Talanta 132 (2015) 727–732
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Living cells imaging for copper and hydrogen sulfide by a selective “on–off–on” fluorescent probe Yong Qian n, Jie Lin 1, Tianbao Liu 1, Hailiang Zhu n State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 July 2014 Received in revised form 11 October 2014 Accepted 15 October 2014 Available online 24 October 2014
A novel highly selective and sensitive fluorescent probe (NJ1) had been designed and synthesized for Cu2 þ detection by fluorescence quenching mechanism, and then the enhancement of fluorescence intensity with the addition of hydrogen sulfide in complex NJ1Cu aqueous solution at physiological conditions were described. This “on–off–on” type fluorescence recognition system was a reversible process, which could be utilized to monitor copper ion and hydrogen sulfide based on a complex NJ1Cu formation–Cu2 þ displacement approach. Importantly, this real-time recognition process of Cu2 þ and hydrogen sulfide exhibited excellent anti-interference ability. In addition, this new fluorescent probe also has potential utility for Cu2 þ and hydrogen sulfide detection in living cells, providing a potential tool for investigating copper ion and hydrogen sulfide in living systems or environment. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Copper ion Hydrogen sulfide Displacement approach Imaging
1. Introduction Developing a sensitive, reliable and selective detection technique for various chemically and biologically pertinent metal cations and anions has attracted many research interests [1]. As the third most abundant transition metal in the human body, copper plays an important role in various physiological processes of organism [2]. However, the excessive exposure to these Cu2 þ sources could cause imbalance in cellular processes, resulting in various diseases such as Alzheimer's disease and Parkinson's disease [3]. Additionally, copper has also been widely used in the industrial process, which is potentially toxic to environment. Therefore, there is an increasing need to monitor the existence of Cu2 þ in the live biological samples or aquatic environment [4]. Fluorescent probes have become an important diagnostic tool for biological and environmental concern considering their sensitivity, special selectivity, and real-time monitoring with fast response time [5]. Owning to the notorious paramagnetic nature of Cu2 þ , it usually leads to fluorescence quenching effect of the bound fluorophore [6]. More interestingly, this Cu2 þ complex would also be potentially applied to monitor other substances, such as sulfide anion [7]. Hydrogen sulfide (H2S) is a toxic gas best known for its rotten egg smell. It is slightly soluble in water, giving the hydrosulfide anion HS (pKa ¼6.9) and the sulfide dianion S2 (pKa 414). H2S has been utilized as a reactant or produced as a by-product in n
Corresponding authors. Tel.: þ 86 025 8359 2672. E-mail addresses: [email protected] (Y. Qian), [email protected] (H. Zhu). 1 These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.talanta.2014.10.034 0039-9140/& 2014 Elsevier B.V. All rights reserved.
industrial process, which is widely spread in the environment. However, the role of H2S in living organisms has recently received increasing attention. It has been suggested as the third signaling gasotransmitter, along with nitric oxide and carbon monoxide [8]. It is shown to act as a vascular relaxant, an energy regulator, and to be associated with cardiovascular regulation and the inflammation response [9]. Altering the level of hydrogen sulfide in human body or exposure to high-level sulfide can cause various physiological and biochemical problems. Therefore, the detection of hydrogen sulfide in industry, environment, and biological samples has attracted great attention. Several conventional detection strategies have been developed for hydrogen sulfide [10], such as electrochemical methods and ion chromatography. However, these analytic methods usually need complicated equipment and trou blesome sample pretreatment that are unsuitable for the analysis of sulfide in real time. For this reason, more convenient detection tools, such as fluorescent probes, have received significant attention as their easy operability and high sensitivity. To the best of our knowledge, several hydrogen sulfide selective probes based on copper ion displacement approach have been reported [7]. However, most of these probes have some limitations such as poor water solubility, complicated synthesis, poor membrane permeability, and detection systems required much organic co-solvents [7c], which would significantly restrict their practical applications in physiological condition. So far, real-time sulfide selective probes that are applicable in aqueous solution are still limited. It is thus desirable to develop simple and effective fluorescent probes for detection of sulfide in aqueous media that displays high sensitivity and selectivity as well as enables easier accessibility.
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Y. Qian et al. / Talanta 132 (2015) 727–732
Scheme 1. Synthesis of NJ1 and the proposed mechanism of the monitoring system.
With this in mind, we designed and synthesized a novel small fluorescent probe NJ1 (Scheme 1). NJ1 demonstrated obvious fluorescence quenching after the addition of copper ion to form new complex NJ1Cu. This new complex displayed high specificity for hydrogen sulfide that induced copper ion to be released from the dye in the aqueous media, resulting in fluorescence enhancement. Herein, we reported the synthesis, photophysical characterization of NJ1 that was highly sensitive and selective for Cu2 þ . The subsequent complex NJ1Cu could be developed as a platform for hydrogen sulfide real-time detection that could be potentially utilized in industry, environment, or biological requirements. Furthermore, the imaging of copper ion and hydrogen sulfide in living cells were also successfully developed.
