An effective fluorescent probe to detect glutathione from other sulfhydryl compounds in aqueous solution and its living cell imaging

Abstract

We designed and synthesized a two-photon fluorescent probe, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-yl acrylate (PPN) with improved properties based on the naphthalene–pyrazoline fluorophore. The probe exhibits high sensitivity to glutathione (GSH) in PBS–CTAB buffer solution with a low detection limit of 1.5 × 10⁻⁸ M and a response time less than 10 min. According to the HRMS and fluorescence spectra analysis, the detection mechanism was confirmed to be a Michael addition reaction induced by the sulfhydryl of GSH. In addition, probe PPN has very good selectivity and is able to discriminate GSH from cysteine (Cys), homocysteine (Hcy) and other sulfhydryl compounds with bright two-photon-excited fluorescence. Moreover, we have successfully applied PPN to calf serum samples and living cell imaging with good effect.

---

Introduction

Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play various crucial roles in physiological processes.¹ As the most abundant intracellular nonprotein thiols, GSH has numerous cellular functions, including gene regulation, intracellular signalling, intracellular redox activities and xenobiotic metabolism.² However, abnormal levels of GSH likely lead to cancer, cardiac failure, and hepatic and renal failure.³ Hence, the detection and quantification of GSH, in biological systems in particular, is of vital importance.

Fluorescent probe has a wide range of application due to its operational simplicity, good specificity, high sensitivity, real-time monitoring and ability of intracellular imaging.⁴ So far, only a few fluorescent probes can discriminate GSH, and the different mechanisms include Michael addition,⁵ cleavage reaction,⁶ nucleophilic substitution of halogen,⁷ redox reaction,⁸ metal complexes^9,10 and others.¹¹ Nevertheless, previous reported probes have still some drawbacks. For instance, a mitochondria-targeted reversible fluorescent probe toward ONOO⁻/GSH redox status could only be used successfully in organic–aqueous system (acetonitrile 10%, v/v), rather than aqueous solution.⁸ Moreover, this kind of probes could not directly monitor GSH, which should actually be regarded as an ONOO⁻ probe. Recently, a coumarin–hemicyanine dye containing three potential reaction sites was reported as a new fluorescent probe for biothiols, which elicited three different chemical reactions toward Cys, GSH and Hcy, respectively.¹² Although this probe can efficiently detect Cys and GSH from two emission channels respectively, it requires a long response time over 60 min to become stable and could not detect GSH and Cys in living cells accurately. In recent years, two-photon excitation dyes have attracted much attention.¹³ However, there is limited number of two-photon fluorescence probes for thiols, especially for GSH.¹⁴ Thus, the development of quick response two-photon probes for GSH detection with high sensitivity and selectivity in aqueous solution is still in need.¹⁵

Hereby, we designed and synthesized a highly sensitive and selective fluorescent probe, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-yl acrylate (PPN) with an acrylate group and naphthalene–pyrazoline fluorophore (Scheme S1†), which was characterized by X-ray single crystal diffraction (Fig. S1†). Also, naphthalene is a common two-photon excitation dye with good solubility and two-photon absorption cross-section.¹⁶ Therefore, PPN can selectively detect GSH from other sulfhydryl compounds within 10 min in aqueous solution (containing CTAB micelles) with either one-photon or two-photon. It was also been successfully applied to calf serum.

Results and discussion

Mechanism studies

Acrylate group is an ideal functional trigger to detect thiols in recent years.¹⁷ Cys, Hcy and GSH with subtle structure distinction react with acrylate derivative to yield different reaction products (Scheme S2†). Most of the reported fluorescent probes with acrylate group could detect Cys and Hcy over other amino acids based on the conjugate addition/cyclization mechanism.¹⁸ The better phenomena were that fluorescent probes can discriminate Cys from most thiols.¹⁹ Up to now, only one reported probe using an acrylate derivative could detect GSH over Cys and Hcy by the Michael addition product.²⁰ However, it had a long response time of 12 h in 30% EtOH aqueous solution. Hence, we undertake to design a probe that can distinguish GSH from Cys/Hcy based on the different reaction product.

