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A schiff-base dual emission ratiometric fluorescent chemosensor for Hg2+ ions and its application in cellular imaging
Accepted Manuscript Title: A Schiff-base Dual Emission Ratiometric Fluorescent Chemosensor for Hg2+ Ions and Its Application in Cellular Imaging Authors: Yang Jiao, Xing Liu, Lu Zhou, Haiyang He, Peng Zhou, Chunying Duan PII: DOI: Reference:
S0925-4005(17)30131-4 http://dx.doi.org/doi:10.1016/j.snb.2017.01.124 SNB 21649
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-10-2016 12-1-2017 20-1-2017
Please cite this article as: Yang Jiao, Xing Liu, Lu Zhou, Haiyang He, Peng Zhou, Chunying Duan, A Schiff-base Dual Emission Ratiometric Fluorescent Chemosensor for Hg2+ Ions and Its Application in Cellular Imaging, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.124 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Schiff-base Dual Emission Ratiometric Fluorescent Chemosensor for Hg2+ Ions and Its Application in Cellular Imaging Yang Jiao1, Xing Liu1, Lu Zhou2 , Haiyang He2, Peng Zhou1, Chunying Duan2,* 1. College of Chemistry, Dalian University of Technology, Dalian 116024, China 2.State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China E-mail: [email protected]
Higlights ●The chemosensor CA high selectively detects Hg2+ ions in the presence of other biologically important and environmentally relevant metal ions at physiological pH. ●The ratiometric response of CA at 490 nm and 415 nm respectively were based on the C=N bond hydrolysis reaction mechanism along with the cancelation of a PET process. ●Chemosensor CA could be applied in cellular imaging at nano molarity by dual fluorescent emission.
Abstract A schiff-base dual emission ratiometric fluorescent chemosensor which includes coumarin aldehyde and 5-aminoisophthalic acid methyl ester has been designed and synthesized. The chemosensor CA high selectively detects Hg2+ ions in the presence of other biologically important and environmentally relevant metal ions at physiological pH. The double bond of schiff-base was broken accompanied by fluorescence enhancement when the chemosensor interacts with Hg2+ ions, and the coumarin aldehyde and 5-aminoisophthalic acid methyl ester shows strong ration fluorescent emission respectively. And the chemosensor could be applied in cellular imaging at nano molarity by dual fluorescent emission.
Keywords: Chemosensor; Hg2+ ions; Ratiometric Fluorescent
1.Introduction Fluorescent chemosensors are regarded as powerful tools in recent years due to their easy operational techniques, real-time response, high selectivity and high sensitivity. [1-6] Especially, the synthesis and development of fluorescent chemosensors targeting heavy and transition metal ions is very important because these metal ions play indispensable roles in biological,[7-9] environmental[10-12] and industrial processes. Hg2+ ions is considered to be one of the most extremely toxic metal ions and deadly to the environment and humans even at very low concentration, [13, 14] and it could easily release into the environment[15, 16] and pass through the skin, respiratory, and gastrointestinal tissues into the human body. [17, 18] Hg2+ ions has potential damage on the brain, lungs, kidneys, immune system, heart and central nervous system in humans and animals at all ages,[19] therefore, the design and synthesis of monitor and quantitative determine Hg2+ ions at low concentration has become a vital as well as essential need for our healthy society. [20] A number of satisfactory fluorescent chemosensors for Hg2+ ions have been reported.[21-27] Most of these fluorescent chemosensors only show fluorescence intensity changes, and few of them associate with spectral shifts in either absorption or emission spectra. This property could allow a kind of ratiometric analyses. However, ratiometric fluorescent sensors are more attractive because they could increase the selectivity and sensitivity of a measurement. Ratiometric fluorescent sensors could provide built-in correction by simultaneously measuring two different emission signals and eliminate most of the possible interference. [28-31] Ratiometric fluorescent sensors could be designed and synthesized to function following four mechanisms: intramolecular charge transfer (ICT),[32,33] chemistry reaction, [34,35] excimer[36] and fluorescence resonance energy transfer (FRET).[37] ICT sensors could adjust the spectral shifts of absorption or emission through binding of the target ions promotes or inhibits ICT interactions. [38] With a specific chemical reaction, conjugated structure of chemistry reaction sensors could be
changed, and the
emission spectra show ratio changes. [39] Because of theΠ-Π interaction of
excimer sensors, it has charge transfer and show ratio changes.[40] FRET sensors have ratio changes through the energy transfer of two fluorophores. [41] Ratiometric fluorescent sensors have been frequently reported. In addition, some of ratiometric fluorescent sensors work well under physiological conditions and suit for cellular imaging.[42,43] A quantitative determination of low concentration could be realized by ratiometric measurement that it could reduce extraneous influence factors including membrane permeability, temperature, and incubation time for biological systems.[44] However, to the best of our knowledge, currently cellular imaging of mercury ions is only at micro molarity,[45] and few of cellular imaging could be at the hundreds of nano molarity.[46] Hence, it is highly desirable to propose a ratiometric fluorescence sensor for Hg2+ ions which could detect Hg2+ ions at nano molarity in cellular imaging. In this work, we present a novel ratiometric fluorescent sensor CA on the basis of the C=N bond hydrolysis reaction mechanism. The ratiometric fluorescent sensor CA having both coumarin aldehyde and 5-aminoisophthalic acid methyl ester fluorophore was described to detect Hg2+ ions(Scheme 1). Coumarin dyes and 5-aminoisophthalic acid have been extensively used in fluorescent sensor for their excellent photochemical and photophysical properties.[47] The sensor was consisting of a schiff-base C=N site that could interact with Hg2+ ions, and the free sensor CA displayed weak fluorescence due to a photoinduced electron transfer (PET) process. However, dual enhanced emission of coumarin aldehyde and 5-aminoisophthalic acid methyl ester fluorophore exhibited based on the C=N bond hydrolysis reaction mechanism as Hg2+ ions existed, and the sensor showed ratiometric detection for Hg2+ ions that the fluorescence of CA before the hydrolysis of C=N bond was as an interior label. At the same time, the sensor CA could be applied in cellular imaging at nano molarity of Hg2+ ions.
2. Experiment 2.1. Materials and instruments All chemicals used were of reagent grade or obtained from commercial sources and used without further purification. The chemical structures of the sensor CA and all its precursors are confirmed by 1H NMR spectroscopy on a Varian INOVA 400M spectrometer and ESI mass spectrometry on a HPLC-Q-Tof MS spectrometer using acetonitrile as mobile phase. The fluorescent spectra were measured on EDINBURGHFS 920, both excitation and emission slit widths were 3nm. UV–vis spectra were measured on a TU1900 spectrometer at room temperature. Crystallographic data of the chemosensor CA was collected on a Bruker APEX CCD diffractometer with graphite-monochromated Mo–Kα (λ=0.71073 A˚). The structure was refined on by full-matrix F2 least squares methods with SHELXTL version 6.1. 2.2 Synthesis The new fluorescent chemosensor CA was synthesized by three steps. The synthesis route was displayed in Scheme 2 .
Synthesis of 7-diethylaminocoumarin(2): 4-Diethylaminosalicyl-aldehyde (1.93 g, 10 mmol), diethylmalonate (3.2 g, 20 mmol) and piperidine (1 mL) were combined in an absolute ethanol solution (30 mL) and stirred for 6 hours under a refluxing condition. After ethanol was removed under reduced pressure, glacial acetic acid (20 mL) and concentrated HCl (20 mL) were added to hydrolyze the reaction with stirring for 6 hours. The solution was cooled to room temperature and poured into 100 mL ice water. NaOH solution (40%) was added drop wise to modulate pH of the solution to ~5, and a pale precipitate formed immediately. After stirring for 30 minutes, the mixture
was filtered, washed with water, dried, then recrystallized with toluene giving compound 2 (1.74 g, 8.0 mmol) in a yield of 80.1%. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.55 (d, 1H, J = 9.5 Hz), 7.23 (d, 1H, J = 8.8 Hz), 6.59 (d, 1H, J = 8.8 Hz), 6.51 (s, 1H), 6.06 (d, 1H, J = 9.2 Hz), 3.42(q, 4H, J = 7.1 Hz), 1.21 (t, 6H, J = 7.1 Hz).
Synthesis of 7-diethylaminocoumarin-3-aldehyde(3): Under nitrogen, fresh distilled DMF (2 mL) was added drop wise to POCl3 (2 mL) at 20-50ºC and stirred for 30 minutes to yield a red solution. Then a portion of 2 (1.50 g, 6.91 mmol, dissolved in 10 mL DMF) was added drop wise to the above solution and yield a scarlet suspension. The mixture was stirred at 60ºC for 12 hours and then poured into 100 mL of ice water. NaOH solution (20%) was added to adjust the pH of the mixture to obtain a large amount of precipitate. The crude product was filtered, washed with water, dried and recrystallized with absolute ethanol giving compound 3 (1.20 g, 4.89 mmol) in a yield of 70.8%.1H NMR (400 MHz, CDCl3): δ (ppm) 10.13 (s, 1H), 8.26 (s, 1H), 7.43 (d, 1H, J = 9.0 Hz), 6.67 (d, 1H, J = 9.0Hz), 6.50 (s, 1H), 3.49 (q, 4H, J = 7.2 Hz), 1.25 (t, 6H, J = 7.2 Hz).
