Y., J. Cell. Comp. Physiol. 1962,59,223-239.
[2] Tsien, R. Y., Annual review of biochemistry 1998, 67, 509-544.
[3] Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C., Science 1994, 263, 802-805.
[4] Belardinelli, J. M.; Jackson, M., Tuberculosis 2017, 105, 13-17.
[5] Deroo, B. J.; Archer, T. K., Journal of Biological Chemistry 2002, 277 , 20120-20123.
[6] Yang, F.; Moss, L. G.; Phillips, G. N., Nature biotechnology 1996, 14, 1246-1251.
[7] Moberg, A. The Nobel Prize in Chemistry.
[8] Zimmer, M., Chemical reviews 2002, 102, 759-782.
[9] Morise, H.; Shimomura, O.; Johnson, F. H.; Winant, J., Biochemistry 1974, 13 , 2656-2662.
[10] Stafforst, T.; Diederichsen, U., European Journal of Organic Chemistry 2007, 2007 , 899-911.
[11] Prachayasittikul, V.; Nantasenamat, C.; Isarankura-Na-Ayudhya, C.; Tansila, N.; Naenna, T.; Journal of Computational Chemistry 2007, 28 , 1275-1289.
[12] Follenius-Wund, A.; Bourotte, M.; Schmitt, M.; Iyice, F.; Lami, H.; Bourguignon, J.-J.; Haiech, J.; Pigault, C., Biophysical journal 2003, 85, 1839-1850.
[13] Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G., Proceedings of the National Academy of Sciences 1996, 93, 8362-8367.
[14] Yang, J.-S.; Huang, G.-J.; Liu, Y.-H.; Peng, S.-M., Chemical communications 2008, 1344-1346.
[15] Sung, R.; Sung, K., Journal of Luminescence 2018, 202, 163-167.
[16] Chen, K.-Y.; Cheng, Y.-M.; Lai, C.-H.; Hsu, C.-C.; Ho, M.-L.; Lee, G.-H.; Chou, P.-T., Journal of the American Chemical Society 2007, 129, 4534-4535.
[17] Chen, Y.-H.; Lo, W.-J.; Sung, K., The Journal of organic chemistry 2013, 78, 301-310.
[18] Patil, A.; Salunke-Gawali, S., Inorganica Chimica Acta 2018, 482, 99-112.
[19] Sahoo, S. K.; Kim, G.-D.; Choi, H.-J., Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2016, 27, 30-53.
[20] Sedgwick, A. C.; Wu, L.; Han, H.-H.; Bull, S. D.; He, X.-P.; James, T. D.; Sessler, J. L.; Tang, B. Z.; Tian, H.; Yoon, J., Chemical Society Reviews 2018, 47 , 8842-8880.
[21] Zhang, W.; Ma, Z.; Du, L.; Li, M., Analyst 2014, 139, 2641-2649.
[22] Wu, L.; Huang, C.; Emery, B. P.; Sedgwick, A. C.; Bull, S. D.; He, X.-P.; Tian, H.; Yoon, J.; Sessler, J. L.; James, T. D., Chemical Society Reviews 2020, 49 , 5110-5139.
[23] Gui, R.; Jin, H.; Bu, X.; Fu, Y.; Wang, Z.; Liu, Q., Coordination Chemistry Reviews 2019, 383, 82-103.
[24] Cho, E. J.; Ryu, B. J.; Lee, Y. J.; Nam, K. C., Organic letters 2005, 7 , 2607-2609.
[25] Jo, Y.; Chidalla, N.; Cho, D.-G., The Journal of organic chemistry 2014, 79 , 9418-9422.
[26] Saravanan, C.; Easwaramoorthi, S.; Hsiow, C.-Y.; Wang, K.; Hayashi, M.; Wang, L., Organic letters 2014, 16 , 354-357.
[27] Wade, C. R.; Broomsgrove, A. E.; Aldridge, S.; Gabbai, F. P., Chemical reviews 2010, 110 , 3958-3984.
[28] Renny, J. S.; Tomasevich, L. L.; Tallmadge, E. H.; Collum, D. B., Angewandte Chemie International Edition 2013, 52 , 11998-12013.
[29] Grynkiewicz, G.; Poenie, M.; Tsien, R. Y., Journal of biological chemistry 1985, 260 , 3440-3450.
[30] Gómez, D. E.; Fabbrizzi, L.; Licchelli, M.; Monzani, E., Organic & biomolecular chemistry 2005, 3 , 1495-1500.