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A novel benzopyran-based colorimetric and near-infrared fluorescent sensor for Hg2+ and its imaging in living cell and zebrafish
Dyes and Pigments 172 (2020) 107658
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A novel benzopyran-based colorimetric and near-infrared fluorescent sensor for Hg2+ and its imaging in living cell and zebrafish
T
Hehong Lva, Gang Yuana, Ganbing Zhanga, Ziqi Rena, Hanping Hea,∗, Qi Sunb,∗∗, Xiuhua Zhanga, Shengfu Wanga a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials & Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei University, Youyi Road 368, Wuchang, Wuhan, Hubei, 430062, PR China b Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, 430205, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hg2+ Near-infrared fluorescent probe Colorimetric High specificity Imaging of living cell and zebrafish
A new near-infrared fluorescent probe (OTA-DCM), which contained a conjugated dicyanomethylene-benzopyran structure as the near-infrared fluorophore and dithia-dioxa-monoaza crown ether moiety as the receptor, was developed as a “naked-eye” indicator for Hg2+ with rapid response, good binding constant (1.05 × 104 M−1), high selectivity and sensitivity. Moreover, OTA-DCM was successfully employed in real aqueous samples and fluorescent imaging for detection of Hg2+ in living cells and zebrafish larvae with low cytotoxicity.
1. Introduction As one of the most toxic metal ions in the environment, mercury and its derivatives are lethal threats to the ecosystem and human health because of their durability and high biological accumulation [1]. Mercury can exist in inorganic, organic or elemental forms and each species displays a different level of toxicity. The detection of inorganic mercury, a precursor of organic or elemental mercury, has attracted much attention. In recent years, myriads of rapid and facile Hg2+ probes for selective signaling or visualization, have been developed, and applied to the analyses of biological and environmental samples [2–12]. The Hg2+-induced mechanisms are mainly based on chelation and fluorometric chemo-dosimetry, such as cyclization [2], hydrolysis [3], ring opening of rhodamine spirolactams [4] and desulphurization reactions [5], and so on. In these probes, the used fluorophores were mainly BODIPY, coumarin, rhodamine, benzothizole, quinoline, naphthalimide and fluorescein, etc. [13]. Unfortunately, the emission maxima of most chemosensors were below 600 nm. By contrast, nearinfrared fluorescent probes are more attractive in practical applications due to minimum photodamage to biomolecules, low light scattering, and negligible low background autofluorescence of biomolecules [14], which attracted lots of researchers. However, near-infrared fluorescent probes for detecting Hg2+ aren't
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much reported [15], to the best of our knowledge. The NIR fluorescent dye for Hg2+ mainly included BODIPY [15b,], coumarin [15e], rhodamine [15 g], and so on [15]. The novel near-infrared fluorophores with colorimetric change, excellent selectivity and high sensitivity for detection of Hg2+ are still in high demand. Recently, dicyanomethylene-4H-chromene (DCM) and its derivatives have attracted considerable interest since they emit light in the red or near infrared (NIR) region [16]. Moreover, the DCM fluorophore has better photostability and larger Stokes shift (about 100 nm) [17]. The chromophore DCM with donor-π-acceptor structure had been designed as a colorimetric fluorophore for detection of various analytes [18]. However, to the best our knowledge, the derivatives based on DCM moiety had been rarely reported for the detection of mercury with high selectivity and sensitivity. Among selective chemsensors for Hg2+, the traditional structure with crown ether skeleton is chosen [19], because the receptor unit would result in good selectivity for Hg2+ as a soft acid [20]. Hence, dithia-dioxa-aza cyclopentadecane [21,22] is chosen as the receptor of Hg2+ in the work. Herein, a novel colorimetric fluorescent probe was designed and synthesized for the detection of Hg2+ with excellent selectivity, which employed dicyanomethylene-4H-chromene as the fluorophore and dithia-dioxa-monoaza crown ether as the recognized site (OTA-DCM in Fig. 1a). The probe showed colorimetric, excellent selectivity and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. He), [email protected] (Q. Sun).
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https://doi.org/10.1016/j.dyepig.2019.107658 Received 19 March 2019; Received in revised form 12 June 2019; Accepted 21 June 2019 Available online 01 August 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (a) Synthetic route for OTA-DCM chemosensors. (b) Fluorescence responses of probe OTA-DCM (10 μM) upon treatment with various metal ions (10 eq.) (c) Naked eyes colorimetric changes of the probe with different metal ions in CH3CN–H2O (1:1, v/v). (d) Fluorescent Photographs under UV–vis lamp (365 nm). (e and f) The reversibility of OTA-DCM by alternately adding Hg2+ and Na2S.
sensitivity toward Hg2+ with no significant interference from other competitive metal ions and anions. Moreover, the near-infrared probe was successfully used in real aqueous samples and fluorescent imaging for Hg2+ in living cells and zebrafish larvae with low cytotoxicity.