Synthesis of CA: (0.729g, 3 mmol) coumarin aldehyde was added to 20mL of absolute dried ethanol, over heated to dissolve, then add (0.627g, 3 mmol) dimethyl 5-aminoisophthalate, and five drops of glacial acetic acid was added dropwise , stirred at 90 ℃, reflux for 12 hours. Hot filtered and the precipitate was washed several times with ethanol, get orange product. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.77 (s, 1H), 8.54 (s, 2H), 8.09 (s, 2H), 7.43 (d, 1H, J = 8.0Hz), 6.64 (dd, 1H, J = 8.8, 2.0 Hz), 6.51 (d, 1H, J = 4 Hz ), 3.95 (s, 6H), 3.47 (q, 4H, J = 7.0 Hz), 1.25 (t, 6H, J = 7.0 Hz). 2.3. General Spectrophotometric experiments The fluorescence titration spectra as well as other fluorescent spectra were recorded by FS 920 luminescence spectrometer. The salts of metal ions used in stock solutions were KClO4,
Ca(ClO4)2·4H2O,NaClO4·H2O,Mg(ClO4)2·6H2O,AgClO4·6H2O, Cu(ClO4)2·6H2O, Fe(ClO4)2·2H2O, Zn(ClO4)2·6H2O,Mn(ClO4)2·6H2O,Cd(ClO4)2·6H2O,Co(ClO4)2·6H2O,Ni(ClO4)2·6H2O,Pb(ClO4)2·6 H2O, Hg(ClO4)2·3H2O. Inorganic salts were dissolved in distilled water to afford 1.0×10-2 M aqueous solution. High concentration of the stock solutions CA (1.0×10-3 M) were prepared in acetonitrile. Aliquots of stock solution of CA was diluted to 2 mL acetonitrile to make the final concentration of 2 μM. The fluorescence spectra of CA (2 μM) in the presence of various competitive species under the same conditions were recorded at 415 nm and 490 nm(Excitation was provided at 315 nm and 440 nm) in acetonitrile. 2.4. Crystallography In this study, crystallographic data of the chemsensor CA was collected on a Bruker APEX-II CCD diffractometer with graphite-monochromated Mo–Kα (λ=0.71073 A˚). The structure was refined on by full-matrix F2 least squares methods with SHELXTL version 6.1. All non–hydrogen atoms were treated anisotropically. Hydrogen atoms were fixed geometrically at calculated distances and allowed to ride on the parent non-hydrogen atoms with the isotropic displacement.
3. Results and discussion Compound CA can be obtained in a three-step procedure via condensation of coumarin dyes and 5-aminoisophthalic acid methyl ester by schiff-base reaction with ethanoic acid (Scheme 2). The overall yield is 25% for the three steps. Orange crystals which were suitable for X-ray diffraction study were obtained by diffusion of diethyl ether in dichloromethane solution of CA at room temperature. X-ray diffraction single crystal structure analysis indicated that CA crystallized in triclinic system, as shown in Fig1. The dihedral angle between 5-aminoisophthalic acid methyl ester and coumarin groups was 66.2 °,which indicated that the molecular planarity is not good and there is a certain degree of distortion. Bond lengths of C=N (1.278(3) Å) is longer than typical C=N double bond length. The molecular structure of CA and crystal data are listed in Figure S4 and
Table S1. These data indicate that there is a certain extent of conjugation between coumarin and 5-aminoisophthalic acid methyl ester. Bond angles of C(11)-N(1)-C(1) and N(1)-C(11)-C(12) are 118.3(2)° and 120.8(3)°, respectively, which revealed long-pairs of nitrogen caused the photoinduced electron transfer (PET) process and leaded to the fluorescence quenching of CA. < Fig 1 is here> Spectroscopic measurements of CA were investigated in CH3CN/H2O=8/2 medium at physiological pH(0.1 M KClO4 buffer, pH = 7.34) under ambient conditions. The metal-free dye CA(2×10-6 M) upon excitation at 440 nm gave only a weak fluorescence at 530 nm due to highly efficient photoinduced electron transfer (PET). However, addition of the perchlorate salt of Hg2+ elicited a significant enhancement of the fluorescence intensity with a maximum wavelength from 530nm to 490nm with the excitation at 440nm (Fig 2).The fluorescence emission of 490nm was supposed to be assigned to coumarin aldehy degenerating from the breaking of CA. Fluorescent spectra reached plateau with 20-fold Hg2+ added. The detection limit of the sensor for Hg2+ ions was proceeded to be determined. As shown in Fig 2 inset, upon titration with Hg2+ ions (1 nM), the fluorescent intensity at 490 nm increased by about ten percent. The plots of the I490nm/I530nm ratio against the concentrations of Hg2+ ranging from 0 to 12 nM displayed a good linear relationship. Hence, the linear curve of the I490nm/I530nm ratio allows for the convenient quantitative detection of Hg2+ ions over this concentration range. And at the same time, with the excitation at 315 nm, addition of the perchlorate salt of Hg2+ ions elicited a significant enhancement of the fluorescence intensity (60-fold) with a maximum wavelength from 530nm to 415 nm (Fig 3), the fluorescence emission of 415nm came from 5-aminoisophthalic acid methyl ester which was also ascribed to the bond-breaking reaction of CA. The detection limit of the sensor for Hg2+ ions was determined (Excitation at 315 nm). As shown in Fig 3 inset, upon titration with Hg2+ ions (1 nM), the fluorescent intensity at 415 nm increased by about ten percent. The plots of the I415nm/I530nm ratio against the concentrations of Hg2+ ions ranging from 0 to 12 nM also displayed a good linear
relationship. Such a unique emission indicated CA was a promising dual emission ratiometric probe for Hg2+ with a 20-fold fluorescence intensity (530 nm to 490 nm) and 60-fold ratio enhancement (530 nm to 415 nm). The fluorescence enhancement at 490 nm and at 415 nm is due to the hydrolysis reaction of C=N bond. < Fig 2 is here> < Fig 3 is here> Once CA was found to exhibit fluorescence enhancement in the presence of Hg2+ ions, its selectivity profile was checked through competition experiments. With excitation at 440 nm, the sensor CA (2 μM) was first mixed with 20 equiv of metal ions of interest that included alkali and alkaline-earth metal ions (Na+, K+, Mg2+, Ca2+, Cd2+), transition metal ions (Mn2+, Fe2+, Co2+, Ni2+,Cu2+, Zn2+), and heavy metal ions (Ag+, Pb2+), followed by addition of 20 equiv of Hg2+ ions. The various fluorescent response experiments revealed that the Hg2+ ions induced luminescence enhancement was unaffected in the presence of relevant alkali-, alkaline-earth metals, transition metal ions and heavy metal ions (Fig 4). First row transition metal ions did not interfere with the Hg2+ ions induced fluorescence enhancement (the concentration of Hg2+ ions is 40 μM), confirming the remarkable selectivity of the sensor CA (2 μM) for Hg2+ ions over other metal ions. When the excitation was changed to 315 nm, the emission profile did not change to any noticeable extent in terms of both intensity and peak positions in the presence of alkali, alkaline earth, transition, Ag+, or Pb2+ metal ions (Fig 5). The above results indicated the remarkably high selectivity of sensor CA toward Hg2+ ions over other analytes, which suggested that CA could be used for specific detection of Hg2+ ions. < Fig 4 is here> < Fig 5 is here> The ultraviolet-visible absorption spectra of CA were tested as well. The absorption spectrum of CA (2×10-5M) in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.34) exhibited absorbance at 465
nm (Fig 6). Addition of a perchlorate salt of a biologically relevant alkali, alkaline earth, transition, or toxic heavy metal ion such as Ag+ or Pb2+ did not give any discernible change in the absorption spectral profile of the sensor. This suggested there was no interaction between the fluorophore and the added metal ions. However, in the presence of Hg2+ ion, prominent changes in the UV−vis spectra were observed. The absorbance moved from 465nm to 445nm. As the same time, there was a isoabsorptive point at about 290 nm and 310 nm, and upon the addition of Hg2+, the absorption at 300nm disappeared which was the peak of C=N bond. It was further confirmed that the addition of mercury(Ⅱ) ions leaded to the hydrolysis reaction of C=N bond and thereby changed the absorbance spectra of CA. < Fig 6 is here> The binding stoichiometry was further studied to understand the fracture mechanism of CA by electronspray ionization (ESI)mass spectral studies in Figure 7. The ESI-MS showed an peak with m/z=437.16 in acetonitrile, which could be assigned to [CA+H]+ in referring to the exact comparison of the intense peak with the simulation on the basis of natural isotopic abundances. Upon the addition of Hg2+ ions, the ESI-MS of the CA-Hg solution showed an intense peak with m/z=210.07 and 246.11 in acetonitrile which could be assigned to 5-aminoisophthalic acid methyl ester [C10H11NO4+H]+ and coumarin aldehyde [C14H15O3N+H]+.At the same time the peak at m/z = 437.16 disappeared. This result was due to the addition of mercury(Ⅱ) ions which leaded to the hydrolysis reaction of C=N bond. Besides, crystals of CA with addition of Hg2+ were measured and the crystals of coumarin aldehyde was found. Both mass spectrometry and X-ray crystallography structural proved there is hydrolysis reaction of C=N bond after the addition of mercury(Ⅱ) ions. We believed that mercury(Ⅱ) ions have interaction with lone pair electrons of the N. It make the C=N carbon electropostitve and favors nucleophilic attack by H2O to get coumarin aldehyde and 5-aminoisophthalic acid methyl ester. < Fig 7 is here>
The effect of pH on the emission responses of the system was evaluated to tackle various pH values of samples. The emission intensity against the pH value in the absence and presence of Hg2+ ions at 490nm and 415nm were measured respectively. The metal-free sensor did not show any significant emission at 490nm over a wide pH range of 5.5 to 9.0 with excitation at 440nm (Fig 8). However, the emission intensity at 490nm of CA-Hg2+ increased dramatically and remained unchanged from pH 5.5 to 9.0.Similarly, with excitation at 315 nm, sensor CA itself is stable in the pH range of 5.5 to 9.0 (Fig 9).After addition of 20 eq of Hg2+ ions, the emission intensity at 415 nm enhanced and remained unchanged from pH 5 to 9.0, implying that CA could be used in a wide pH range including physiological conditions. The result showed the capacity of potentially detection Hg2+ ions in living systems. < Fig 8 is here> < Fig 9 is here> Encouraged by the above favorable properties of the sensor CA for monitor Hg2+ ions, including excellent sensitivity, high selectivity and stability in the physiological pH range, the ability of the biosensor in tracking Hg2+ ions in the living cells were performed to sensor. The ability of CA to track Hg2+ ions in living cells through confocal microscopy. MCF-7 cells was incubated with 1nM CA for up to 20 min at 37 °C and negligible intracellular fluorescence signals showed as we expected, while none fluorescence could be found from the green channel from the range of 410-470 nm and the red channel from the range of 490-590 nm (Fig10a, b and c). On the contrary, while cells stained by CA exposed to 2 nM of Hg2+ for 20 min at 37 °C, obvious intracellular fluorescence signals displayed as predicted (Fig10d, e and f). The sensor CA was proved to have remarkable member permeability and the results clearly demonstrated that the proposed sensor was low toxicity to cultured cells at the concentration of 1nM for 20 min under the experimental conditions. Hence, colocalization experiment clearly confirmed that the sensor CA could respond toward the changing in intracellular Hg2+ ions (n mol/L levels) in living cells.
