β-Cyclodextrin as the Vehicle for Forming Ratiometric Mercury Ion

Oct 19, 2010 - Camacho , C.; Matías , J. C.; Cao , R.; Matos , M.; Chico , B.; Hernandez , J.; Longo , M. A.; Sanroman , M. A.; Villalonga , R. Langm...
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β-Cyclodextrin as the Vehicle for Forming Ratiometric Mercury Ion Sensor Usable in Aqueous Media, Biological Fluids, and Live Cells Gang Fang, Meiyun Xu, Fang Zeng,* and Shuizhu Wu* College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, China Received August 24, 2010. Revised Manuscript Received October 3, 2010 The selective and sensitive detection methods for toxic transition-metal ions, which are rapid, facile, and applicable to the environmental and biological milieus, are of great importance. In this study, we designed a β-CD-based ratiometric sensor for detecting mercury ions in aqueous media, some biological fluids, and live cells. In this sensing platform, the thiocarbamido-containing probe dye was covalently linked onto the hydrophilic β-CD rim, which is conducive to complexing with metal ion, while the donor dye was anchored inside hydrophobic β-CD cavity via the adamantyl moiety, which is good for avoiding self-aggregation and enhancing the quantum yield of the donor dye. Upon associating with mercury ion, the probe dye undergoes ring-opening process and serves as the energy acceptor and constitutes the FRET system with the donor dye; by this way ratiometric detection of mercury ion in water can be realized with the detection limit of 10 nM. The cyclodextrin plays a crucial role for the sensing system; it not only accommodates both the donor dye and the probe dye which can form FRET system upon addition of Hg2þ but also makes the sensor water-soluble and cell membrane permeable. This nontoxic sensing platform can be used for mercury ion detection in aqueous medium, biological fluids, and live cells (L929 and Hela). We also found that, upon being taken up by L929 cells, the sensor exhibited no cytotoxicity, and the cell proliferation was not affected.

1. Introduction Mercury pollution is a global problem, and a major source of human exposure stems from contaminated natural waters. As estimated by US Environmental Protection Agency (EPA), the annual total global mercury emissions from all sources, both natural and human-generated, reaches nearly 7500 tons per year.1 Thus, obtaining new methods for detecting mercury and other toxic metal ions which are cost-effective, rapid, facile, and applicable to the environmental and biological milieus is an important goal.2-10 Nowadays, fluorescence techniques have become powerful tools for sensing and imaging trace amounts of metal ions because of its simplicity, sensitivity, and real-time monitoring with fast response time. Many fluorescent sensing systems for metal ions function by fluorescence enhancement or quenching.11,12 However, as the change in fluorescence intensity is the only detection signal, some factors such as instrumental efficiency, dye concentration, and environmental conditions will interfere with signal output.11,12 On the other hand, ratiometric *To whom correspondence should be addressed. Phone: (þ86)-2022236262. Fax: (þ86)-20-22236363. E-mail: [email protected] (S.W.); [email protected] (F.Z.). (1) US EPA, Regulatory Impact Analysis of the Clean Air Mercury Rule, EPA452/R-05-003, Research Triangle Park, NC, 2005. (2) Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Angew. Chem., Int. Ed. 2007, 46, 6824–6828. (3) Yoon, S.; Albers, A. E.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16030–16031. (4) Wu, J. S.; Hwang, I. C.; Kim, K. S.; Kim, J. S. Org. Lett. 2007, 9, 907–910. (5) Xu, M.; Wu, S.; Zeng, F.; Yu, C. Langmuir 2010, 26, 4529–4534. (6) Yu, C. J.; Tseng, W. L. Langmuir 2008, 24, 12717–12722. (7) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760–16761. (8) Ma, B.; Zeng, F.; Wu, S. Sens. Actuators, B 2010, 145, 451–456. (9) Yoon, S.; Miller, E. W.; He, Q.; Do, P. H.; Chang, C. J. Angew. Chem., Int. Ed. 2007, 46, 6658–6661. (10) Ma, B.; Wu, S.; Zeng, F.; Luo, Y.; Zhao, J.; Tong, Z. Nanotechnology 2010, 21, 195501. (11) Lee, S. H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10482–10489. (12) Chen, J.; Zeng, F.; Wu, S.; Su, J.; Zhao, J.; Tong, Z. Nanotechnology 2009, 20, 365502.

