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DOI: 10.1039/C5AN00119F (Communication) Analyst, 2015, 140, 7165-7169

Imaging of living cells and zebrafish in vivo using a ratiometric fluorescent probe for hydrogen sulfide†

Tianbao Liu , Jie Lin , Zhen Li , Lin Lin , Yuning Shen , Hailiang Zhu * and Yong Qian *
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, No.163 Xianlin Road, Nanjing 210023, China. E-mail: yongqian@nju.edu.cn; zhuhl@nju.edu.cn

Received 19th January 2015 , Accepted 13th September 2015

First published on 15th September 2015

Abstract

We have developed a novel colorimetric and ratiometric fluorescence probe for the selective and sensitive monitoring of hydrogen sulfide based on a dicyanoisophorone platform. An excellent linear relationship of fluorescence intensity ratio (I637/I558) (R2 = 0.9867) versus hydrogen sulfide concentration in the range of 1–12 μM was obtained. This probe exhibited a remarkable fluorescence response to hydrogen sulfide over other physiological thiols or biological species, which fluoresces in the red region with a large Stokes shift (172 nm). This probe was successfully utilized to monitor H2S under in vitro physiological conditions and for imaging H2S in living cells and living zebrafish in vivo.

Hydrogen sulfide, an important endogenous gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO), plays important roles in a number of biological processes.1 It has been established to regulate intracellular redox status and mediate fundamental biological and physiological processes, including anti-oxidation, anti-inflammation, anti-apoptosis, intervention of neurotransmission, regulation of vascular pressure etc.2 In mammals, the production of endogenous hydrogen sulfide is mediated by the enzymatic process of cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), as well as the coordinated action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST).3 Abnormal levels of hydrogen sulfide correlate with many diseases, such as Alzheimer's disease, Down syndrome, and liver cirrhosis.4 Therefore, a detection method for hydrogen sulfide in a selective and sensitive manner in living biological systems is important for better understanding its biological and physiological functions.

Fluorescence imaging has attracted much attention given its excellent sensitivity, selectivity, and capability of detecting biomolecules in live biological specimens.5 In order to meet the urgent need for selective visualization tools for the detection of hydrogen sulfide in live biosystems, the development of fluorescent probes for hydrogen sulfide has recently made rapid advances.6 Early work in this area from our laboratory as well as from Xian et al. has developed a series of fluorescent turn-on probes based on dual nucleophilic reaction.6a,b Nagano et al. and Zeng et al. have reported copper sulfide precipitation strategies to remove quencher from fluorescent probes used for hydrogen sulfide detection.6c,d,7 Chang and Wang et al. exploited selective fluorescent turn-on probes based on hydrogen sulfide induced specific azide reduction;6e,f this approach has recently been widely adopted.8 These fluorescent probes have been successfully utilized for detecting exogenous or endogenous hydrogen sulfide in living cells, blood and tissue samples, and in vitro enzyme assays, and most of them display a response time varying from 20 min to 2 hours. However, many of these fluorescent probes need high amounts of organic cosolvent (≥10%, v/v)6a,d,7,8g,9 and short-wavelength excitation.8f,g,9 Moreover, most of these probes are based on a fluorescence off–on response mechanism, which is easily influenced by probe concentration, the microenvironment, photobleaching etc. As a result, their biological application in complicated living systems is limited as the strong absorption and autofluorescence of background biomolecules would lead to a low signal-to-noise ratio.10

We are now interested in designing and developing fluorescent probes to detect and image hydrogen sulfide in living cells and in small animal models in vivo, based on ratiometric measurements rather than simple fluorescence intensity-based off–on processes. There is increasing interest in developing ratiometric probes for hydrogen sulfide owing to their self-calibration effect which will reduce most of the aforementioned interference. Recently, a few ratiometric fluorescent probes for hydrogen sulfide have been reported.8b,d,11 Nevertheless, some of these ratiometric probes needed pure organic solvent to work,11a or the excitation and emission wavelengths of the probe were relatively short for biological applications,11b,d or the low fluorescence quantum yield and poor stability would limit their application.8d Thus, ratiometric fluorescent probes with good solubility, large Stokes shift, longer excitation wavelength, and high quantum yield are still urgently needed for hydrogen sulfide imaging in living cells and in vivo.

