Highly Sensitive and Selective Colorimetric and Off−On Fluorescent

Jul 27, 2009 - Design Strategies for Water-Soluble Small Molecular Chromogenic and Fluorogenic Probes. Xiaohua Li , Xinghui Gao , Wen Shi , and Huimin...
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Anal. Chem. 2009, 81, 7022–7030

Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Chemosensor for Cu2+ in Aqueous Solution and Living Cells Yan Zhao,† Xiao-Bing Zhang,*,† Zhi-Xiang Han,† Li Qiao,† Chun-Yan Li,‡ Li-Xin Jian,† Guo-Li Shen,† and Ru-Qin Yu† State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China, and Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, PR China The design and synthesis of a novel rhodamine spirolactam derivative and its application in fluorescent detections of Cu2+in aqueous solution and living cells are reported. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. It exhibits a highly sensitive “turn-on” fluorescent response toward Cu2+ in aqueous solution with an 80-fold fluorescence intensity enhancement under 10 equiv of Cu2+added.Thisindicatesthatthesynthesizedchemosensor effectively avoided the fluorescence quenching for the paramagnetic nature of Cu2+ via its strong binding capability toward Cu2+. With the experimental conditions optimized, the probe exhibits a dynamic response range for Cu2+ from 8.0 × 10-7 to 1.0 × 10-5 M, with a detection limit of 3.0 × 10-7 M. The response of the chemosensor for Cu2+ is instantaneous and reversible. Most importantly, both the color and fluorescence changes of the chemosensor are remarkably specific for Cu2+ in the presence of other heavy and transition metal ions (even those that exist in high concentration), which meet the selective requirements for biomedical and environmental monitoring application. The proposed chemosensor has been used for direct measurement of Cu2+ content in river water samples and imaging of Cu2+ in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems. Copper plays an important role in the areas of biological, environmental, and chemical systems. It is an essential trace element for both plants and animals, including humans. Besides zinc and iron, copper ranks the third in abundance in human bodies among the essential heavy metals.1 However, copper is highly toxic to some organisms such as many bacteria and * To whom correspondence should be addressed. E-mail: [email protected]. Tel: +86-731-8821903. Fax: +86-731-8821916. † Hunan University. ‡ Xiangtan University. (1) Barceloux, D. G. J. Toxicol., Clin. Toxicol. 1999, 37, 217–230.

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viruses.2 Owing to its toxicity to bacteria, elevated concentrations of copper hamper the self-purification capability of the sea or rivers and destroy the biological reprocessing systems in water. It is also found to be harmful to humans at high concentrations. Copper has been suspected to cause infant liver damage in recent years. Both Indian childhood cirrhosis (ICC)3 and non-Indian childhood cirrhosis (NICC)4 were proved to be associated with an excessive intake of copper ions. Alteration in the cellular homeostasis of copper ions was reported to be connected with some serious neurodegenerative diseases such as Alzheimer’s disease and prion diseases.5 Accordingly, facile techniques, enabling professionals to monitor the concentration of copper ions in environmental water samples and visualize its subcellular distribution in physiological processes, are of considerable significance for environment protection and human health. Several methods for the detection of copper ions at trace quantity levels in various samples have been proposed. They include atomic absorption spectrometry,6 inductively coupled plasma mass spectroscopy (ICPMS),7 inductively coupled plasmaatomic emission spectrometry (ICP-AES),8 and voltammetry.9 In the past decade, considerable attention has been focused on the (2) Barranguet, C.; van den Ende, F. P.; Rutgers, M.; Breure, A. M.; Greijdanus, M.; Sinke, J. J.; Admiraal, W. Environ. Toxicol. Chem. 2003, 22, 1340– 1349. (3) Hahn, S. H.; Tanner, M. S.; Danke, D. M.; Gahl, W. A. Biochem. Mol. Med. 1995, 54, 142–145. (4) Zietz, B. P.; de Vergara, J. D.; Dunkelberg, H. Environ. Res. 2003, 92, 129–138. (5) (a) Brown, D. R.; Kozlowski, H. Dalton Trans. 2004, 1907–1917. (b) Leach, S. P.; Salman, M. D.; Hamar, D. Anim. Health Res. Rev. 2006, 7, 97–105. (c) Barnham, K. J.; Bush, A. I. Curr. Opin. Chem. Biol. 2008, 12, 222– 228. (d) Crichton, R. R.; Dexter, D. T.; Ward, R. J. Coord. Chem. Rev. 2008, 252, 1189–1199. (e) Brewer, G. J. Cell Biol. Toxicol. 2008, 24, 423–426. (6) (a) Gonzales, A. P. S.; Firmino, M. A.; Nomura, C. S.; Rocha, F. R. P.; Oliveira, P. V.; Gaubeur, I. Anal. Chim. Acta 2009, 636, 198–204. (b) Pourreza, N.; Hoveizavi, R. Anal. Chim. Acta 2005, 549, 124–128. (c) Lin, T. W.; Huang, S. D. Anal. Chem. 2001, 73, 4319–4325. (7) (a) Becker, J. S.; Matusch, A.; Depboylu, C.; Dobrowolska, J.; Zoriy, M. V. Anal. Chem. 2007, 79, 3208–3216. (b) Becker, J. S.; Zoriy, M. V.; Pickhardt, C.; Palomero-Gallagher, N.; Zilles, K. Anal. Chem. 2005, 77, 6074–6080. (8) (a) Liu, Y.; Liang, P.; Guo, L. Talanta 2005, 68, 25–30. (b) Otero-Romani, J.; Moreda-Pineiro, A.; Bermejo-Barrera, A.; Bermejo-Barrera, P. Anal. Chim. Acta 2005, 536, 213–218. (9) (a) Shtoyko, T.; Conklin, S.; Maghasi, A. T.; Richardson, J. N.; Piruska, A.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2004, 76, 1466–1473. (b) Beni, V.; Ogurtsov, V. I.; Bakunin, N. V. Anal. Chim. Acta 2005, 552, 190– 200. (c) Ensafi, A. A.; Khayamian, T.; Benvidi, A. Anal. Chim. Acta 2006, 561, 225–232. 10.1021/ac901127n CCC: $40.75  2009 American Chemical Society Published on Web 07/27/2009