2. Experimental 2.1. Materials and instrumentations All chemicals were purchased from commercial suppliers and used without further purification. Chromatographic purification of products was accomplished by using forced-flow chromatography on silica gel (300–400 mesh). Thin layer chromatography was performed on EM Science silica gel 60 F254 plates (250 μm). Visualization of the developed chromatogram was accomplished using UV lamp. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX-400 operating at 100 MHz for 1H NMR and 13C NMR; residual protio solvent signals were used as internal standards for calibration purposes. Data for 1H NMR are reported as follows: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet), integration, coupling constant (Hz). TOF EI mass was performed by Mass Spectrometry Facility at Nanjing University. All fluorescence measurements were recorded on a Varian Cary Eclipse Fluorescence Spectrophotometer and Hitachi Fluorescence Spectrophotometer F-7000. The pH measurements were carried out on a HI 2221 calibration check pH/ORP meter. 2.2. General procedure for analysis Stock solution of metal ions and other anions were prepared in ultrapure H2O, stock solution of NJ1 (1 mM) were prepared in DMSO, which was diluted to the required concentration for measurement. All fluorescence measurements were carried out at room temperature on a Hitachi Fluorescence Spectrophotometer F-7000.
The samples were excited at 360 nm; the emission spectrum was scanned from 425 nm to 650 nm at 1200 nm/min. 2.3. Cell culture and fluorescence imaging HeLa cells were grown up in DMEM medium with 10% fetal bovine serum/penicillin/streptomycin in a 5% CO2 atmosphere at 37 1C. Cells were then seeded on Coverglass-Bottom confocaldish and continuously incubated at 37 1C in a 5% CO2 atmosphere for 24 h. After cells were incubated with 1 μL, 10 mM NJ1(final concentration: 10 μM) for 30 min at 37 1C, media was removed, and cells were washed with HEPES buffer three times again to remove any probe left in solution to optimize the background signal. Then, cells were added to fresh media and incubated with Cu2 þ (0 μM, 10 μM) for another 20 min at 37 1C before imaging; or NaHS (10 μM) was added into the cells with NJ1 (10 μM) and Cu (10 μM) for another 20 min at 37 1C before imaging. All imaging experiments were performed on a fixed cell DSU spinning confocal microscope (Olympus), excitation at 405 nm. Imaging was performed using X40 objective and captured using Slidebook software. For all experiments, solution of NJ1 was prepared in DMSO (10 mM) and diluted into DMEM to the desired working concentration (10 μM). Cu2 þ , and NaHS were diluted into ddH2O to the desired working concentrations (10 μM) from a 10-mM stock solution. 2.4. Synthesis of probe NJ1 2.4.1. (E)-7-(dimethylamino)-3-((2-(pyridine-2-yl)hydrazono) methyl)naphthalene-2-ol To a mixture compounds 3- (645 mg, 3 mmol) and 2hydrazinylpyridine (360 mg, 3.3 mmol) was added ethanol under argon. The mixture was stirred at 80 1C for 1 h. The solvent was then evaporated under reduced pressure and the obtained residue was purified by silica gel column chromatography (eluent: 30 ethyl acetate/petroleum ether). (E)-7-(dimethylamino)-3-((2-(pyridine2-yl)hydrazono)methyl)naphthalene-2-ol (NJ1) was obtained as a brown solid (421 mg, 46). 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1 H), 9.72 (s, 1 H), 8.54 (s, 1 H), 8.49 (d, J¼ 2.3 Hz, 1 H), 8.12 (d, J¼ 3.8 Hz, 1 H), 7.73 (d, J ¼8.8 Hz, 1 H), 7.69 (d, J ¼8.8 Hz, 1 H), 7.73 (d, J ¼7.4 Hz, 1 H), 7.30 (d, J ¼8.4 Hz, 1 H), 7.20 (d, J ¼8.8 Hz, 1 H), 6.96–6.99 (m, 2 H), 2.80 (s, 6 H).13C NMR (100 MHz, DMSOd6): δ 157.8, 157.2, 152.7, 148.2, 140.8, 138.3, 133.1, 130.24, 130.22, 125.0, 119.2, 116.9 115.6, 114.8, 107.8, 106.9, 45.6; Ms (TOF MS EI þ ): (m/z): 304.2.