17764 DOI: 10.1021/la103368z

measurement uses the ratio of two fluorescent peaks instead of the absolute intensity of one peak, and the advantage of the ratiometric sensors is that factors such as excitation source fluctuations and sensor concentration will not affect the ratio between the two fluorescence intensities. Thus, the ratiometric fluorescent sensors can eliminate most ambiguities in the detection by self-calibration of two emission bands.13,14 In particular, the fluorescence resonance energy transfer (FRET)-based sensing technique, which does not directly produce redox-active ions that could lead to photodamage or other undesirable processes, has recently attracted considerable attention due to its simplicity, high sensitivity, and its ratiometric detection capability;14-16 moreover, the use of FRET can allow us to excite the detection system with the light of shorter wavelength compared to the acceptor emission wavelength, thus avoiding any influence of excitation backscattering effects on fluorescence detection.14,16 At present, some FRET-based sensors for metal ions have been developed, including the spriolactam rhodamine derivatives.7,17,18 As the chromophore probes, the spirolactam rhodamine fluorochrome has attracted considerable interest from chemists due to its excellent photophysical properties. The spirolactam rhodamine derivatives have been used to construct OFF-ON fluorescent chemosensors for metal ions due to its particular structural property. The rhodamine derivatives with spirolactam structure are nonfluorescent, whereas ring-opening of the spirolactam gives rise to a strong fluorescence emission.7,17,18 However, many of the current FRET-based sensors are created in the form of a (13) Kashiwada, A.; Nakamura, Y.; Matsuda, K. Sens. Actuators, B 2005, 108, 845–850. (14) Cejas, M. A.; Raymo, F. M. Langmuir 2005, 21, 5795–5802. (15) Merzlyakov, M.; Li, E.; Casas, R.; Hristova, K. Langmuir 2006, 22, 6986– 6992. (16) Cicchetti, G.; Biernacki, M.; Farquharson, J.; Allen, P. G. Biochemistry 2004, 43, 1939–1949. (17) Zhang, X.; Xiao, Y.; Qian, X. Angew. Chem., Int. Ed. 2008, 47, 8025–8029. (18) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008, 37, 1465–1472.

Published on Web 10/19/2010

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small molecular dyad or triad; they usually need synthetic efforts to covalently link the donor with acceptor through a spacer.19 In addition, for the dyad or triad systems, some exhibit low water solubility, making them only usable in organic solvent(s); for example, at present many of the spirolactam rhodamine derivatives for metal ion sensing can only be used in organic solvents such as methyl cyanide or ethanol/water mixed solvent.17,18 Thus, the quest to develop ratiometric sensors, which are usable in aqueous media and easy to fabricate, is of great importance. In recent years, the water-dispersible FRET-based systems have been constructed within colloidal nanoparticles, such as ligandcapped quantum dots,20-22 dye-doped silica,23-25 and polymer particles,26-34 and these particle-based FRET systems were found by some researchers and our group to exhibit high brightness and improved photostability.20-34 On the other hand, it is well-known that cyclodextrin (CD) has a unique configuration which makes its outer surface hydrophilic and inner surface (cavity) hydrophobic; thus, it can form supramolecular inclusion complexes with hydrophobic small molecular fluorophores that fit into its 5-8 A˚ cavity,35-41 and the fluorescence intensity, water solubility, biocompatibility, and photostability of the guest fluorophores could be enhanced.36-43 The (19) Guo, X.; Zhang, D.; Zhou, Y.; Zhu, D. J. Org. Chem. 2003, 68, 5681–5687. (20) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nature Mater. 2005, 4, 435–446. (21) Tomasulo, M.; Giordani, S.; Raymo, F. M. Adv. Funct. Mater. 2005, 15, 787–793. (22) Medintz, I. L.; Trammell, S. A.; Mattoussi, H.; Mauro, J. M. J. Am. Chem. Soc. 2004, 126, 30–31. (23) Brasola, E.; Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Chem. Commun. 2003, 3026–3027. (24) Corrie, S. R.; Lawrie, G. A.; Trau, M. Langmuir 2006, 22, 2731–2737. (25) Achatz, D. E.; Mezo, G.; Kele, P.; Wolfbeis, O. S. ChemBioChem 2009, 10, 2316–2320. (26) Meallet-Renault, R.; Herault, A.; Vachon, J. J.; Pansu, R. B.; AmigoniGerbier, S.; Larpent, C. Photochem. Photobiol. Sci. 2006, 5, 300–310. (27) Gouanve, F.; Schuster, T.; Allard, E.; Meallet-Renault, R.; Larpent, C. Adv. Funct. Mater. 2007, 17, 2746–2756. (28) Frigoli, M.; Ouadahi, K.; Larpent, C. Chem.;Eur. J. 2009, 15, 8319–8330. (29) Chen, J.; Zeng, F.; Wu, S.; Chen, Q.; Tong, Z. Chem.;Eur. J. 2008, 14, 4851–4860. (30) Chen, J.; Zeng, F.; Wu, S.; Su, J.; Tong, Z. Small 2009, 5, 970–978. (31) Chen, J.; Zeng, F.; Wu, S.; Zhao, J.; Chen, Q.; Tong, Z. Chem. Commun. 2008, 5580–5582. (32) Li, C. H.; Zhang, Y. X.; Hu, J. M.; Cheng, J. J.; Liu, S. Y. Angew. Chem., Int. Ed. 2010, 49, 5120–5124. (33) Hu, J. M.; Li, C. H.; Liu, S. Y. Langmuir 2010, 26, 724–729. (34) Wu, T.; Zhang, Y. F.; Wang, X. F.; Liu, S. Y. Chem. Mater. 2008, 20, 101– 109. (35) Wu, S.; Luo, Y.; Zeng, F.; Chen, J.; Tong, Z. Angew. Chem., Int. Ed. 2007, 46, 7015–7018. (36) Dondon, R.; Fery-Forgues, S. J. Phys. Chem. B 2001, 105, 10715–10722. (37) Jagt, R. B. C.; Gomez-Biagi, R. F.; Nitz, M. Angew. Chem., Int. Ed. 2009, 48, 1995–1997. (38) Deng, W.; Yamaguchi, H.; Takashima, Y.; Harada, A. Angew. Chem., Int. Ed. 2007, 46, 5144–5147. (39) Murphy, R. S.; Barros, T. C.; Mayer, B.; Marconi, G.; Bohne, C. Langmuir 2000, 16, 8780–8788. (40) Onclin, S.; Mulder, A.; Huskens, J.; Ravoo, B. J.; Reinhoudt, D. N. Langmuir 2004, 20, 5460–5466. (41) Tomatsu, I.; Hashidzume, A.; Harada, A. Angew. Chem., Int. Ed. 2006, 45, 4605–4608. (42) Miyauchi, M.; Harada, A. J. Am. Chem. Soc. 2004, 126, 11418–11419. (43) Harada, A.; Hashidzume, A.; Yamaguchi, H.; Takashima, Y. Chem. Rev. 2009, 109, 5974–6023. (44) Yana, D.; Takahashi, T.; Mihara, H.; Ueno, A. Macromol. Rapid Commun. 2004, 25, 577–581. (45) Freeman, R.; Finder, T.; Bahshi, L.; Willner, I. Nano Lett. 2009, 9, 2073– 2076. (46) Ogoshi, T.; Harada, A. Sensors 2008, 8, 4961–4982. (47) Kang, X.; Cheley, S.; Guan, X.; Bayley, H. J. Am. Chem. Soc. 2006, 128, 10684–10685. (48) Zhao, Y. L.; Hu, L.; Gruner, G.; Stoddart, J. F. J. Am. Chem. Soc. 2008, 130, 16996–7003. (49) Pagliari, S.; Corradini, R.; Galaverna, G.; Sforza, S.; Dossena, A.; Montalti, M.; Prodi, L.; Zaccheroni, N.; Marchelli, R. Chem.;Eur. J. 2004, 10, 2749–2758.