design of fluorescent chemosensors for Cu2+ ion due to the highly sensitive, quick, and nondestructive advantages of the fluorescent method.10 In terms of sensitivity concerns, chemosensors exhibiting fluorescence enhancement (fluorescence “turnon”) upon Cu2+ ion complexation are favored over those showing fluorescence quenching (fluorescence “turn-off”) under Cu2+ binding. Moreover, fluorescence “turn-off” probes may report false positive results caused by other quenchers in practical samples and are undesirable for practical analytical applications. However, for most of the reported Cu2+ ion fluorescent chemosensors, the binding of the metal ion causes a quenching of the fluorescence emission arising from the paramagnetic nature of Cu2+ and gives a “turn-off” signal.11 The past five years have seen increasing interest in design and synthesis of fluorescent chemosensors with a Cu2+ induced “turn-on” fluorescence signal.12 Among these chemosensors, unfortunately, those that can be applied in aqueous solutions at neutral pH (as most copper containing samples are nearneutral aqueous systems) are still rare due to the strong hydration ability of Cu2+ in aqueous solution. Moreover, most of these fluorescent chemosensors are driven by UV light excitation, which might suffer from the influence of background fluorescence. Lu et al. have reported a functional oligonucleotide-based “turn-on” fluorescent probe for detection of Cu2+ in aqueous solution with satisfying sensitivity and selectivity, although its application in living cells was not investigated.13 As to visualizing subcellular distribution of copper ion in physiological processes, fluorescence bioimaging technology seems to be the best choice by virtue of its highly sensitive, high(10) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515– 1566. (b) Jiang, P.; Guo, Z. Coord. Chem. Rev. 2004, 248, 205–229. (c) Lim, M. H.; Lippard, S. J. Acc. Chem. Res. 2007, 40, 41–51. (11) (a) Fabrizzi, L.; Licchelli, M.; Pallavicini, P.; Perotti, A.; Sacchi, D. Angew. Chem., Int. Ed. Engl. 1994, 33, 1975–1977. (b) Fabrizzi, L.; Licchelli, M.; Pallavicini, P.; Perotti, A.; Taglietti, A.; Sacchi, D. Chem.sEur. J. 1996, 2, 75–82. (c) Torrado, A.; Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1998, 120, 609–610. (d) Zheng, Y.; Huo, Q.; Kele, P.; Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. Org. Lett. 2001, 3, 3277–3280. (e) Li, Y.; Yang, C. M. Chem. Commun. 2003, 2884–2885. (f) Shao, N.; Zhang, Y.; Cheung, S. M.; Yang, R. H.; Chan, W. H.; Mo, T.; Li, K. A.; Liu, F. Anal. Chem. 2005, 77, 7294–7303. (g) Kim, S. H.; Kim, J. S.; Park, S. M.; Chang, S.-K. Org. Lett. 2006, 8, 371–374. (h) Choi, J. K.; Kim, S. H.; Yoon, J.; Lee, K.-H.; Bartsch, R. A.; Kim, J. S. J. Org. Chem. 2006, 71, 8011–8015. (i) Park, S. M.; Kim, M. H.; Choe, J.-I.; No, K. T.; Chang, S.-K. J. Org. Chem. 2007, 72, 3550– 3553. (j) Xie, J.; Menand, M.; Maisonneuve, S.; Metivier, R. J. Org. Chem. 2007, 72, 5980–5985. (k) Wen, Y. Q.; Yue, F.; Zhong, Y. R.; Ye, B. H. Inorg. Chem. 2007, 46, 7749–7755. (l) Khatua, S.; Choi, S. H.; Lee, J.; Huh, J. O.; Do, Y.; Churchill, D. G. Inorg. Chem. 2009, 48, 1799–1801. (12) (a) Wu, Q.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 14682–14683. (b) Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, 1612– 1613. (c) Xu, Z. C.; Xiao, Y.; Qian, X. H.; Cui, J. N.; Cui, D. W. Org. Lett. 2005, 7, 889–892. (d) Wen, Z. C.; Yang, R. H.; He, H.; Jiang, Y. B. Chem. Commun. 2006, 106–108. (e) Martinez, R.; Zapata, F.; Caballero, A.; Espinosa, A.; Tarraga, A.; Molina, P. Org. Lett. 2006, 8, 3235–3238. (f) Zheng, L.; Miller, E. W.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 10–11. (g) Choi, S. H.; Pang, K.; Kim, K.; Churchill, D. G. Inorg. Chem. 2007, 46, 10564–10577. (h) Li, G. K.; Xu, Z.; Chen, X. C. F.; Huang, Z. T. Chem. Commun. 2008, 1774–1776. (i) Shao, N.; Jin, J. Y.; Wang, H.; Zhang, Y.; Yang, R. H.; Chan, W. H. Anal. Chem. 2008, 80, 3466–3475. (j) Lin, W. Y.; Yuan, L.; Tan, W.; Feng, J. B.; Long, L. L. Chem.sEur. J. 2009, 15, 1030–1035. (k) Khatua, S.; Choi, S. H.; Lee, J.; Huh, J. O.; Do, Y.; Churchill, D. G. Inorg. Chem. 2009, 48, 1799–1801. (13) Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 9838–9839.

