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Fluorescein-based fluorescent sensor with high selectivity for mercury and its imaging in living cells
Inorganic Chemistry Communications 89 (2018) 46–50
Contents lists available at ScienceDirect
Inorganic Chemistry Communications journal homepage: www.elsevier.com/locate/inoche
Short communication
Fluorescein-based fluorescent sensor with high selectivity for mercury and its imaging in living cells Daying Liu a,b,⁎, Yajie Wang a, Ruina Wang c, Bangchen Wang c, Hexi Chang c, Jiatong Chen b, Guangming Yang b, Huarui He c,⁎⁎ a b c
Department of Applied Chemistry, College of Basic Science, Chemistry Experiment Teaching Center, Tianjin Agricultural University, Tianjin, China Department of Chemistry, Department of Biochemistry and Molecular Biology, Key Laboratory of Advanced Energy Materials Chemistry (Ministry of Education), Nankai University, Tianjin, China Heowns Biochem Technologies LLC, Tianjin, China
a r t i c l e
i n f o
Article history: Received 7 November 2017 Received in revised form 15 January 2018 Accepted 17 January 2018 Keywords: Fluorescein Photoinduced electron transfer (PET) Mercury Fluorescent sensor
a b s t r a c t A novel fluorescein-based fluorescent Hg2+ sensor (Sensor-Hg) with N-Ethylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methyl-Phenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, was developed and applied successfully to image Hg2+ in living cells. It demonstrates high selectivity and sensitivity for sensing Hg2+ with about 51-fold enhancement in aqueous solution, with a characteristic emission band of fluorescein at 539 nm. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Mercury is well known as one of the most toxic metals and is widespread in air, water, and soil [1]. As it can cause strong damage to the central nervous system, accumulation of mercury in the human body can lead to various cognitive and motor disorders, and Minamata disease [2]. Mercury pollution becomes a global problem [3]. Therefore, there is a great need for methods of detecting and monitoring mercury levels in the food, environment and water. Current techniques for mercury screening, including atomic absorption/emission spectroscopy and inductively coupled plasma mass spectrometry, often require expensive and sophisticated instrumentation or sample preparation [4]. Fluorescent detection of Hg2+ offers a promising approach for simple and rapid tracking of mercury ion in biological, toxicological and environmental monitoring. Fluorescent sensors are useful and powerful tools in the detection of metal ions, because of their simplicity, high sensitivity, good selectivity and high response speed [5]. The most applied mechanisms of fluorescence signal transduction in the design of
⁎ Correspondence to: D.Y. Liu, Department of Applied Chemistry, College of Basic Science, Chemistry Experiment Teaching Center, Tianjin Agricultural University, Tianjin, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (D. Liu), [email protected] (H. He).
https://doi.org/10.1016/j.inoche.2018.01.016 1387-7003/© 2018 Elsevier B.V. All rights reserved.
fluorescent chemosensors are photoinduced electron transfer (PET) and intermolecular charge transfer (ICT) [6]. For a PET chemosensor, a fluorophore is usually connected via a spacer to a receptor containing a relatively high-energy non-bonding electron pair, such as nitrogen atom, which can transfer an electron to the excited fluorophore and as a result quench the fluorescence. Fluorescent PET (Photoinduced Electron Transfer) sensors are the potent analytical tools for detection of metal ions [7]. On the other hand, the ICT mechanism has been widely used in the design of ratiometric fluorescent chemosensors. When a fluorophore, without a spacer, is directly connected to a receptor (usually an amino group) to form a p-electron conjugation system with electron rich and electron poor terminals, then ICT from the electron donor to receptor would be enhanced upon excitation by light. When a receptor (playing the role of an electron donor within the fluorophore) interacts with a cation, it reduces the electrondonating character of the receptor and a blue shift of the emission spectrum is expected. In the same way, if a cation receptor plays the role of an electron receptor, the interaction between the receptor and the cation would further strengthen the push–pull effects. Then a red shift in emission would be observed. Most ratiomeric fluorescent sensors based on ICT mechanism are reported [8]. Therefore, highly selective and sensitive, PETbased fluorescent Hg2+ sensors are reported more and more [9]. And some examples of fluorescent sensors for mercury have been reported available in living cells [10]. Moreover, for the selective recognition of such a soft heavy metal ion, a sulfur-based functional group should be considered and introduced [11].