Y. Qian et al. / Talanta 132 (2015) 727–732
3. Results and discussion 3.1. Synthesis The preparation of the fluorescent probe (NJ1) and the possible reaction mechanism of NJ1 toward Cu2 þ to form NJ1Cu complex, which could be utilized to monitor hydrogen sulfide, are shown in Scheme 1. The synthesis is quite straightforward through a threestep route with 2, 7-dihydroxynaphthalene (1) as the starting material. 7-(Dimethylamino)naphthalen-2-ol (2) was then synthesized by a Bucherer reaction using dimethylamine. The formylation of compound 2 was achieved using POCl3/DMF reaction to afford aldehyde 3 in 83% yields. Finally, the target compound NJ1 was obtained by refluxing with 2-hydrazinylpyridine in EtOH. The final probe was characterized using NMR spectroscopy and mass spectrometry (see the supporting information). 3.2. Effects of the absorption and emission spectroscopic properties of NJ1 With this novel probe in hand, we first investigated its absorption and emission spectra in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO, 25 1C), which could be utilized for studying the
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binding mechanism of NJ1 with Cu2 þ . As described in Fig. 1, upon the addition of Cu2 þ into the solution of NJ1, the absorption bands at 250 and 350 nm decreased, and new absorption bands at 300 and 450 nm emerged simultaneously. We then evaluated the change of fluorescence emission spectra after the addition of Cu2 þ into the solution of NJ1. The free probe displayed a strong fluorescence emission at physiological pH. The introduction of Cu2 þ (1.0 equiv.) to the solution of NJ1 would lead to almost complete fluorescence quenching, revealing that NJ1 could be recognized Cu2 þ through fluorescence “on–off” behavior. The pH effect studies suggested that the significant fluorescent signals could be observed at the physiological pH (Fig. S1). We next evaluated the sensitivity of NJ1 for Cu2 þ through varying concentrations of Cu2 þ (0–10 mM) (Fig. 2a). The continuous addition of Cu2 þ resulted in a gradual decrease of fluorescence intensity at 492 nm, and reached the saturation state when 1.0 equiv. of Cu2 þ was added into NJ1 solution; the detection limit was found to be 0.5 mM. The effective fluorescence quenching of NJ1 was attributed to the coordination to a paramagnetic copper ion to form NJ1–Cu complex. The linear relationship of the fluorescence titration displayed that NJ1 responded to copper ion in 1:1 stoichiometry (Fig. 2b). This result had also been confirmed using TOF EI-MS (see the supporting information).
Fig. 1. (a) Absorption spectra of NJ1 (5 mM) in the absence and presence of Cu2 þ (5 mM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (b) Fluorescence spectra of NJ1 (5 mM) in the absence and presence of Cu2 þ (5 mM) at 25 1C. Excitation wavelength¼360 nm. The data represent the average of three independent experiments.
Fig. 2. (a) The fluorescence spectra of NJ1 (5 mM) incubated with different concentrations of Cu2 þ (0.5–10 mM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (b) The fluorescence intensity of NJ1 at 492 nm versus different concentrations of Cu2 þ (0.5–10 mM). Excitation: 360 nm, emission: 425–650 nm. The data represent the average of three independent experiments.
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Then, the specificity of NJ1 toward Cu2 þ was investigated by monitoring the change of the emission signal of NJ1 upon addition of other metal ions (represented by K þ , Na þ , Ag þ , Cu þ , Pb2 þ , Pd2 þ , Zn2 þ , Mg2 þ , Mn2 þ , Fe2 þ , Cd2 þ , Al3 þ , Cr3 þ , Fe3 þ , Cu2 þ ). All other metal ions had little quenching effect on the fluorescence spectra of NJ1, which did not induce significant fluorescence spectral changes. Only Cu2 þ could cause a remarkable fluorescence quenching of NJ1, which demonstrated that NJ1 have high selectivity toward Cu2 þ ions. To examine the intereference of different metal ions on the Cu2 þ recognition of NJ1, antiinterference of NJ1 was also carried out. As displayed in Fig. 3, in the absence of Cu2 þ , other potential competitive metal ions failed to cause significantly fluorescent quenching of NJ1, whereas it would be dramatically quenched after addition of Cu2 þ ions. Taken together, all these results showed that NJ1 possessed high selectivity toward Cu2 þ ; the other coexiting metal ions could not lead to significant intereference on Cu2 þ recognition.
Subsequently, the “off–on” property of NJ1Cu toward H2S was investigated. As H2S is a small gas molecule and has a pKa of around 6.9, we chose NaHS as the sulfide sources. The fluorescence recovered quickly upon the addition of NaHS to the solution of NJ1Cu, so this probe was potentially suitable for real-time monitoring of hydrogen sulfide levels in biological samples. The high selectivity was an important quality for an excellent fluorescent sensor; then selective fluorescence response of NJ1Cu to various anions or sulfur species had also been investigated in physiological conditions. Interestingly, NJ1 could only be regenerated by adding hydrogen sulfide to the NJ1Cu solution. Other anions or sulfur species, such as Cl , Br , I , HSO3 , HSO4 , S2O5 , SO24 , SO23 , lys, cys, could not result in the significant change of the fluorescence intensity and corresponding fluorescence regeneration. The competitive experiment of NJ1Cu system was also carried out for the selectivity of NJ1Cu toward hydrogen sulfide in the presence of other anions. As shown is Fig. 4a, there were nearly no difference in the absence and presence of other anions; this system indicated remarkably selective “turn on” behavior toward H2S. Furthermore, an excellent linear response of NJ1Cu versus the concentration of NaHS in the low concentration region (varying between 0 and 5 mM) was observed (Fig. 4b). Thus, the above results strongly proved that NJ1Cu system was a selective and sensitive detection method toward hydrogen sulfide that could not be significantly interfered by the commonly coexistent anions. 3.3. Cellular imaging experiment
Fig. 3. Fluorescence emission spectra of NJ1 (5 μM) and different metal ions (5 μM) in the absence (gray bar) and presence (orange bar) of Cu2 þ (5 μM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (1) K þ , (2)Na þ , (3) Ag þ , (4) Cu þ , (5) Pb2 þ , (6) Pd2 þ , (7) Zn2 þ , (8) Mg2 þ , (9) Mn2 þ , (10) Fe2 þ , (11) Cd2 þ , (12) Al3 þ , (13) Cr3 þ , (14) Fe3 þ , (15) Cu2 þ . All the data represent the fluorescence intensity at 492 nm, excitation: 360 nm. The data represent the average of three independent experiments.