According to previous experience, we deduced that the reaction mechanism of PPN with GSH/Cys took place as illustrated in Scheme 1.²¹ Initially, the fluorometric detection of compound 4, PPN and PPN with GSH or Cys in CTAB–PBS buffer solution was summarized in Fig. S2.† Obvious fluorescence enhancement was observed when GSH was added to the solution of PPN, implying the Michael addition product 6. Afterwards, it's difficult to form a ring, and only got conjugate addition product (fluorescence quantum yield Φ = 0.499). In contrary, PPN with Cys showed weak fluorescence (fluorescence quantum yield Φ = 0.072), similar to compound 4 and PPN (fluorescence quantum yield Φ = 0.024 and Φ = 0.018), implying the conjugate addition/intramolecular cyclization reaction. The reaction product of PPN with GSH/Cys was subjected to electrospray ionization mass spectral analyses. The peaks at m/z 726.2483 and 176.0935 were consistent with compound 6 and 8 (Fig. S3 and S4†).

Scheme 1 Synthesis of probe PPN (compound 5) and reaction mechanism of probe PPN with GSH and Cys.

Noteworthy, the change of fluorescence of PPN with Cys did not show expected trend. At first, the fluorescent intensity of adduct should be increased, then decreased after intramolecular cyclization reaction. Compared with literature,²⁰ reaction time might be the crucial reason. Time-dependent spectra of PPN with GSH/Cys, were investigated (Fig. S5†). The fluorescent intensity at 480 nm increased to the maximum within 10 min for GSH and can remain slight changes for a long time, but that for Cys is 2 min. Therefore, we only detected the fluorescent intensity of equilibrium state. In order to further study the effect of time on the fluorescent intensity, we measured the time-dependent fluorescent intensity changes of probe PPN towards GSH and Cys in PBS–CH₃CN (1 : 1, v/v) buffer (Fig. S6†). Miraculously, with the addition of Cys, fluorescent intensity initially increased, and decreased after 100 min and reached steady state at 300 min. GSH induced initially a lower fluorescent intensity and needed a longer response time (400 min) to reach maximum than that in PBS–CTAB buffer solution. Fig. S6† shows the same trend as that of literature,²⁰ which means that organic solvent cannot improve the reaction rate. Except one reported probe had 5 min response time,⁹ other biothiols probes always need more than 30 min response time.²² Cationic surfactant CTAB could build a micellar system to absorb GSH, and then the higher GSH concentration should be resulted around CTAB micelle.²³ Obviously, GSH has weak response to PPN without CTAB in PBS buffer (Fig. S7†). On the contrary, a higher peak was raised when CTAB added to buffer. The results show that cationic surfactant CTAB added to PBS buffer solution accelerated the solubility, sensitivity and spectral response of the probe.

Fluorescent studies of probe PPN

In view of the merits of cationic surfactant CTAB, we recorded the all samples in PBS buffer (pH 7.4, 1 mM CTAB). Our buffer system composed of aqueous solution to avoid toxic organic reagent as compared with other reports.²⁴ The selectivity of PPN was investigated by the detection of amino acids (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, threonine, tryptophan, tyrosine), metal ions (K⁺, Ca²⁺, Na⁺, Mg²⁺, Zn²⁺, Fe³⁺), Na₂S, H₂O₂ and glucose in CTAB–PBS system. Obviously, GSH induced a fluorescent enhancement at 480 nm up to 27-fold, which exhibited a strong blue fluorescence (Fig. 1). Other analytes including similar structure molecule (Cys, Hcy) and Na₂S induced negligible change. Competitive experiments investigated by treating PPN with GSH in the presence of some biologically relevant analytes (20 equiv.) showed scarce interference (Fig. S8†). Furthermore, we explored the effects of more sulfhydryl compounds on PPN (Fig. 2A). Aliphatic thiols (2-mercaptoethanol, 6-mercaptohexan-1-ol) and aromatic thiol (4-aminobenzenethiol) also cannot induce strong fluorescence. Under the same conditions, two-photon fluorescence spectra of PPN with the sulfhydryl analytes were measured (Fig. 2B), which is similar to one-photon fluorescence spectra. Two-photon excitation fluorescence intensity at 480 nm enhanced 24-fold after GSH addition, and the others induced weak fluorescent intensity changes (Fig. S9†). The results foreboded that probe PPN was able to efficiently discriminate GSH from not only Cys/Hcy, but also other aliphatic thiols and aromatic thiols. In order to demonstrate its specificity, GSH was added to the aqueous solution with PPN and N-methylmaleimide (NEM). Obviously, PPN had no optical response because of the deficiency of GSH caused by NEM (Fig. S10†). Therefore, GSH was the only factor that could induce fluorescent enhancement, which is a major advantage over most reported thiols probes.²⁵