D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
Based on those in mind, herein, a novel, highly selective and sensitive, fluorescein-PET-based fluorescent Hg2+ sensor, with NEthylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methylPhenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, was developed and applied successfully to image Hg2+ in living cells. Photo-induced electron transfer (PET) is an electron transfer which occurs when certain photoactive materials interact with light. The general design of a PET-type fluoro-ionophore is the “fluorophore–spacer– receptor (ionophore)” format. A fluorescent moiety (fluorophore) is covalently linked to an ion receptor by means of a non-π-electron-conjugating spacer group, e.g. arkyl group with one to four carbons. Typically, the ionophore will contain a tertiary amine the electrons of which can ligate the cation. The selection of a suitable ionophore was driven by several design criteria. First, the ionophore must contain tertiary nitrogen that can act as an electron donor and will also interact with a bound mercury cation. Additionally, the ionophore's binding properties should be insensitive to pH changes so as to minimize undesirable pH interference to the measurement of mercury. Finally, the ionophore should preferentially bind mercury in the aqueous medium, while also in the presence of other cations. Anilines have been proven to be efficient ionophores. In order to enhance the binding strength, another tertiary amine is introduced by phenolic hydroxyl group. Therefore, we chose to use fluorescein as the fluorophore reporter in designing Sensor Hg, and the introduction of multiple sulfur-based functional groups greatly increased the affinity of the sensor for Hg2+.
2. Experimental section 2.1. Material, measurements and methods Unless otherwise noted, all materials were obtained from Heowns Biochem Technologies LLC and were used without further purification. Flash chromatography was carried out on silica gel (300–400 mesh). 1 H NMR spectra were recorded using Varian 300 MHz. Absorption spectra and fluorescence spectra were measured on the Gangdong A-230 photometer and F-280 fluorometer, respectively.
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2.2. Synthesis Now, we present our design and synthesis of a new fluoresceinbased fluorescent sensor with N-Ethylthioethyl-N-[N′, N′-(2′Diethylthioethylamino)-5′-methyl- Phen oxyethyl]-2-Methoxy Aniline (EDPMA) as receptor. Scheme 1 explains the synthetic route of Sensor Hg. The detailed characterization of the new compounds are described in the Supporting Information. The compounds 1, 3 and 4 were synthesized according to a previously reported procedure [7c]. Synthesis of 3 A suspension of 80 g (307.59 mmol) compound 1, 25 g (205.06 mmol) compound 2, 56 g (410.12 mol) K2CO3 and 33.5 g (205.06 mol) KI in 300 mL acetonitrile was heated under reflux for 20 h. The progress was monitored by TLC (PE: EA = 5:1). After the reaction was completed, the mixture was cooled and solvent was evaporated. The residue was purified by flash column chromatography, to obtain product 44 g. 1HNMR (CDCl3) δ 7.80 (d, J = 8.2 Hz, 1H), 6.86 (ddd, J = 22.3, 12.4, 4.9 Hz, 4H), 6.75–6.66 (m, 2H), 4.30 (t, J = 5.3 Hz, 2H), 3.85 (s, 3H), 3.62 (t, J = 5.3 Hz. 2H), 2.38 (s, 3H). Synthesis of 4:10.0 g (33.08 mmol) compound 3 was dissolved in 50 mL mixed solvent (DCM: MeOH = 1:5), 1 g 10% palladium on activated carbon was added. This suspension was hydrogenated at 2.2 atm. For 18 h, till no more hydrogen uptake was observed. The progress was monitored by TLC (PE: EA = 4:1).The catalyst was filtered off and the solvent was evaporated, to obtain product 7.7 g. 1HNMR (CDCl3) δ 2.17(s, 3H), 3.51(m, 2H), 3.54(s, 2H), 3.76(s, 3H), 4.12(t, 2H), 4.24(s, 1H), 6.5–7.17(m, 7H). Synthesis of 5: A suspension of 0.5 g (1.84 mmol) of compound 4, 3.2 mL (27.54 mmol) of 2-chloroethyl ethyl sulfide, 9.1 mL (55.08 mmol) N, N-diisopropylethyl -amine and 4.57 g (27.54 mmol) of KI in DMF (10 mL) was heated at 110 °C for 20 h under nitrogen atmosphere. The progress was monitored by TLC (PE: EA = 4:1). After the reaction was complete, the mixture was cooled and poured into water. The resultant precipitate was filtered, dissolved in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4, filtered and evaporated to get 0.75 g crude product, which was purified by flash column chromatography, to afford product 295 mg. 1HNMR (CDCl3) δ 7.06 (dd, J = 8.2, 1.6
Scheme 1. The synthetic route of Sensor Hg.
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D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
Fig. 1. (a) Fluorescence spectra of Sensor Hg (25 μM, λex = 470 nm) in the presence of various metal ions in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v); the inset shows the fluorescence change (left) before and (right) after the addition of Hg2+ ions. (b) The relative fluorescence intensity of Sensor Hg (25 μM) in the presence of various metal ions in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v).