< Fig 10 is here> 4. Conclusions To conclude, we have described here the synthesis, photophysical properties, and cellular applications of a ratiometric sensor where coumarin aldehyde and 5-aminoisophthalicacid methyl ester are covalently linked with an schiff-base reaction. The sensor features visible excitation and emission profiles and selective turn-on fluorescence response to Hg2+ ion in the presence of a large excess of biologically relevant metal ions at physiological pH. The ratiometric response of CA at 490 nm and 415 nm respectively were based on the C=N bond hydrolysis reaction mechanism along with the cancelation of a PET process. Because of its usability at physiological pH, the sensor CA was successfully applied to track Hg2+ ions at nano molarity levels in living cells. Acknowledgments This work was partly supported by the National Natural Science Foundation of China (21501020), Project Funded by China Postdoctoral Science Foundation (20105M571295) and the Fundamental Research Funds for the Central Universities (DUT16LK11). Supporting Information Available X-ray structural data of CA CIF format. This material is available free of charge via the Internet at XXXXXXXX. References [1] G. L. Wang, K. L. Liu, Y. M. Dong, Z. J. Li and C. Zhang, In situ formation of p–n junction: A novel principle for photoelectrochemical sensor and its application for mercury(II) ion detection, Anal. Chim. Acta 827 (2014) 34–39. [2] G. V. Guerreiro, A. J. Zaitouna and R. Y. Lai, Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry, Anal. Chim. Acta 810 (2014) 79–85.
[3] M. Vendrell, D. Zhai, J. C. Er, Y. T. Chang, Combinatorial strategies in fluorescent probe development, Chem. Rev. 112 (2012) 4391–4420. [4] M. Wang, J. Wen, Z. H. Qin, H. M. Wang, A new coumarin–rhodamine FRET system as an efficient ratiometric fluorescent probe for Hg2+ in aqueous solution and in living cells, Dyes Pigment. 120 (2015) 208–212. [5] P. Srivastava, S. S. Razi, R. Ali, Selective naked-eye detection of Hg2+ through an efficient turn-on photoinduced electron transfer fluorescent probe and its real applications, Anal. Chem. 86 (2014) 8693–8699. [6] W. Lin,X. Cao, Y. Ding, A highly selective and sensitive fluorescent probe for Hg2+ imaging in live cells based on a rhodamine–thioamide–alkyne scaffold, Chem. Commun. 46 (2010) 3529–3531. [7] S. Cao, Q. Y. Jin, L. Geng, L. Y. Mua, S. L. Dong, A ―turn-on‖ fluorescent probe for the detection of Cu2+ in living cells based on a signaling mechanism of N=N isomerization, New J. Chem. 40 (2016) 6264–6269. [8] X. Wang, J. Zhao, C. Guo, M. Pei, G. Zhang, Simple hydrazide-based fluorescent sensors for highly sensitive and selective optical signaling of Cu2+ and Hg2+ in aqueous solution, Sens. Actuators, B. 193 (2014) 157–165. [9] S. Lee, B. A. Rao, Y. A. Son, Colorimetric and ―turn-on‖ fluorescent determination of Hg2+ ions based on a rhodamine-pyridine derivative, Sens. Actuators, B. 196 (2014) 388–397. [10] A. A. A. Aziz, S. H. Sed, Detection of trace amounts of Hg2+ in different real samples based on immobilization of novel unsymmetrical tetradentate Schiff base within PVC membrane, Sens. Actuators, B. 197 (2014) 155–163. [11] W. J. Qu, G.Y. Gao, B. B. Shi, T.B. Wei, Y.M. Zhang, Q. Lin, H. Yao, A highly selective and sensitive fluorescent chemosensor for mercury ions based on the mechanism of supramolecular self-assembly, Sens. Actuators, B. 204 (2014) 368–374.
[12] Elizabeth M. Nolan, Stephen J. Lippard, Tools and tactics for the optical detection of mercuric ion, Chem. Rev. 108 (2008) 3443–3480. [13] P. Zhuang, M. McBride, H. Xia, N. Li, Z. Li, Health risk from heavy metals via consumption of food crops in the vicinity of dabaoshan mine, South China, Sci. Total Environ. 407 (2009) 1551−1561. [14] E. M. Nolan, S. Lippard, Tools and tactics for the optical detection of mercuric ion, ChemInform. 108 (2008) 3443−3480. [15] T. Agusa, T. Kunito, H. Iwata, I. Monirith, T. S. Tana, A. Subramanian, S. Tanabe, Mercury contamination in human hair and fish from cambodia: levels, specific accumulation and risk assessment, Environ. Pollut. 134 (2005) 79–86. [16] J. P. Bourdineaud, R. Rossignol, D. Brèthes, Zebrafish: A model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics, Int. J. Biochem. Cell Biol. 45 (2013) 16–22. [17] Y. Wei, B. Li, X. Wang and Y. Duan, A nano-graphite–DNA hybrid sensor for magnified fluorescent detection of mercury(II) ions in aqueous solution, Analyst 139 (2014) 1618–1621. [18] Q. Wang, D. Kim, D. D. Dionysiou, G. A. Sorial, Sources and remediation for mercury contamination in aquatic systems—a literature review, Environ. Pollut. 131 (2004) 323−336. [19] H. S. So, B. A. Rao, J. Hwang, K. Yesudas and Y. A. Son, Synthesis of novel squaraine–bis(rhodamine-6G): a fluorescent chemosensor for the selective detection of Hg2+, Sens. Actuators B 202 (2014) 779–787. [20] R. Eisler, Health Risks of Gold Miners: A synoptic review, Environ. Geochem. Health 25 (2003) 325−345. [21] S. B. Maity, S. Banerjee, K. Sunwoo, J. S. Kim, and P. K. Bharadwaj, A fluorescent chemosensor for Hg2+ and Cd2+ ions in aqueous medium under physiological pH and its applications in imaging living cells, Inorg. Chem. 54 (2015) 3929−3936.
[22] K. Bera, A. K. Das, M. Nag, S. Basak, Development of a rhodamine–rhodanine-based fluorescent mercury sensor and its use to monitor real-time uptake and distribution of inorganic mercury in live zebrafish larvae, Anal. Chem. 86 (2014) 2740–2746. [23] G. Chen, Z. Guo, G. Zeng and L. Tang, Fluorescent and colorimetric sensors for environmental mercury detection, Analyst 140 (2015) 5400–5443. [24] P. Mahato, S, Saha, P, Das, H, Agarwalla and A, Das, An overview of the recent developments on Hg2+ recognition, RSC Adv. 4 (2014) 36140–36174. [25] Y. Xu, Z. Jiang, Y. Xiao, T. T. Zhang, J. Y. Miao, B. X. Zhao, A new fluorescent turn-on chemodosimeter for mercury ions in solution and its application in cells and organisms, Anal. Chim. Acta 807 (2014) 126–134. [26] W. Y. Liu, S. L. Shen, H. Y. Li, J. Y. Miao, B. X. Zhao, Fluorescence turn-on chemodosimeter for rapid detection of mercury (II) ions in aqueous solution and blood from mice with toxicosis, Anal. Chim. Acta 791 (2013) 65–71. [27] H. Ye, F. Ge, X. C. Chen, Y. Li, H. Zhang, B. X. Zhao, J. Y. Miao, A new probe for fluorescent recognition of Hg2+ in living cells and colorimetric detection of Cu2+ in aqueous solution, Sensors and Actuators B 182 (2013)273–279. [28] P. Ashokkumar, V. T. Ramakrishnan, and P. Ramamurthy, Photoinduced rlectron transfer (PET) Based Zn2+ fluorescent probe: transformation of turn-on sensors into ratiometric ones with dual emission in acetonitrile, J. Phys. Chem. A 115 (2011) 14292–14299. [29] Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen, Z. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angew. Chem. Int. Ed. 52 (2013) 1688−1691. [30] H. T. Zhang, R. C. Liu, Y. Tan, H. W. Xie, H. P. Lei, H. Y. Cheung, H. Y. Sun, A FRET-based ratiometric fluorescent probe for nitroxyl detection in living cells, ACS Appl. Mater. Interfaces 7 (2015) 5438−5443.