We further investigated the potential application of NJ1 in living cells. Hela cells were incubated with 10 μM NJ1 in culture medium for 30 min at 37 1C; then live-cell imaging of the cells were carried out using confocal fluorescence microscopy. As shown in Fig. 5, strong fluorescence in the cells was observed after the cells were incubated with this developed probe. Next, the cells were added to 10 μM Cu2 þ and further incubated for another 20 min, the fluorescence of the cells was quickly quenched and showed almost no fluorescence. However, the fluorescence of cells could be quickly recovered after incubation with 10 μM NaHS for 20 min at 37 1C. Therefore, these results revealed that NJ1 has the potential to use for the detection of copper ion in living cells and their complex NJ1Cu would also be an excellent monitoring system to visualize sulfide in living cells.
Fig. 4. (a) Fluorescence spectra of NJ1Cu (5 μM) upon addition of different anions (5 μM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. The gray bars represent the fluorescence intensity of NJ1Cu in the presence of 5 μM anions; the orange bars represent the fluorescence intensity of the above solution on further addition of 5 μM NaHS. (1) Cl , (2) Br , (3) I , (4) HClO4 , (5) CH3COO , (6) HPO24 , (7) HSO3 , (8) HSO4 , (9) NO2 , (10) NO3 , (11) OCN , (12) S2O5 , (13) SO24 , (14) SO23 , (15) lys, (16) cys, (17) HS . (b) Fluorescence spectra of NJ1Cu (5 μM) upon addition of different amounts of HS ions (0–5 μM). All the data represent the fluorescence intensity at 492 nm, excitation: 360 nm. The data represent the average of three independent experiments.
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Fig. 5. Confocal fluorescent images of HeLa cells: Cells incubated with NJ1 (10 μM) for 30 min (a–c); Cells incubated with NJ1 (10 μM) for 30 min and subsequent treatment of the cells with Cu2 þ (10 μM) for 20 min (e–f); Cells incubated with NJ1 (10 μM), Cu2 þ (10 μM) for 30 min and subsequent treatment of the cells with NaHS (10 μM) for another 20 min (g–i). (a, d, and g) Bright-field images; (b, e, and h) fluorescent images; (c, f, and i) merged images. λex ¼ 405 nm, scare bar ¼ 50 μM.
4. Conclusions In summary, we have designed and synthesized a novel highly selective and sensitive fluorescent probe NJ1 for Cu2 þ detection by fluorescence quenching mechanism. The enhancement of fluorescence intensity with the addition of hydrogen sulfide by Cu2 þ released from the core of the complex NJ1Cu in aqueous solution at physiological conditions were also described. This “on–off–on” type fluorescence recognition system was a reversible process, which could be utilized to detect copper ion and hydrogen sulfide based on a complex NJ1Cu formation–Cu2 þ displacement approach. More importantly, this real-time recognition process of Cu2 þ and hydrogen sulfide exhibited excellent antiinterference ability. Furthermore, Preliminary imaging experiments demonstrated that NJ1 also had the potential to be used for imaging in living cells. Therefore, this new, simple, sensitive, and selective fluorescent probe has potential application in biological or environment systems for Cu2 þ and hydrogen sulfide detection. The development of more specific fluorescent probes for optical imaging in living cells, tissues, and animals is our current interest and ongoing in our laboratory.
Acknowledgments This work is financially supported by grants of the National Natural Science Foundation of China (21302094 to Y.Q.), the
Jiangsu Natural Science Foundation (BK20130552 to Y.Q.), the Fundamental Research Funds for the Central Universities (20620140214 to Y.Q.), the Research Fund for the Doctoral Program of Higher Education of China (20130091120036 to Y.Q.), and Jiangsu Province large scientific instruments shared services platform (BZ201307 to Y.Q.).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.10. 034. References [1] J. Du, M. Hu, J. Fan, X. Peng, Chem. Soc. Rev. 41 (2012) 4511–4535. [2] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 106 (2006) 1995–2044. [3] K.J. Barnham, C.L. Masters, A.I. Bush, Nat. Rev. Drug Discov. 3 (2004) 205–214. [4] T.Q. Duong, J.S. Kim, Chem. Rev. 110 (2010) 6280–6301. [5] (a) Y. Qian, J. Karpus, O. Kabil, S.Y. Zhang, H.L. Zhu, R. Banerjee, J. Zhao, C. He, Nat. Commun. 2 (2011) 495; (b) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, J. Am. Chem. Soc. 133 (2011) 18003–18005. [6] S. Sirilaksanapong, M. Sukwattanasinitt, P. Rashatasakhon, Chem. Commun. (Camb) 48 (2012) 293–295.