speed spatial analysis and less cell damage.14 The development of highly sensitive, selective, and cell membrane-penetrable probes that exhibit a visible “turn-on” fluorescent emission in aqueous media is often declared the bottleneck for fluorescence imaging metal ions in living cells. Several fluorescence “turn-on” probes for imaging various intracellular heavy metal ions such as Zn2+,14h,15 Hg2+,16 Cd2+,17 Pb2+,18 and Cu+ 19 have been successfully designed and synthesized. However, few such probes have been proposed for imaging intracellular Cu2+. On the basis of a Rhodamine B derivative containing a highly electron-rich S atom, Li and Huang have reported a “turn-on” chemodosimeter for imaging Cu2+ in living HeLa cells with high sensitivity and fast response time.20 This probe, however, shows an irreversible fluorescence “turn-on” response toward Cu2+, cannot distinctly recognize Cu2+ from Hg2+, and thereby, is not suitable for monitoring the concentration of copper ions in complicated environmental samples. More recently, Shin and Yoon have proposed a monoboronic acid-conjugated rhodaminebased probe for Cu2+ with a reversible fluorescence “turn-on” response and high selectivity, and they successfully applied it (14) (a) Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nat. Biotechnol. 2003, 21, 1369–1377. (b) Stephens, D. J.; Allan, V. J. Science 2003, 300, 82–86. (c) Lichtman, J.; Conchello, J. A. Nat. Methods 2005, 2, 910–919. (d) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217–224. (e) Que, E. L.; Domaille, D. W.; Chang, C. J. Chem. Rev. 2008, 108, 1517–1549. (f) Thoumine, O.; Ewers, H.; Heine, M.; Groc, L.; Frischknecht, R.; Giannone, G.; Poujol, C.; Legros, P.; Lounis, B.; Cognet, L.; Choquet, D. Chem. Rev. 2008, 108, 1565–1587. (g) Fernandez-Suarez, M.; Ting, A. Y. Nat. Rev. Mol. Cell Biol. 2008, 9, 929–943. (h) Nolan, E. M.; Lippard, S. J. Acc. Chem. Res. 2009, 42, 193–203. (15) (a) Lim, N. C.; Freake, H. C.; Bruckner, C. Chem.sEur. J. 2005, 11, 38– 49. (b) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 10197–10204. (c) Nolan, E. M.; Ryu, J. W.; Jaworski, J.; Feazell, R. P.; Sheng, M.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 15517–15528. (d) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447–13454. (e) Zhang, X. A.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10780– 10785. (f) Zhang, L.; Clark, R. J.; Zhu, L. Chem.sEur. J. 2008, 14, 2894– 2903. (g) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Bio. 2008, 4, 168–175. (h) Xue, L.; Liu, C.; Jiang, H. Chem. Commun. 2009, 1061– 1063. (16) (a) Zhang, Z. C.; Wu, D.; Guo, X. F.; Qian, X. H.; Lu, Z.; Xu, Q.; Yang, Y. Y.; Duan, L. P.; He, Y. K.; Feng, Z. Chem. Res. Toxicol. 2005, 18, 1814–1820. (b) Ko, S. K.; Yang, Y. K.; Tae, J. S.; Shin, I. J. J. Am. Chem. Soc. 2006, 128, 14150–14155. (c) Yoon, S. H.; Miller, E. W.; He, Q. W.; Do, P. H.; Chang, C. J. Angew. Chem., Int. Ed. 2007, 46, 6658–6661. (d) Yang, H.; Zhou, Z. G.; Huang, K. W.; Yu, M. X.; Li, F. Y.; Yi, T.; Huang, C. H. Org. Lett. 2007, 9, 4729–4732. (e) Zhang, X. L.; Xiao, Y.; Qian, X. H. Angew. Chem., Int. Ed. 2008, 47, 8025–8029. (f) Tang, B.; Cui, L. J.; Xu, K. H. ChemBioChem 2008, 9, 1159–1164. (17) (a) Peng, X. J.; Du, J. J.; Fan, J. L.; Wang, J. Y.; Wu, Y. K.; Zhao, J. Z.; Sun, S. G.; Xu, T. J. Am. Chem. Soc. 2007, 129, 1500–1501. (b) Liu, W. M.; Xu, L. W.; Sheng, R. L.; Wang, P. F.; Li, H. P.; Wu, S. K. Org. Lett. 2007, 9, 3829–3832. (c) Taki, M.; Desaki, M.; Ojida, A.; Iyoshi, S.; Hirayama, T.; Hamachi, I.; Yamamoto, Y. J. Am. Chem. Soc. 2008, 130, 12564–12565. (d) Cheng, T.; Xu, Y.; Zhang, S.; Zhu, W.; Qian, X.; Duan, L. J. Am. Chem. Soc. 2008, 130, 16160–16161. (18) (a) He, Q. W.; Miller, E. W.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 9316–9317. (b) Miller, E. W.; He, Q. W.; Chang, C. J. Nat. Protoc. 2008, 3, 777–783. (c) Shete, V. S.; Benson, D. E. Biochemistry 2009, 48, 462–470. (19) (a) Yang, L. C.; Mcrae, R.; Henary, M. M.; Patel, R.; Lai, B.; Vogt, S.; Fahrni, C. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11179–11184. (b) Zeng, L.; Miller, E. W.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 10–11. (c) Miller, E. W.; Zeng, L.; Domaille, D. W.; Chang, C. J. Nat. Protoc. 2006, 1, 824–827. (20) Yu, M.; Shi, M.; Chen, Z.; Li, F.; Li, X.; Gao, Y.; Xu, J.; Yang, H.; Zhou, Z.; Yi, T.; Huang, C. Chem.sEur. J. 2008, 14, 6892–6900.