[31] Z. Guo, G. Kim, J. Yoon, Synthesis of a highly Zn2+-selective cyanine-based probe and its use for tracing endogenous zinc ions in cells and organisms, Nat. Protoc. 9 (2014) 1245−1254. [32] L. Yuan, W. Y. Lin, Y. T. Yang, J. Z. Song, J. L. Wang, Rational design of a highly reactive ratiometric fluorescent probe for cyanide, Org. Lett. 13 (2011) 3730–3733. [33] X. S. Zhou, X. Lv, J. S. Hao, D. S. Liu, W. Guo, Coumarin-indanedione conjugate as a doubly activated Michael addition type probe for the colorimetric and ratiometric fluorescent detection of cyanide, Dyes Pigment. 95 (2012) 168–173. [34] X . Lv, J. Liu, Y. L. Liu, Y. Zhao, Y. Q. Sun, P. Wang, W. Guo, Ratiometric fluorescence detection of cyanide based on a hybrid coumarin-hemicyanine dye: the large emission shift and the high selectivity, Chem. Commun. 47 (2011) 12843–12845. [35] Z. P. Liu, X. Q. Wang, Z. H. Yang, W. J. He, Rational design of a dual chemosensor for cyanide Anion Sensing Based on dicyanovinyl-substituted benzofurazan, J. Org. Chem. 76 (2011) 10286–10290. [36] J. N. Weng, Q. B. Mei, Q. D. Ling, Q. L. Fan, W. Huang, A new colorimetric and fluorescent ratiometric sensor for Hg2+ based on 4-pyren-l-yl-pyrimidine, Tetrahedron 68 (2012) 3129–3134. [37] A. Coskun, U. Akkaya, Signal ratio amplification via modulation of resonance energy transfer; Proof of principle in an emission ratiometric Hg (II) sensor, J. Am. Chem. Soc. 128 (2006) 14474–14475. [38] J. Jiang , W. Liu , J. Cheng, L. Z. Yang, H. Jiang, D. C. Bai, W. S. Liu, A sensitive colorimetric and ratiometric fluorescent probe for mercury species in aqueous solution and living cells, Chem. Commun. 48 (2012) 8371–8373. [39] N. DiCesare, J. R. Lakowicz, Fluorescent probe for monosaccharides based on a functionalized boron-dipyrromethene with a boronic acid group, Tetrahedron Lett. (2001) 9105–9108.
[40] Z. C. Xu, J. Yoon, D. R. Spring, A selective and ratiometric Cu2+ fluorescent probe based on naphthalimide excimer-monomer switching, Chem. Commun. 46 (2010) 2563–2565. [41] A. E. Albers, V. S. Okreglak, C. J. Chang, A FRET-Based approach to ratiometric fluorescence detection of hydrogen peroxide, J. Am. Chem. Soc. 128 (2006) 9640–9641. [42] K. Ghosh, T. Sarkara and A. Samadderb, A rhodamine appended tripodal receptor as a ratiometric probe for Hg2+ ions, Org. Biomol. Chem. 10 (2012) 3236–3243. [43] Y. R. Zhang, N. Meng, J. Y. Miao, B. X. Zhao. A ratiometric fluorescent probe based on a Through-Bond Energy Transfer (TBET) system for imaging HOCl in living cells, Chem. Eur. J. 21 (2015) 19058–19063. [44] Y. J. Gong, X. B. Zhang, C. C. Zhang, A. L. Luo, T. Fu, W. H. Tan, G. L. Shen, R. Q. Yu, Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications, Anal. Chem.84 (2012) 10777–10784. [45] H. B. Yu, Y. Xiao, H. Y. Guo, X. H. Qian, Convenient and efficient FRET platform featuring a rigid biphenyl spacer between rhodamine and BODIPY: transformation of 'turn-on' sensors into ratiometric ones with dual emission, Chem. Eur. J. 17 (2011) 3179–3191. [46] H. H. Wang, L. Xue, C. L. Yu, Y. Y. Qian, H. Jiang, Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells, Dyes Pigment. 91 (2011) 350–355. [47] Y. Gong, X. Zhang, C. Zhang, A. L. Luo, T. Fu, W. H. Tan, G. L. Shen, R. Q. Yu, Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications, Anal. Chem. 84 (2012) 10777–10784. [48] Crystal structure analysis: The data was collected at temperature of 296±2K on a Bruker Smart APEX Ⅱ X–diffractometer equipped with graphite monochromated Mo–Kα radiation (λ = 0.71073 Å) using the SMART and SAINT programs. Indexing and unit cell refinement were based on all observed reflections from those 72 frames. The structure was solved in the P1 space group by direct method and refined by the full–matrix least–squares fitting on F2 using
SHELXTL 6.1. All non–hydrogen atoms were treated anisotropically. Hydrogen atoms of organic ligands were generated geometrically, fixed isotropic thermal parameters, and included in the structure factor calculations. Crystal data for CA: C24H24N2O6, M = 436.45, Triclinic, P1, a = 7.0594(3) Å, b = 9.9418(4) Å, c = 16.8535(7) Å, α= 95.809(3) °, β = 99.209(3)°, γ= 109.521(3)°, V = 1085.15(8) Å3, Z = 2, Dc = 1.336 g·cm–3, μ = 0.097 mm–1.8827 reflections were collected (Rint = 0.0867). The final refinement gave R1 = 0.0486 and wR2 = 0.1206 with I 2(I). The structures were solved by direct methods and refined on F2by full-matrix least-squares methods with SHELXTL version 6.1.Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge, CB21EZ, UK (fax: +44–1223–336–033; e-mail: [email protected])
Biography
Dr. Yang Jiao is a Doctor at Dalian University of Technology, China. She research interests are focused on fluorescent sensors and their applications in biological, medical diagnostics, environmental monitoring; dye molecular structure and function regulation. She published articles in the journal Chem. Commun., Chem. Eur. J. and Talanta, etc. [email protected] Prof Chunying Duan is a Professor at Dalian University of Technology, China. His research interests are in the fields of biological molecular probes and fluorescence imaging; photoelectric functional inorganic material and molecular devices; the chiral porous skeleton structure and asymmetric catalysis. He has published over 100 academic articles. [email protected]
Scheme1 Mechanism of Sensor Interaction with Hg2+ Ions
Scheme 2 Synthetic Scheme for the Sensor CA
Fig 1 Molecular structure of CA with the atomic-numbering scheme showing the schiff-base structural fashion (Hydrogen atoms were omitted for clarity, the C, O and N atoms were drawn in gray, red and blue.)
1.0
2+
[Hg ]
I490/I530
20
F/F0
15
0.9 0.8 0.7 0.6 2
10
4
6
8
10
12
2+
[Hg ](ppb)
5
0 450
500
550
600
650
700
Wavelength(nm)
Fig 2 Fluorescence spectra of CA (2×10-6 M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH=7.34;The fluorescence intensities were measured at 490 nm. Excitation was at 440 nm. Both emission and excitation slit widths were 3 nm)Inset: Fluorescence intensities at 490 nm of complex CA as a function of the mercury concentration (2–10 ppb).
60
2.2
55 45
2.1
[Hg2+]
F/F0
40
I415/I530
50
2.0 1.9 1.8 1.7 1.6
35
1.5
30
1.4 2
25
4
6 8 2+ [Hg ](ppb)
10
12
20 15 10 5 0 350
400
450
500
550
600
Wavelength(nm)
Fig 3 Fluorescence spectra of CA (2×10-6 M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.34; The fluorescence intensities were measured at 415 nm. Excitation was at 315nm. Emission/excitation = 3 nm, 3 nm) Inset: Fluorescence intensities at 415 nm of complex CA as a function of the mercury concentration (2–10 ppb).
2.5
I490/I530
2.0 1.5 1.0 0.5 0.0
+
2+
2+
2+
2+
+ + K Na Ag Ca2+Co2+ Zn2+ Ni2+Fe2+Cu2+Cd Mg Mn Pb2+Hg
Fig 4 Fluorescence responses of CA to various cations in an optimized CH3CN/H2O=8/2 solution (0.1 M KClO4 buffer, pH = 7.34; [CA] = 2μM). The left bars in each group represent the emission intensities of CA in the presence of 40μM of Na+, K+, Ca2+ and Mg2+ and 40μM of the other cations of interest, respectively. The right bars in each group represent the change in emission that occurs upon subsequent addition of 40μM of Hg2+ ions to the above solution. Excitation was provided at 440 nm, and the emission was recorded at 490 nm.
20
I415/I530
15
10
5
0 2+
2+
+ 2+ + 2+ + 2+ 2+ 2+ 2+ 2+ K Na Ag Ca2+Co Zn Ni2+Fe Cu Cd Mg Mn Pb Hg
Fig 5 Fluorescence responses of CA to various cations in an optimized CH3CN/H2O=8/2 solution (0.1 M KClO4 buffer, pH = 7.34; [CA] = 2μM). The left bars in each group represent the emission intensities of CA in the presence of 40μM of Na+, K+, Ca2+ and Mg2+ and 40μM of the other cations of interest, respectively. The right bars in each group represent the change in emission that occurs upon subsequent addition of 40μM of Hg2+ ions to the above solution. Excitation was provided at 315 nm, and the emission was recorded at 415 nm.
1.2
Absorbance
1.0
Hg
2+
Other metal ions
0.8 0.6 0.4 0.2 0.0 200
300
400
500
600
700
Wavelength(nm)
Fig 6 UV-vis spectra of CA (2×10-5M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 MKClO4 buffer, pH = 7.34)
Fig 7 ESI-MS of the CA in acetonitrile (top) and CA in the presence of 1 equivalents molar ration of Hg2+ ions in acetonitrile (bottom).
20
F/F0
15
10
5
0 6
7
8
9
pH
Fig 8. Emission intensities of CA and CA-Hg2+ at various pH values in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.4). Excitation wavelength= 440 nm.[CA] = 2μM, [Hg2+] = 40μM.
60 50
F/F0
40 30 20 10 0 6
7
8
9
pH
Fig 9. Emission intensities of CA and CA-Hg2+ at various pH values in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.4). Excitation wavelength= 315nm.[CA] = 2μM, [Hg2+] = 40μM.
Figure 10. Confocal microscopy images of MCF-7 cells. MCF-7 cells were incubated with 1nM CA and observed under bright field (a),green channel (b) and red channel (c), then further incubation with Hg2+ ions (2nM) for 20 min at 37 ℃ and observed under bright field (d), green channel (e), red channel (f).Cells were incubated at 37 °C under humidified atmosphere 5% CO2. The excitation wavelength was 405 nm(green channel) and 458nm(red channel), and images were collected at 410-470 nm(green channel) and 490-590nm(red channel).