peak showed a slight hypsochromic shift from 517 to 498 nm. Consistent with the significant change in UV absorption, the change in colour of the solution was obvious after addition of Hg2+. In Fig. 1c, the free OTA-DCM can be seen as bright rose red coloured solution. The colour of the solution underwent change from rose red to yellow in the presence of Hg2+, however other metal ions did not bring about any obvious change in the colour of the solution. So, OTA-DCM can be used as visual indictor for Hg2+. This technique is therefore superior to other analytical techniques because it has ability to detect Hg2+ by nakedeye. The fluorescence emission spectrum of OTA-DCM showed a strong emission at 645 nm and excellent selectivity. When Hg2+ was added, the strong fluorescence was almost completely quenched (Fig. 1b). Since complexation of Hg2+ with dithia-dioxa-monoaza crown weakened electron-donating ability of the recognition site, which resulted in the blocking of intermolecular charge transfer (ICT) process. However, other metal ions couldn't result in the obvious fluorescence changes of OTA-DCM, except that Ag+ brought about with slight quenching. This may be due to the different electrical properties and
2. Results and discussion 2.1. Specificity of OTA-DCM to Hg2+ Firstly, the binding behavior of OTA-DCM probe with metal ions was investigated by UV absorption and fluorescence spectroscopy in CH3CN/H2O (1:1, v/v). In the absence of Hg2+, the free OTA-DCM exhibited a main absorption peaks at 517 nm and other small peaks at around 392 nm and 415 nm. After the addition of Hg2+, the absorption peaks at 517 nm disappeared gradually, whereas peaks at 435 nm and 415 nm increased with increasing concentrations of Hg2+ (Fig. S8). Other metal ions such as K+, Na+, Ni2+, Co2+, Zn2+, Pb2+, Mg2+, Cu2+, Mn2+, Fe2+, Fe3+, Cr3+ and Al3+ did not show any significant changes. However, upon addition of Ag+, the maximum absorption 2
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Fig. 2. (a) Fluorescence spectra of OTA-DCM (10 μM) upon incremental addition of Hg2+ (0–2 eqv.). Insert: The ratio of fluorescence intensity (F/F0) of OTA-DCM in the presence of increasing Hg2+ concentration. (b) The linearity between F/F0 and Hg2+ concentration. F0: fluorescence intensities at 645 nm without Hg2+. F: fluorescence intensities at 645 nm with Hg2+. (c) Benesi-Hildebrand plot based on a 1:1 binding stoichiometry between OTA-DCM and Hg2+.
show that OTA-DCM has high selectivity for Hg2+, and can be used an excellent visual probe. Moreover, the response of OTA-DCM to Hg2+ was rapid (only 1min, Fig. S9). The fluorescence quenching was very stable over a wide range of pH (2.0–10.0) (Fig. S10). The rapid and stable response of OTA-DCM to Hg2+ was very useful for the detection of real samples with portable device. In order to further verify the specificity, competitive experiments were carried using other interfering cations and anions as shown in Fig. S11. It was found that fluorescence quenching was clearly caused by the mixture containing Hg2+, whereas the fluorescence intensity remained unchanged with metal ions other than Hg2+. Moreover, different anions, such as F−, OH−, CO32−, HCO3−, HSO3−, NO2−, NO3−, HPO42−, H2PO4−, SO32−, SO42− and CH3COO−, showed no obvious interferences. However, the Hg2+-triggered quenching efficiency was affected in the presence of Br−, Cl− and S2−, probably due to precipitation reaction between Hg2+ and Br−, Cl−, and also the strong binding ability of S2− towards Hg2+ ions. These results strongly suggested that OTA-DCM exhibited excellent selectivity toward Hg2+.
2.2. The reversibility of OTA-DCM Talking into account the binding of S2− with Hg2+, the binding ability and reversibility of OTA-DCM to Hg2+ on the fluorescence were examined in the presence of Na2S. It was observed that on addition of Na2S to a solution of OTA-DCM–Hg2+ complex, the fluorescence intensity was immediately restored (Fig. 1e). This was evident from the colour changes of the solution from yellow to pleasantly bright rose red (Fig. 1f). This could be attributed to the fact that the addition of S2− would liberate Hg2+ from the metal-ligand complex, because of the strong binding ability of S2− towards Hg2+ ions, resulting in the revival of the free probe [23]. The fluorescence was quenched after the addition of Hg2+ again, which was accompanied by colour change. The sequence of quenching and revival of fluorescence continued approximately for more than five cycles without loss of sensitivity (Fig. 1f).
Fig. 3. The optimized geometric structures based on B3LYP and SMD salvation model for OTA-DCM and OTA-DCM Hg2+ in mixture solvents, water/acetonitrile (1/1, volume ratio), where the bond lengths are in the unit of Å. Here, both top views and side views are shown.