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to image Cu2+ in living cells and organisms.21 However, it shows moderate sensitivity with only about a 6.4-fold fluorescence intensity enhancement upon 100 equiv of Cu2+ added (with relative quantum yields changing from 0.07 to 0.45 in the presence of Cu2+), which might limit its application in detection of Cu2+ in a water sample with a very low concentration. In the course of our preparation of this manuscript, Lee, Joo, and Kim have reported a 2-picolyl functionalized coumarinbased fluorogenic probe for imaging Cu2+ in living cells.22 However, it showed a “turn-off” fluorescence signal with incubation of Cu2+. Therefore, the search for highly sensitive, selective, reversible, and “turn-on” fluorescent probes for monitoring Cu2+ in environmental water samples and in living cells is still very active and poses a significant challenge for chemical research. Rhodamine derivatives possess excellent spectroscopic properties such as a large molar extinction coefficient, high fluorescence quantum yield, and visible light excitation as well as longwavelength emission. Moreover, rhodamine derivatives with a spirolactam form are nonfluorescent, whereas a ring-opened amide form gives rise to a strong fluorescence emission. So, the rhodamine framework seems to be an ideal model to construct “turn-on” fluorescent chemosensors. However, very few research interests were drawn to such chemosensors until the first rhodamine-based fluorescent chemodosimeter for Cu2+ was reported by Czarnik’s group.23 Their chemodosimeter was based on a Rhodamine B hydrazide which showed an irreversible fluorescence enhancement toward Cu2+ in water with high selectivity. Inspired by their successful work, increasing attention has focused on rhodamine spirolactam-based fluorescent chemosensors and chemodosimeters for various metal ions,20,21,24 and other biologically important species25 in recent years. In the context of our long-term interests in searching for fluorescent chemosensors for heavy metal ions,26 we try to design a rhodamine spirolactam-based chemosensor for “turn-on” detection of metal ions in aqueous samples. Herein, we report the synthesis and application of our novel design, rhodamine spirolactam-based (21) Swamy, K. M. K.; Ko, S.-K.; Kwon, S. K.; Lee, H. N.; Mao, C.; Kim, J.-M.; Lee, K.-H.; Kim, J.; Shin, I.; Yoon, J. Chem. Commun. 2008, 5915–5917. (22) Jung, H. S.; Kwon, P. S.; Lee, J. W.; Kim, J. I.; Hong, C. S.; Kim, J. W.; Yan, S.; Lee, J. Y.; Lee, J. H.; Joo, T.; Kim, J. S. J. Am. Chem. Soc. 2009, 131, 2008–2012. (23) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 7386– 7387. (24) (a) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760– 16761. (b) Kwon, J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.; Nam, W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, 10107–10111. (c) Zheng, H.; Qian, Z. H.; Xu, L.; Yuan, F. F.; Lan, L. D.; Xu, J. G. Org. Lett. 2006, 8, 859–861. (d) Xiang, Y.; Tong, A. Org. Lett. 2006, 8, 1549–1552. (e) Xiang, Y.; Tong, A. J.; Jin, P. Y.; Ju, Y. Org. Lett. 2006, 8, 2863–2866. (f) Zhang, X.; Shiraishi, Y.; Hirai, T. Org. Lett. 2007, 9, 5039–5042. (g) Wu, D.; Huang, W.; Duan, C.; Lin, Z.; Meng, Q. Inorg. Chem. 2007, 46, 1538–1540. (h) Shi, W.; Ma, H. Chem. Commun. 2008, 5915–5917. (i) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008, 37, 1465– 1472. (j) Lee, M. H.; Kim, H. J.; Yoon, S.; Park, N.; Kim, J. S. Org. Lett. 2008, 10, 213–216. (k) Liu, W. M.; Xu, L. W.; Zhang, H. Y.; You, J. J.; Zhang, X. L.; Wang, P. F. Org. Biomol. Chem. 2009, 7, 660–664. (l) Huang, W.; Song, C.; He, C.; Lv, G.; Hu, X.; Zhu, X.; Duan, C. Inorg. Chem. 2009, 48, 5061–5072. (25) (a) Zheng, H.; Shang, G. Q.; Yang, S. Y.; Gao, X.; Xu, J. G. Org. Lett. 2008, 10, 2357–2360. (b) Chen, X. Q.; Wang, X. C.; Wang, S. J.; Shi, W.; Wang, K.; Ma, H. M. Chem.sEur. J. 2008, 14, 4719–4724. (c) Yang, Y. K.; Cho, H. J.; Lee, J.; Shin, I.; Tae, J. Org. Lett. 2009, 11, 859–861.

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Scheme 1. Chemical Structure and Synthetic Route of Compounds 1, 2, 3, and 4

chemosensor 1 (Scheme 1). It shows a reversible “turn-on” fluorescent response for Cu2+ in neutral aqueous solution with remarkably high sensitivity and selectivity. Furthermore, the proposed chemosensor has been used for direct measurement of Cu2+ content in river water samples and imaging of Cu2+ in living cells with satisfying results. EXPERIMENTAL SECTION General. CH2Cl2 was distilled from calcium hydride and stored over molecular sieves, and tetrahydrofuran (THF) was desiccated by sodium metal and indicated by benzophenone. Rhodamine 6G, 2-(chloromethyl)pyridine hydrochloride and 2-pyridinecarboxaldehyde were purchased from Sigma-Aldrich; all other chemicals were of analytical reagent grade, purchased from Shanghai Chemical Reagent Corporation (Shanghai, China), and used without further purification. Twice-distilled water was used throughout all experiments. Solutions of Cu2+, Fe2+, Mn2+, Na+, K+, and Ca2+ were prepared from their chloride salts; solutions of Zn2+, Cd2+, Fe3+, Pb2+, Co2+, Ni2+, Ag+, and Mg2+ Hg2+, were prepared from their nitrate salts. Thin layer chromatography (TLC) was carried out using silica (26) (a) Zhang, X. B.; Guo, C.C.; Li, Z. Z.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2002, 74, 821–825. (b) Zhang, X. B.; Peng, J.; He, C. L.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2006, 567, 189–195. (c) Li, C. Y.; Zhang, X. B.; Jin, Z.; Han, R.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2006, 580, 143–148. (d) Luo, H. Y.; Jiang, J. H.; Zhang, X. B.; Li, C. Y.; Shen, G. L.; Yu, R. Q. Talanta 2007, 72, 575–581. (e) Li, C. Y.; Zhang, X. B.; Dong, Y. Y.; Ma, Q. J.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2008, 616, 214–221.