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Article

selective association of organic molecules to the hydrophobic cavity of β-cyclodextrin (β-CD) was used to develop different sensors by Harada and other researchers.44-53 For example, Ueno et al. prepared various chromophore-appended cyclodextrins, and through complexation with guest molecules the fluorescence intensity of the chromophore could be decreased or increased; thus, the “turn-off” or “turn-on” fluorescent chemical sensors for several guest molecules are obtained based on the competitive inclusion of different compounds with β-CD.44 Willner et al. reported quantum-dot-appended cyclodextrin and used it as the FRET sensor for organic compound such as 1-adamantanecarboxylic acid due to the strong binding between adamantyl group and β-CD and the corresponding displacement of the acceptor dye from cyclodextrin cavity.45 In these cyclodextrin-based sensors, they require that the analyte (e.g., adamantanecarboxylic acid) associates to β-CD cavity with a larger binding constant that allows the displacement of the probing dye. Liu’s group constructed the cyclodextrin-based sensors for metal ion sensing, the metal-ion-binding ligand was appended onto cyclodextrin rim, and the emission intensity of the fluorescent ligand was enhanced or quenched upon metal ion binding.50-53 Previously, we constructed a FRET-based cyclodextrin sensor system for sensing ferric ion.5 In that system the energy transfer donor dye was linked onto the rim of β-CD, and the probe dye was included into the cavity through adamantyl moiety, acting as the energy acceptor upon complexing with ferric ion. The advantage of this system is that a single cyclodextrin molecule can serve as a water-soluble vehicle for a FRET system, and there is no need to covalently link the donor and acceptor together. However, since the donor dye was linked onto the hydrophilic β-CD rim, the quantum yield of the donor dye could be affected by unfavorable interactions of the dye with the water. Moreover, the probe dye included into the hydrophobic β-CD cavity is not favorable for complexing with metal ion. In the present work, also by using β-CD as the vehicle, we constructed a FRET-based ratiometric sensing platform for Hg2þ. In this sensing platform, the probe dye was covalently linked onto the hydrophilic β-CD rim, which is conducive to complexing with metal ion; the donor dye was anchored inside hydrophobic β-CD cavity through the adamantyl moiety, which is good for avoiding self-aggregation and enhancing the quantum yield of the donor dye. The thiocarbamido-containing probe dye selectively associates with mercury ion, and upon associating with mercury ion, the probe dye undergoes ring-opening process. As a result, it acts as the energy acceptor and constitutes the FRET system with the donor dye; by this way ratiometric detection of mercury ion can be realized, as shown in Scheme 1. In this study, we also investigated the system’s sensing capability in some biological fluids and in live cells as well as the cytotoxicity of this β-CD-based system.

