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A tetraphenylimidazole-based fluorescent probe for the detection of hydrogen sulfide and its application in living cells Biao Gu, Naxiu Mi, Youyu Zhang, Pen Yin, Haitao Li * , Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A sensitive and selective fluorescent probe for sensing H2S was developed. The sensing mechanism of the synthesized probe was based on ESIPT. Upon treatment with H2S, fluorescence of the probe shows a large Stokes shift. The probe can be used for fluorescence imaging of H2S in living cells.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2014 Received in revised form 16 February 2015 Accepted 10 March 2015 Available online xxx
A novel probe based on the fluorescence off–on strategy was prepared to optically detect hydrogen sulfide (H2S) via an excited state intramolecular proton transfer (ESIPT) mechanism. The probe shows high sensitivity and excellent selectivity to H2S. It also displays a large Stokes shift (140 nm) and a remarkable quantum yield enhancement (K = 0.412) after interaction with H2S. Moreover, the cellular imaging experiment demonstrated that it has potential utility for H2S sensing in biological sciences. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Probe Hydrogen sulfide ESIPT Cellular imaging
1. Introduction As one of the most important gaseous signaling molecule [1], hydrogen sulfide (H2S) plays an important role in many biological
* Corresponding author. Tel.: +86 731 88865515; fax: +86 731 88865515. E-mail address: [email protected] (H. Li).
processes such as the relaxation of blood vessels [2], neural signal transmission [3], cell growth regulation [4] and inflammation [5]. Scientific evidence has demonstrated that H2S is present at levels of about 10–100 mM in blood [6] and may reach 600 mM in the brains of human [7]. However, the abnormal H2S level in cells could be related to many health issues such as Alzheimer’s disease [8], congenital down low intelligence [9], diabetes [10] and liver cirrhosis [11], as H2S is involved in the chemical regulation of many
http://dx.doi.org/10.1016/j.aca.2015.03.017 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
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biological processes. It has also been reported that H2S can act as a neuromodulator in the brain [7] and a scavenger for endogenous oxidants such as peroxynitrite, superoxide and hydrogen peroxide [12,13]. Therefore, the development of efficient methods for H2S detection and direct indication of its contribution in living systems is of considerable significance for the related biomedical research and disease diagnosis. Up to now, a number of analytical methods for the detection of H2S have been developed, including colorimetric method [14], electrochemical analysis [15], chromatography [16], metal-induced precipitation [17] and fluorescence spectroscopy [18]. Among these methods, fluorescence probes are more favorable due to their high selectivity, simplicity, real-time spatial imaging and non-destructive advantage [19,20]. In the past few years, there have been several kinds of probes reported for detecting H2S. In general, these probes were designed by taking advantage of chemical properties of H2S, such as binding affinity with copper ion [21–23], nucleophilic reaction [24–26] and good reducing property [27–29]. The probes based on copper sulfide precipitation for H2S showed good sensitivity and selectivity, but the poor live cell permeability of the probes and toxicity of Cu2+ limited their biological applications [30]. The probe based on nucleophilic substitution suffered the possible interference from biological thiols [31,32], such as cysteine, glutathione and homocysteine, because of their similar reactivity of thiolcontaining molecules and the biological thiols are typically higher in cellular levels than H2S in living cells [33,34]. Recently, the probe based on H2S reductive property have been developed through either intramolecular charge transfer (ICT) or photoinduced electron transfer (PET) processes. Nevertheless, some of them exhibited relatively weak fluorescence intensity and small Stokes shift, which might decrease their sensing performance. In comparison with ICT and PET processes, excited state intramolecular proton transfer (ESIPT) process generally has large Stokes shift [35,36], which offers an advantage that minimizes the selfabsorption