gel 60 F254, and column chromatography was conducted over silica gel (200-300 mesh), both of which were obtained from the Qingdao Ocean Chemicals (Qingdao, China). 1H and 13C NMR spectra were recorded on a Bruker DRX-400 spectrometer operating at 400 and 100 MHz, respectively. All chemical shifts are reported in the standard δ notation of parts per million. LC-MS analyses were performed using an Agilent 1100 HPLC/MSD spectrometer; UV-vis absorption spectra were recorded with a Shimadzu MultiSpec-1501 spectrophotometer. All fluorescence measurements were carried out on a PerkinElmer LS55 luminescence spectrometer with excitation slit set at 10.0 nm and emission at 5.0 nm. The pH measurements were carried out on a Mettler-Toledo Delta 320 pH meter. Synthesis of Compound 1. Rhodamine 6G hydrazide was synthesized according to a reported method.27 It was then reacted with 2-pyridinecarboxaldehyde following the literature procedure to give compound 2 (Scheme 1).24f Their structures were confirmed by MS data and NMR spectra. To a stirred solution of 2 (1.00 g, 1.93 mmol) in anhydrous CH2Cl2 (20 mL) cooled to 0 °C was carefully added solid sodium borohydride (0.37 g, 9.65 mmol) in portions. The resulting mixture was stirred at room temperature for 48 h and was quenched with 1 M HCl (10 mL) at 0 °C. After stirring at room temperature for another 2 h, the reaction mixture was adjusted to neutral with 1 M NaHCO3. The organic phase was removed, and the aqueous phase was extracted three times with CHCl3 (3 × 20 mL); all CHCl3 extracts were combined and filtered. The CHCl3 solution was reduced to a smaller volume by rotary evaporation, washed with water and brine, and dried over anhydrous sodium sulfate. Purification with silica flash chromatography (silica, CH2Cl2/ C2H5OH ) 50:1, v/v) afforded 1 (0.23 g, 23%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ (ppm): 1.32 (t, J ) 7.2 Hz, 6H), 1.86 (s, 6H), 3.20 (m, 4H), 3.47 (s, 2H), 3.92 (d, J ) 6.0 Hz, 2H), 4.77 (t, 1H), 6.21(s, 2H), 6.34 (s, 2H), 7.00 (m, 1H), 7.05 (m, 1H), 7.14 (d, J ) 7.6 Hz, 1H), 7.45 (m, 3H), 7.94 (m, 1H), 8.37 (d, J ) 4.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 14.75, 16.69, 38.33, 56.39, 65.59, 96.69, 106.14, 117.55, 121.61, 122.40, 122.81, 123.96, 128.05, 128.26, 130.10, 132.69, 135.97, 147.25, 148.44, 151.89, 152.09, 158.12, 166.84. ESI-MS m/z: (1 + H)+ calcd for C32H34N5O2, 520.3; found, 520.2. Synthesis of Compound 3. Rhodamine B hydrazide was synthesized following a literature procedure and characterized by NMR spectra and mass data.27 To a stirred suspension of NaH (60% dispersion in oil, 0.060 g, 1.5 mmol) in anhydrous THF (3 mL) was added a solution of Rhodamine B hydrazide (0.231 g, 0.5 mmol) in anhydrous THF (2 mL), and the resulting mixture was stirred at room temperature for 0.5 h. A solution of 2-(chloromethyl)pyridine hydrochloride (0.123 g, 0.75 mmol) in anhydrous THF (2 mL) was then added, and the mixture was heated to reflux under a nitrogen atmosphere for 3 h (Scheme 1). The mixture was cooled, filtered, and diluted with CHCl3 (30 mL). It was then washed with water and brine and dried over anhydrous sodium sulfate. Crude product was dried under reduced pressure and purified by column chromatography (CH2Cl2/C2H5OH ) 100:1, v/v). Evaporation of the solvent afforded 3 (0.182 g, 67%) as a colorless solid. 1H NMR (CDCl3, (27) Anthoni, U.; Christophersen, C.; Nielsen, P.; Puschl, A.; Schaumburg, K. Struct. Chem. 1995, 3, 161–165.

400 MHz) δ (ppm): 1.15 (t, J ) 7.2 Hz, 12H), 3.33 (q, J ) 7.2 Hz, 8H), 4.05 (s, 2H), 4.56 (s, 1H), 6.31 (d, J ) 8.4, 2H), 6.46 (s, 2H), 6.49 (s, 2H), 7.07-7.10 (m, 1H),7.16 (s, 1H), 7.24 (d, J ) 7.6 Hz, 1H), 7.44-7.49 (m, 2H), 7.62 (m, 1H), 7.90-7.92 (m, 1H), 8.4 (d, J ) 4.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 12.36, 14.06, 22.63, 29.307, 29.63, 31.86, 44.96, 55.30, 65.44, 99.09, 108.66, 122.29, 122.90, 123.29, 123.95, 128.24, 128.62, 129.98, 132.80, 138.02, 153.53, 157.24, 166.60. ESI-MS m/z: (3 + H)+ calc. for C34H38N5O2, 548.3; found, 548.3. Synthesis of Compound 4. Compound 4 was synthesized following the similar procedure of compound 3 by the reaction of Rhodamine B hydrazide with benzyl chloride. Purification with silica flash chromatography (silica, CH2Cl2/C2H5OH ) 100:1, v/v) afforded 4 (0.142, 53%) as colorless solid. 1H NMR (CDCl3, 400 MHz) δ (ppm): 1.15(t, J ) 6.8 Hz, 12H), 3.30 (q, J ) 7.2 Hz, 8H), 3.72 (s, 2H), 4.24 (s, 1H), 6.23 (d, J ) 8.8 Hz, 2H), 6.41 (s, 2H), 6.43 (s, 2H), 7.14-7.18 (m, 6H), 7.47-7.50 (m, 2H), 7.94 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 12.567, 29.655, 44.303, 55.090, 65.534, 97.796, 106.051, 107.645, 122.689, 124.093, 127.083, 127.991, 128.128, 128.548, 129.074, 130.989, 132.530, 137.664, 148.688, 151.038, 154.074, 166.341. ESI-MS m/z: (4 + H)+ calc. for C35H39N4O2, 547.3; found, 547.2. Procedures for Metal Ion Sensing. A 50 µM stock solution of 1 was prepared by dissolving 1 in absolute ethanol. A standard stock solution of Cu2+ (10 mM) was prepared by dissolving an appropriate amount of copper chloride in water and adjusting the volume to 500 mL in a volumetric flask. This was further diluted to 1 mM-0.1 µM stepwise. The complex solution of Cu2+/1 was prepared by adding 2.0 mL of the stock solution of 1 and 1.0 mL of the stock solution of Cu2+ in a 10 mL volumetric flask. Then, the mixture was diluted to 10 mL with Tris-HNO3 buffer solution. In the solution, thus obtained, the concentrations were 10 µM of 1 and 1 mM-0.01 µM of Cu2+. The solution was protected from light and kept at 4 °C for further use. Blank solution of 1 was prepared under the same conditions without Cu2+. Stock solutions of other metal ions were prepared in water with a similar procedure. For all measurements of fluorescence spectra, excitation was fixed at 500 nm with excitation slit set at 10.0 nm and emission at 5.0 nm. Cell Incubation and Imaging. The living HeLa cells were provided by the biomedical engineering center of Hunan University (China). Immediately before the experiments, the cells were washed with phosphate-buffered saline (PBS), followed by incubating with 10 µM of compound 1 (in the culture medium containing 0.5% EtOH) for 30 min at 37 °C and then by washing with PBS three times. After incubating with 10 µM of CuCl2 for another30 min at 37 °C, the HeLa cells were rinsed with PBS three times again. Fluorescence imaging of intracellular Cu2+ in HeLa cells was observed under a Nikon Eclipse TE-300 fluorescence microscope with excitation wavelength fixed at 500 nm. RESULTS AND DISCUSSION Synthesis and Characteristics of Rhodamine Spirolactams. To synthesize compound 1, compound 2 was first synthesized as a starting material from Rhodamine 6G hydrazide and 2-pyridinecarboxaldehyde following the literature procedure.24f Compound 1 was then easily synthesized by a simple reduction Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 1. Change in color (top) and fluorescence (bottom) of 1 (10 µM) in buffered (Tris-HCl, pH ) 7.10) water/C2H5OH (8:2, v/v) with metal ions: blank, Hg2+, Pb2+, Ni2+, Co2+, Fe2+, Fe3+, and Cu2+(10 equiv, from left to right).