2. Experimental Section 2.1. Materials. Rhodamine B (RhB), 1,2-ethylenediamine, β-cyclodextrin (β-CD), chloride salts of metal ion (Mg2þ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ, Pb2þ), nitrate salt of metal ion (Agþ), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich Co. p-Toluenesulfonyl (50) Liu, Y.; Zhang, N.; Chen, Y.; Wang, L. Org. Lett. 2007, 9, 315–318. (51) Zhang, Y. M.; Chen, Y.; Li, Z. Q.; Li, N.; Liu, Y. Bioorg. Med. Chem. 2010, 18, 1415–1420. (52) Zhang, N.; Chen, Y.; Yu, M.; Liu, Y. Chem.;Asian J. 2009, 4, 1697–1702. (53) Liu, Y.; Yu, M.; Chen, Y.; Zhang, N. Bioorg. Med. Chem. 2009, 17, 3887– 3891.

DOI: 10.1021/la103368z

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Article Scheme 1. Formation of FRET-Based Ratiometric Sensing System for Hg2þ with β-CD as Vehicle

chloride (TSO-Cl) and triethylamine were obtained from Acros. 1-Aminoadamantane (AD-NH2), mercury(II) perchlorate hydrate, and carbon disulfide were obtained from Alfa. The culture medieum RPMI1640 was obtained from Invitrogen. Fetal bovine serum was supplied by Hangzhou Sijiqing Biological Engineering Materials Co. Ltd. N,N-Dimethylformamide (DMF) was dried by CaH2 and vacuum distilled. Triethylamine and ethyl acetate were dried by molecular sieve and vacuum distilled. Ethanol (EtOH) was distilled over magnesium, and tetrahydrofuran (THF) was distilled over sodium. Methanol, dichloroethane, and dichloromethane were analytically pure solvents and distilled before use.

2.2. Synthesis of the Probe-Containing β-Cyclodextrin: Thiocarbamido-SRhB-β-CD. First, the spirolactam rhodamine amine (SRhB) was synthesized as follows: under nitrogen, rhodamine B (10.46 mmol) was dissolved in anhydrous ethanol (180 mL), then ethylenediamine (0.209 mol) was added as soon as possible at 40 °C, and the temperature was raised to 85 °C slowly. After 24 h, the solvent was evaporated under reduced pressure, then CH2Cl2 (100 mL) and water (200 mL) were added, and the organic layer was separated, washed five times with water, and dried over anhydrous Mg2SO4. After filtration of sodium sulfate, the solvent was removed under reduced pressure to give orange powder and purified by silica gel column chromatography (CH2Cl2/EtOH/ Et3N, 5:1:0.1) to give the white product SRhB. Yield: 90%. Second, mono-6-deoxy-6-aminoethyl-β-CD (6-NH2-β-CD) was synthesized as follows: mono-6-O-(p-tolylsulfonyl)-β-cyclodextrin (6-TSO-β-CD) was prepared as previously reported.5 Under nitrogen, the 6-TSO-β-CD (0.776 mmol) was dissolved in 1,2-ethylenediamine (5 mL). The reaction mixture was heated to 75 °C and stirred for 8 h at this temperature. Afterward, the reaction mixture was cooled down to room temperature, then the solution was treated with acetone (50 mL), and a white crystalline precipitate was formed. The crude product was recrystallized from 1:1 water-ethanol mixture. Yield: 92%. Thiocarbamido-SRhB-β-CD was synthesized as follows: The reaction vessel was wrapped with aluminum foil to ensure the reaction took place in the dark. Under nitrogen, SRhB (0.62 mmol) was dissolved in 10 mL of anhydrous THF, the solution was cooled down to -15 °C, triethylamine (0.7 mL) was added, then CS2 (12.4 mmol) was added dropwise, and the mixture was stirred for 24 h. KOH solution was used to absorb any gas resulting from 17766 DOI: 10.1021/la103368z