S0925-4005(17)30131-4 http://dx.doi.org/doi:10.1016/j.snb.2017.01.124 SNB 21649
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-10-2016 12-1-2017 20-1-2017
Please cite this article as: Yang Jiao, Xing Liu, Lu Zhou, Haiyang He, Peng Zhou, Chunying Duan, A Schiff-base Dual Emission Ratiometric Fluorescent Chemosensor for Hg2+ Ions and Its Application in Cellular Imaging, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.124 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Schiff-base Dual Emission Ratiometric Fluorescent Chemosensor for Hg2+ Ions and Its Application in Cellular Imaging Yang Jiao1, Xing Liu1, Lu Zhou2 , Haiyang He2, Peng Zhou1, Chunying Duan2,* 1. College of Chemistry, Dalian University of Technology, Dalian 116024, China 2.State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China E-mail: [email protected]
Higlights ●The chemosensor CA high selectively detects Hg2+ ions in the presence of other biologically important and environmentally relevant metal ions at physiological pH. ●The ratiometric response of CA at 490 nm and 415 nm respectively were based on the C=N bond hydrolysis reaction mechanism along with the cancelation of a PET process. ●Chemosensor CA could be applied in cellular imaging at nano molarity by dual fluorescent emission.
Abstract A schiff-base dual emission ratiometric fluorescent chemosensor which includes coumarin aldehyde and 5-aminoisophthalic acid methyl ester has been designed and synthesized. The chemosensor CA high selectively detects Hg2+ ions in the presence of other biologically important and environmentally relevant metal ions at physiological pH. The double bond of schiff-base was broken accompanied by fluorescence enhancement when the chemosensor interacts with Hg2+ ions, and the coumarin aldehyde and 5-aminoisophthalic acid methyl ester shows strong ration fluorescent emission respectively. And the chemosensor could be applied in cellular imaging at nano molarity by dual fluorescent emission.
Keywords: Chemosensor; Hg2+ ions; Ratiometric Fluorescent
1.Introduction Fluorescent chemosensors are regarded as powerful tools in recent years due to their easy operational techniques, real-time response, high selectivity and high sensitivity. [1-6] Especially, the synthesis and development of fluorescent chemosensors targeting heavy and transition metal ions is very important because these metal ions play indispensable roles in biological,[7-9] environmental[10-12] and industrial processes. Hg2+ ions is considered to be one of the most extremely toxic metal ions and deadly to the environment and humans even at very low concentration, [13, 14] and it could easily release into the environment[15, 16] and pass through the skin, respiratory, and gastrointestinal tissues into the human body. [17, 18] Hg2+ ions has potential damage on the brain, lungs, kidneys, immune system, heart and central nervous system in humans and animals at all ages,[19] therefore, the design and synthesis of monitor and quantitative determine Hg2+ ions at low concentration has become a vital as well as essential need for our healthy society. [20] A number of satisfactory fluorescent chemosensors for Hg2+ ions have been reported.[21-27] Most of these fluorescent chemosensors only show fluorescence intensity changes, and few of them associate with spectral shifts in either absorption or emission spectra. This property could allow a kind of ratiometric analyses. However, ratiometric fluorescent sensors are more attractive because they could increase the selectivity and sensitivity of a measurement. Ratiometric fluorescent sensors could provide built-in correction by simultaneously measuring two different emission signals and eliminate most of the possible interference. [28-31] Ratiometric fluorescent sensors could be designed and synthesized to function following four mechanisms: intramolecular charge transfer (ICT),[32,33] chemistry reaction, [34,35] excimer[36] and fluorescence resonance energy transfer (FRET).[37] ICT sensors could adjust the spectral shifts of absorption or emission through binding of the target ions promotes or inhibits ICT interactions. [38] With a specific chemical reaction, conjugated structure of chemistry reaction sensors could be
changed, and the
emission spectra show ratio changes. [39] Because of theΠ-Π interaction of
excimer sensors, it has charge transfer and show ratio changes.[40] FRET sensors have ratio changes through the energy transfer of two fluorophores. [41] Ratiometric fluorescent sensors have been frequently reported. In addition, some of ratiometric fluorescent sensors work well under physiological conditions and suit for cellular imaging.[42,43] A quantitative determination of low concentration could be realized by ratiometric measurement that it could reduce extraneous influence factors including membrane permeability, temperature, and incubation time for biological systems.[44] However, to the best of our knowledge, currently cellular imaging of mercury ions is only at micro molarity,[45] and few of cellular imaging could be at the hundreds of nano molarity.[46] Hence, it is highly desirable to propose a ratiometric fluorescence sensor for Hg2+ ions which could detect Hg2+ ions at nano molarity in cellular imaging. In this work, we present a novel ratiometric fluorescent sensor CA on the basis of the C=N bond hydrolysis reaction mechanism. The ratiometric fluorescent sensor CA having both coumarin aldehyde and 5-aminoisophthalic acid methyl ester fluorophore was described to detect Hg2+ ions(Scheme 1). Coumarin dyes and 5-aminoisophthalic acid have been extensively used in fluorescent sensor for their excellent photochemical and photophysical properties.[47] The sensor was consisting of a schiff-base C=N site that could interact with Hg2+ ions, and the free sensor CA displayed weak fluorescence due to a photoinduced electron transfer (PET) process. However, dual enhanced emission of coumarin aldehyde and 5-aminoisophthalic acid methyl ester fluorophore exhibited based on the C=N bond hydrolysis reaction mechanism as Hg2+ ions existed, and the sensor showed ratiometric detection for Hg2+ ions that the fluorescence of CA before the hydrolysis of C=N bond was as an interior label. At the same time, the sensor CA could be applied in cellular imaging at nano molarity of Hg2+ ions.
2. Experiment 2.1. Materials and instruments All chemicals used were of reagent grade or obtained from commercial sources and used without further purification. The chemical structures of the sensor CA and all its precursors are confirmed by 1H NMR spectroscopy on a Varian INOVA 400M spectrometer and ESI mass spectrometry on a HPLC-Q-Tof MS spectrometer using acetonitrile as mobile phase. The fluorescent spectra were measured on EDINBURGHFS 920, both excitation and emission slit widths were 3nm. UV–vis spectra were measured on a TU1900 spectrometer at room temperature. Crystallographic data of the chemosensor CA was collected on a Bruker APEX CCD diffractometer with graphite-monochromated Mo–Kα (λ=0.71073 A˚). The structure was refined on by full-matrix F2 least squares methods with SHELXTL version 6.1. 2.2 Synthesis The new fluorescent chemosensor CA was synthesized by three steps. The synthesis route was displayed in Scheme 2 .
Synthesis of 7-diethylaminocoumarin(2): 4-Diethylaminosalicyl-aldehyde (1.93 g, 10 mmol), diethylmalonate (3.2 g, 20 mmol) and piperidine (1 mL) were combined in an absolute ethanol solution (30 mL) and stirred for 6 hours under a refluxing condition. After ethanol was removed under reduced pressure, glacial acetic acid (20 mL) and concentrated HCl (20 mL) were added to hydrolyze the reaction with stirring for 6 hours. The solution was cooled to room temperature and poured into 100 mL ice water. NaOH solution (40%) was added drop wise to modulate pH of the solution to ~5, and a pale precipitate formed immediately. After stirring for 30 minutes, the mixture
was filtered, washed with water, dried, then recrystallized with toluene giving compound 2 (1.74 g, 8.0 mmol) in a yield of 80.1%. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.55 (d, 1H, J = 9.5 Hz), 7.23 (d, 1H, J = 8.8 Hz), 6.59 (d, 1H, J = 8.8 Hz), 6.51 (s, 1H), 6.06 (d, 1H, J = 9.2 Hz), 3.42(q, 4H, J = 7.1 Hz), 1.21 (t, 6H, J = 7.1 Hz).
Synthesis of 7-diethylaminocoumarin-3-aldehyde(3): Under nitrogen, fresh distilled DMF (2 mL) was added drop wise to POCl3 (2 mL) at 20-50ºC and stirred for 30 minutes to yield a red solution. Then a portion of 2 (1.50 g, 6.91 mmol, dissolved in 10 mL DMF) was added drop wise to the above solution and yield a scarlet suspension. The mixture was stirred at 60ºC for 12 hours and then poured into 100 mL of ice water. NaOH solution (20%) was added to adjust the pH of the mixture to obtain a large amount of precipitate. The crude product was filtered, washed with water, dried and recrystallized with absolute ethanol giving compound 3 (1.20 g, 4.89 mmol) in a yield of 70.8%.1H NMR (400 MHz, CDCl3): δ (ppm) 10.13 (s, 1H), 8.26 (s, 1H), 7.43 (d, 1H, J = 9.0 Hz), 6.67 (d, 1H, J = 9.0Hz), 6.50 (s, 1H), 3.49 (q, 4H, J = 7.2 Hz), 1.25 (t, 6H, J = 7.2 Hz).