inappropriate diameters of different metal ions. Fig. 1d showed the photographs of fluorescent response of OTA-DCM probe treated with different metal ions under UV–Vis light. The fluorescence quenching occurred obviously in case of Hg2+, but the other metal ions, including Ag+, did not cause significant changes in fluorescence. These results 3
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Fig. 4. Fluorescence imaging of A549 cells incubated with OTS-DCM (10 μM) for 30min (A–C), and then with Hg2+ (100 μM) for another 30min (E–F) and 60min (G–I). (Left) bright filed image, (Medium) red channel (590–650 nm), (Right) Merge image, (J) average intensity ratios in red channel fluorescent image. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the protons in –CH2- adjacent to the O atoms (Ha and Hb) were shifted slightly downfield (0.08 ppm). The proton (He) also shifted downfield from δ 3.13 to δ 3.52 ppm. Concurrently, the chemical shifts of protons in aromatic shifted low field (0.06–0.45 ppm). These results indicated that S, N, and O in crown ether acted as electron donors for coordination to Hg2+, especially S and N. These coordination weakened the electron-donating ability of recognition site (N, S) to fluorophore, which resulted in blocking of ICT process. In addition, The ESI-MS spectrum of OTA-DCM in the presence of 10 equivalents of Hg2+ had some peaks at m/z 779.1154, 780.1151, 781.1144, 782.1151, 783.1150, 784.1161, which are be assignable to calculated [OTADCM + Hg + Cl]+ ([C30H31ClHgN3O3S2]+, m/z: 779.1172, 780.1173, 781.1193, 782.1196, 783.1230, 784.1167) as shown in Fig. S14. The data further proved the complex formation with a stoichiometric ratio of 1:1. Based on the above studies, a possible structure of 1:1 complex of OTA-DCM with Hg2+ was proposed, which was explained by computational study. The optimized geometries of OTA-DCM, and OTA-DCMHg2+ are shown in Fig. 3, which were consistent with the above observations. The theoretical calculations combined the SMD salvation
2.3. The binding studies The binding properties of OTA-DCM with Hg2+ were studied by fluorescence titration experiments firstly. As shown in Fig. 2, when the concentration of Hg2+ ions increased to 15 μM, no further restoring fluorescence was found, which meant that the fluorescence response had reached its maximum. There was a sensitive linear fluorescence response for Hg2+ in the range from 0 μM to 5 μM with the detection limit of 0.14 μM, based on the equation DL = 3σ/S [23] (Fig. 2b). The binding constant of OTA-DCM and Hg2+ was determined to be 1.05 × 104 M−1 from the slope of the linear (Fig. 2c) according to Benesi-Hildebrand equation, details of which can be seen in the supporting information [24]. The binding stoichiometry and possible pattern recognition were investigated to further understand the binding process. Analysis of Job's plot showed a stoichiometric ratio of 1:1 for the complex formation between OTA-DCM and Hg2+ (Fig. S12), which was consistent with reported literature [22]. 1H NMR studies supported the complexation of Hg2+ with OTA-DCM (Fig. S13). Hc and Hd protons showed a big downfield shifts with 0.43 ppm and 0.42 ppm respectively. Moreover, 4
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Fig. 5. Fluorescence imaging of zebrafish larvae treated with OTA-DCM (10 μM) for 0 h (A–C), 0.5 h (B–F), 1 h (G–I),3 h (J–K), and further treated with Hg2+ (100 μM, M − O) for another 4 h. (Up) bright field imaging, (Medium) red channel (590–650 nm), (Down) merge image. (P) average intensity ratios. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cytotoxicity and excellent biocompatibility for cancer and normal cells. Furthermore, experiments for imaging of intracellular Hg2+ in A549 cells were carried out using OTA-DCM as the fluorescent probe. As shown in Fig. 4, incubation of the cell with OTA-DCM for 30 min produced a significant fluorescence from the intracellular region, which could be observed clearly. After treatment with Hg2+ for 30 min, the fluorescence intensity decreased. After incubating for 30min further, the fluorescent intensity became very low dim and was then quenched. These implied that the intracellular uptake of Hg2+ ions resulted in form of complex of OTA-DCM and Hg2+ to yield a non-fluorescent ensemble. These findings showed that OTA-DCM probe was biocompatible and can be used for detection of Hg2+ by fluorescent imaging in living cells. Importantly, fluorescence imaging in zebrafish were performed in order to further explore the application of OTA-DCM in living body. As shown in Fig. 5, bright fluorescence was observed in the digestive system of the zebrafish larvae, and the fluorescence intensity increased obviously with increasing incubation time. After incubation for 3 h, the remaining OTA-DCM in the zebrafish was removed by three times washing, and a further incubation was continued for another 4 h in the presence of Hg2+. It was found that the fluorescence intensity was much weaker and quenched in the digestive system of the zebrafish larvae. The results showed that OTA-DCM was organism-permeable and was admirably sensitive for detection of Hg2+ in living body.