Hz, 1H), 7.00 (dd, J = 7.3, 2.1 Hz, 1H), 6.90 (dd, J = 10.7, 4.6 Hz, 3H), 6.66 (d, J = 8.9 Hz, 1H), 6.61 (s, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.86 (s, 3H), 3.60 (t, J = 6.5 Hz, 2H), 3.48–3.41 (m, 2H), 3.34–3.25 (m, 4H), 2.68–2.47 (m, 12H), 2.25 (s, 3H), 1.22 (ddd, J = 10.0, 7.0, 3.3 Hz, 9H). Synthesis of 6: The Vilsmeier reagent was prepared by adding POCl3 (1 mL) dropwise to ice-cold dry DMF (2 mL) under stirring. After 30 min, to the above Vilsmeier reagent was added compound 5 (250 mg, 0.465 mmol) as a solution in DMF (1.0 mL). Then the mixture was further heated at 65 °C. The progress was monitored by TLC (PE: EA = 4:1). After the reaction was complete, the mixture was cooled to room temperature, then poured into ice-water and the pH of the aqueous mixture was adjusted to 8–9 with Na2CO3 saturated solution. The product was extracted into twice. The combined organic extracts were dried over anhydrous Na2SO4, filtered. The organic layer was removed under reduced pressure, and then the residue was purified by flash column chromatography, to obtain product 200 mg. 1HNMR (CDCl3) δ 9.780 (s, 1H), 7.06 (dd, J = 8.2, 1.6 Hz, 1H), 7.00 (dd, J = 7.3, 2.1 Hz, 1H), 6.90 (dd, J = 10.7, 4.6 Hz, 3H), 6.66 (d, J = 8.9 Hz, 1H), 6.61 (s, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.86 (s, 3H), 3.60 (t, J = 6.5 Hz, 2H), 3.48–3.41 (m, 2H), 3.34–3.25 (m, 4H), 2.68–2.47 (m, 12H), 2.25 (s, 3H), 1.22 (ddd, J = 10.0, 7.0, 3.3 Hz, 9H). Synthesis of Sensor Hg: A solution of compound 6 (100 mg, 0.17 mmol), 4-Chlororesorcinol (50 mg, 0.35 mmol) and methanesulfonic acid (1 mL) were placed in a round bottom flask dissolved in 1 mL mixture of dichloromethane-ether (v:v) and stirred at room temperature overnight. After reaction was complete monitored by TLC, the reaction mixture was poured into water. The resulting suspension was extracted with ethyl acetate (10 mL × 3). The combined organic extracts were dried over MgSO4 and evaporated, and purification by preparative thin-layer chromatography gave pure compound 7. A solution of 2,3dichloro-5,6-dicyano-1,4- benzoquinone (DDQ, 137 mg, 0.60 mmol) in 1:1 AcOH/benzene (2 mL) was added dropwise into a solution of compound 2 (251 mg, 0.30 mmol) in 2 mL of 1:1 AcOH/benzene at room temperature. The resulting mixture was stirred 3 h at room temperature. The progress was monitored by TLC. After the reaction was complete, the reaction was concentrated in vacuo, and purification by preparative thin-layer chromatography gave pure Sensor Hg. 1HNMR (CDCl3) δ 1.21(m,15H), 2.25(s, 3H), 2.55(m, 15H), 3.27(m, 4H), 3.53(t, 2H), 3.78(t, 2H), 3.84(s, 3H), 4.18(t, 2H), 6.68–7.43(m, 10H).
wavelength was set at 470 nm, it exhibited a characteristic emission band of fluorescein at 529 nm, a weak green color emission [12]. To obtain insight into the sensing properties of Sensor Hg toward metal ions, the emission changes were examined with different ions such as Mn2+, Fe2+, Fe3+,Co2+, Cr3+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Ag+, Mg2+, Ca2+, K+, Na+, La3+, Eu3+, Er3+ in HEPES buffer solution (20 mM, pH = 7.4). The fluorescence changes of Sensor Hg are depicted in Fig. 1. As shown in Fig. 1, the addition of Hg2+ to a solution of Sensor Hg induced a significant enhancement of fluorescence (ca. 51-fold) with the emission maximum at 539 nm. However, under identical conditions, nearly no fluorescence intensity changes were observed in emission spectra with the other ions. The results indicated that Sensor Hg was a highly selective fluorescent sensor for Hg2+ in aqueous solution. Achieving high selectivity toward Hg2+ over the other competitive species coexisting is a very important feature to evaluate the performance of the fluorescent Sensor Hg. Therefore, the competition experiments were also conducted for Sensor Hg. Fig. 2 shows that when Hg2+ was added into the solution of Sensor Hg in the presence of other ions, the emission spectra displayed a similar pattern at 529 nm to that with Hg2 + only. It indicated that the selectivity against other metals remained. The responses of Sensor Hg to increasing Hg2+ concentration were investigated shown in Fig. 3. Upon the addition of Hg2+, the
3. Results and discussion 3.1. Fluorescent properties The fluorescence properties of Sensor Hg were evaluated in aqueous solution (20 mM HEPES buffer, pH = 7.4). When the excitation
Fig. 2. The fluorescent intensity at 529 nm of Sensor Hg (25 μM) with 125 μM of various metal ions (black bar), and then added 125 μM of Hg2+ (red bar) in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v).