Fang et al. the reaction. Afterward, TSO-Cl (1.3 mmol) was added; the reaction mixture was gradually heated to room temperature and kept at this temperature for 1 h. The reaction mixture was filtered, solvent was evaporated under vacuum, and the obtained powder was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 3:1) to give the light-yellow crystalline SRhBNCS. Then SRhB-NCS was dissolved in 10 mL of anhydrous DMF, 6-NH2-β-CD (0.359 mmol) was added, and the solution was kept at room temperature for 24 h. Afterward, acetone was added into the solution, and precipitate formed. Upon filtration, the precipitate was recrystallized from an acetone-water mixture. The white crystal product was obtained. Yield: 70.4%. Elem. Anal.: calcd for C75H110N6O36S: C, 52.87; H, 6.51; N, 4.93; found: C, 52.56; H, 6.56; N, 4.89. 1H NMR (300 MHz, DMSO-d6) δ: 7.81 (d, 1H), 7.49 (m, 2H), 7.02-6.99 (t, 1H), 6.35-6.27 (m, 6H), 5.86-5.62 (m, 14H), 4.89-4.73 (m, 6H), 4.43 (m, 6H), 4.2 (m, 1H), 3.63-3.13 (m, 21 H), 1.15-1.07 (t, 12H). ESI MS m/z [M þ H]þ 1703.8. 2.3. Synthesis of the Donor Dye (FITC-AD). Under nitrogen, FITC (0.534 mmol) was dissolved in 5 mL of anhydrous DMF. To this solution 200 μL of triethylamine was added, and then AD-NH2 (0.534 mmol) was added; the reaction mixture was stirred and kept at room temperature for 10 h. Then most of the solvent was removed under vacuum, the solution was extracted with dichloromethane/water, and the organic layer was separated. After the solvent was removed under vacuum, the solid was purified by silica gel column chromatography (petroleum ether/ ethyl acetate, 2:1) to give the light-orange powder. Yield: 75.9%. Elem. Anal.: calcd. for C31H30N2O5S: C, 68.61; H, 5.57; N, 5.16; found: C, 68.50; H, 5.60; N, 5.13. 1H NMR (300 MHz, DMSO-d6) δ: 8.30 (s, 1H), 7.74-7.68 (t, 2H), 7.15-7.12 (d, 1H), 6.87 (s, 2H), 6.68-6.54 (m, 6H), 3.62-3.58 (s, 1H), 2.25-1.54 (m, 15H, adamantyl). ESI MS m/z [M þ H]þ 542.2.

2.4. Preparation of the β-CD Inclusion Complex Aqueous Solution. Thiocarbamido-SRhB-β-CD (4  10-5 mol) in

40 mL of deionized water was stirred at 45 °C until the solution was clear. Then, the solution of the donor dye (FITC-AD) (4  10-5 mol) in 10 mL of THF (or ethanol) was added slowly. The solution was stirred for 5 days at room temperature. Then THF and water were evaporated under reduced pressure, and the solid inclusion complex was obtained. Afterward, the solid inclusion complex was dissolved in HEPES buffer solution to give the sensor stock solution (2  10-4 M). When preparing the solutions for spectral measurement, the sensor stock solution was first added into the flask, then metal ion dissolved in HEPES buffer solution was added, and the system was stirred for 15 min; finally, the fluorescence and absorption measurements were performed. As for the determination of mercury ions in tap water sample and the biological fluid samples, the solid supramolecular complex was dissolved into tap water containing HEPES buffer concentrate; afterward, mercury ion in HEPES buffer solution was added, the solutions were shaken for 15 min, and then fluorescence spectra were recorded. Two kinds of biological fluids were used to confirm the applicability of this sensor: fetal bovine serum and human urine. Human urine was from a healthy female. The biological fluid samples were prepared as follows: A certain amount of mercury ion was added into urine or fetal bovine serum under stirring, then the urine solution was 50-fold diluted by HEPES buffer solution (pH = 7.0), and the fetal bovine serum solution was 100-fold diluted. Afterward, the solid supramolecular complex was added, and the solutions were shaken for 15 min, and finally fluorescence spectra were recorded. 2.5. Cell Incubation and Imaging. Two cell lines, Hela (human cervical cancer cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS, Invitrogen). One day before imaging, cells were passed and plated on 30 mm glass culture dishes. Before the experiments, cells were washed with Langmuir 2010, 26(22), 17764–17771