and reduces the interference from auto-fluorescence for in vivo application. Therefore, it is very meaningful to develop a novel probe based on ESIPT process for detecting H2S in living cells. Recently, Skonieczny et al. reported the synthesis and optical properties of imidazole derivatives and their analogues, an interesting new class of fluorophores [37]. A large Stokes shift, strong fluorescence and high fluorescence quantum yield of 2-(1(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2-yl)aniline prompted us to select it as the active fluorophore. We envisioned that the conversion of the amino group into an azido, which could occlude the ESIPT process, would result in quenching of fluorescence. Due to the reducing ability of H2S, the azido group could be converted into an amino group and consequently turn on the ESIPT, which could restore the fluorescence properties of the fluorophore. Therefore, the strategy should be meaningful to the new design of tetraphenylimidazole-based ESIPT molecular probe (P-N3). Indeed, P-N3 as a new probe for H2S has many advantages including the wide concentration range for linear response, the larger Stokes shift and the high selectivity and sensitivity for H2S. Further more, cell imaging experiments suggested that the biological application of the probe is prospective. 2. Experimental 2.1. Materials and instruments Phenanthrenequinone, p-toluidine, and o-nitrobbenzaldehyde were obtained from Sigma–Aldrich Company. Ethanol, dichloromethane and sodium hydrosulfide were purchased from Sinopharm Chemical Reagent Company. All other chemicals used in this work were of analytical grade. The detection buffer was PBS buffer
(0.01 M, pH 7.4). Milli-Q ultrapure water (Millipore, 18 MV cm) was used throughout. Except the specific statement, solvents were purified by distillation. Silica gel 300–400 mesh (37–54 mm) was used for column chromatography. The thin-layer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. Bruker AVB-500 spectrometer, ZQ2000 mass spectrometer (Manchester, UK), Nexus 670 Fourier Transform Infrared Spectrometer (Nicolet Co., USA) and F-4500 FL spectrophotometer (Hitachi Co., Japan) were used to characterize the probe P-N3. Fluorescence images of HeLa cells were performed by fluorescence microscopy (Nikon, Eclipse Ti-S). 2.2. Synthetic procedure 2.2.1. Synthesis of 2-(2-nitrophenyl)-1-(p-tolyl)-1H-phenanthro[9,10d]imidazole (1) Phenanthrenequinone (416.4 mg, 2.00 mmol) and ammonium acetate (770.8 mg, 10.00 mmol) were added to a solution of o-nitrobbenzaldehyde (302.2 mg, 2.00 mmol) and p-toluidine (321.5 mg, 3.00 mmol) in glacial acetic acid. The reaction mixture was refluxed for 12 h and then cooled to room temperature. Subsequently, the reaction mixture was poured into 80 mL water, and then extracted with ethyl acetate. The organic layer was washed with brine, dried with Na2SO4, filtered and concentrated. An orange crystalline solid (790.2 mg, 92%) was obtained from crystallization and characterized by NMR. 1H NMR (500 MHz, CDCl3): d 8.78 (d, J = 8.4 Hz, 1H), 8.75 (dd, J = 7.9, 0.8 Hz, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.71 (dd, J = 11.0, 3.9 Hz, 1H), 7.68–7.60 (m, 3H), 7.54 (tt, J = 8.4, 2.5 Hz, 2H), 7.32–7.27 (m, 3H), 7.24 (d, J = 8.0 Hz, 3H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3): d 148.87, 147.70, 139.81, 134.46, 133.51, 132.99, 130.51, 130.47, 129.35, 128.27, 127.36, 127.11, 126.66, 126.34, 125.68, 125.17, 124.66, 124.10, 123.15, 122.89, 122.71, 120.96, 22.40. 2.2.2. Synthesis of 2-(1-(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2yl)aniline (2) Compound 1 (584.1 mg, 1.36 mmol) was dissolved in 40 mL ethyl acetate and reduced with hydrogen (1 atm) on 10% Pd-C (40 mg) as a catalyst. After the reaction completed, the catalyst was filtered off. Then, the crude product was purified by crystallization from ethyl acetate to afford a dark green crystalline solid (523.0 mg, 96%) and the solid was measured with NMR. 1H NMR (500 MHz, CDCl3): d 8.80 (dd, J = 7.9, 0.9 Hz, 1H), 8.77 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 7.78–7.70 (m, 1H), 7.68–7.62 (m, 1H), 7.51 (ddd, J = 8.3, 6.7, 1.6 Hz, 1H), 7.40–7.32 (m, 4H), 7.31–7.26 (m, 2H), 7.15–6.98 (m, 1H), 6.88 (dd, J = 7.8, 1.3 Hz, 1H), 6.78 (dd, J = 8.1, 0.7 Hz, 1H), 6.45 (td, J = 7.9, 1.1 Hz, 1H), 5.32 (d, J = 28.1 Hz, 2H), 2.51 (s, 3H). 