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[7] (a) F. Hou, J. Cheng, P. Xi, F. Chen, L. Huang, G. Xie, Y. Shi, H. Liu, D. Bai, Z. Zeng, Dalton Trans. 41 (2012) 5799–5804; (b) F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie, Y. Shi, F. Cheng, X. Yao, D. Bai, Z. Zeng, Inorg. Chem. 51 (2012) 2454–2460; (c) M. Xu, C. Yin, F. Huo, Y. Zhang, J. Chao, Sensor. Actuator. B. 204 (2014) 18. [8] D.J. Lefer, Proc. Natl. Acad. Sci. U S A 104 (2007) 17907–17908. [9] (a) G.A. Benavides, G.L. Squadrito, R.W. Mills, H.D. Patel, T.S. Isbell, R.P. Patel, V.M. Darley-Usmar, J.E. Doeller, D.W. Kraus, Proc. Natl. Acad. Sci. U S A 104
(2007) 17977–17982; (b) M. Fu, W. Zhang, L. Wu, G. Yang, H. Li, R. Wang, Proc. Natl. Acad. Sci. U S A 109 (2012) 2943–2948. [10] (a) B. Divjak, W. Goessler, J. Chromatogr. A. 582 (1999) 267–274; (b) B. Vallejo, P. Richter, I. Toral, C. Tapia, M.D. Anal. Chim. Acta 436 (2001) 301–307.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Living cells imaging for copper and hydrogen sulfide by a selective “on–off–on” fluorescent probe Yong Qian n, Jie Lin 1, Tianbao Liu 1, Hailiang Zhu n State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 July 2014 Received in revised form 11 October 2014 Accepted 15 October 2014 Available online 24 October 2014
A novel highly selective and sensitive fluorescent probe (NJ1) had been designed and synthesized for Cu2 þ detection by fluorescence quenching mechanism, and then the enhancement of fluorescence intensity with the addition of hydrogen sulfide in complex NJ1Cu aqueous solution at physiological conditions were described. This “on–off–on” type fluorescence recognition system was a reversible process, which could be utilized to monitor copper ion and hydrogen sulfide based on a complex NJ1Cu formation–Cu2 þ displacement approach. Importantly, this real-time recognition process of Cu2 þ and hydrogen sulfide exhibited excellent anti-interference ability. In addition, this new fluorescent probe also has potential utility for Cu2 þ and hydrogen sulfide detection in living cells, providing a potential tool for investigating copper ion and hydrogen sulfide in living systems or environment. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Copper ion Hydrogen sulfide Displacement approach Imaging
1. Introduction Developing a sensitive, reliable and selective detection technique for various chemically and biologically pertinent metal cations and anions has attracted many research interests [1]. As the third most abundant transition metal in the human body, copper plays an important role in various physiological processes of organism [2]. However, the excessive exposure to these Cu2 þ sources could cause imbalance in cellular processes, resulting in various diseases such as Alzheimer's disease and Parkinson's disease [3]. Additionally, copper has also been widely used in the industrial process, which is potentially toxic to environment. Therefore, there is an increasing need to monitor the existence of Cu2 þ in the live biological samples or aquatic environment [4]. Fluorescent probes have become an important diagnostic tool for biological and environmental concern considering their sensitivity, special selectivity, and real-time monitoring with fast response time [5]. Owning to the notorious paramagnetic nature of Cu2 þ , it usually leads to fluorescence quenching effect of the bound fluorophore [6]. More interestingly, this Cu2 þ complex would also be potentially applied to monitor other substances, such as sulfide anion [7]. Hydrogen sulfide (H2S) is a toxic gas best known for its rotten egg smell. It is slightly soluble in water, giving the hydrosulfide anion HS (pKa ¼6.9) and the sulfide dianion S2 (pKa 414). H2S has been utilized as a reactant or produced as a by-product in n
Corresponding authors. Tel.: þ 86 025 8359 2672. E-mail addresses: [email protected] (Y. Qian), [email protected] (H. Zhu). 1 These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.talanta.2014.10.034 0039-9140/& 2014 Elsevier B.V. All rights reserved.
industrial process, which is widely spread in the environment. However, the role of H2S in living organisms has recently received increasing attention. It has been suggested as the third signaling gasotransmitter, along with nitric oxide and carbon monoxide [8]. It is shown to act as a vascular relaxant, an energy regulator, and to be associated with cardiovascular regulation and the inflammation response [9]. Altering the level of hydrogen sulfide in human body or exposure to high-level sulfide can cause various physiological and biochemical problems. Therefore, the detection of hydrogen sulfide in industry, environment, and biological samples has attracted great attention. Several conventional detection strategies have been developed for hydrogen sulfide [10], such as electrochemical methods and ion chromatography. However, these analytic methods usually need complicated equipment and trou blesome sample pretreatment that are unsuitable for the analysis of sulfide in real time. For this reason, more convenient detection tools, such as fluorescent probes, have received significant attention as their easy operability and high sensitivity. To the best of our knowledge, several hydrogen sulfide selective probes based on copper ion displacement approach have been reported [7]. However, most of these probes have some limitations such as poor water solubility, complicated synthesis, poor membrane permeability, and detection systems required much organic co-solvents [7c], which would significantly restrict their practical applications in physiological condition. So far, real-time sulfide selective probes that are applicable in aqueous solution are still limited. It is thus desirable to develop simple and effective fluorescent probes for detection of sulfide in aqueous media that displays high sensitivity and selectivity as well as enables easier accessibility.