Fang et al. RPMI1640, incubated in RPMI1640 medium containing 5 μM of the CD-based sensor (Thiocarbamido-SRhB-β-CD/FITC-AD) at 37 °C under 5% CO2 for 2 h, washed with PBS, and then treated with a drop of 0.4% trypan blue solution to quench the fluorescence of the probe adsorbed on the outer cell membrane such that the observed fluorescence emission comes from the intracellular probe. After that, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD (blue light excitation). Experiments to assess mercury uptake were performed as follows: the cells stained with the sensor were incubated in the HEPES balanced saline with 1 ppm of mercury(II) perchlorate hydrate for 15 min and washed with HEPES balanced saline for three times prior to cell imaging. 2.6. MTT Cell Viability Assays. The L929 cell line was incubated in RPMI-1640 supplemented with 10% FBS and then seeded in 96-well plates at the cell population of 5000 cells/well. After 48 h of incubation in 96-well plates at 37 °C, cells were washed with prewarmed PBS buffer and incubated with prewarmed fresh media for 30 min before addition of the CD-based sensor. Cells were incubated with the sensor (with the concentration of 5 μM) for 2, 24, and 48 h at 37 °C and then washed two times with PBS. Cell viability was assessed colorimetrically with the MTT reagent (ATCC) following the standard protocol provided by the manufacturer. The absorbance was read with a Thermo MK3 ELISA reader at 570 nm. The control experiments were performed with the same procedure except that no CD-based sensor was added into the culture medium. 2.7. Measurements. 1H NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer. UV-vis spectra were recorded on a Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. The mass spectrum was obtained through Bruker Esquire HCT Plus mass spectrometer. Fluorescence emission lifetime was measured on an Edinburgh FL920 fluorescence lifetime spectrometer. The 2D NMR ROESY spectrum was measured on a Bruker Avance 400 MHz NMR spectrometer.

3. Results and Discussion 3.1. Synthesis, Characterization, and Formation of Supramolecular Donor-Acceptor System. The probe-containing β-CD (thiocarbamido-SRhB-β-CD) was synthesized by the reaction of mono-6-deoxy-6-aminoethyl-β-CD and spirolactam rhodamine isothiocyanate. The donor dye (FITC-AD) was synthesized by the reaction of fluorescein isothiocyanate and 1-aminoadamantane. The molecular structures of the probecontaining β-cyclodextrin (thiocarbamido-SRhB-β-CD) and the donor dye (FITC-AD) were confirmed by NMR and MS spectra (Figures S1-S4). The formation of the supramolecular complex between thiocarbamido-SRhB-β-CD and FITC-AD was verified by the 2D ROESY NMR spectrum (Figure S5). The ROESY spectrum of the supramolecular complex consisting of thiocarbamidoSRhB-β-CD with FITC-AD displays the clear NOE correlations between the H-3 protons of thiocarbamido-SRhB-β-CD and the protons of adamantyl groups in FITC-AD. Since the H-3 protons are located inside the cyclodextrin cavity and near the secondary side (wide side) of cavity, these NOE correlations indicate that the adamantyl part of FITC-AD is accommodated inside the cavity of thiocarbamido-SRhB-β-CD. The donor group (FITC) was included into the cavity of β-CD via an adamantyl group (AD), which is a well-known guest that forms stable supramolecular inclusion complex with β-CD, and the association constant (binding constant) between adamantyl (54) Silva, O. F.; Fernandez, M. A.; Pennie, S. L.; Gil, R. R.; de Rossi, R. H. Langmuir 2008, 24, 3718–3726.

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Figure 1. Absorption spectra of the thiocarbamido-SRhB-β-CD/ FITC-AD aqueous solution (1.4  10-4 M) upon addition of mercury ion (pH = 7.0, HEPES buffer solution). Concentration of added mercury ion: 0, 1  10-8, 5  10-7, 5  10-6, 2  10-5, 3  10-5, 6  10-5, 8  10-5, 1  10-4, 1.2  10-4, and 1.4  10-4 M.

group and β-CD can reach as high as 105 M-1.41,54-56 In this study, the binding constant for FITC-AD and thiocarbamidoSRhB-β-CD has been determined to be 2.77  104 M-1 (Figure S6), indicating that FITC-AD bound strongly with thiocarbamido-SRhB-β-CD. Therefore, in this sensing platform, the acceptor (the probe dye) is located on the cyclodextrin rim, while the donor is located inside the cyclodextrin cavity through the adamantyl moiety; this can keep the donor and acceptor within the effective distance of the F€orster energy transfer. This donor-acceptor system (as shown in Scheme 1) can also avoid any undesirable interaction between the donor and acceptor, which might exist if they were in the unbound form, for example, the π-π interactions between donor and acceptor (both the donor and acceptor are aromatic), which could lead to dye aggregation through the stacking of aromatic units and accordingly changes in spectral property. 3.2. Detection of Mercury Ion by Using Supramolecular Complex-Based FRET System. To ensure high selectivity for metal ion sensing, the key is to tune the metal ion affinity of the probe dye. In this study, we introduced the thiocarbamido moiety into the probe dye spirolactam rhodamine, so as to design the probe dye that selectively associates with mercury ion. The underlying concept is to place a coordinating ligand (thiocarbamido group) near the spirolactam rhodamine that would be expected to alter the electron density and thus the polarizability and ion affinity of the probe dye. The thiocarbamido-containing β-CD and the donor dye (FITC-AD) formed supramolecular complex in water. Figure 1 shows the UV/vis spectra of the system in water in the absence and presence of Hg2þ. It can be seen that, in the absence of Hg2þ, the system showed only the absorption profile of the donor (FITC), which has a maximum around 490 nm. Addition of Hg2þ ions immediately induced an increase in the absorption intensity around 560 nm, which corresponds to the absorption of rhodamine, as shown in Figure 1. At the same time, a significant color change from green to pink could be observed easily by eye, as shown in Figure 2. This confirms that the addition of Hg2þ ions can lead to the ring-opening of the spirolactam rhodamine. Figure 3 shows the fluorescence spectra of the thiocarbamidoSRhB-β-CD/FITC-AD system upon addition of varied amount of mercury ions. Before addition of the mercury ion, excitation of (55) Camacho, C.; Matı´ as, J. C.; Cao, R.; Matos, M.; Chico, B.; Hernandez, J.; Longo, M. A.; Sanroman, M. A.; Villalonga, R. Langmuir 2008, 24, 7654–7657. (56) Blomberg, E.; Kumpulainen, A.; David, C.; Amiel, C. Langmuir 2004, 20, 10449–10454.