reaction of compound 2 with NaBH4 as a reducer (Scheme 1). Its structure was confirmed by MS data and 1H NMR and 13C NMR spectra. Similar to other rhodamine spirolactam derivatives, compound 1 forms a nearly colorless and fluorescence inactive solution in either Tris-HCl aqueous buffer solution or pure organic solvent, indicating that the spirolactam form predominantly exists. This is also confirmed by a distinctive spirocycle carbon chemical shift at 65.6 ppm in the 13C NMR spectrum of 1.27 Compounds 3 and 4 were synthesized in a different and simpler two-step strategy with Rhodamine B as the starting material (Scheme 1). It was first converted to a hydrazide derivative following a reported procedure.28 Different from Rhodamine 6G hydrazide, there is no other active proton except the protons on the hydrazine section on Rhodamine B hydrazide. We could then directly couple it with 2-picolyl chloride or benzyl chloride under basic conditions to produce 3 and 4 with good or satisfying yields, respectively. These compounds also form nearly colorless and fluorescence inactive solutions in either Tris-HCl aqueous buffer solution or pure organic solvent, indicating their existence in spirolactam forms. Similarly, distinctive spirocycle carbon chemical shifts at about 65 ppm are found in their 13C NMR spectrum. Metal Ions Sensing of 1. Compound 2 was reported by Duan et al. to show a remarkable and specific Hg2+-induced fluorescence enhancement,24f while compound 1 shows neither the color nor the fluorescence (excited at 500 nm) characteristics of rhodamine with the addition of Hg2+, even with high concentration (Figure 1). Interestingly, compound 1 emitted strong fluorescence near 550 nm (excited at 500 nm) with the addition of Cu2+, accompanied with an obvious orange color appearance (Figure 1). However, other heavy metal ions that usually show an interfering effect on Cu2+ ion assays, such as Pb2+, Ni2+, Co2+, Fe2+, and Fe3+, show neither a color nor a fluorescence change after they were added to buffered solution of 1 (Figure 1). Figure 2 showed the absorption spectra variation of 1 on the gradual addition Cu2+. The colorless free 1 in buffered (Tris-HCl, pH ) 7.1) water/C2H5OH (8:2, v/v) exhibited almost no (28) Yang, X. F.; Guo, X. Q.; Zhao, Y. B. Talanta 2002, 57, 883–890.

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Figure 2. UV-vis spectra of 1 (10 µM) with gradual addition of different amounts of Cu2+ (from bottom to top: 0, 0.5, 1, 2, 3, 10, 20, 30, 40, 50, 100, 200, 500, and 1000 µM).

absorption near 530 nm. Upon the gradual addition of Cu2+ to the solution, a new absorption peak at 529 nm emerged with increasing intensity, and the solution displayed a clear change from colorless to orange simultaneously, which can be ascribed to variation from a spirocyclic form to a ring-opened amide form. Job’s method for the absorbance was applied to determine the stoichiometry of the 1-Cu2+ complex, by keeping the sum of the initial concentration of copper ion and 1 at 6.0 × 10-5 M and the molar ratio of copper ion changing from 0 to 1.29 The absorbance of 1 in the absence (A0) and presence (A) of copper ion was determined, respectively. A plot of (A-A0)/A0 versus the molar fraction of copper ion is provided in Figure 3A. It shows that the (A-A0)/A0 value goes through a maximum at a molar fraction of about 0.5, indicating a 1:1 stoichiometry of the Cu2+ to 1 in the complex. The association constant for 1 binding to Cu2+ was determined from the absorption titration data. If 1 binds with Cu2+ to form a complex with a complexing ratio of 1:1, one can describe the equilibrium as follows: K

R + Cu2+ y\z [RCu]2+

(1)

Here, R and [RCu]2+ denote the rhodamine spirolactam 1 and 1-Cu2+ complex, respectively, and K denotes the association constant. The relative absorbance R is defined as the ratio of free R, [R]f, to the total amount of R, [R]t in the buffered water/ C2H5OH. It can be experimentally determined by measuring the absorbance values in the presence of different concentrations of Cu2+:

R)

At - A [R]f ) [R]t At - A0

(2)

Here, A0 and At are the limiting absorbance values for R ) 1 (in the absence of Cu2+) and R ) 0 (1 is completely complexed (29) (a) Job, P. Ann. Chim. 1928, 9, 113–116. (b) Vosburgh, W. C.; Cooper, G. R. J. Am. Chem. Soc. 1941, 63, 437–442.

Figure 4. Fluorescence emission of 1 (10 µM) with gradual addition of different amounts of Cu2+ (from bottom to top: 0, 1, 2, 4, 5, 10, 20, 30, 40, 50, and 100 µM) and a plot of log[(F - F0)/F0] as a function of the log[Cu2+] (inset).

Figure 3. (A) Job plot for determining the stoichiometry of 1 and Cu2+. The total concentration of 1 and copper was 6.0 × 10-5 M. Molar fraction was given by [Cu2+]/([Cu2+] + [1]). (B) Relative absorbance R as a function of log[Cu2+] calculated from eq 3: •, data points experimentally obtained.

with Cu2+), respectively. According to the derivation following the mass law reported elsewhere,26a the relationship between the R and the Cu2+ concentration can be represented as follows: R 1 ) 1-R K[Cu2+]

(3)

It is apparent from eq 3 that the relative absorbance R has a distinct functional relationship with the concentration of Cu2+ and the association constant K, which provides the basis for the detection of the K value. The experimental data were fitted to eq 3 by adjusting the K value. Figure 3B shows the fitted curve to incorporate the experimental data for Cu2+, which gives an association constant K value of 2.08 × 10 4 M-1 for 1 binding to Cu2+, corresponding to a stronger binding capability toward Cu2+ in comparison with a tren/dansyl-appended rhodaminebased FRET probe for Cu2+ (with a K value of 7 × 10 3 M-1)24i or a rhodamine spirolactam derivative-based probe for Fe3+ (with a K value of 3.2 × 10 3 M-1).24d Fluorescent Sensing of Copper Ions. The fluorescence titration of the Cu2+ ion was carried out using a solution of 10