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Y. Qian et al. / Talanta 132 (2015) 727–732
Scheme 1. Synthesis of NJ1 and the proposed mechanism of the monitoring system.
With this in mind, we designed and synthesized a novel small fluorescent probe NJ1 (Scheme 1). NJ1 demonstrated obvious fluorescence quenching after the addition of copper ion to form new complex NJ1Cu. This new complex displayed high specificity for hydrogen sulfide that induced copper ion to be released from the dye in the aqueous media, resulting in fluorescence enhancement. Herein, we reported the synthesis, photophysical characterization of NJ1 that was highly sensitive and selective for Cu2 þ . The subsequent complex NJ1Cu could be developed as a platform for hydrogen sulfide real-time detection that could be potentially utilized in industry, environment, or biological requirements. Furthermore, the imaging of copper ion and hydrogen sulfide in living cells were also successfully developed.
2. Experimental 2.1. Materials and instrumentations All chemicals were purchased from commercial suppliers and used without further purification. Chromatographic purification of products was accomplished by using forced-flow chromatography on silica gel (300–400 mesh). Thin layer chromatography was performed on EM Science silica gel 60 F254 plates (250 μm). Visualization of the developed chromatogram was accomplished using UV lamp. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX-400 operating at 100 MHz for 1H NMR and 13C NMR; residual protio solvent signals were used as internal standards for calibration purposes. Data for 1H NMR are reported as follows: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet), integration, coupling constant (Hz). TOF EI mass was performed by Mass Spectrometry Facility at Nanjing University. All fluorescence measurements were recorded on a Varian Cary Eclipse Fluorescence Spectrophotometer and Hitachi Fluorescence Spectrophotometer F-7000. The pH measurements were carried out on a HI 2221 calibration check pH/ORP meter. 2.2. General procedure for analysis Stock solution of metal ions and other anions were prepared in ultrapure H2O, stock solution of NJ1 (1 mM) were prepared in DMSO, which was diluted to the required concentration for measurement. All fluorescence measurements were carried out at room temperature on a Hitachi Fluorescence Spectrophotometer F-7000.
The samples were excited at 360 nm; the emission spectrum was scanned from 425 nm to 650 nm at 1200 nm/min. 2.3. Cell culture and fluorescence imaging HeLa cells were grown up in DMEM medium with 10% fetal bovine serum/penicillin/streptomycin in a 5% CO2 atmosphere at 37 1C. Cells were then seeded on Coverglass-Bottom confocaldish and continuously incubated at 37 1C in a 5% CO2 atmosphere for 24 h. After cells were incubated with 1 μL, 10 mM NJ1(final concentration: 10 μM) for 30 min at 37 1C, media was removed, and cells were washed with HEPES buffer three times again to remove any probe left in solution to optimize the background signal. Then, cells were added to fresh media and incubated with Cu2 þ (0 μM, 10 μM) for another 20 min at 37 1C before imaging; or NaHS (10 μM) was added into the cells with NJ1 (10 μM) and Cu (10 μM) for another 20 min at 37 1C before imaging. All imaging experiments were performed on a fixed cell DSU spinning confocal microscope (Olympus), excitation at 405 nm. Imaging was performed using X40 objective and captured using Slidebook software. For all experiments, solution of NJ1 was prepared in DMSO (10 mM) and diluted into DMEM to the desired working concentration (10 μM). Cu2 þ , and NaHS were diluted into ddH2O to the desired working concentrations (10 μM) from a 10-mM stock solution. 2.4. Synthesis of probe NJ1 2.4.1. (E)-7-(dimethylamino)-3-((2-(pyridine-2-yl)hydrazono) methyl)naphthalene-2-ol To a mixture compounds 3- (645 mg, 3 mmol) and 2hydrazinylpyridine (360 mg, 3.3 mmol) was added ethanol under argon. The mixture was stirred at 80 1C for 1 h. The solvent was then evaporated under reduced pressure and the obtained residue was purified by silica gel column chromatography (eluent: 30 ethyl acetate/petroleum ether). (E)-7-(dimethylamino)-3-((2-(pyridine2-yl)hydrazono)methyl)naphthalene-2-ol (NJ1) was obtained as a brown solid (421 mg, 46). 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1 H), 9.72 (s, 1 H), 8.54 (s, 1 H), 8.49 (d, J¼ 2.3 Hz, 1 H), 8.12 (d, J¼ 3.8 Hz, 1 H), 7.73 (d, J ¼8.8 Hz, 1 H), 7.69 (d, J ¼8.8 Hz, 1 H), 7.73 (d, J ¼7.4 Hz, 1 H), 7.30 (d, J ¼8.4 Hz, 1 H), 7.20 (d, J ¼8.8 Hz, 1 H), 6.96–6.99 (m, 2 H), 2.80 (s, 6 H).13C NMR (100 MHz, DMSOd6): δ 157.8, 157.2, 152.7, 148.2, 140.8, 138.3, 133.1, 130.24, 130.22, 125.0, 119.2, 116.9 115.6, 114.8, 107.8, 106.9, 45.6; Ms (TOF MS EI þ ): (m/z): 304.2.