Synthesis of CA: (0.729g, 3 mmol) coumarin aldehyde was added to 20mL of absolute dried ethanol, over heated to dissolve, then add (0.627g, 3 mmol) dimethyl 5-aminoisophthalate, and five drops of glacial acetic acid was added dropwise , stirred at 90 ℃, reflux for 12 hours. Hot filtered and the precipitate was washed several times with ethanol, get orange product. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.77 (s, 1H), 8.54 (s, 2H), 8.09 (s, 2H), 7.43 (d, 1H, J = 8.0Hz), 6.64 (dd, 1H, J = 8.8, 2.0 Hz), 6.51 (d, 1H, J = 4 Hz ), 3.95 (s, 6H), 3.47 (q, 4H, J = 7.0 Hz), 1.25 (t, 6H, J = 7.0 Hz). 2.3. General Spectrophotometric experiments The fluorescence titration spectra as well as other fluorescent spectra were recorded by FS 920 luminescence spectrometer. The salts of metal ions used in stock solutions were KClO4,
Ca(ClO4)2·4H2O,NaClO4·H2O,Mg(ClO4)2·6H2O,AgClO4·6H2O, Cu(ClO4)2·6H2O, Fe(ClO4)2·2H2O, Zn(ClO4)2·6H2O,Mn(ClO4)2·6H2O,Cd(ClO4)2·6H2O,Co(ClO4)2·6H2O,Ni(ClO4)2·6H2O,Pb(ClO4)2·6 H2O, Hg(ClO4)2·3H2O. Inorganic salts were dissolved in distilled water to afford 1.0×10-2 M aqueous solution. High concentration of the stock solutions CA (1.0×10-3 M) were prepared in acetonitrile. Aliquots of stock solution of CA was diluted to 2 mL acetonitrile to make the final concentration of 2 μM. The fluorescence spectra of CA (2 μM) in the presence of various competitive species under the same conditions were recorded at 415 nm and 490 nm(Excitation was provided at 315 nm and 440 nm) in acetonitrile. 2.4. Crystallography In this study, crystallographic data of the chemsensor CA was collected on a Bruker APEX-II CCD diffractometer with graphite-monochromated Mo–Kα (λ=0.71073 A˚). The structure was refined on by full-matrix F2 least squares methods with SHELXTL version 6.1. All non–hydrogen atoms were treated anisotropically. Hydrogen atoms were fixed geometrically at calculated distances and allowed to ride on the parent non-hydrogen atoms with the isotropic displacement.
3. Results and discussion Compound CA can be obtained in a three-step procedure via condensation of coumarin dyes and 5-aminoisophthalic acid methyl ester by schiff-base reaction with ethanoic acid (Scheme 2). The overall yield is 25% for the three steps. Orange crystals which were suitable for X-ray diffraction study were obtained by diffusion of diethyl ether in dichloromethane solution of CA at room temperature. X-ray diffraction single crystal structure analysis indicated that CA crystallized in triclinic system, as shown in Fig1. The dihedral angle between 5-aminoisophthalic acid methyl ester and coumarin groups was 66.2 °,which indicated that the molecular planarity is not good and there is a certain degree of distortion. Bond lengths of C=N (1.278(3) Å) is longer than typical C=N double bond length. The molecular structure of CA and crystal data are listed in Figure S4 and
Table S1. These data indicate that there is a certain extent of conjugation between coumarin and 5-aminoisophthalic acid methyl ester. Bond angles of C(11)-N(1)-C(1) and N(1)-C(11)-C(12) are 118.3(2)° and 120.8(3)°, respectively, which revealed long-pairs of nitrogen caused the photoinduced electron transfer (PET) process and leaded to the fluorescence quenching of CA. < Fig 1 is here> Spectroscopic measurements of CA were investigated in CH3CN/H2O=8/2 medium at physiological pH(0.1 M KClO4 buffer, pH = 7.34) under ambient conditions. The metal-free dye CA(2×10-6 M) upon excitation at 440 nm gave only a weak fluorescence at 530 nm due to highly efficient photoinduced electron transfer (PET). However, addition of the perchlorate salt of Hg2+ elicited a significant enhancement of the fluorescence intensity with a maximum wavelength from 530nm to 490nm with the excitation at 440nm (Fig 2).The fluorescence emission of 490nm was supposed to be assigned to coumarin aldehy degenerating from the breaking of CA. Fluorescent spectra reached plateau with 20-fold Hg2+ added. The detection limit of the sensor for Hg2+ ions was proceeded to be determined. As shown in Fig 2 inset, upon titration with Hg2+ ions (1 nM), the fluorescent intensity at 490 nm increased by about ten percent. The plots of the I490nm/I530nm ratio against the concentrations of Hg2+ ranging from 0 to 12 nM displayed a good linear relationship. Hence, the linear curve of the I490nm/I530nm ratio allows for the convenient quantitative detection of Hg2+ ions over this concentration range. And at the same time, with the excitation at 315 nm, addition of the perchlorate salt of Hg2+ ions elicited a significant enhancement of the fluorescence intensity (60-fold) with a maximum wavelength from 530nm to 415 nm (Fig 3), the fluorescence emission of 415nm came from 5-aminoisophthalic acid methyl ester which was also ascribed to the bond-breaking reaction of CA. The detection limit of the sensor for Hg2+ ions was determined (Excitation at 315 nm). As shown in Fig 3 inset, upon titration with Hg2+ ions (1 nM), the fluorescent intensity at 415 nm increased by about ten percent. The plots of the I415nm/I530nm ratio against the concentrations of Hg2+ ions ranging from 0 to 12 nM also displayed a good linear
relationship. Such a unique emission indicated CA was a promising dual emission ratiometric probe for Hg2+ with a 20-fold fluorescence intensity (530 nm to 490 nm) and 60-fold ratio enhancement (530 nm to 415 nm). The fluorescence enhancement at 490 nm and at 415 nm is due to the hydrolysis reaction of C=N bond. < Fig 2 is here> < Fig 3 is here> Once CA was found to exhibit fluorescence enhancement in the presence of Hg2+ ions, its selectivity profile was checked through competition experiments. With excitation at 440 nm, the sensor CA (2 μM) was first mixed with 20 equiv of metal ions of interest that included alkali and alkaline-earth metal ions (Na+, K+, Mg2+, Ca2+, Cd2+), transition metal ions (Mn2+, Fe2+, Co2+, Ni2+,Cu2+, Zn2+), and heavy metal ions (Ag+, Pb2+), followed by addition of 20 equiv of Hg2+ ions. The various fluorescent response experiments revealed that the Hg2+ ions induced luminescence enhancement was unaffected in the presence of relevant alkali-, alkaline-earth metals, transition metal ions and heavy metal ions (Fig 4). First row transition metal ions did not interfere with the Hg2+ ions induced fluorescence enhancement (the concentration of Hg2+ ions is 40 μM), confirming the remarkable selectivity of the sensor CA (2 μM) for Hg2+ ions over other metal ions. When the excitation was changed to 315 nm, the emission profile did not change to any noticeable extent in terms of both intensity and peak positions in the presence of alkali, alkaline earth, transition, Ag+, or Pb2+ metal ions (Fig 5). The above results indicated the remarkably high selectivity of sensor CA toward Hg2+ ions over other analytes, which suggested that CA could be used for specific detection of Hg2+ ions. < Fig 4 is here> < Fig 5 is here> The ultraviolet-visible absorption spectra of CA were tested as well. The absorption spectrum of CA (2×10-5M) in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.34) exhibited absorbance at 465
nm (Fig 6). Addition of a perchlorate salt of a biologically relevant alkali, alkaline earth, transition, or toxic heavy metal ion such as Ag+ or Pb2+ did not give any discernible change in the absorption spectral profile of the sensor. This suggested there was no interaction between the fluorophore and the added metal ions. However, in the presence of Hg2+ ion, prominent changes in the UV−vis spectra were observed. The absorbance moved from 465nm to 445nm. As the same time, there was a isoabsorptive point at about 290 nm and 310 nm, and upon the addition of Hg2+, the absorption at 300nm disappeared which was the peak of C=N bond. It was further confirmed that the addition of mercury(Ⅱ) ions leaded to the hydrolysis reaction of C=N bond and thereby changed the absorbance spectra of CA. < Fig 6 is here> The binding stoichiometry was further studied to understand the fracture mechanism of CA by electronspray ionization (ESI)mass spectral studies in Figure 7. The ESI-MS showed an peak with m/z=437.16 in acetonitrile, which could be assigned to [CA+H]+ in referring to the exact comparison of the intense peak with the simulation on the basis of natural isotopic abundances. Upon the addition of Hg2+ ions, the ESI-MS of the CA-Hg solution showed an intense peak with m/z=210.07 and 246.11 in acetonitrile which could be assigned to 5-aminoisophthalic acid methyl ester [C10H11NO4+H]+ and coumarin aldehyde [C14H15O3N+H]+.At the same time the peak at m/z = 437.16 disappeared. This result was due to the addition of mercury(Ⅱ) ions which leaded to the hydrolysis reaction of C=N bond. Besides, crystals of CA with addition of Hg2+ were measured and the crystals of coumarin aldehyde was found. Both mass spectrometry and X-ray crystallography structural proved there is hydrolysis reaction of C=N bond after the addition of mercury(Ⅱ) ions. We believed that mercury(Ⅱ) ions have interaction with lone pair electrons of the N. It make the C=N carbon electropostitve and favors nucleophilic attack by H2O to get coumarin aldehyde and 5-aminoisophthalic acid methyl ester. < Fig 7 is here>
The effect of pH on the emission responses of the system was evaluated to tackle various pH values of samples. The emission intensity against the pH value in the absence and presence of Hg2+ ions at 490nm and 415nm were measured respectively. The metal-free sensor did not show any significant emission at 490nm over a wide pH range of 5.5 to 9.0 with excitation at 440nm (Fig 8). However, the emission intensity at 490nm of CA-Hg2+ increased dramatically and remained unchanged from pH 5.5 to 9.0.Similarly, with excitation at 315 nm, sensor CA itself is stable in the pH range of 5.5 to 9.0 (Fig 9).After addition of 20 eq of Hg2+ ions, the emission intensity at 415 nm enhanced and remained unchanged from pH 5 to 9.0, implying that CA could be used in a wide pH range including physiological conditions. The result showed the capacity of potentially detection Hg2+ ions in living systems. < Fig 8 is here> < Fig 9 is here> Encouraged by the above favorable properties of the sensor CA for monitor Hg2+ ions, including excellent sensitivity, high selectivity and stability in the physiological pH range, the ability of the biosensor in tracking Hg2+ ions in the living cells were performed to sensor. The ability of CA to track Hg2+ ions in living cells through confocal microscopy. MCF-7 cells was incubated with 1nM CA for up to 20 min at 37 °C and negligible intracellular fluorescence signals showed as we expected, while none fluorescence could be found from the green channel from the range of 410-470 nm and the red channel from the range of 490-590 nm (Fig10a, b and c). On the contrary, while cells stained by CA exposed to 2 nM of Hg2+ for 20 min at 37 °C, obvious intracellular fluorescence signals displayed as predicted (Fig10d, e and f). The sensor CA was proved to have remarkable member permeability and the results clearly demonstrated that the proposed sensor was low toxicity to cultured cells at the concentration of 1nM for 20 min under the experimental conditions. Hence, colocalization experiment clearly confirmed that the sensor CA could respond toward the changing in intracellular Hg2+ ions (n mol/L levels) in living cells.