D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
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Fig. 3. (a) Fluorescence spectra of Sensor Hg (25 μM, λex = 470 nm) upon the titration of Hg2+ (0–80 μM) in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v); (b) Fluorescence intensity of Sensor Hg (25 μM) versus increasing concentrations;
fluorescence emission intensity of Sensor Hg gradually increased by about 51-fold without change in the emission spectra. The results indicate that Sensor Hg is highly sensitive to Hg2+ and can be potentially used to quantitatively detect Hg2+ concentration. These results clearly demonstrate that the Sensor Hg has excellent affinity for Hg2+ over other metal ions and can be used as a fluorescent sensor with high selectivity and sensitivity for mercury in aqueous solution. Moreover, to obtain the detection limit of the Hg sensors, the emission intensity of ion-free Hg sensors was measured 10 times and the standard deviation of blank measurements was determined. The fluorescence intensity of Hg sensors (2.5 × 10−5 mol·L−1) at 539 nm was found to increase linearly with the concentration of Hg2+ in range of 1.0–4.0 × 10−5 mol·L−1 (R2 = 0.9990) (Fig. S1). The detection limit was then calculated with the equation [13]: Detection limit ¼ 3σ=k
Fig. 4. Job's plot for Sensor Hg (forms 1:1 complexes) in 20 mM pH = 7.4 HEPES buffer solution (buffer/methanol = 95/5, v/v). The total concentration of Sensor Hg and Hg2+ is 10 μM.
ð1Þ
where σ is the standard deviation of blank measurement, and k is the slope of the intensity versus Hg2+ concentration. The detection limit of Hg2+ in HEPES buffer solution (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v) was measured to be 1.10 × 10−7 mol·L−1 or 22.06ppb for Hg2+. Compared with the previously reported Hg2+-fluorescent sensors, the value of detection limit of the Hg sensors is lower or
Fig. 5. Fluorescence images and their corresponding bright-field transmission images: (a) HeLa cells incubated with Sensor Hg; (b) HeLa cells incubated with Sensor Hg for 24 h, then washed three times, and then further incubated with 10 μM Hg2+ for 4 h.
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D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
in the same range [14] and it is low enough to detect mercury levels in environmental sources and water [15]. The Job's plot, which exhibited a maximum at the 0.5 M fraction of Hg2+, indicated the 1:1 binding ratio between Hg2+ and Sensor Hg (Fig. 4).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.inoche.2018.01.016. References
3.2. Fluorescent detection of Hg2+ in living cells To further demonstrate the practical biological application of Sensor Hg, fluorescence imaging experiments were carried out in living cells. As indicated in Fig. 5, when HeLa cells were only treated with 25 μM of Sensor Hg, no fluorescence was observed. In contrast, upon treatment of 10 μM of Hg2+ to the cells pre-treated with Sensor Hg, strong green fluorescence emission was observed. The bright-field transmission and fluorescence images revealed that the fluorescence signal resulted from the intracellular region. The green fluorescence of the cells is similar to that of Sensor Hg in solution, which indicates that Sensor Hg is membrane permeable. Taken together, these results show that Sensor Hg has good membrane permeability and can be used as a sensor for detecting intracellular Hg2+ in living cells. 4. Conclusion In conclusion, a novel highly selective and sensitive, fluoresceinbased fluorescent Hg2+ sensor, with N-Ethylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methyl -Phenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, has been developed and applied successfully to image Hg2+ in living cells. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 20941004, 21071084, 90922032, and 21404082) and the MOE (IRT-0927), Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, National Students' Program for Innovation and Entrepreneurship Training (No. 201610061080), Tianjin Agricultural University Science Development Fund (2016NZD06), Natural Science Foundation of Tianjin (No. 15JCQNJC05900). The authors acknowledge the helpful discussions and collaboration from co-workers within Heowns Biochem Technologies LLC.