Fig. 1 Fluorescence spectra of probe PPN (2 μM) with various analytes (40 μM) in PBS buffer (CTAB 1 mM, pH 7.4, λ_ex = 390 nm).

Fig. 2 Fluorescence spectra (A) one-photon excitation λ_ex = 390 nm and (B) two-photon excitation λ_ex = 800 nm of probe PPN (2 μM) with various sulfhydryl analytes (40 μM) in PBS buffer (CTAB 1 mM, pH 7.4).

To research the possibility of precise quantitative detection of GSH, PPN was treated with various concentrations of GSH in CTAB–PBS. Fluorescent intensity at 480 nm increased upon increasing addition of GSH, which was linearly proportional to GSH in the range of 0 to 1.8 μM (Fig. 3). Under the optimized conditions, the detection limit (LOD) was calculated to be 1.5 × 10⁻⁸ M (σ = 15.955, k = 6472.993, R² = 0.994, Fig. S11†). The two-photon fluorescent titration spectra were shown in Fig. S12.† Fluorescent intensity increases with the concentrations of GSH (0–10 μM). Similarly, based on the linearly proportional relation, the LOD was 7.5 × 10⁻⁸ M (σ = 36.397, k = 2969.076, R² = 0.987, Fig. S13†). In comparison, we measured the LOD of probe PPN towards GSH in PBS–CH₃CN (1 : 1, v/v) buffer is 29 × 10⁻⁸ M (σ = 9.759, k = 504.801, R² = 0.989, Fig. S14†), which shows that again organic solvent could reduce the sensitivity of the probe. All the results indicate that PPN can detect GSH qualitatively and quantitatively by the fluorescence spectrometry method no matter one-photon or two-photon excited.²⁶

Fig. 3 Fluorescence spectra of probe PPN (2 μM) with GSH (0–10 equiv.) in PBS buffer (CTAB 1 mM, λ_ex = 390 nm, pH 7.4). Inset is the plot of fluorescence intensity of probe PPN vs. equivalents of GSH. Data are mean ± SE (bars) (n = 3).

Application of probe PPN

As the hydrolysis of ester group, the test can potentially be affected by the pH of the media. Therefore, pH effect on the detection of GSH with PPN was measured (Fig. S15†). The fluorescent intensity of PPN in the absence and presence of GSH was stable and was negligible influenced by pH in the range from 5.53 to 9.10. The results indicate that PPN is suitable to be utilized in both diseased cells and normal living cells. Thus, we chose physiological pH 7.4 as the test condition.

We incubated PPN (5 μM) with calf serum (CS) samples at 37 °C to explore its practical application. Fluorescent intensity increased with increasing concentration of CS, which had exhibited excellent blue fluorescence (Fig. 4). According to the reaction mechanism, we declared that the fluorescence was generated by probe PPN and GSH in the CS. Moreover, we got fluorescent images of various concentrations of CS without probe PPN (Fig. S16A†). Although CS has weak autofluorescence at higher concentration, probe PPN can detect CS at lower concentrations (0–20%) by the quantified data of fluorescent intensity deducted background fluorescence (Fig. S16B†).

Fig. 4 Fluorescence images of PPN (5 μM) with different concentrations of calf serum. Calf serum was diluted 100 times (1%), 50 times (2%), 20 times (5%), 10 times (10%), 5 times (20%), 2 times (50%), 4/3 times (75%) with PBS and not diluted (100%) (n > 3).