< Fig 10 is here> 4. Conclusions To conclude, we have described here the synthesis, photophysical properties, and cellular applications of a ratiometric sensor where coumarin aldehyde and 5-aminoisophthalicacid methyl ester are covalently linked with an schiff-base reaction. The sensor features visible excitation and emission profiles and selective turn-on fluorescence response to Hg2+ ion in the presence of a large excess of biologically relevant metal ions at physiological pH. The ratiometric response of CA at 490 nm and 415 nm respectively were based on the C=N bond hydrolysis reaction mechanism along with the cancelation of a PET process. Because of its usability at physiological pH, the sensor CA was successfully applied to track Hg2+ ions at nano molarity levels in living cells. Acknowledgments This work was partly supported by the National Natural Science Foundation of China (21501020), Project Funded by China Postdoctoral Science Foundation (20105M571295) and the Fundamental Research Funds for the Central Universities (DUT16LK11). Supporting Information Available X-ray structural data of CA CIF format. This material is available free of charge via the Internet at XXXXXXXX. References [1] G. L. Wang, K. L. Liu, Y. M. Dong, Z. J. Li and C. Zhang, In situ formation of p–n junction: A novel principle for photoelectrochemical sensor and its application for mercury(II) ion detection, Anal. Chim. Acta 827 (2014) 34–39. [2] G. V. Guerreiro, A. J. Zaitouna and R. Y. Lai, Characterization of an electrochemical mercury sensor using alternating current, cyclic, square wave and differential pulse voltammetry, Anal. Chim. Acta 810 (2014) 79–85.
[3] M. Vendrell, D. Zhai, J. C. Er, Y. T. Chang, Combinatorial strategies in fluorescent probe development, Chem. Rev. 112 (2012) 4391–4420. [4] M. Wang, J. Wen, Z. H. Qin, H. M. Wang, A new coumarin–rhodamine FRET system as an efficient ratiometric fluorescent probe for Hg2+ in aqueous solution and in living cells, Dyes Pigment. 120 (2015) 208–212. [5] P. Srivastava, S. S. Razi, R. Ali, Selective naked-eye detection of Hg2+ through an efficient turn-on photoinduced electron transfer fluorescent probe and its real applications, Anal. Chem. 86 (2014) 8693–8699. [6] W. Lin,X. Cao, Y. Ding, A highly selective and sensitive fluorescent probe for Hg2+ imaging in live cells based on a rhodamine–thioamide–alkyne scaffold, Chem. Commun. 46 (2010) 3529–3531. [7] S. Cao, Q. Y. Jin, L. Geng, L. Y. Mua, S. L. Dong, A ―turn-on‖ fluorescent probe for the detection of Cu2+ in living cells based on a signaling mechanism of N=N isomerization, New J. Chem. 40 (2016) 6264–6269. [8] X. Wang, J. Zhao, C. Guo, M. Pei, G. Zhang, Simple hydrazide-based fluorescent sensors for highly sensitive and selective optical signaling of Cu2+ and Hg2+ in aqueous solution, Sens. Actuators, B. 193 (2014) 157–165. [9] S. Lee, B. A. Rao, Y. A. Son, Colorimetric and ―turn-on‖ fluorescent determination of Hg2+ ions based on a rhodamine-pyridine derivative, Sens. Actuators, B. 196 (2014) 388–397. [10] A. A. A. Aziz, S. H. Sed, Detection of trace amounts of Hg2+ in different real samples based on immobilization of novel unsymmetrical tetradentate Schiff base within PVC membrane, Sens. Actuators, B. 197 (2014) 155–163. [11] W. J. Qu, G.Y. Gao, B. B. Shi, T.B. Wei, Y.M. Zhang, Q. Lin, H. Yao, A highly selective and sensitive fluorescent chemosensor for mercury ions based on the mechanism of supramolecular self-assembly, Sens. Actuators, B. 204 (2014) 368–374.
[12] Elizabeth M. Nolan, Stephen J. Lippard, Tools and tactics for the optical detection of mercuric ion, Chem. Rev. 108 (2008) 3443–3480. [13] P. Zhuang, M. McBride, H. Xia, N. Li, Z. Li, Health risk from heavy metals via consumption of food crops in the vicinity of dabaoshan mine, South China, Sci. Total Environ. 407 (2009) 1551−1561. [14] E. M. Nolan, S. Lippard, Tools and tactics for the optical detection of mercuric ion, ChemInform. 108 (2008) 3443−3480. [15] T. Agusa, T. Kunito, H. Iwata, I. Monirith, T. S. Tana, A. Subramanian, S. Tanabe, Mercury contamination in human hair and fish from cambodia: levels, specific accumulation and risk assessment, Environ. Pollut. 134 (2005) 79–86. [16] J. P. Bourdineaud, R. Rossignol, D. Brèthes, Zebrafish: A model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics, Int. J. Biochem. Cell Biol. 45 (2013) 16–22. [17] Y. Wei, B. Li, X. Wang and Y. Duan, A nano-graphite–DNA hybrid sensor for magnified fluorescent detection of mercury(II) ions in aqueous solution, Analyst 139 (2014) 1618–1621. [18] Q. Wang, D. Kim, D. D. Dionysiou, G. A. Sorial, Sources and remediation for mercury contamination in aquatic systems—a literature review, Environ. Pollut. 131 (2004) 323−336. [19] H. S. So, B. A. Rao, J. Hwang, K. Yesudas and Y. A. Son, Synthesis of novel squaraine–bis(rhodamine-6G): a fluorescent chemosensor for the selective detection of Hg2+, Sens. Actuators B 202 (2014) 779–787. [20] R. Eisler, Health Risks of Gold Miners: A synoptic review, Environ. Geochem. Health 25 (2003) 325−345. [21] S. B. Maity, S. Banerjee, K. Sunwoo, J. S. Kim, and P. K. Bharadwaj, A fluorescent chemosensor for Hg2+ and Cd2+ ions in aqueous medium under physiological pH and its applications in imaging living cells, Inorg. Chem. 54 (2015) 3929−3936.
[22] K. Bera, A. K. Das, M. Nag, S. Basak, Development of a rhodamine–rhodanine-based fluorescent mercury sensor and its use to monitor real-time uptake and distribution of inorganic mercury in live zebrafish larvae, Anal. Chem. 86 (2014) 2740–2746. [23] G. Chen, Z. Guo, G. Zeng and L. Tang, Fluorescent and colorimetric sensors for environmental mercury detection, Analyst 140 (2015) 5400–5443. [24] P. Mahato, S, Saha, P, Das, H, Agarwalla and A, Das, An overview of the recent developments on Hg2+ recognition, RSC Adv. 4 (2014) 36140–36174. [25] Y. Xu, Z. Jiang, Y. Xiao, T. T. Zhang, J. Y. Miao, B. X. Zhao, A new fluorescent turn-on chemodosimeter for mercury ions in solution and its application in cells and organisms, Anal. Chim. Acta 807 (2014) 126–134. [26] W. Y. Liu, S. L. Shen, H. Y. Li, J. Y. Miao, B. X. Zhao, Fluorescence turn-on chemodosimeter for rapid detection of mercury (II) ions in aqueous solution and blood from mice with toxicosis, Anal. Chim. Acta 791 (2013) 65–71. [27] H. Ye, F. Ge, X. C. Chen, Y. Li, H. Zhang, B. X. Zhao, J. Y. Miao, A new probe for fluorescent recognition of Hg2+ in living cells and colorimetric detection of Cu2+ in aqueous solution, Sensors and Actuators B 182 (2013)273–279. [28] P. Ashokkumar, V. T. Ramakrishnan, and P. Ramamurthy, Photoinduced rlectron transfer (PET) Based Zn2+ fluorescent probe: transformation of turn-on sensors into ratiometric ones with dual emission in acetonitrile, J. Phys. Chem. A 115 (2011) 14292–14299. [29] Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen, Z. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angew. Chem. Int. Ed. 52 (2013) 1688−1691. [30] H. T. Zhang, R. C. Liu, Y. Tan, H. W. Xie, H. P. Lei, H. Y. Cheung, H. Y. Sun, A FRET-based ratiometric fluorescent probe for nitroxyl detection in living cells, ACS Appl. Mater. Interfaces 7 (2015) 5438−5443.