Herein, we report a novel ratiometric fluorescent probe (SFP-GR) based on a dicyanoisophorone electron-accepting group for imaging hydrogen sulfide in living biosystems. In this probe, a reactive azide group was introduced into the dicyanoisophorone electron-accepting group.12 Upon reduction of hydrogen sulfide, we anticipate that the electron-withdrawing azide group will be converted into an electron-donating amine group resulting in a red shift in emission,6e,8b which may provide a ratiometric detection of hydrogen sulfide. As expected, a selective and sensitive fluorescent probe for hydrogen sulfide has been developed. This probe exhibits a colorimetric and ratiometric fluorescence response to hydrogen sulfide over other physiological thiols or biological species, which fluoresces in the red region with a large Stokes shift (172 nm, Fig. S1†). We successfully used this fluorescent probe to visualize hydrogen sulfide in living HeLa cells and in small model animal zebrafish.

The preparation of the fluorescent probe (SFP-GR) and the possible reaction mechanism of SFP-GR toward hydrogen sulfide are shown in Scheme 1. We synthesized SFP-GR conveniently in three simple steps. The detailed synthetic steps and characterization of SFP-GR are described in the ESI.†


image file: c5an00119f-s1.tif
Scheme 1 Design and synthesis of the fluorescent probe (SFP-GR) for H2S detection.

With this fluorescent probe in hand, we firstly examined the spectral properties and reactivity of SFP-GR (5 μM) (Φ = 0.47) with Na2S (20 μM) as an aqueous sulfide source in HEPES buffer (20 mM, pH 7.4, 0.5% DMSO) with 1 mM CTAB at 25 °C (Fig. 1 and Fig. S2†). The free SFP-GR displayed one major absorption peak at around 413 nm. Upon addition of hydrogen sulfide, the azide group of the probe was reduced to an amine group. Consequently, the maximum absorption peak exhibited a 64 nm red shift to 477 nm and the color of the solution changed from colorless to tan (Fig. 1 inset). So SFP-GR could serve as a visual probe for hydrogen sulfide, which would allow the colorimetric detection of hydrogen sulfide directly with the naked-eye.


image file: c5an00119f-f1.tif
Fig. 1 The absorption (a) and fluorescence emission (b) spectra of SFP-GR (5 μM) before and after the addition of Na2S (20 μM) in HEPES buffer (20 mM, pH 7.4, 0.5% DMSO) with 1 mM CTAB. The inset shows the photographs of the solution of SFP-GR (5 μM) in the absence and presence of Na2S under visible light or irradiation with a UV (365 nm) lamp.

We next investigated the fluorescence emission spectra. In the absence of hydrogen sulfide, SFP-GR only exhibited a weak emission peak around 558 nm. Interestingly, SFP-GR showed a fast and significant increase of the fluorescence intensity at 637 nm when excited at 466 nm after the addition of hydrogen sulfide, which was completed within 5 min (see ESI, Fig. S3†). With this large red shift (79 nm) in emission, the color changed from colorless to red under irradiation with a UV (365 nm) lamp. We also monitored the change of this chemical reaction before and after the addition of hydrogen sulfide in SFP-GR solution using liquid chromatography-mass spectrometry (HPLC-MS), and the results revealed that the reaction of SFP-GR with hydrogen sulfide would reduce the azide group of the probe to an amine group (see ESI, Fig. S4†). The pH effect studies suggested that the significant fluorescence signals could be observed between pH 7.0 and 10.0, indicating the good suitability of SFP-GR under physiological conditions (see ESI, Fig. S5†).

To evaluate the sensitivity of SFP-GR toward hydrogen sulfide, we then investigated the change in fluorescence spectra via varying concentrations of Na2S (1–40 μM) under simulated physiological conditions (Fig. 2a). With a continuously increasing concentration of sulfide in SFP-GR (5 μM) solution, the fluorescence intensity decreased at 558 nm and gradually increased around 637 nm (excitation wavelength at 466 nm), resulting in the increase of fluorescence intensity ratio (I637/I558) from 0.72 to 10.6. Moreover, an excellent linear relationship of fluorescence intensity ratio (I637/I558) (R2 = 0.9867) or emission intensity at 637 nm (R2 = 0.9977) versus hydrogen sulfide concentration in the range of 1–12 μM was also obtained (Fig. 2b and S6†). The detection limit of SFP-GR for hydrogen sulfide was 77 nM (see ESI†). These results demonstrated that SFP-GR could not only monitor hydrogen sulfide qualitatively but could also provide a sensitive and quantitative detection method for hydrogen sulfide based on the excellent linear relationship between concentration and fluorescence intensity.