µM of compound 1 in buffered (Tris-HCl, pH ) 7.1) water/ C2H5OH (8:2, v/v). Upon the addition of increasing concentrations of Cu2+, the fluorescence intensity in the 510-630 nm range showed a linear enhancement. With the concentration of Cu2+ up to 10 equiv of compound 1, an 80-fold fluorescence enhancement was observed. Meanwhile, the maximum emission wavelength undergoes a slight red shift from 550 to 552 nm (Figure 4). All of this supports our expectation that compound 1 could serve as a sensitive fluorescent switcher as well as a naked-eye chemosensor for Cu2+. For optimization purposes, compound 3 was designed and synthesized using a Rhodamine B fluorophore instead of Rhodamine 6G in compound 1, as the fluorescent emission of Rhodamine B (575 nm) is located at a longer wavelength than that of Rhodamine 6G (550 nm). However, comparing to compound 1, compound 3 shows lower fluorescence sensitivity toward Cu2+, which shows a 33-fold fluorescence enhancement with the addition of 10 equiv of Cu2+. Therefore, compound 1 was chosen as a Cu2+ fluorescent chemosensor for further investigation. The fluorescent response of compound 1 toward the Cu2+ ion was calculated to cover a linear range from 8.0 × 10-7 to 1.0 × 10-5 M (Figure 4, inset), with a detection limit of 3.0 × 10-7 M (based on S/N ) 3). Therefore, our proposed chemosensor was sensitive enough to detect Cu2+ in environmental water samples, even in drinking water, which has a limit of 20 µM defined by the U.S. Environmental Protection Agency. To study the practical applicability, the effects of pH on the fluorescence response to Cu2+ of the new chemosensor 1 were also investigated. Experimental results shows that for free 1, at acidic conditions (pH < 3), an obvious fluorescence Off-On (Figure 5) appeared due to the formation of the open-ring state because of the strong protonation, and it shows a fluorescence quenching effect upon addition of Cu2+. In the pH range from 4.0 to 10.0, neither the color nor the fluorescence (excited at 500 nm) characteristics of rhodamine could be observed for 1, suggesting that the spirocyclic form was still preferred in this range. However, in the presence of the Cu2+ ion, there was an obvious fluorescence Off-On change of 1 with different Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 5. Fluorescence intensity (552 nm) of free 1 (10 µM) (×) and after addition of 100 µM Cu2+ (•) in Tris-HCl buffers as a function of different pH values.

fluorescence enhancement efficiency under different pH values in a pH range from 4.0 to 8.0. Chemosensor 1 shows the highest fluorescence response toward the Cu2+ ion under a pH 7.1, therefore, this pH value was chosen as an optimum experimental condition. A short response time is necessary for a fluorescent chemosensor to monitor Cu2+ in aqueous samples and in living cells in real time. The time dependence of the response of 1 to Cu2+ was investigated by recording the change of fluorescence intensity at 552 nm with time under different concentrations of Cu2+ (Figure S1 in the Supporting Information). The results revealed that the response time of the chemosensor toward Cu2+ increases with the increase of Cu2+ concentration. The reaction of 10 µM of compound 1 and 100 µM of Cu2+ was completed within 1 min. Moreover, an instantaneous response toward Cu2+ was observed with its concentration less than 10 µM, indicating that our fluorescent chemosensor could meet the response time requirements for real-time monitoring of Cu2+ in practical samples. This experimental result also shows that the 1-Cu2+ complex is photostable. Irradiating the probe solution with visible light did not liberate metal ion with a regeneration change of the fluorescence intensity. We subsequently studied the chemical reversibility behavior of the binding of 1 and Cu2+ in the ethanol-water solution. Because of the high stability constant of the EDTA-Cu2+ complex, it was anticipated that addition of EDTA will sequester Cu2+ of the metal complex, liberating the free 1. With this intention, excess of EDTA was added to the Cu2+ complex of 1. The color and fluorescence of the solution disappeared instantly upon the addition of EDTA, whereas readdition of excess Cu2+ could recover the fluorescence signal (Figure S2 in the Supporting Information), demonstrating that the binding of 1 with Cu2+ is chemically reversible. Selectivity is a very important parameter to evaluate the performance of a fluorescence chemosensor. The interference of a number of common metal ions for detection of Cu2+ with 1 was carried out with excitation fixed at 500 nm and emission at 552 nm. Figure 6 (the black bar portion) illustrated the fluorescence response of 1 to different metal ions of interest; no significant fluorescence intensity changes were observed with 7028

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Figure 6. Fluorescence response of 1 (10 µM) to 10 µM of Cu2+ or 100 µM of other metal ions (the black bar portion) and to the mixture of 100 µM of other divalent metal ions with 10 µM of Cu2+ (the gray bar portion).

common interferences such as alkali, alkaline earth, and transitional metal ions, indicating that our proposed sensor exhibits high selectivity to Cu2+ over other metal ions. In certain environmental samples, such as river water and seawater, the concentrations of some other contaminating metal ions, such as Zn2+, Ni2+, or Pb2+, are significantly higher than that of Cu2+; selective detection of Cu2+ in the presence of these metal ions with high concentration is a challenge to the application of most common sensors. To test practical applicability of our fluorescent chemosensor for Cu2+, competition experiments were also carried out. Ten times the concentration of above-mentioned metal ions (100 µM) are added to 10 µM of Cu2+ in Tris-HCl buffered water/C2H5OH, and the fluorescence response of the chemosensor is detected and then compared with that of Tris-HCl buffered water/C2H5OH containing only 10 µM of Cu2+. Results are also shown in Figure 6 (the gray bar portion). Our sensor showed almost unchanged responses to Cu2+ before and after addition of other interfering metal ions. These experimental results show that the response of the sensor to Cu2+ is unaffected by the presence of the other possible contaminating metal ions, even existing in a concentration 10 times higher than that of Cu2+. All these selective results indicate that our proposed chemosensor could meet the selective requirements for biomedical and environmental application. Sensing Mechanism. Similar to many reported rodaminespirolactam-based fluorescent chemosensors, the fluorescence enhancement response of chemosensor 1 and 3 toward Cu2+ is most likely the result of the spiro ring-opening mechanism rather than an ion-catalyzed hydrolysis reaction. The abovementioned EDTA experiment could serve as experimental evidence to support this reversible spiro ring-opening mechanism. More direct evidence was obtained by comparing the ESI mass spectra of 1 and 1-Cu2+ (Figures S3 and S4 in the Supporting Information). In Figure S3 (in the Supporting Information), the unique peak at m/z ) 520.2 (calcd ) 520.3) corresponded to [1 + H]+. While in Figure S4 (in the Supporting Information), with excess Cu2+ added to 1, the peak at m/z )