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Contents lists available at ScienceDirect
Inorganic Chemistry Communications journal homepage: www.elsevier.com/locate/inoche
Short communication
Fluorescein-based fluorescent sensor with high selectivity for mercury and its imaging in living cells Daying Liu a,b,⁎, Yajie Wang a, Ruina Wang c, Bangchen Wang c, Hexi Chang c, Jiatong Chen b, Guangming Yang b, Huarui He c,⁎⁎ a b c
Department of Applied Chemistry, College of Basic Science, Chemistry Experiment Teaching Center, Tianjin Agricultural University, Tianjin, China Department of Chemistry, Department of Biochemistry and Molecular Biology, Key Laboratory of Advanced Energy Materials Chemistry (Ministry of Education), Nankai University, Tianjin, China Heowns Biochem Technologies LLC, Tianjin, China
a r t i c l e
i n f o
Article history: Received 7 November 2017 Received in revised form 15 January 2018 Accepted 17 January 2018 Keywords: Fluorescein Photoinduced electron transfer (PET) Mercury Fluorescent sensor
a b s t r a c t A novel fluorescein-based fluorescent Hg2+ sensor (Sensor-Hg) with N-Ethylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methyl-Phenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, was developed and applied successfully to image Hg2+ in living cells. It demonstrates high selectivity and sensitivity for sensing Hg2+ with about 51-fold enhancement in aqueous solution, with a characteristic emission band of fluorescein at 539 nm. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Mercury is well known as one of the most toxic metals and is widespread in air, water, and soil [1]. As it can cause strong damage to the central nervous system, accumulation of mercury in the human body can lead to various cognitive and motor disorders, and Minamata disease [2]. Mercury pollution becomes a global problem [3]. Therefore, there is a great need for methods of detecting and monitoring mercury levels in the food, environment and water. Current techniques for mercury screening, including atomic absorption/emission spectroscopy and inductively coupled plasma mass spectrometry, often require expensive and sophisticated instrumentation or sample preparation [4]. Fluorescent detection of Hg2+ offers a promising approach for simple and rapid tracking of mercury ion in biological, toxicological and environmental monitoring. Fluorescent sensors are useful and powerful tools in the detection of metal ions, because of their simplicity, high sensitivity, good selectivity and high response speed [5]. The most applied mechanisms of fluorescence signal transduction in the design of
⁎ Correspondence to: D.Y. Liu, Department of Applied Chemistry, College of Basic Science, Chemistry Experiment Teaching Center, Tianjin Agricultural University, Tianjin, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (D. Liu), [email protected] (H. He).
https://doi.org/10.1016/j.inoche.2018.01.016 1387-7003/© 2018 Elsevier B.V. All rights reserved.
fluorescent chemosensors are photoinduced electron transfer (PET) and intermolecular charge transfer (ICT) [6]. For a PET chemosensor, a fluorophore is usually connected via a spacer to a receptor containing a relatively high-energy non-bonding electron pair, such as nitrogen atom, which can transfer an electron to the excited fluorophore and as a result quench the fluorescence. Fluorescent PET (Photoinduced Electron Transfer) sensors are the potent analytical tools for detection of metal ions [7]. On the other hand, the ICT mechanism has been widely used in the design of ratiometric fluorescent chemosensors. When a fluorophore, without a spacer, is directly connected to a receptor (usually an amino group) to form a p-electron conjugation system with electron rich and electron poor terminals, then ICT from the electron donor to receptor would be enhanced upon excitation by light. When a receptor (playing the role of an electron donor within the fluorophore) interacts with a cation, it reduces the electrondonating character of the receptor and a blue shift of the emission spectrum is expected. In the same way, if a cation receptor plays the role of an electron receptor, the interaction between the receptor and the cation would further strengthen the push–pull effects. Then a red shift in emission would be observed. Most ratiomeric fluorescent sensors based on ICT mechanism are reported [8]. Therefore, highly selective and sensitive, PETbased fluorescent Hg2+ sensors are reported more and more [9]. And some examples of fluorescent sensors for mercury have been reported available in living cells [10]. Moreover, for the selective recognition of such a soft heavy metal ion, a sulfur-based functional group should be considered and introduced [11].