Cytotoxicity is an essential factor for the application of PPN as a probe in biological systems. Thus, we performed sulforhodamine B assays of probe PPN in A549 cells (Fig. S17†). The cellular viability exhibited no significant reduction after incubation for 6 h with 10 μM probe PPN. Furthermore, we applied probe PPN for the fluorescence imaging of GSH in living cells. Living A549 cells were incubated with PPN at different concentrations and times (Fig. 5A). Obviously, living cells showed blue fluorescence at 1 μM PPN after 0.5 h. Moreover, the fluorescent intensity increased with time and concentration of probe PPN. We can get bright blue fluorescence after 1 h, even 1 μM of PPN. The quantified data of fluorescent intensity obtained by using ImageJ exhibited wonderful time and concentration dependent manner (Fig. 5B).

Fig. 5 (A) Fluorescence images of probe PPN in A549 cells at different concentrations and times. (B) Fluorescence intensity quantification. Data are mean ± SE (n > 3; *p < 0.05, **p < 0.01 vs. control).

Conclusions

We have developed a high selective and sensitive two-photon fluorescent probe PPN for qualitative and quantitative detection of GSH from other amino acids and sulfhydryl compounds under physiological pH condition. The addition of GSH to the aqueous solution of PPN induces a 27-fold fluorescence intensity enhancement and the detection limit was calculated to be 1.5 × 10⁻⁸ M. Similarly, PPN got a linearly proportional to GSH in the range of 0 to 2.0 μM with two-photon excited, and the detection limit was 7.5 × 10⁻⁸ M. The fluorescence quantum efficiency enlarged and reached its maximum within 10 min based on the Michael addition. Moreover, PPN had potential biological significance and had good application in monitoring GSH in calf serum and living cell imaging.

Acknowledgements

This study was supported by the National Basic Research Program of China (2010CB933504) and the National Natural Science Foundation of China (91313303).