image file: c5an00119f-f2.tif
Fig. 2 Fluorescence spectra of SFP-GR (5 μM) in HEPES buffer (20 mM, pH 7.4, 0.5% DMSO) with 1 mM CTAB. (a) Incubated with different concentrations of Na2S (0–40 μM) for 10 min at 25 °C. Inset: fluorescence intensity changes at 637 nm of SFP-GR with the amount of Na2S. (b) The linear relationship of fluorescence intensity ratio (I637/I558) with the concentration of Na2S (0, 1, 2, 4, 6, 8, 10, 12 μM). Excitation wavelength: 466 nm, emission: 450–750 nm, excitation and emission slit widths = 5 nm. The data represent an average of three independent experiments.

To examine the selectivity of SFP-GR, we subsequently treated various biologically relevant species with SFP-GR, including various anions, metal ions, amino acids, and thiols. As shown in Fig. 3, SFP-GR showed high selectivity for hydrogen sulfide over other competing analytes. Especially, no obvious change in fluorescence signal was detected in the presence of biologically relevant thiols such as glutathione (1 mM), cysteine (1 mM), and homocysteine (10 mM) (Fig. 3 and S7†). As a comparison, upon the addition of Na2S (20 μM) to the probe, a large fluorescence signal (13-fold) enhancement at 637 nm and a notable change of fluorescence intensity ratio (I637/I558) (10.6) was observed (Fig. S7†). To further demonstrate the ability to monitor hydrogen sulfide in the presence of other competitive biological thiols, the anti-interference of SFP-GR was also investigated (Fig. 3b and S8†). As the physiological concentrations of thiols are in the millimolar region (0.1–0.25 mM Cys, 5–10 mM GSH, and 0.010–0.012 mM Hcy), therefore, the selectivity of SFP-GR was checked at 0.2 mM Cys, 10 mM GSH, and 0.01 mM Hcy. As shown in Fig. 3b, the fluorescence emission enhancement induced by other thiols is very limited compared to that induced by hydrogen sulfide. Additionally, exposing the probe to a mixture of other thiols and Na2S still yields a significant fluorescence signal increase. The above results indicate that SFP-GR is able to detect hydrogen sulfide without any distinct interference from other biological thiols. Taken together, the in vitro selectivity experiments demonstrate that this selective fluorescent probe SFP-GR can be used for the fluorescence detection of hydrogen sulfide under physiological conditions.


image file: c5an00119f-f3.tif
Fig. 3 Fluorescence response of SFP-GR (5 μM) to various biologically relevant species in HEPES buffer (20 mM, pH 7.4, 0.5% DMSO) with 1 mM CTAB. (a) Control (probe alone); AcO− (200 μM); Cl − (200 μM); CO32− (200 μM); F− (200 μM); HCO3− (200 μM); HSO4− (200 μM); N3− (200 μM); NO2− (200 μM); NO3− (200 μM); SCN− (200 μM); SO42− (200 μM); Al3+ (1 mM); Ca2+ (1 mM); K+ (1 mM); Mg2+ (1 mM); Na+ (1 mM); Cys (1 mM); GSH (1 mM); Lys (1 mM); Pro (1 mM); Hcy (10 mM); Na2S (20 μM). Excitation: 466 nm; emission: 637 nm. (b) Gray bars correspond to free SFP-GR or with Na2S (20 μM) or other biological thiols (0.2 mM Cys, 10 mM GSH, 0.01 mM Hcy); purple bars correspond to SFP-GR in the presence of both Na2S (20 μM) and the other biological thiols (0.2 mM Cys, 10 mM GSH, 0.01 mM Hcy).