model provided valuable information regarding the structures, properties and plausible recognition patterns through the B3LYP and NBO analyses and TD-CAM-B3LYP and NTO analyses. These calculations supported blocking of ICT mechanism of the fluorescence quenching by Hg2+. The details are provided in supporting information (Fig. S15, Tables S1 and S2). 2.4. Practical application The major source of mercury pollution is from contaminated natural waters, which is a global problem. In order to evaluate the feasibility of OTA-DCM to detect Hg2+ practically, tap water and lake water (Shahu Lake in Wuhan) samples were collected. Results in Table S3, showed that Hg2+ was absent in three blank samples. And the detected concentrations were close to the concentration of added Hg2+. The relative standard deviations (RSD) were less than 2% based on three measurements. The recovery was in the range from 97% to 103%. These results suggested that OTA-DCM could be satisfactorily applied in the detection of Hg2+ in real water. 2.5. Imaging of living cell and zebrafish Herein the feasibility of OTA-DCM for imaging Hg2+ in living cells was investigated. First, MTT assay with A549 cells and normal cells was used to determine the cytotoxicity of OTA-DCM. The data showed that the cellular viability was estimated to be more than 82% after 24 h (Figs. S16 and S17), which indicated that free OTA-DCM has low 5
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3. Conclusions In summary, we were successful in designing and synthesizing a near-infrared fluorescent probe (OTA-DCM), employing dicy-anomethylene-4H-chromene as the NIR fluorescence group and a dithiadioxa-monoaza crown ether moiety as a highly selective recognition receptor for Hg2+. Compared to other reported work (Table S4), this probe demonstrated superior analytical performance such as a remarkably large Stokes shift (120 nm), rapid response, high selectivity and sensitivity, and good binding constant (1.05 × 104 M−1) with the formation of 1:1 complexes of Hg2+. The detection limit was found to be 0.14 μM by fluorescence titration. Importantly, the solution colour changed from rose red to yellow after the addition of Hg2+, which meant that the probe can be used as a “naked-eye” visual indictor. The binding and plausible recognition patterns were derived from computational calculations. Moreover, OTA-DCM could be successfully applied in the detection of Hg2+ in real aqueous samples. Impressively, OTA-DCM was applied in fluorescent imaging of Hg2+ with excellent cell-membrane-permeability in living cells and zebrafish model. Overall, this probe can be used as an excellent turn-off sensor candidate with colorimetric and NIR fluorescence for Hg2+, which holds a great promise for the detection of Hg2+ in complex living samples. Acknowledgements This work was supported by National Natural Science Foundation of China (21575035, 21804102), the Hubei Provincial Natural Science Foundation of China (2017CFB222) and Foreign Science and Technology Cooperation Fund of Hubei province, China (2015BHE025). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107658. References [1] (a) Aragay G, Pons J, Merkoçi A. Recent trends in macro-, micro-, and nanomaterial-based tools and strategies for heavy-metal detection. Chem Rev 2011;111:3433–58; (b) Foster KL, Stern GA, Pazerniuk MA, Hickie B, Walkusz W, Wang F, Macdonald RW. Mercury biomagnification in marine zooplankton food webs in hudson bay. Environ Sci Technol 2012;46:2952–12959; (c) Diaz de Grenu B, Garcia-Calvo J, Cuevas J, Garcia-Herbosa G, Garcia B, Busto N, lbeas N, Torroba T, Torroba B, Herrera A, Pons S. Chemical speciation of MeHg+ and Hg2+ in aqueous solution and HEK cells nuclei by means of DNA interacting fluorogenic probes. Chem Sci 2015;6:3757–64. [2] Ru J, Tang X, Ju Z, Zhang G, Dou W, Mi X, Wang C, Liu W. Exploitation and application of a highly sensitive Ru(II) complex-based phosphorescent chemodosimeter for Hg2+ in aqueous solutions and living cells. ACS Appl Mater Interfaces 2015;7:4247–56. [3] Zhu B, Wang W, Liu L, Jiang H, Du B, Wei Q. A highly selective colorimetric and long-wavelength fluorescent probe for Hg2+. Sens Actuators B Chem 2014;191:605–11. [4] Tripathi K, Rai A, Yadav AK, Srikrishna S, Kumari N, Mishra L. Fluorescein hydrazone-based supramolecular architectures, molecular recognition, sequential logic operation and cell imaging. RSC Adv 2017;7:2264–72. [5] Ding J, Li H, Wang C, Yang J, Xie Y, Peng Q, Li Q, Li Z. “Turn-On” fluorescent probe for mercury(II): high selectivity and sensitivity and new design approach by the adjustment of the π-bridge. ACS Appl Mater Interfaces 2015;7:11369–76. [6] Liu K, Zhao X, Liu Q, Huo J, Li Z, Wang X. A novel multifunctional BODIPY-derived probe for the sequential recognition of Hg2+ and I−, and the fluorometric detection of Cr3+. Sens Actuators B Chem 2017;239:883–9. [7] Singh G, Sanchita, Rani S, Sharma G, Kalra P, Singh N, Verma V. Coumarin–derived Organosilatranes: functionalization at magnetic silica surface and selective recognition of Hg2+ ion. Sens Actuators B Chem 2018;266:861–72. [8] Fang Y, Li X, Li J-Y, Wang G-Y, Zhou Y, Xu N-Z, Hu Y, Yao C. Thiooxo-Rhodamine B hydrazone derivatives bearing bithiophene group as fluorescent chemosensors for detecting mercury (II) in aqueous media and living HeLa cells. Sens Actuators B Chem 2018;255:1182–90. [9] Xu D, Tang L, Tian M, He P, Yan X. A benzothizole-based fluorescent probe for Hg2+ recognition utilizing ESIPT coupled AIE characteristics. Tetrahedron Lett 2017;58:3654–7.