Notes and references

1. H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin and J. M. Scott, Clin. Chem., 2004, 50, 3; Z. A. Wood, E. Schroder, J. R. Harris and L. B. Poole, Trends Biochem. Sci., 2003, 28, 32.
2. H. J. Forman, H. Zhang and A. Rinna, Mol. Aspects Med., 2009, 30, 1; L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275.
3. S. K. Biswas and I. Rahman, Mol. Aspects Med., 2009, 30, 60; R. Obeid and W. Herrmann, FEBS Lett., 2006, 580, 2994.
4. D. Jeyanthi, M. Iniya, K. Krishnaveni and D. Chellappa, RSC Adv., 2013, 3, 20984; G. Sivaraman, B. Vidya and D. Chellappa, RSC Adv., 2014, 4, 30828; A. Barve, M. Lowry, J. O. Escobedo, K. T. Huynh, L. Hakuna and R. M. Strongin, Chem. Commun., 2014, 50, 8219; H. Zheng, X. Q. Zhan, Q. N. Bian and X. J. Zhang, Chem. Commun., 2013, 49, 429; C. Yin, F. Huo, J. Zhang, R. Martinez-Manez, Y. Yang, H. Lv and S. Li, Chem. Soc. Rev., 2013, 42, 6032; H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019; N. Boens, V. Leen and W. Dehaen, Chem. Soc. Rev., 2012, 41, 1130; X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120; N. Kaur, P. Kaur and K. Singh, RSC Adv., 2014, 4, 29340; S. J. Balkrishna, A. S. Hodage, S. Kumar, P. Panini and S. Kumar, RSC Adv., 2014, 4, 11535 (Chin. J. Org. Chem., 2014, 34, 1717–1729).
5. G. J. Kim, D. H. Yoon, M. Y. Yun, H. Kwon, H. J. Ha and H. J. Kim, RSC Adv., 2014, 4, 18731; B. K. McMahon and T. Gunnlaugsson, J. Am. Chem. Soc., 2012, 134, 10725; A. Agostini, I. Campos, M. Milani, S. Elsayed, L. Pascual, R. Martinez-Manez, M. Licchelli and F. Sancenon, Org. Biomol. Chem., 2014, 12, 1871.
6. D. Zhai, S. C. Lee, S. W. Yun and Y. T. Chang, Chem. Commun., 2013, 49, 7207; L. Wang, H. Chen, H. Wang, F. Wang, S. Kambam, Y. Wang, W. Zhao and X. Chen, Sens. Actuators, B, 2014, 192, 708; J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu and J. Yoon, J. Am. Chem. Soc., 2014, 136, 5351; X. F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedo and R. M. Strongin, Chem. Sci., 2014, 5, 2177.
7. L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and Q. Z. Yang, J. Am. Chem. Soc., 2012, 134, 18928.
8. F. Yu, P. Li, B. Wang and K. Han, J. Am. Chem. Soc., 2013, 135, 7674.
9. Z. Chen, Z. Wang, J. Chen and X. Chen, Biosens. Bioelectron., 2012, 38, 202.
10. W. Liang, Z. Zhao, Y. Zhang, Q. Wang, X. Zhao and J. Ouyang, J. Lumin., 2012, 132, 1160.
11. Y. Guo, H. Wang, Y. Sun and B. Qu, Chem. Commun., 2012, 48, 3221; Z. Yao, X. Feng, C. Li and G. Shi, Chem. Commun., 2009, 5886; R. Gui, X. An, H. Su, W. Shen, L. Zhu, X. Ma, Z. Chen and X. Wang, Talanta, 2012, 94, 295.
12. J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi and W. Guo, J. Am. Chem. Soc., 2014, 136, 574.
13. H. M. Kim and B. R. Cho, Oxid. Med. Cell. Longevity, 2013, 2013, 323619; F. Miao, W. Zhang, Y. Sun, R. Zhang, Y. Liu, F. Guo, G. Song, M. Tian and X. Yu, Biosens. Bioelectron., 2014, 55, 423; D. Kim, H. G. Ryu and K. H. Ahn, Org. Biomol. Chem., 2014, 12, 4550; W. Yang, P. S. Chan, M. S. Chan, K. F. Li, P. K. Lo, N. K. Mak, K. W. Cheah and M. S. Wong, Chem. Commun., 2013, 49, 3428.
14. S. Singha, D. Kim, A. S. Rao, T. Wang, K. H. Kim, K. H. Lee, K. T. Kim and K. H. Ahn, Dyes Pigm., 2013, 99, 308; Z. Yang, N. Zhao, Y. Sun, F. Miao, Y. Liu, X. Liu, Y. Zhang, W. Ai, G. Song, X. Shen, X. Yu, J. Sun and W. Y. Wong, Chem. Commun., 2012, 48, 3442; L. Polavarapu, M. Manna and Q. H. Xu, Nanoscale, 2011, 3, 429; C. S. Lim, G. Masanta, H. J. Kim, J. H. Han, H. M. Kim and B. R. Cho, J. Am. Chem. Soc., 2011, 133, 11132; J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem. Soc., 2010, 132, 1216; X. Zhang, X. Ren, Q. H. Xu, K. P. Loh and Z. K. Chen, Org. Lett., 2009, 11, 1257; M. Zhang, M. Yu, F. Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu, J. Zhang, D. Zhang, T. Yi and C. Huang, J. Am. Chem. Soc., 2007, 129, 10322; M. Zhang, M. Li, Q. Zhao, F. Li, D. Zhang, J. Zhang, T. Yi and C. Huang, Tetrahedron Lett., 2007, 48, 2329; X. Sun, A. Y. Shih, H. C. Johannssen, H. Erb, P. Li and T. H. Murphy, J. Biol. Chem., 2006, 281, 17420.