[31] Z. Guo, G. Kim, J. Yoon, Synthesis of a highly Zn2+-selective cyanine-based probe and its use for tracing endogenous zinc ions in cells and organisms, Nat. Protoc. 9 (2014) 1245−1254. [32] L. Yuan, W. Y. Lin, Y. T. Yang, J. Z. Song, J. L. Wang, Rational design of a highly reactive ratiometric fluorescent probe for cyanide, Org. Lett. 13 (2011) 3730–3733. [33] X. S. Zhou, X. Lv, J. S. Hao, D. S. Liu, W. Guo, Coumarin-indanedione conjugate as a doubly activated Michael addition type probe for the colorimetric and ratiometric fluorescent detection of cyanide, Dyes Pigment. 95 (2012) 168–173. [34] X . Lv, J. Liu, Y. L. Liu, Y. Zhao, Y. Q. Sun, P. Wang, W. Guo, Ratiometric fluorescence detection of cyanide based on a hybrid coumarin-hemicyanine dye: the large emission shift and the high selectivity, Chem. Commun. 47 (2011) 12843–12845. [35] Z. P. Liu, X. Q. Wang, Z. H. Yang, W. J. He, Rational design of a dual chemosensor for cyanide Anion Sensing Based on dicyanovinyl-substituted benzofurazan, J. Org. Chem. 76 (2011) 10286–10290. [36] J. N. Weng, Q. B. Mei, Q. D. Ling, Q. L. Fan, W. Huang, A new colorimetric and fluorescent ratiometric sensor for Hg2+ based on 4-pyren-l-yl-pyrimidine, Tetrahedron 68 (2012) 3129–3134. [37] A. Coskun, U. Akkaya, Signal ratio amplification via modulation of resonance energy transfer; Proof of principle in an emission ratiometric Hg (II) sensor, J. Am. Chem. Soc. 128 (2006) 14474–14475. [38] J. Jiang , W. Liu , J. Cheng, L. Z. Yang, H. Jiang, D. C. Bai, W. S. Liu, A sensitive colorimetric and ratiometric fluorescent probe for mercury species in aqueous solution and living cells, Chem. Commun. 48 (2012) 8371–8373. [39] N. DiCesare, J. R. Lakowicz, Fluorescent probe for monosaccharides based on a functionalized boron-dipyrromethene with a boronic acid group, Tetrahedron Lett. (2001) 9105–9108.
[40] Z. C. Xu, J. Yoon, D. R. Spring, A selective and ratiometric Cu2+ fluorescent probe based on naphthalimide excimer-monomer switching, Chem. Commun. 46 (2010) 2563–2565. [41] A. E. Albers, V. S. Okreglak, C. J. Chang, A FRET-Based approach to ratiometric fluorescence detection of hydrogen peroxide, J. Am. Chem. Soc. 128 (2006) 9640–9641. [42] K. Ghosh, T. Sarkara and A. Samadderb, A rhodamine appended tripodal receptor as a ratiometric probe for Hg2+ ions, Org. Biomol. Chem. 10 (2012) 3236–3243. [43] Y. R. Zhang, N. Meng, J. Y. Miao, B. X. Zhao. A ratiometric fluorescent probe based on a Through-Bond Energy Transfer (TBET) system for imaging HOCl in living cells, Chem. Eur. J. 21 (2015) 19058–19063. [44] Y. J. Gong, X. B. Zhang, C. C. Zhang, A. L. Luo, T. Fu, W. H. Tan, G. L. Shen, R. Q. Yu, Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications, Anal. Chem.84 (2012) 10777–10784. [45] H. B. Yu, Y. Xiao, H. Y. Guo, X. H. Qian, Convenient and efficient FRET platform featuring a rigid biphenyl spacer between rhodamine and BODIPY: transformation of 'turn-on' sensors into ratiometric ones with dual emission, Chem. Eur. J. 17 (2011) 3179–3191. [46] H. H. Wang, L. Xue, C. L. Yu, Y. Y. Qian, H. Jiang, Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells, Dyes Pigment. 91 (2011) 350–355. [47] Y. Gong, X. Zhang, C. Zhang, A. L. Luo, T. Fu, W. H. Tan, G. L. Shen, R. Q. Yu, Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications, Anal. Chem. 84 (2012) 10777–10784. [48] Crystal structure analysis: The data was collected at temperature of 296±2K on a Bruker Smart APEX Ⅱ X–diffractometer equipped with graphite monochromated Mo–Kα radiation (λ = 0.71073 Å) using the SMART and SAINT programs. Indexing and unit cell refinement were based on all observed reflections from those 72 frames. The structure was solved in the P1 space group by direct method and refined by the full–matrix least–squares fitting on F2 using
SHELXTL 6.1. All non–hydrogen atoms were treated anisotropically. Hydrogen atoms of organic ligands were generated geometrically, fixed isotropic thermal parameters, and included in the structure factor calculations. Crystal data for CA: C24H24N2O6, M = 436.45, Triclinic, P1, a = 7.0594(3) Å, b = 9.9418(4) Å, c = 16.8535(7) Å, α= 95.809(3) °, β = 99.209(3)°, γ= 109.521(3)°, V = 1085.15(8) Å3, Z = 2, Dc = 1.336 g·cm–3, μ = 0.097 mm–1.8827 reflections were collected (Rint = 0.0867). The final refinement gave R1 = 0.0486 and wR2 = 0.1206 with I 2(I). The structures were solved by direct methods and refined on F2by full-matrix least-squares methods with SHELXTL version 6.1.Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge, CB21EZ, UK (fax: +44–1223–336–033; e-mail: [email protected])
Biography
Dr. Yang Jiao is a Doctor at Dalian University of Technology, China. She research interests are focused on fluorescent sensors and their applications in biological, medical diagnostics, environmental monitoring; dye molecular structure and function regulation. She published articles in the journal Chem. Commun., Chem. Eur. J. and Talanta, etc. [email protected] Prof Chunying Duan is a Professor at Dalian University of Technology, China. His research interests are in the fields of biological molecular probes and fluorescence imaging; photoelectric functional inorganic material and molecular devices; the chiral porous skeleton structure and asymmetric catalysis. He has published over 100 academic articles. [email protected]
Scheme1 Mechanism of Sensor Interaction with Hg2+ Ions
Scheme 2 Synthetic Scheme for the Sensor CA
Fig 1 Molecular structure of CA with the atomic-numbering scheme showing the schiff-base structural fashion (Hydrogen atoms were omitted for clarity, the C, O and N atoms were drawn in gray, red and blue.)
1.0
2+
[Hg ]
I490/I530
20
F/F0
15
0.9 0.8 0.7 0.6 2
10
4
6
8
10
12
2+
[Hg ](ppb)
5
0 450
500
550
600
650
700
Wavelength(nm)
Fig 2 Fluorescence spectra of CA (2×10-6 M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH=7.34;The fluorescence intensities were measured at 490 nm. Excitation was at 440 nm. Both emission and excitation slit widths were 3 nm)Inset: Fluorescence intensities at 490 nm of complex CA as a function of the mercury concentration (2–10 ppb).
60
2.2
55 45
2.1
[Hg2+]
F/F0
40
I415/I530
50
2.0 1.9 1.8 1.7 1.6
35
1.5
30
1.4 2
25
4
6 8 2+ [Hg ](ppb)
10
12
20 15 10 5 0 350
400
450
500
550
600
Wavelength(nm)
Fig 3 Fluorescence spectra of CA (2×10-6 M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.34; The fluorescence intensities were measured at 415 nm. Excitation was at 315nm. Emission/excitation = 3 nm, 3 nm) Inset: Fluorescence intensities at 415 nm of complex CA as a function of the mercury concentration (2–10 ppb).
2.5
I490/I530
2.0 1.5 1.0 0.5 0.0
+
2+
2+
2+
2+
+ + K Na Ag Ca2+Co2+ Zn2+ Ni2+Fe2+Cu2+Cd Mg Mn Pb2+Hg
Fig 4 Fluorescence responses of CA to various cations in an optimized CH3CN/H2O=8/2 solution (0.1 M KClO4 buffer, pH = 7.34; [CA] = 2μM). The left bars in each group represent the emission intensities of CA in the presence of 40μM of Na+, K+, Ca2+ and Mg2+ and 40μM of the other cations of interest, respectively. The right bars in each group represent the change in emission that occurs upon subsequent addition of 40μM of Hg2+ ions to the above solution. Excitation was provided at 440 nm, and the emission was recorded at 490 nm.
20
I415/I530
15
10
5
0 2+
2+
+ 2+ + 2+ + 2+ 2+ 2+ 2+ 2+ K Na Ag Ca2+Co Zn Ni2+Fe Cu Cd Mg Mn Pb Hg
Fig 5 Fluorescence responses of CA to various cations in an optimized CH3CN/H2O=8/2 solution (0.1 M KClO4 buffer, pH = 7.34; [CA] = 2μM). The left bars in each group represent the emission intensities of CA in the presence of 40μM of Na+, K+, Ca2+ and Mg2+ and 40μM of the other cations of interest, respectively. The right bars in each group represent the change in emission that occurs upon subsequent addition of 40μM of Hg2+ ions to the above solution. Excitation was provided at 315 nm, and the emission was recorded at 415 nm.
1.2
Absorbance
1.0
Hg
2+
Other metal ions
0.8 0.6 0.4 0.2 0.0 200
300
400
500
600
700
Wavelength(nm)
Fig 6 UV-vis spectra of CA (2×10-5M) with addition of various concentrations of Hg2+ ions in CH3CN/H2O=8/2 (0.1 MKClO4 buffer, pH = 7.34)
Fig 7 ESI-MS of the CA in acetonitrile (top) and CA in the presence of 1 equivalents molar ration of Hg2+ ions in acetonitrile (bottom).
20
F/F0
15
10
5
0 6
7
8
9
pH
Fig 8. Emission intensities of CA and CA-Hg2+ at various pH values in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.4). Excitation wavelength= 440 nm.[CA] = 2μM, [Hg2+] = 40μM.
60 50
F/F0
40 30 20 10 0 6
7
8
9
pH
Fig 9. Emission intensities of CA and CA-Hg2+ at various pH values in CH3CN/H2O=8/2 (0.1 M KClO4 buffer, pH = 7.4). Excitation wavelength= 315nm.[CA] = 2μM, [Hg2+] = 40μM.
Figure 10. Confocal microscopy images of MCF-7 cells. MCF-7 cells were incubated with 1nM CA and observed under bright field (a),green channel (b) and red channel (c), then further incubation with Hg2+ ions (2nM) for 20 min at 37 ℃ and observed under bright field (d), green channel (e), red channel (f).Cells were incubated at 37 °C under humidified atmosphere 5% CO2. The excitation wavelength was 405 nm(green channel) and 458nm(red channel), and images were collected at 410-470 nm(green channel) and 490-590nm(red channel).