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A novel benzopyran-based colorimetric and near-infrared fluorescent sensor for Hg2+ and its imaging in living cell and zebrafish
T
Hehong Lva, Gang Yuana, Ganbing Zhanga, Ziqi Rena, Hanping Hea,∗, Qi Sunb,∗∗, Xiuhua Zhanga, Shengfu Wanga a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials & Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei University, Youyi Road 368, Wuchang, Wuhan, Hubei, 430062, PR China b Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, 430205, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hg2+ Near-infrared fluorescent probe Colorimetric High specificity Imaging of living cell and zebrafish
A new near-infrared fluorescent probe (OTA-DCM), which contained a conjugated dicyanomethylene-benzopyran structure as the near-infrared fluorophore and dithia-dioxa-monoaza crown ether moiety as the receptor, was developed as a “naked-eye” indicator for Hg2+ with rapid response, good binding constant (1.05 × 104 M−1), high selectivity and sensitivity. Moreover, OTA-DCM was successfully employed in real aqueous samples and fluorescent imaging for detection of Hg2+ in living cells and zebrafish larvae with low cytotoxicity.
1. Introduction As one of the most toxic metal ions in the environment, mercury and its derivatives are lethal threats to the ecosystem and human health because of their durability and high biological accumulation [1]. Mercury can exist in inorganic, organic or elemental forms and each species displays a different level of toxicity. The detection of inorganic mercury, a precursor of organic or elemental mercury, has attracted much attention. In recent years, myriads of rapid and facile Hg2+ probes for selective signaling or visualization, have been developed, and applied to the analyses of biological and environmental samples [2–12]. The Hg2+-induced mechanisms are mainly based on chelation and fluorometric chemo-dosimetry, such as cyclization [2], hydrolysis [3], ring opening of rhodamine spirolactams [4] and desulphurization reactions [5], and so on. In these probes, the used fluorophores were mainly BODIPY, coumarin, rhodamine, benzothizole, quinoline, naphthalimide and fluorescein, etc. [13]. Unfortunately, the emission maxima of most chemosensors were below 600 nm. By contrast, nearinfrared fluorescent probes are more attractive in practical applications due to minimum photodamage to biomolecules, low light scattering, and negligible low background autofluorescence of biomolecules [14], which attracted lots of researchers. However, near-infrared fluorescent probes for detecting Hg2+ aren't
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much reported [15], to the best of our knowledge. The NIR fluorescent dye for Hg2+ mainly included BODIPY [15b,], coumarin [15e], rhodamine [15 g], and so on [15]. The novel near-infrared fluorophores with colorimetric change, excellent selectivity and high sensitivity for detection of Hg2+ are still in high demand. Recently, dicyanomethylene-4H-chromene (DCM) and its derivatives have attracted considerable interest since they emit light in the red or near infrared (NIR) region [16]. Moreover, the DCM fluorophore has better photostability and larger Stokes shift (about 100 nm) [17]. The chromophore DCM with donor-π-acceptor structure had been designed as a colorimetric fluorophore for detection of various analytes [18]. However, to the best our knowledge, the derivatives based on DCM moiety had been rarely reported for the detection of mercury with high selectivity and sensitivity. Among selective chemsensors for Hg2+, the traditional structure with crown ether skeleton is chosen [19], because the receptor unit would result in good selectivity for Hg2+ as a soft acid [20]. Hence, dithia-dioxa-aza cyclopentadecane [21,22] is chosen as the receptor of Hg2+ in the work. Herein, a novel colorimetric fluorescent probe was designed and synthesized for the detection of Hg2+ with excellent selectivity, which employed dicyanomethylene-4H-chromene as the fluorophore and dithia-dioxa-monoaza crown ether as the recognized site (OTA-DCM in Fig. 1a). The probe showed colorimetric, excellent selectivity and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. He), [email protected] (Q. Sun).
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https://doi.org/10.1016/j.dyepig.2019.107658 Received 19 March 2019; Received in revised form 12 June 2019; Accepted 21 June 2019 Available online 01 August 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (a) Synthetic route for OTA-DCM chemosensors. (b) Fluorescence responses of probe OTA-DCM (10 μM) upon treatment with various metal ions (10 eq.) (c) Naked eyes colorimetric changes of the probe with different metal ions in CH3CN–H2O (1:1, v/v). (d) Fluorescent Photographs under UV–vis lamp (365 nm). (e and f) The reversibility of OTA-DCM by alternately adding Hg2+ and Na2S.
sensitivity toward Hg2+ with no significant interference from other competitive metal ions and anions. Moreover, the near-infrared probe was successfully used in real aqueous samples and fluorescent imaging for Hg2+ in living cells and zebrafish larvae with low cytotoxicity.