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3. Results and discussion 3.1. Synthesis The preparation of the fluorescent probe (NJ1) and the possible reaction mechanism of NJ1 toward Cu2 þ to form NJ1Cu complex, which could be utilized to monitor hydrogen sulfide, are shown in Scheme 1. The synthesis is quite straightforward through a threestep route with 2, 7-dihydroxynaphthalene (1) as the starting material. 7-(Dimethylamino)naphthalen-2-ol (2) was then synthesized by a Bucherer reaction using dimethylamine. The formylation of compound 2 was achieved using POCl3/DMF reaction to afford aldehyde 3 in 83% yields. Finally, the target compound NJ1 was obtained by refluxing with 2-hydrazinylpyridine in EtOH. The final probe was characterized using NMR spectroscopy and mass spectrometry (see the supporting information). 3.2. Effects of the absorption and emission spectroscopic properties of NJ1 With this novel probe in hand, we first investigated its absorption and emission spectra in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO, 25 1C), which could be utilized for studying the
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binding mechanism of NJ1 with Cu2 þ . As described in Fig. 1, upon the addition of Cu2 þ into the solution of NJ1, the absorption bands at 250 and 350 nm decreased, and new absorption bands at 300 and 450 nm emerged simultaneously. We then evaluated the change of fluorescence emission spectra after the addition of Cu2 þ into the solution of NJ1. The free probe displayed a strong fluorescence emission at physiological pH. The introduction of Cu2 þ (1.0 equiv.) to the solution of NJ1 would lead to almost complete fluorescence quenching, revealing that NJ1 could be recognized Cu2 þ through fluorescence “on–off” behavior. The pH effect studies suggested that the significant fluorescent signals could be observed at the physiological pH (Fig. S1). We next evaluated the sensitivity of NJ1 for Cu2 þ through varying concentrations of Cu2 þ (0–10 mM) (Fig. 2a). The continuous addition of Cu2 þ resulted in a gradual decrease of fluorescence intensity at 492 nm, and reached the saturation state when 1.0 equiv. of Cu2 þ was added into NJ1 solution; the detection limit was found to be 0.5 mM. The effective fluorescence quenching of NJ1 was attributed to the coordination to a paramagnetic copper ion to form NJ1–Cu complex. The linear relationship of the fluorescence titration displayed that NJ1 responded to copper ion in 1:1 stoichiometry (Fig. 2b). This result had also been confirmed using TOF EI-MS (see the supporting information).
Fig. 1. (a) Absorption spectra of NJ1 (5 mM) in the absence and presence of Cu2 þ (5 mM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (b) Fluorescence spectra of NJ1 (5 mM) in the absence and presence of Cu2 þ (5 mM) at 25 1C. Excitation wavelength¼360 nm. The data represent the average of three independent experiments.
Fig. 2. (a) The fluorescence spectra of NJ1 (5 mM) incubated with different concentrations of Cu2 þ (0.5–10 mM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (b) The fluorescence intensity of NJ1 at 492 nm versus different concentrations of Cu2 þ (0.5–10 mM). Excitation: 360 nm, emission: 425–650 nm. The data represent the average of three independent experiments.
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Then, the specificity of NJ1 toward Cu2 þ was investigated by monitoring the change of the emission signal of NJ1 upon addition of other metal ions (represented by K þ , Na þ , Ag þ , Cu þ , Pb2 þ , Pd2 þ , Zn2 þ , Mg2 þ , Mn2 þ , Fe2 þ , Cd2 þ , Al3 þ , Cr3 þ , Fe3 þ , Cu2 þ ). All other metal ions had little quenching effect on the fluorescence spectra of NJ1, which did not induce significant fluorescence spectral changes. Only Cu2 þ could cause a remarkable fluorescence quenching of NJ1, which demonstrated that NJ1 have high selectivity toward Cu2 þ ions. To examine the intereference of different metal ions on the Cu2 þ recognition of NJ1, antiinterference of NJ1 was also carried out. As displayed in Fig. 3, in the absence of Cu2 þ , other potential competitive metal ions failed to cause significantly fluorescent quenching of NJ1, whereas it would be dramatically quenched after addition of Cu2 þ ions. Taken together, all these results showed that NJ1 possessed high selectivity toward Cu2 þ ; the other coexiting metal ions could not lead to significant intereference on Cu2 þ recognition.