D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
Based on those in mind, herein, a novel, highly selective and sensitive, fluorescein-PET-based fluorescent Hg2+ sensor, with NEthylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methylPhenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, was developed and applied successfully to image Hg2+ in living cells. Photo-induced electron transfer (PET) is an electron transfer which occurs when certain photoactive materials interact with light. The general design of a PET-type fluoro-ionophore is the “fluorophore–spacer– receptor (ionophore)” format. A fluorescent moiety (fluorophore) is covalently linked to an ion receptor by means of a non-π-electron-conjugating spacer group, e.g. arkyl group with one to four carbons. Typically, the ionophore will contain a tertiary amine the electrons of which can ligate the cation. The selection of a suitable ionophore was driven by several design criteria. First, the ionophore must contain tertiary nitrogen that can act as an electron donor and will also interact with a bound mercury cation. Additionally, the ionophore's binding properties should be insensitive to pH changes so as to minimize undesirable pH interference to the measurement of mercury. Finally, the ionophore should preferentially bind mercury in the aqueous medium, while also in the presence of other cations. Anilines have been proven to be efficient ionophores. In order to enhance the binding strength, another tertiary amine is introduced by phenolic hydroxyl group. Therefore, we chose to use fluorescein as the fluorophore reporter in designing Sensor Hg, and the introduction of multiple sulfur-based functional groups greatly increased the affinity of the sensor for Hg2+.
2. Experimental section 2.1. Material, measurements and methods Unless otherwise noted, all materials were obtained from Heowns Biochem Technologies LLC and were used without further purification. Flash chromatography was carried out on silica gel (300–400 mesh). 1 H NMR spectra were recorded using Varian 300 MHz. Absorption spectra and fluorescence spectra were measured on the Gangdong A-230 photometer and F-280 fluorometer, respectively.
47
2.2. Synthesis Now, we present our design and synthesis of a new fluoresceinbased fluorescent sensor with N-Ethylthioethyl-N-[N′, N′-(2′Diethylthioethylamino)-5′-methyl- Phen oxyethyl]-2-Methoxy Aniline (EDPMA) as receptor. Scheme 1 explains the synthetic route of Sensor Hg. The detailed characterization of the new compounds are described in the Supporting Information. The compounds 1, 3 and 4 were synthesized according to a previously reported procedure [7c]. Synthesis of 3 A suspension of 80 g (307.59 mmol) compound 1, 25 g (205.06 mmol) compound 2, 56 g (410.12 mol) K2CO3 and 33.5 g (205.06 mol) KI in 300 mL acetonitrile was heated under reflux for 20 h. The progress was monitored by TLC (PE: EA = 5:1). After the reaction was completed, the mixture was cooled and solvent was evaporated. The residue was purified by flash column chromatography, to obtain product 44 g. 1HNMR (CDCl3) δ 7.80 (d, J = 8.2 Hz, 1H), 6.86 (ddd, J = 22.3, 12.4, 4.9 Hz, 4H), 6.75–6.66 (m, 2H), 4.30 (t, J = 5.3 Hz, 2H), 3.85 (s, 3H), 3.62 (t, J = 5.3 Hz. 2H), 2.38 (s, 3H). Synthesis of 4:10.0 g (33.08 mmol) compound 3 was dissolved in 50 mL mixed solvent (DCM: MeOH = 1:5), 1 g 10% palladium on activated carbon was added. This suspension was hydrogenated at 2.2 atm. For 18 h, till no more hydrogen uptake was observed. The progress was monitored by TLC (PE: EA = 4:1).The catalyst was filtered off and the solvent was evaporated, to obtain product 7.7 g. 1HNMR (CDCl3) δ 2.17(s, 3H), 3.51(m, 2H), 3.54(s, 2H), 3.76(s, 3H), 4.12(t, 2H), 4.24(s, 1H), 6.5–7.17(m, 7H). Synthesis of 5: A suspension of 0.5 g (1.84 mmol) of compound 4, 3.2 mL (27.54 mmol) of 2-chloroethyl ethyl sulfide, 9.1 mL (55.08 mmol) N, N-diisopropylethyl -amine and 4.57 g (27.54 mmol) of KI in DMF (10 mL) was heated at 110 °C for 20 h under nitrogen atmosphere. The progress was monitored by TLC (PE: EA = 4:1). After the reaction was complete, the mixture was cooled and poured into water. The resultant precipitate was filtered, dissolved in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4, filtered and evaporated to get 0.75 g crude product, which was purified by flash column chromatography, to afford product 295 mg. 1HNMR (CDCl3) δ 7.06 (dd, J = 8.2, 1.6
Scheme 1. The synthetic route of Sensor Hg.
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Fig. 1. (a) Fluorescence spectra of Sensor Hg (25 μM, λex = 470 nm) in the presence of various metal ions in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v); the inset shows the fluorescence change (left) before and (right) after the addition of Hg2+ ions. (b) The relative fluorescence intensity of Sensor Hg (25 μM) in the presence of various metal ions in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v).