peak showed a slight hypsochromic shift from 517 to 498 nm. Consistent with the significant change in UV absorption, the change in colour of the solution was obvious after addition of Hg2+. In Fig. 1c, the free OTA-DCM can be seen as bright rose red coloured solution. The colour of the solution underwent change from rose red to yellow in the presence of Hg2+, however other metal ions did not bring about any obvious change in the colour of the solution. So, OTA-DCM can be used as visual indictor for Hg2+. This technique is therefore superior to other analytical techniques because it has ability to detect Hg2+ by nakedeye. The fluorescence emission spectrum of OTA-DCM showed a strong emission at 645 nm and excellent selectivity. When Hg2+ was added, the strong fluorescence was almost completely quenched (Fig. 1b). Since complexation of Hg2+ with dithia-dioxa-monoaza crown weakened electron-donating ability of the recognition site, which resulted in the blocking of intermolecular charge transfer (ICT) process. However, other metal ions couldn't result in the obvious fluorescence changes of OTA-DCM, except that Ag+ brought about with slight quenching. This may be due to the different electrical properties and
2. Results and discussion 2.1. Specificity of OTA-DCM to Hg2+ Firstly, the binding behavior of OTA-DCM probe with metal ions was investigated by UV absorption and fluorescence spectroscopy in CH3CN/H2O (1:1, v/v). In the absence of Hg2+, the free OTA-DCM exhibited a main absorption peaks at 517 nm and other small peaks at around 392 nm and 415 nm. After the addition of Hg2+, the absorption peaks at 517 nm disappeared gradually, whereas peaks at 435 nm and 415 nm increased with increasing concentrations of Hg2+ (Fig. S8). Other metal ions such as K+, Na+, Ni2+, Co2+, Zn2+, Pb2+, Mg2+, Cu2+, Mn2+, Fe2+, Fe3+, Cr3+ and Al3+ did not show any significant changes. However, upon addition of Ag+, the maximum absorption 2
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Fig. 2. (a) Fluorescence spectra of OTA-DCM (10 μM) upon incremental addition of Hg2+ (0–2 eqv.). Insert: The ratio of fluorescence intensity (F/F0) of OTA-DCM in the presence of increasing Hg2+ concentration. (b) The linearity between F/F0 and Hg2+ concentration. F0: fluorescence intensities at 645 nm without Hg2+. F: fluorescence intensities at 645 nm with Hg2+. (c) Benesi-Hildebrand plot based on a 1:1 binding stoichiometry between OTA-DCM and Hg2+.
show that OTA-DCM has high selectivity for Hg2+, and can be used an excellent visual probe. Moreover, the response of OTA-DCM to Hg2+ was rapid (only 1min, Fig. S9). The fluorescence quenching was very stable over a wide range of pH (2.0–10.0) (Fig. S10). The rapid and stable response of OTA-DCM to Hg2+ was very useful for the detection of real samples with portable device. In order to further verify the specificity, competitive experiments were carried using other interfering cations and anions as shown in Fig. S11. It was found that fluorescence quenching was clearly caused by the mixture containing Hg2+, whereas the fluorescence intensity remained unchanged with metal ions other than Hg2+. Moreover, different anions, such as F−, OH−, CO32−, HCO3−, HSO3−, NO2−, NO3−, HPO42−, H2PO4−, SO32−, SO42− and CH3COO−, showed no obvious interferences. However, the Hg2+-triggered quenching efficiency was affected in the presence of Br−, Cl− and S2−, probably due to precipitation reaction between Hg2+ and Br−, Cl−, and also the strong binding ability of S2− towards Hg2+ ions. These results strongly suggested that OTA-DCM exhibited excellent selectivity toward Hg2+.
2.2. The reversibility of OTA-DCM Talking into account the binding of S2− with Hg2+, the binding ability and reversibility of OTA-DCM to Hg2+ on the fluorescence were examined in the presence of Na2S. It was observed that on addition of Na2S to a solution of OTA-DCM–Hg2+ complex, the fluorescence intensity was immediately restored (Fig. 1e). This was evident from the colour changes of the solution from yellow to pleasantly bright rose red (Fig. 1f). This could be attributed to the fact that the addition of S2− would liberate Hg2+ from the metal-ligand complex, because of the strong binding ability of S2− towards Hg2+ ions, resulting in the revival of the free probe [23]. The fluorescence was quenched after the addition of Hg2+ again, which was accompanied by colour change. The sequence of quenching and revival of fluorescence continued approximately for more than five cycles without loss of sensitivity (Fig. 1f).
Fig. 3. The optimized geometric structures based on B3LYP and SMD salvation model for OTA-DCM and OTA-DCM Hg2+ in mixture solvents, water/acetonitrile (1/1, volume ratio), where the bond lengths are in the unit of Å. Here, both top views and side views are shown.