Subsequently, the “off–on” property of NJ1Cu toward H2S was investigated. As H2S is a small gas molecule and has a pKa of around 6.9, we chose NaHS as the sulfide sources. The fluorescence recovered quickly upon the addition of NaHS to the solution of NJ1Cu, so this probe was potentially suitable for real-time monitoring of hydrogen sulfide levels in biological samples. The high selectivity was an important quality for an excellent fluorescent sensor; then selective fluorescence response of NJ1Cu to various anions or sulfur species had also been investigated in physiological conditions. Interestingly, NJ1 could only be regenerated by adding hydrogen sulfide to the NJ1Cu solution. Other anions or sulfur species, such as Cl , Br , I , HSO3 , HSO4 , S2O5 , SO24 , SO23 , lys, cys, could not result in the significant change of the fluorescence intensity and corresponding fluorescence regeneration. The competitive experiment of NJ1Cu system was also carried out for the selectivity of NJ1Cu toward hydrogen sulfide in the presence of other anions. As shown is Fig. 4a, there were nearly no difference in the absence and presence of other anions; this system indicated remarkably selective “turn on” behavior toward H2S. Furthermore, an excellent linear response of NJ1Cu versus the concentration of NaHS in the low concentration region (varying between 0 and 5 mM) was observed (Fig. 4b). Thus, the above results strongly proved that NJ1Cu system was a selective and sensitive detection method toward hydrogen sulfide that could not be significantly interfered by the commonly coexistent anions. 3.3. Cellular imaging experiment
Fig. 3. Fluorescence emission spectra of NJ1 (5 μM) and different metal ions (5 μM) in the absence (gray bar) and presence (orange bar) of Cu2 þ (5 μM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. (1) K þ , (2)Na þ , (3) Ag þ , (4) Cu þ , (5) Pb2 þ , (6) Pd2 þ , (7) Zn2 þ , (8) Mg2 þ , (9) Mn2 þ , (10) Fe2 þ , (11) Cd2 þ , (12) Al3 þ , (13) Cr3 þ , (14) Fe3 þ , (15) Cu2 þ . All the data represent the fluorescence intensity at 492 nm, excitation: 360 nm. The data represent the average of three independent experiments.
We further investigated the potential application of NJ1 in living cells. Hela cells were incubated with 10 μM NJ1 in culture medium for 30 min at 37 1C; then live-cell imaging of the cells were carried out using confocal fluorescence microscopy. As shown in Fig. 5, strong fluorescence in the cells was observed after the cells were incubated with this developed probe. Next, the cells were added to 10 μM Cu2 þ and further incubated for another 20 min, the fluorescence of the cells was quickly quenched and showed almost no fluorescence. However, the fluorescence of cells could be quickly recovered after incubation with 10 μM NaHS for 20 min at 37 1C. Therefore, these results revealed that NJ1 has the potential to use for the detection of copper ion in living cells and their complex NJ1Cu would also be an excellent monitoring system to visualize sulfide in living cells.
Fig. 4. (a) Fluorescence spectra of NJ1Cu (5 μM) upon addition of different anions (5 μM) in 20 mM HEPES buffer (pH 7.4, 0.5% DMSO) at 25 1C. The gray bars represent the fluorescence intensity of NJ1Cu in the presence of 5 μM anions; the orange bars represent the fluorescence intensity of the above solution on further addition of 5 μM NaHS. (1) Cl , (2) Br , (3) I , (4) HClO4 , (5) CH3COO , (6) HPO24 , (7) HSO3 , (8) HSO4 , (9) NO2 , (10) NO3 , (11) OCN , (12) S2O5 , (13) SO24 , (14) SO23 , (15) lys, (16) cys, (17) HS . (b) Fluorescence spectra of NJ1Cu (5 μM) upon addition of different amounts of HS ions (0–5 μM). All the data represent the fluorescence intensity at 492 nm, excitation: 360 nm. The data represent the average of three independent experiments.
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Fig. 5. Confocal fluorescent images of HeLa cells: Cells incubated with NJ1 (10 μM) for 30 min (a–c); Cells incubated with NJ1 (10 μM) for 30 min and subsequent treatment of the cells with Cu2 þ (10 μM) for 20 min (e–f); Cells incubated with NJ1 (10 μM), Cu2 þ (10 μM) for 30 min and subsequent treatment of the cells with NaHS (10 μM) for another 20 min (g–i). (a, d, and g) Bright-field images; (b, e, and h) fluorescent images; (c, f, and i) merged images. λex ¼ 405 nm, scare bar ¼ 50 μM.
4. Conclusions In summary, we have designed and synthesized a novel highly selective and sensitive fluorescent probe NJ1 for Cu2 þ detection by fluorescence quenching mechanism. The enhancement of fluorescence intensity with the addition of hydrogen sulfide by Cu2 þ released from the core of the complex NJ1Cu in aqueous solution at physiological conditions were also described. This “on–off–on” type fluorescence recognition system was a reversible process, which could be utilized to detect copper ion and hydrogen sulfide based on a complex NJ1Cu formation–Cu2 þ displacement approach. More importantly, this real-time recognition process of Cu2 þ and hydrogen sulfide exhibited excellent antiinterference ability. Furthermore, Preliminary imaging experiments demonstrated that NJ1 also had the potential to be used for imaging in living cells. Therefore, this new, simple, sensitive, and selective fluorescent probe has potential application in biological or environment systems for Cu2 þ and hydrogen sulfide detection. The development of more specific fluorescent probes for optical imaging in living cells, tissues, and animals is our current interest and ongoing in our laboratory.
Acknowledgments This work is financially supported by grants of the National Natural Science Foundation of China (21302094 to Y.Q.), the
Jiangsu Natural Science Foundation (BK20130552 to Y.Q.), the Fundamental Research Funds for the Central Universities (20620140214 to Y.Q.), the Research Fund for the Doctoral Program of Higher Education of China (20130091120036 to Y.Q.), and Jiangsu Province large scientific instruments shared services platform (BZ201307 to Y.Q.).
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