Scheme 2. Proposed Binding Mechanism of Cu2+ with 1

Table 1. Results of Cu2+ Detection in River Water Samples samples

concentrationa (M)

concentrationb (M)

relative error (%)

river water 1 (10.62 ± 0.09) × 10-6 (10.24 ± 0.12) × 10-6 river water 2 (7.64 ± 0.11) × 10-6 (7.39 ± 0.10) × 10-6 river water 3 (14.09 ± 0.11) × 10-6 (14.47 ± 0.09) × 10-6

520.2 disappeared, and the main peak at m/z ) 615.1 (calcd ) 614.2) corresponding to [1 + Cu2++ MeOH - H]+ was clearly observed, which indicated that 1 was not cleaved in the process of binding with Cu2+. Duan et al. have reported compound 2 as a chemosensor which shows a remarkable Hg2+-induced fluorescence enhancement with no interference from Cu2+. Compound 2 holds a rigid double bond of CdN, which inhibits the binding of Cu2+ with the oxygen atom of carbonyl. As was indicated by Duan et al., the carbonyl group was not concerned with the coordination of 2 with Hg2+.24f Different from 2, compounds 1 and 3 possess a flexible single bond of C-N, which increases the flexibility of the whole molecule and is favorable for the oxygen atom of carbonyl, nitrogen atom of amido, and nitrogen atom of 2-picolyl to coordinate with Cu2+ under a suitable geometry conformation and switch the spirocyclic form of 1 and 3 to the ring-opened form. The affinity of carbonyl and amido of 1 and 3 with Cu2+ was proved by Czarnik et al. To study the role of the groups of 1 and 3 for the binding of Cu2+, compound 4 was designed and synthesized using a phenyl group instead of the pyridyl group in compound 3. Experimental results show that compound 4 shows a slower and lower fluorescence sensitivity response toward Cu2+ than that of compounds 1 and 3 (a 19-fold fluorescence enhancement with the concentration of Cu2+ up to 10 equiv of 4), which indicated that the 2-picolyl group in compounds 1 and 3 indeed plays an important role in the course of binding with Cu2+. The proposed binding mechanism of 1 with Cu2+ was shown in Scheme 2. Similar tridentate ligand model including oxygen-coordination was also proposed by Lee et al. for their 2-picolyl functionalized coumarin-based fluorogenic probe for Cu2+.22 We found that for their probe the recognition unit is almost the same as ours. The binding model as well as the sensing mechanism were carefully studied by them with crystal structure, time-resolved laser spectroscopy, and quantum calculation. Detection of Cu2+ in Aqueous Samples and Living Cells. The practical applications of the designed chemosensor were first evaluated by detection of Cu2+ in river water samples (obtained from different locations of Xiangjiang River, Changsha, with a concentration of Cu2+ ranging from 5 to 15 µM, corresponding to a typical Cu2+ level for surface water in China), and the results were compared with those given by the atomic absorption spectrometry reference method. In order to reduce the pH influence in the detection, 4 mL of Tris-HCl buffered water/C2H5OH containing 1 was added to 1 mL of the water samples to keep the pH value at 7.1, and then its fluorescence intensity change was detected. For calculation of the Cu2+ concentration in river water samples, a calibration curve of 1 to Cu2+ was first prepared with the addition of different concentrations of Cu2+ (Figure S5 in the Supporting Informa-

-3.77 -3.27 2.70

a Detected by atomic absorption spectrometry. b Average of three detections found by the proposed method.

Figure 7. Fluorescence images of Cu2+ in HeLa cells with 1. (a) Bright-field transmission image of HeLa cells incubated with 1 (10 µM) for 30 min. (b) Fluorescence image of HeLa cells incubated with 1 (10 µM) for 30 min (λex ) 500 nm). (c) Fluorescence image of HeLa cells incubated with 1 (10 µM) for 30 min, washed three times, and then further incubated with 10 µM Cu2+ for 30 min (λex ) 500 nm).

tion). The analytical results are shown in Table 1. All the measurements were performed three times. The results obtained with the proposed chemosensor were in good agreement with that obtained by atomic absorption spectrometry with a relative error less than 4%, which confirmed that the proposed sensor is applicable for practical Cu2+ detection. These results demonstrated that our proposed fluorescent chemosensor could meet the sensitivity as well as selectivity requirements for environmental water samples monitoring applications. To further demonstrate the practical applicability of the probe in biological samples, fluorescence imaging experiments were carried out in living cells on a Nikon Eclipse TE-300 fluorescence microscope. HeLa cells were incubated with 1 (10 µM) for 0.5 h at 37 °C, then followed by the addition of Cu2+ (10 µM) and incubated for another 0.5 h. The cells were washed with PBS buffer solution, and their fluorescence images were recorded before and after addition of Cu2+ (figure 7). In the absence of Cu2+, free 1 showed no detectable fluorescence signal in living cells. After incubation with Cu2+, a bright fluorescence was observed in living cells. The results suggest that probe 1 can penetrate the cell membrane and can be applied for in vitro imaging of Cu2+ in living cells and potentially in vivo. CONCLUSIONS In summary, we have developed a new rhodamine spirolactambased fluorescence chemosensor 1. It exhibits a fluorescence response toward Cu2+ in aqueous solution with high sensitivity and selectivity. The fluorescence intensity was significantly increased about 80-fold with 10 equiv of Cu2+ added. Moreover, its fluorescence intensity was enhanced in a linear fashion with a Cu2+ concentration from 8.0 × 10-7 to 1.0 × 10-5 M, with a detection limit of 3.0 × 10-7 M, and thus, the chemosensor can be used for quantification of Cu2+ in aqueous samples. Most Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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importantly, both the color and fluorescence changes of the chemosensor are remarkably specific for Cu2+ in the presence of other heavy and transition metal ions (even in high concentration), which meet the selective requirements for biomedical and environmental monitoring application. The living cell imaging experiments further demonstrate its value in the practical applications in biological systems.

20775023), “973” National Key Basic Research Program of China (2007CB310500), Ministry of Education of China (NCET-07-0272), and Hunan Natural Science Foundation (06JJ4010, 07JJ3025).

ACKNOWLEDGMENT This work was supported.by the National Natural Science Foundation of China (Grant 20505008, 20605007, 20675028, and

Received for review May 22, 2009. Accepted July 17, 2009.

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC901127N