inappropriate diameters of different metal ions. Fig. 1d showed the photographs of fluorescent response of OTA-DCM probe treated with different metal ions under UV–Vis light. The fluorescence quenching occurred obviously in case of Hg2+, but the other metal ions, including Ag+, did not cause significant changes in fluorescence. These results 3
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Fig. 4. Fluorescence imaging of A549 cells incubated with OTS-DCM (10 μM) for 30min (A–C), and then with Hg2+ (100 μM) for another 30min (E–F) and 60min (G–I). (Left) bright filed image, (Medium) red channel (590–650 nm), (Right) Merge image, (J) average intensity ratios in red channel fluorescent image. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the protons in –CH2- adjacent to the O atoms (Ha and Hb) were shifted slightly downfield (0.08 ppm). The proton (He) also shifted downfield from δ 3.13 to δ 3.52 ppm. Concurrently, the chemical shifts of protons in aromatic shifted low field (0.06–0.45 ppm). These results indicated that S, N, and O in crown ether acted as electron donors for coordination to Hg2+, especially S and N. These coordination weakened the electron-donating ability of recognition site (N, S) to fluorophore, which resulted in blocking of ICT process. In addition, The ESI-MS spectrum of OTA-DCM in the presence of 10 equivalents of Hg2+ had some peaks at m/z 779.1154, 780.1151, 781.1144, 782.1151, 783.1150, 784.1161, which are be assignable to calculated [OTADCM + Hg + Cl]+ ([C30H31ClHgN3O3S2]+, m/z: 779.1172, 780.1173, 781.1193, 782.1196, 783.1230, 784.1167) as shown in Fig. S14. The data further proved the complex formation with a stoichiometric ratio of 1:1. Based on the above studies, a possible structure of 1:1 complex of OTA-DCM with Hg2+ was proposed, which was explained by computational study. The optimized geometries of OTA-DCM, and OTA-DCMHg2+ are shown in Fig. 3, which were consistent with the above observations. The theoretical calculations combined the SMD salvation
2.3. The binding studies The binding properties of OTA-DCM with Hg2+ were studied by fluorescence titration experiments firstly. As shown in Fig. 2, when the concentration of Hg2+ ions increased to 15 μM, no further restoring fluorescence was found, which meant that the fluorescence response had reached its maximum. There was a sensitive linear fluorescence response for Hg2+ in the range from 0 μM to 5 μM with the detection limit of 0.14 μM, based on the equation DL = 3σ/S [23] (Fig. 2b). The binding constant of OTA-DCM and Hg2+ was determined to be 1.05 × 104 M−1 from the slope of the linear (Fig. 2c) according to Benesi-Hildebrand equation, details of which can be seen in the supporting information [24]. The binding stoichiometry and possible pattern recognition were investigated to further understand the binding process. Analysis of Job's plot showed a stoichiometric ratio of 1:1 for the complex formation between OTA-DCM and Hg2+ (Fig. S12), which was consistent with reported literature [22]. 1H NMR studies supported the complexation of Hg2+ with OTA-DCM (Fig. S13). Hc and Hd protons showed a big downfield shifts with 0.43 ppm and 0.42 ppm respectively. Moreover, 4
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Fig. 5. Fluorescence imaging of zebrafish larvae treated with OTA-DCM (10 μM) for 0 h (A–C), 0.5 h (B–F), 1 h (G–I),3 h (J–K), and further treated with Hg2+ (100 μM, M − O) for another 4 h. (Up) bright field imaging, (Medium) red channel (590–650 nm), (Down) merge image. (P) average intensity ratios. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cytotoxicity and excellent biocompatibility for cancer and normal cells. Furthermore, experiments for imaging of intracellular Hg2+ in A549 cells were carried out using OTA-DCM as the fluorescent probe. As shown in Fig. 4, incubation of the cell with OTA-DCM for 30 min produced a significant fluorescence from the intracellular region, which could be observed clearly. After treatment with Hg2+ for 30 min, the fluorescence intensity decreased. After incubating for 30min further, the fluorescent intensity became very low dim and was then quenched. These implied that the intracellular uptake of Hg2+ ions resulted in form of complex of OTA-DCM and Hg2+ to yield a non-fluorescent ensemble. These findings showed that OTA-DCM probe was biocompatible and can be used for detection of Hg2+ by fluorescent imaging in living cells. Importantly, fluorescence imaging in zebrafish were performed in order to further explore the application of OTA-DCM in living body. As shown in Fig. 5, bright fluorescence was observed in the digestive system of the zebrafish larvae, and the fluorescence intensity increased obviously with increasing incubation time. After incubation for 3 h, the remaining OTA-DCM in the zebrafish was removed by three times washing, and a further incubation was continued for another 4 h in the presence of Hg2+. It was found that the fluorescence intensity was much weaker and quenched in the digestive system of the zebrafish larvae. The results showed that OTA-DCM was organism-permeable and was admirably sensitive for detection of Hg2+ in living body.