DOI: 10.1021/la103368z

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Figure 2. Photographs of the thiocarbamido-SRhB-β-CD/FITC-AD FRET system aqueous solution (1.4  10-4 M) in the presence of various metal ions (1  10-4 M) under ambient light (absorption change) and under UV light (fluorescence change).

Figure 3. Fluorescence spectra (λexc: 495 nm) of the thiocarbamido-

SRhB-β-CD/FITC-AD aqueous solution (1.4  10-4 M) upon addition of mercury ion (pH = 7.0, HEPES). Mercury ion: 0, 1  10-8, 5  10-8, 1  10-7, 5  10-7, 1 10-6, 3  10-6, 5  10-6, 7  10-6, 1  10-5, 2  10-5, 3  10-5, 4  10-5, 5  10-5, 6  10-5, 8  10-5, 1  10-4, 1.1  10-4, 1.2  10-4, 1.4  10-4, and 2.5  10-4 M.

the system at 495 nm resulted in the emission of FITC at 518 nm. Upon addition of Hg2þ ions, the donor emission at 518 nm decreased, and a new emission band with a maximum at 586 nm (rhodamine) appeared; as the amount of Hg2þ ion was increased, the donor emission decreased, while the acceptor emission increased gradually. Accordingly, the ratio of the emission intensities at 586 and 518 nm (I586/I518) increased steadily as the concentration of mercury ion was increased, affording the system a sensitive ratiometric fluorescent sensor for Hg2þ (Figure S7). The color of the fluorescence of the system changed from green to orange-red (Figure 2). It is possible that the FRET process was switched on by Hg2þ ions as excitation of the donor FITC at 495 nm resulted in the emission of rhodamine with a maximum of 586 nm. The response time of the sensor system is about 15 min (Figure S8), and the binding constant of mercury ion with the sensor is 5.3  107 M-1 (Figure S9). In addition, the emission lifetime of the thiocarbamido-SRhBβ-CD/FITC-AD FRET system aqueous solution was measured. In the absence of mercury ion, the emission lifetime of the donor in the supramolecular-complex aqueous solution was 3.58 ns. Upon addition of mercury ion into the supramolecular-complex aqueous solution, in the decay curve of the donor, fast decay components appear, indicating the quenching of FITC emission by the RhB molecules resulted from the ring-opening process induced by mercury ion, and the corresponding lifetime was 17768 DOI: 10.1021/la103368z

Figure 4. Fluorescence intensity ratio (I586/I518) of the aqueous solution of β-CD based sensor (1.4  10-4 M) upon addition of different metal ions (1.1  10-4 M) (λexc = 495 nm, pH = 7.0, HEPES).

0.38 ns (Figure S10). The emission lifetime of the donor decreased from 3.58 to 0.38 ns, thus providing additional evidence that in this system the FRET process was turned on by mercury ion, since FRET process usually leads to a fluorescence lifetime decrease of the donor directly induced by energy transfer from the donor to the acceptor.57,58 3.3. Selectivity of the Detection System. The thiocarbamidoSRhB-β-CD/FITC-AD system showed high selectivity toward Hg2þ ions. From Figure 4, it can be seen that, Hg2þ induced a prominent fluorescence increment, whereas other metal ions such as Kþ, Mg2þ, Ca2þ, Mn2þ, Co2þ, Ni2þ, Zn2þ, Cd2þ, Hg2þ, Cr3þ, Pb2þ, Fe3þ, and Agþ gave negligible response. In addition, further experiments for Hg2þ-selective sensing were performed with the supramolecular-complex aqueous solution in the presence of multifarious cations including Kþ, Mg2þ, Ca2þ, Cr3þ, Mn2þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ, Hg2þ, Pb2þ, Fe3þ, and Agþ (1 equiv), as shown in Figure 5. Most of the coexisting metal ions have a negligible interfering effect with Hg2þ sensing by the thiocarbamido-SRhB-β-CD/FITC-AD system, even when Mn2þ, Mg2þ, Pb2þ, Agþ, Co2þ, Ni2þ, Zn2þ, Cd2þ, and Ca2þ were (57) Domingo, B.; Sabariegos, R.; Picazo, F.; Llopis, J. Microsc. Res. Tech. 2007, 70, 1010–1021. (58) Biskup, C.; Zimmer, T.; Kelbauskas, L.; Hoffmann, B.; Klocker, N.; Becker, W.; Bergmann, A.; Benndorf, K. Microsc. Res. Tech. 2007, 70, 442–451.