Hz, 1H), 7.00 (dd, J = 7.3, 2.1 Hz, 1H), 6.90 (dd, J = 10.7, 4.6 Hz, 3H), 6.66 (d, J = 8.9 Hz, 1H), 6.61 (s, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.86 (s, 3H), 3.60 (t, J = 6.5 Hz, 2H), 3.48–3.41 (m, 2H), 3.34–3.25 (m, 4H), 2.68–2.47 (m, 12H), 2.25 (s, 3H), 1.22 (ddd, J = 10.0, 7.0, 3.3 Hz, 9H). Synthesis of 6: The Vilsmeier reagent was prepared by adding POCl3 (1 mL) dropwise to ice-cold dry DMF (2 mL) under stirring. After 30 min, to the above Vilsmeier reagent was added compound 5 (250 mg, 0.465 mmol) as a solution in DMF (1.0 mL). Then the mixture was further heated at 65 °C. The progress was monitored by TLC (PE: EA = 4:1). After the reaction was complete, the mixture was cooled to room temperature, then poured into ice-water and the pH of the aqueous mixture was adjusted to 8–9 with Na2CO3 saturated solution. The product was extracted into twice. The combined organic extracts were dried over anhydrous Na2SO4, filtered. The organic layer was removed under reduced pressure, and then the residue was purified by flash column chromatography, to obtain product 200 mg. 1HNMR (CDCl3) δ 9.780 (s, 1H), 7.06 (dd, J = 8.2, 1.6 Hz, 1H), 7.00 (dd, J = 7.3, 2.1 Hz, 1H), 6.90 (dd, J = 10.7, 4.6 Hz, 3H), 6.66 (d, J = 8.9 Hz, 1H), 6.61 (s, 1H), 4.03 (t, J = 6.5 Hz, 2H), 3.86 (s, 3H), 3.60 (t, J = 6.5 Hz, 2H), 3.48–3.41 (m, 2H), 3.34–3.25 (m, 4H), 2.68–2.47 (m, 12H), 2.25 (s, 3H), 1.22 (ddd, J = 10.0, 7.0, 3.3 Hz, 9H). Synthesis of Sensor Hg: A solution of compound 6 (100 mg, 0.17 mmol), 4-Chlororesorcinol (50 mg, 0.35 mmol) and methanesulfonic acid (1 mL) were placed in a round bottom flask dissolved in 1 mL mixture of dichloromethane-ether (v:v) and stirred at room temperature overnight. After reaction was complete monitored by TLC, the reaction mixture was poured into water. The resulting suspension was extracted with ethyl acetate (10 mL × 3). The combined organic extracts were dried over MgSO4 and evaporated, and purification by preparative thin-layer chromatography gave pure compound 7. A solution of 2,3dichloro-5,6-dicyano-1,4- benzoquinone (DDQ, 137 mg, 0.60 mmol) in 1:1 AcOH/benzene (2 mL) was added dropwise into a solution of compound 2 (251 mg, 0.30 mmol) in 2 mL of 1:1 AcOH/benzene at room temperature. The resulting mixture was stirred 3 h at room temperature. The progress was monitored by TLC. After the reaction was complete, the reaction was concentrated in vacuo, and purification by preparative thin-layer chromatography gave pure Sensor Hg. 1HNMR (CDCl3) δ 1.21(m,15H), 2.25(s, 3H), 2.55(m, 15H), 3.27(m, 4H), 3.53(t, 2H), 3.78(t, 2H), 3.84(s, 3H), 4.18(t, 2H), 6.68–7.43(m, 10H).
wavelength was set at 470 nm, it exhibited a characteristic emission band of fluorescein at 529 nm, a weak green color emission [12]. To obtain insight into the sensing properties of Sensor Hg toward metal ions, the emission changes were examined with different ions such as Mn2+, Fe2+, Fe3+,Co2+, Cr3+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Ag+, Mg2+, Ca2+, K+, Na+, La3+, Eu3+, Er3+ in HEPES buffer solution (20 mM, pH = 7.4). The fluorescence changes of Sensor Hg are depicted in Fig. 1. As shown in Fig. 1, the addition of Hg2+ to a solution of Sensor Hg induced a significant enhancement of fluorescence (ca. 51-fold) with the emission maximum at 539 nm. However, under identical conditions, nearly no fluorescence intensity changes were observed in emission spectra with the other ions. The results indicated that Sensor Hg was a highly selective fluorescent sensor for Hg2+ in aqueous solution. Achieving high selectivity toward Hg2+ over the other competitive species coexisting is a very important feature to evaluate the performance of the fluorescent Sensor Hg. Therefore, the competition experiments were also conducted for Sensor Hg. Fig. 2 shows that when Hg2+ was added into the solution of Sensor Hg in the presence of other ions, the emission spectra displayed a similar pattern at 529 nm to that with Hg2 + only. It indicated that the selectivity against other metals remained. The responses of Sensor Hg to increasing Hg2+ concentration were investigated shown in Fig. 3. Upon the addition of Hg2+, the
3. Results and discussion 3.1. Fluorescent properties The fluorescence properties of Sensor Hg were evaluated in aqueous solution (20 mM HEPES buffer, pH = 7.4). When the excitation
Fig. 2. The fluorescent intensity at 529 nm of Sensor Hg (25 μM) with 125 μM of various metal ions (black bar), and then added 125 μM of Hg2+ (red bar) in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v).