model provided valuable information regarding the structures, properties and plausible recognition patterns through the B3LYP and NBO analyses and TD-CAM-B3LYP and NTO analyses. These calculations supported blocking of ICT mechanism of the fluorescence quenching by Hg2+. The details are provided in supporting information (Fig. S15, Tables S1 and S2). 2.4. Practical application The major source of mercury pollution is from contaminated natural waters, which is a global problem. In order to evaluate the feasibility of OTA-DCM to detect Hg2+ practically, tap water and lake water (Shahu Lake in Wuhan) samples were collected. Results in Table S3, showed that Hg2+ was absent in three blank samples. And the detected concentrations were close to the concentration of added Hg2+. The relative standard deviations (RSD) were less than 2% based on three measurements. The recovery was in the range from 97% to 103%. These results suggested that OTA-DCM could be satisfactorily applied in the detection of Hg2+ in real water. 2.5. Imaging of living cell and zebrafish Herein the feasibility of OTA-DCM for imaging Hg2+ in living cells was investigated. First, MTT assay with A549 cells and normal cells was used to determine the cytotoxicity of OTA-DCM. The data showed that the cellular viability was estimated to be more than 82% after 24 h (Figs. S16 and S17), which indicated that free OTA-DCM has low 5
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3. Conclusions In summary, we were successful in designing and synthesizing a near-infrared fluorescent probe (OTA-DCM), employing dicy-anomethylene-4H-chromene as the NIR fluorescence group and a dithiadioxa-monoaza crown ether moiety as a highly selective recognition receptor for Hg2+. Compared to other reported work (Table S4), this probe demonstrated superior analytical performance such as a remarkably large Stokes shift (120 nm), rapid response, high selectivity and sensitivity, and good binding constant (1.05 × 104 M−1) with the formation of 1:1 complexes of Hg2+. The detection limit was found to be 0.14 μM by fluorescence titration. Importantly, the solution colour changed from rose red to yellow after the addition of Hg2+, which meant that the probe can be used as a “naked-eye” visual indictor. The binding and plausible recognition patterns were derived from computational calculations. Moreover, OTA-DCM could be successfully applied in the detection of Hg2+ in real aqueous samples. Impressively, OTA-DCM was applied in fluorescent imaging of Hg2+ with excellent cell-membrane-permeability in living cells and zebrafish model. Overall, this probe can be used as an excellent turn-off sensor candidate with colorimetric and NIR fluorescence for Hg2+, which holds a great promise for the detection of Hg2+ in complex living samples. Acknowledgements This work was supported by National Natural Science Foundation of China (21575035, 21804102), the Hubei Provincial Natural Science Foundation of China (2017CFB222) and Foreign Science and Technology Cooperation Fund of Hubei province, China (2015BHE025). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107658. References [1] (a) Aragay G, Pons J, Merkoçi A. Recent trends in macro-, micro-, and nanomaterial-based tools and strategies for heavy-metal detection. Chem Rev 2011;111:3433–58; (b) Foster KL, Stern GA, Pazerniuk MA, Hickie B, Walkusz W, Wang F, Macdonald RW. Mercury biomagnification in marine zooplankton food webs in hudson bay. Environ Sci Technol 2012;46:2952–12959; (c) Diaz de Grenu B, Garcia-Calvo J, Cuevas J, Garcia-Herbosa G, Garcia B, Busto N, lbeas N, Torroba T, Torroba B, Herrera A, Pons S. Chemical speciation of MeHg+ and Hg2+ in aqueous solution and HEK cells nuclei by means of DNA interacting fluorogenic probes. Chem Sci 2015;6:3757–64. [2] Ru J, Tang X, Ju Z, Zhang G, Dou W, Mi X, Wang C, Liu W. Exploitation and application of a highly sensitive Ru(II) complex-based phosphorescent chemodosimeter for Hg2+ in aqueous solutions and living cells. ACS Appl Mater Interfaces 2015;7:4247–56. [3] Zhu B, Wang W, Liu L, Jiang H, Du B, Wei Q. A highly selective colorimetric and long-wavelength fluorescent probe for Hg2+. Sens Actuators B Chem 2014;191:605–11. [4] Tripathi K, Rai A, Yadav AK, Srikrishna S, Kumari N, Mishra L. Fluorescein hydrazone-based supramolecular architectures, molecular recognition, sequential logic operation and cell imaging. RSC Adv 2017;7:2264–72. [5] Ding J, Li H, Wang C, Yang J, Xie Y, Peng Q, Li Q, Li Z. “Turn-On” fluorescent probe for mercury(II): high selectivity and sensitivity and new design approach by the adjustment of the π-bridge. ACS Appl Mater Interfaces 2015;7:11369–76. [6] Liu K, Zhao X, Liu Q, Huo J, Li Z, Wang X. A novel multifunctional BODIPY-derived probe for the sequential recognition of Hg2+ and I−, and the fluorometric detection of Cr3+. Sens Actuators B Chem 2017;239:883–9. [7] Singh G, Sanchita, Rani S, Sharma G, Kalra P, Singh N, Verma V. Coumarin–derived Organosilatranes: functionalization at magnetic silica surface and selective recognition of Hg2+ ion. Sens Actuators B Chem 2018;266:861–72. [8] Fang Y, Li X, Li J-Y, Wang G-Y, Zhou Y, Xu N-Z, Hu Y, Yao C. Thiooxo-Rhodamine B hydrazone derivatives bearing bithiophene group as fluorescent chemosensors for detecting mercury (II) in aqueous media and living HeLa cells. Sens Actuators B Chem 2018;255:1182–90. [9] Xu D, Tang L, Tian M, He P, Yan X. A benzothizole-based fluorescent probe for Hg2+ recognition utilizing ESIPT coupled AIE characteristics. Tetrahedron Lett 2017;58:3654–7.
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