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Article Table 1. Energy Transfer Efficiency, Quantum Yield, and F€ orster Critical Radius R0 for the Thiocarbamido-SRhB-β-CD/FITC-AD FRET System sample

λabs (nm)a

λem (nm)b

Φf

E (%)c

R0 (nm)

485 518 0.39 without Hg2þ (spirolactam state) 557 586 0.03 2þ 485 518 0.07 90.2 3.55 with Hg (open-ring state) 557 586 0.17 a In the absence of Hg2þ, the system showed no absorption at 557 nm, while in the presence of Hg2þ (1.4  10-4 M), it exhibited absorption at 557 nm. b λex = 495 nm. c Energy transfer E was calculated by E = 1 (IDA/ID), where IDA is the fluorescence intensity of the donor in the presence of acceptor and ID represents the fluorescence intensity of the donor in the absence of acceptor.

Figure 5. Fluorescence intensity ratio (I586/I518) of the thiocarbami

do-SRhB-β-CD/FITC-AD aqueous solution (1.4  10-4 M) upon addition of Hg2þ ion (1.1  10-4 M) only and the aqueous solution containing Hg2þ ion with the addition of Fe3þ, Pb2þ, Mg2þ, Ca2þ, Cr3þ, Mn2þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ, and Agþ (1.1  10-4 M). Mixed ions: 1.1  10-4 M of Hg2þ, Mn2þ, Mg2þ, Pb2þ, Agþ, Co2þ, Ni2þ, Zn2þ, Cd2þ, and Ca2þ ions are present simultaneously (λexc = 495 nm, pH = 7.0).

simultaneously present (Figure 5). The thiocarbamido-SRhB-βCD/FITC-AD system showed very good antidisturbance. 3.4. Energy Transfer from Donor Dye to ThiocarbamidoSRhB/Hg2þ within the Supramolecular Complex. The variation of fluorescence spectra for the β-CD-based supramolecular complex upon titration of Hg2þ suggests that the energy transfer may take place from FITC-AD included inside the cavity of β-CD to thiocarbamido-SRhB/Hg2þ linked on the rim of β-CD. The FRET process requires that the emission band of the donor and the absorption band of the acceptor should overlap significantly and the distance from the donor to the acceptor be within the effective energy transfer radius (generally, 1-10 nm).59,60 In the present case, the emission band (518 nm) of the donor (FITC) well overlaps with the absorption band (560 nm) of the acceptor (thiocarbamido-SRhB/Hg2þ associate) as shown in Figure S11; thus, the energy transfer from the excited state of FITC to the thiocarbamido-SRhB/Hg2þ associate is spectrally possible. On the other hand, in accordance with the F€orster nonradiative energy transfer theory,59,60 the energy transfer efficiency E depends on the F€orster critical radius R0 and on the distance (r) between the donor (FITC) and the acceptor (probe/mercury ion complex), as shown in the following equation: E ¼

R0 6 R0 6 þ r6

The energy transfer is effective over distances in the R0 ( 50% R0 range.59,60 The energy transfer efficiency E can also be measured and calculated by E = 1 - (IDA/ID), where IDA is the fluorescence intensity of the donor in the presence of acceptor and ID represents the fluorescence intensity of the donor in the absence of acceptor. For the thiocarbamido-SRhB-β-CD/FITC-AD system in the current experimental situation, we calculated R0 = 35.5 A˚, and the energy transfer will be effective for 17.75 A˚ e d e 53.25 A˚ (R0 ( 50% R0). The F€orster critical radius R0 and other parameters (59) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1999; pp 443-472. (60) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley-VCH: New York, 2002; pp 247-272.

Langmuir 2010, 26(22), 17764–17771

Figure 6. Effect of sensor concentration for ratiometric and nonratiometric sensor on the fluorescence intensity (or intensity ratio) in the presence of 1.5  10-4 M of mercury ion (λexc: 495 nm). (A) Fluorescence intensity at 586 nm for aqueous solution of nonratiometric thiocarbamido-SRhB-β-CD. (B) Fluorescence intensity ratio (I586/I518) for the aqueous solution of ratiometric thiocarbamido-SRhB-β-CD/FITC-AD supramolecular complex (pH = 7.0).

for the thiocarbamido-SRhB-β-CD/FITC-AD system were calculated and are listed in Table 1. In this system, the F€orster radius R0 is longer than the donor-acceptor separation (