Next, we investigated the potential application of SFP-GR in living biosystems. We tested the ability of SFP-GR to detect hydrogen sulfide in live HeLa cells using confocal microscopy imaging (Fig. 4). Cells were incubated with 10 μM SFP-GR for 30 min, and then green fluorescence was observed, suggesting good cell permeability of SFP-GR. Interestingly, HeLa cells that were treated with 20 μM Na2S for an additional 30 min after the cells were incubated with 10 μM SFP-GR for 30 min at 37 °C led to an obviously increase in red channel fluorescence. Distinct changes of ratiometric fluorescence response in living cells were observed (Fig. 4d and h). These results indicated that this novel developed SFP-GR indeed could be used for ratiometric fluorescence imaging of hydrogen sulfide in living cells. Furthermore, we also performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in the HeLa cells to evaluate the cytotoxicity of SFP-GR (10–50 μM). The results were further indication that SFP-GR was of low toxicity or non-toxic to cultured cells under the experimental conditions at the concentration of 50 μM for 24 hours (Fig. S9†).


image file: c5an00119f-f4.tif
Fig. 4 Confocal microscopy images of H2S detection in live HeLa cells using SFP-GR. (a–d) HeLa cells incubated with SFP-GR (10 μM) for 30 min at 37 °C; (e–h) HeLa cells after treatment with SFP-GR (10 μM) for 30 min and subsequent treatment of the cells with Na2S (20 μM) for another 30 min at 37 °C. (a, e) Bright-field images and (b, f) green channel images were collected in the range of 520–560 nm; (c, g) red channel images were collected in the range of 620–660 nm; (d, h) ratio images generated from (b) and (c), (f) and (g), respectively. Excitation at 488 nm. Scale bars represent 20 μm.

We further investigated if this probe could also be utilized for imaging hydrogen sulfide in vivo, such as in a small animal model, e.g. zebrafish. As shown in Fig. 5, five-day-old zebrafish themselves show no fluorescence (data not shown). However, when the zebrafish were incubated with 10 μM SFP-GR for 30 min at 37 °C, a green fluorescence in the fish was observed (Fig. 5b). Moreover, exposure to 20 μM Na2S followed by treatment with SFP-GR led to a significant increase in the red channel fluorescence of the overall fish (Fig. 5g). More importantly, distinct changes in the ratiometric fluorescence response in zebrafish were also observed. These results further suggested that this sensitive H2S probe SFP-GR can be used for tracing the distribution of hydrogen sulfide in living organisms.


image file: c5an00119f-f5.tif
Fig. 5 Confocal microscopy images of H2S detection in living 5 days-old zebrafish using SFP-GR. (a–d) Zebrafish were treated with SFP-GR (10 μM) for 30 min at 37 °C. (e–h) Zebrafish were treated with SFP-GR (10 μM) for 30 min at 37 °C, then treated with 20 μM Na2S for another 30 min at 37 °C. (a, e) Bright-field images; (b, f) green channel images; (c, g) red channel images; (d, h) ratio images generated from (b) and (c), (f) and (g), respectively. Green channel images were collected in the range of 520–560 nm; red channel images were collected in the range of 620–660 nm. Scale bars represent 100 μm.

In summary, we have rationally designed and synthesized a novel fluorescent probe SFP-GR with high selectivity and sensitivity for hydrogen sulfide. This colorimetric and ratiometric probe could be used for monitoring hydrogen sulfide under physiological conditions in vitro and could also successfully be applied for imaging hydrogen sulfide in living cells and in zebrafish in vivo. Current efforts are focused on developing real-time in situ fluorescence probes for imaging hydrogen sulfide in living cells, tissues and living animals as well as the application of these fluorescence probes to investigate the biological functions and physiological processes of hydrogen sulfide.

Acknowledgements

This work is financially supported by grants from the National Natural Science Foundation of China (21302094), the Jiangsu Natural Science Foundation (BK20130552), the Research Fund for the Doctoral Program of Higher Education of China (20130091120036) and the Fundamental Research Funds for the Central Universities (20620140214).