15. Q. Lin, C. Bao, S. Cheng, Y. Yang, W. Ji and L. Zhu, J. Am. Chem. Soc., 2012, 134, 5052; L. Long, W. Lin, B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47, 893; H. Lee and H. J. Kim, Org. Biomol. Chem., 2013, 11, 5012; P. Wang, J. Liu, X. Lv, Y. Liu, Y. Zhao and W. Guo, Org. Lett., 2012, 14, 520; M. H. Lee, J. H. Han, J. H. Lee, H. G. Choi, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012, 134, 17314.
16. H. M. Kim, M. J. An, J. H. Hong, B. H. Jeong, O. Kwon, J. Y. Hyon, S. C. Hong, K. J. Lee and B. R. Cho, Angew. Chem., Int. Ed., 2008, 47, 2231; S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E. H. Joe, B. R. Cho and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915; G. Masanta, C. H. Heo, C. S. Lim, S. K. Bae, B. R. Cho and H. M. Kim, Chem. Commun., 2012, 48, 3518.
17. N. J. Leonard and R. Y. Ning, J. Org. Chem., 1966, 31, 3928; X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., Int. Ed., 2011, 50, 10690; C. Zhao, X. Li and F. Wang, Chem.–Asian J., 2014, 9, 1777; D. Yu, Q. Zhang, S. Ding and G. Feng, RSC Adv., 2014, 4, 46561.
18. Z. Guo, S. Nam, S. Park and J. Yoon, Chem. Sci., 2012, 3, 2760; L. Wang, Q. Zhou, B. Zhu, L. Yan, Z. Ma, B. Du and X. Zhang, Dyes Pigm., 2012, 95, 275.
19. X. Dai, Q. H. Wu, P. C. Wang, J. Tian, Y. Xu, S. Q. Wang, J. Y. Miao and B. X. Zhao, Biosens. Bioelectron., 2014, 59, 35; D. P. Murale, H. Kim, W. S. Choi and D. G. Churchill, RSC Adv., 2014, 4, 5289.
20. S. Q. Wang, Q. H. Wu, H. Y. Wang, X. X. Zheng, S. L. Shen, Y. R. Zhang, J. Y. Miao and B. X. Zhao, Biosens. Bioelectron., 2014, 55, 386.
21. B. Zhu, Y. Zhao, Q. Zhou, B. Zhang, L. Liu, B. Du and X. Zhang, Eur. J. Org. Chem., 2013, 888.
22. X. Cao, W. Lin and Q. Yu, J. Org. Chem., 2011, 76, 7423; D. Maity and T. Govindaraju, Org. Biomol. Chem., 2013, 11, 2098; Y. Liu, S. Zhang, X. Lv, Y. Q. Sun, J. Liu and W. Guo, Analyst, 2014, 139, 4081.
23. H. Tian, J. Qian, Q. Sun, H. Bai and W. Zhang, Anal. Chim. Acta, 2013, 788, 165; H. Tian, J. Qian, H. Bai, Q. Sun, L. Zhang and W. Zhang, Anal. Chim. Acta, 2013, 768, 136; W. Dong, H. Wen, X. F. Yang and H. Li, Dyes Pigm., 2013, 96, 653.
24. B. Liu, J. Wang, G. Zhang, R. Bai and Y. Pang, ACS Appl. Mater. Interfaces, 2014, 6, 4402; Q. Q. Wu, Z. F. Xiao, X. J. Du and Q. H. Song, Chem.–Asian J., 2013, 8, 2564; T. Anand, G. Sivaraman and D. Chellappa, J. Photochem. Photobiol., A, 2014, 281, 47; S. S. Razi, R. Ali, P. Srivastava, M. Shahid and A. Misra, RSC Adv., 2014, 4, 16999; T. Anand, G. Sivaraman and D. Chellappa, Spectrochim. Acta, Part A, 2014, 123, 18.
25. X. L. Liu, X. Y. Duan, X. J. Du and Q. H. Song, Chem.–Asian J., 2012, 7, 2696.
26. X. F. Yang, Z. Su, C. Liu, H. Qi and M. Zhao, Anal. Bioanal. Chem., 2010, 396, 2667; X. D. Liu, R. Sun, Y. Xu, Y. J. Xu, J. F. Ge and J. M. Lu, Sens. Actuators, B, 2013, 178, 525.

---

Footnote

† Electronic supplementary information (ESI) available: Detail for the materials and methods, synthesis methods, X-ray single crystal diffraction, additional absorption, fluorescence spectra, cell imaging and NMR. CCDC 1000795. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra09712b

---