D. Liu et al. / Inorganic Chemistry Communications 89 (2018) 46–50
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Fig. 3. (a) Fluorescence spectra of Sensor Hg (25 μM, λex = 470 nm) upon the titration of Hg2+ (0–80 μM) in HEPES buffer (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v); (b) Fluorescence intensity of Sensor Hg (25 μM) versus increasing concentrations;
fluorescence emission intensity of Sensor Hg gradually increased by about 51-fold without change in the emission spectra. The results indicate that Sensor Hg is highly sensitive to Hg2+ and can be potentially used to quantitatively detect Hg2+ concentration. These results clearly demonstrate that the Sensor Hg has excellent affinity for Hg2+ over other metal ions and can be used as a fluorescent sensor with high selectivity and sensitivity for mercury in aqueous solution. Moreover, to obtain the detection limit of the Hg sensors, the emission intensity of ion-free Hg sensors was measured 10 times and the standard deviation of blank measurements was determined. The fluorescence intensity of Hg sensors (2.5 × 10−5 mol·L−1) at 539 nm was found to increase linearly with the concentration of Hg2+ in range of 1.0–4.0 × 10−5 mol·L−1 (R2 = 0.9990) (Fig. S1). The detection limit was then calculated with the equation [13]: Detection limit ¼ 3σ=k
Fig. 4. Job's plot for Sensor Hg (forms 1:1 complexes) in 20 mM pH = 7.4 HEPES buffer solution (buffer/methanol = 95/5, v/v). The total concentration of Sensor Hg and Hg2+ is 10 μM.
ð1Þ
where σ is the standard deviation of blank measurement, and k is the slope of the intensity versus Hg2+ concentration. The detection limit of Hg2+ in HEPES buffer solution (20 mM, pH = 7.4, buffer/methanol = 95/5, v/v) was measured to be 1.10 × 10−7 mol·L−1 or 22.06ppb for Hg2+. Compared with the previously reported Hg2+-fluorescent sensors, the value of detection limit of the Hg sensors is lower or
Fig. 5. Fluorescence images and their corresponding bright-field transmission images: (a) HeLa cells incubated with Sensor Hg; (b) HeLa cells incubated with Sensor Hg for 24 h, then washed three times, and then further incubated with 10 μM Hg2+ for 4 h.
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in the same range [14] and it is low enough to detect mercury levels in environmental sources and water [15]. The Job's plot, which exhibited a maximum at the 0.5 M fraction of Hg2+, indicated the 1:1 binding ratio between Hg2+ and Sensor Hg (Fig. 4).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.inoche.2018.01.016. References
3.2. Fluorescent detection of Hg2+ in living cells To further demonstrate the practical biological application of Sensor Hg, fluorescence imaging experiments were carried out in living cells. As indicated in Fig. 5, when HeLa cells were only treated with 25 μM of Sensor Hg, no fluorescence was observed. In contrast, upon treatment of 10 μM of Hg2+ to the cells pre-treated with Sensor Hg, strong green fluorescence emission was observed. The bright-field transmission and fluorescence images revealed that the fluorescence signal resulted from the intracellular region. The green fluorescence of the cells is similar to that of Sensor Hg in solution, which indicates that Sensor Hg is membrane permeable. Taken together, these results show that Sensor Hg has good membrane permeability and can be used as a sensor for detecting intracellular Hg2+ in living cells. 4. Conclusion In conclusion, a novel highly selective and sensitive, fluoresceinbased fluorescent Hg2+ sensor, with N-Ethylthioethyl-N-[N′,N′-(2′Diethylthioethylamino)-5′-methyl -Phenoxyethyl]-2-Methoxy Aniline (EDPMA) as receptor, has been developed and applied successfully to image Hg2+ in living cells. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 20941004, 21071084, 90922032, and 21404082) and the MOE (IRT-0927), Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, National Students' Program for Innovation and Entrepreneurship Training (No. 201610061080), Tianjin Agricultural University Science Development Fund (2016NZD06), Natural Science Foundation of Tianjin (No. 15JCQNJC05900). The authors acknowledge the helpful discussions and collaboration from co-workers within Heowns Biochem Technologies LLC.
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