Notes and references

  1. (a) E. Culotta and D. E. Koshland Jr., Science, 1992, 258, 1862 CAS ; (b) C. Szabo, Nat. Rev. Drug Discovery, 2007, 6, 917 CrossRef CAS PubMed .
  2. (a) J. W. Elrod, J. W. Calvert, J. Morrison, J. E. Doeller, D. W. Kraus, L. Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Kimura, C. W. Chow and D. J. Lefer, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 15560 CrossRef CAS PubMed ; (b) G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang, Science, 2008, 322, 587 CrossRef CAS PubMed ; (c) J. W. Calvert, S. Jha, S. Gundewar, J. W. Elrod, A. Ramachandran, C. B. Pattillo, C. G. Kevil and D. J. Lefer, Circ. Res., 2009, 105, 365 CrossRef CAS PubMed ; (d) H. Kimura, Amino Acids, 2011, 41, 113 CrossRef CAS PubMed .
  3. O. Kabil and R. Banerjee, J. Biol. Chem., 2010, 285, 21903 CrossRef CAS PubMed .
  4. (a) K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura, Biochem. Biophys. Res. Commun., 2002, 293, 1485 CrossRef CAS ; (b) P. Kamoun, M. C. Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-Vekemans, Am. J. Med. Genet., Part A, 2003, 116A, 310 CrossRef PubMed ; (c) S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005, 42, 539 CrossRef CAS PubMed .
  5. H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and Y. Urano, Chem. Rev., 2010, 110, 2620 CrossRef CAS PubMed .
  6. (a) Y. Qian, J. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011, 2, 495 CrossRef PubMed ; (b) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 50, 10327 CrossRef CAS PubMed ; (c) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am. Chem. Soc., 2011, 133, 18003 CrossRef CAS PubMed ; (d) X. Qu, C. Li, H. Chen, J. Mack, Z. Guo and Z. Shen, Chem. Commun., 2013, 49, 7510 RSC ; (e) A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078 CrossRef CAS PubMed ; (f) H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore, D. J. Lefer and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672 CrossRef CAS PubMed ; (g) B. Wang, P. Li, F. Yu, P. Song, X. Sun, S. Yang, Z. Lou and K. Han, Chem. Commun., 2013, 49, 1014 RSC ; (h) Z. Lou, P. Li, Q. Pan and K. Han, Chem. Commun., 2013, 49, 2445 RSC ; (i) X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem. Sci., 2013, 4, 2551 RSC ; (j) L. Yuan, F. Jin, Z. Zeng, C. Liu, S. Luo and J. Wu, Chem. Sci., 2015, 6, 2360 RSC .
  7. F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie, Y. Shi, F. Cheng, X. Yao, D. Bai and Z. Zeng, Inorg. Chem., 2012, 51, 2454 CrossRef CAS PubMed .
  8. (a) S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho, Chem. Commun., 2012, 48, 8395 RSC ; (b) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun., 2013, 49, 502 RSC ; (c) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767 RSC ; (d) F. Yu, P. Li, P. Song, B. Wang, J. Zhao and K. Han, Chem. Commun., 2012, 48, 2852 RSC ; (e) V. S. Lin, A. R. Lippert and C. J. Chang, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 7131 CrossRef CAS PubMed ; (f) B. Chen, W. Li, C. Lv, M. Zhao, H. Jin, J. Du, L. Zhang and X. Tang, Analyst, 2013, 138, 946 RSC ; (g) Z. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Commun., 2012, 48, 10120 RSC .
  9. K. Zheng, W. Lin and L. Tan, Org. Biomol. Chem., 2012, 10, 9683 CAS .
  10. L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He and S. Zhu, J. Am. Chem. Soc., 2012, 134, 13510 CrossRef CAS PubMed .
  11. (a) M. Y. Wu, K. Li, J. T. Hou, Z. Huang and X. Q. Yu, Org. Biomol. Chem., 2012, 10, 8342 RSC ; (b) Z. Xu, L. Xu, J. Zhou, Y. Xu, W. Zhu and X. Qian, Chem. Commun., 2012, 48, 10871 RSC ; (c) Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen and Z. Guo, Angew. Chem., Int. Ed., 2013, 52, 1688 CrossRef CAS PubMed ; (d) L. Zhang, S. Li, M. Hong, Y. Xu, S. Wang, Y. Liu, Y. Qian and J. Zhao, Org. Biomol. Chem., 2014, 12, 5115 RSC .
  12. S. Redon, J. Massin, S. Pouvreau, E. De Meulenaere, K. Clays, Y. Queneau, C. Andraud, A. Girard-Egrot, Y. Bretonniere and S. Chambert, Bioconjugate Chem., 2014, 25, 773 CrossRef CAS PubMed .

Footnote

† Electronic supplementary information (ESI) available: Experimental details, including compliance with ethical guidelines for our experiments with live animals, and supplemental data. See DOI: 10.1039/c5an00119f
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