13C NMR (126 MHz, CDCl3): d 149.57, 147.29, 139.64, 136.59, 135.95, 130.62, 130.36, 129.89, 129.18, 128.73, 128.25, 127.24, 126.98, 126.31, 125.57, 124.86, 124.07, 123.17, 123.07, 122.63, 121.13, 116.77, 116.40, 113.92, 77.34, 77.09, 76.83, 21.47. 2.2.3. Synthesis of 2-(2-azidophenyl)-1-(p-tolyl)-1H-phenanthro [9,10-d]imidazole (P-N3) Compound 2 (259.7 mg, 0.65 mmol) was dissolved slowly in HCl aq. (37.5%, 3 mL) at room temperature. The solution was cooled in an ice bath and 3 mL solution of NaNO2 (67.6 mg, 0.98 mmol) was added. The reaction mixture was stirred for 1 h in an ice bath. This was followed by the addition of potassium acetate to adjust the pH of the resulting solution to 4. Then, NaN3 (126.7 mg, 1.95 mmol) was added in portions. Subsequently, the mixture was stirred at 0–5 C for another 4 h, and it was extracted with ethyl acetate. The organic layer was washed with brine, dried with Na2SO4, filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (15:1, petrol ether:ethyl acetate, v/v) to afford the desired product as an orange solid
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(141.1 mg, 51%). 1H NMR (500 MHz, CDCl3): d 8.94–8.80 (m, 1H), 8.77 (d, J = 8.5 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 7.72 (dd, J = 11.0, 4.0 Hz, 1H), 7.68–7.62 (m, 1H), 7.53 (ddd, J = 8.3, 6.0, 2.3 Hz, 1H), 7.47 (dd, J = 7.6, 1.4 Hz, 1H), 7.40 (td, J = 8.0, 1.5 Hz, 1H), 7.34–7.28 (m, 4H), 7.28–7.20 (m, 4H), 2.44 (s, 3H). 13C NMR (126 MHz, CDCl3): d 148.74, 139.89, 139.40, 137.29, 135.14, 132.84, 130.93, 130.04, 129.30, 128.26, 127.40, 126.23, 125.53, 124.99, 124.42, 124.05, 123.06, 122.79, 121.03, 118.45, 21.36. MS (ESI+) [M + H]: 425.64. IR (KBr, cm1): 2132 (N3). The synthesis processes and structure characterization of intermediates and probe are provided in the Supplementary information (see Scheme S1 and Figs. S1–S8). 2.3. Determination of the fluorescence quantum yield The fluorescence quantum yields of P-N3 and P-NH2 were determined in ethanol at 25 C, using quinine sulfate (F = 0.546 in 1 N H2SO4) as a reference. The quantum yield was calculated using the following equation: 2 AF ns Fs ¼ Fr r s As F r nr 2 where s and r denote sample and reference, respectively, A is the absorbance, F is the relative integrated fluorescence intensity, and n is the refractive index of the solvent. 2.4. Fluorescent detection of H2S For all tests, the experiments were repeated at least three times to ensure the accuracy of the measurements. The fluorescent measurements for H2S were carried out as follows: P-N3 was dissolved in ethanol to prepare a stock solution (1 mM). The fluorescence of P-N3 (10 mM) was detected after incubating 80 min at 37 C in phosphate buffer (0.01 M, pH 7.4) in the presence and absence of H2S (NaHS was used as the hydrogen sulfide source in all experiments). Then, fluorescence spectra were recorded with excitation wavelength at 300 nm. The fluorescence emission intensity at 436 nm was used for quantitative analysis of H2S. 2.5. Fluorescence imaging The HeLa cells were incubated on 96-well plate in the culture medium and allowed to adhere for 24 h at 37 C. Immediately prior to the imaging experiments, the cells were washed with phosphate buffered saline (PBS), incubated with 10 mM P-N3 (in the culture medium containing 1% ethanol) for 40 min at 37 C, then washed with PBS for three times, and imaged. After incubating with 60 mM NaHS for another 80 min at 37 C, the HeLa cells were washed with PBS three times and imaged again. The fluorescence imaging of living HeLa cells was observed and imaged on fluorescence microscopy with a 40 objective lens.
Fig. 1. (a) Absorption spectra, (b) excitation spectra, and (c) emission spectra of probe P-N3 (10 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v).
3.2. Mechanism of the detection of H2S Initially, we proposed the optical probe P-N3 for the detection of H2S based on the tetraphenylimidazole dye. Briefly, the probe P-N3 was designed by combining tetraphenylimidazole moiety as the fluorophore and azido group as the recognition unit. The fluorescence of the fluorophore could be quenched by the azido group due to the hampered ESIPT process. Once reduction of azide to amine was triggered by H2S, the tetraphenylimidazole moiety would shed high fluorescence because of the resulting ESIPT modulated fluorescence off–on response. To verify the proposed mechanism, the fluorescence of P-N3 in the absence and presence of H2S (NaHS was used as a hydrogen sulfide source in all experiments) was investigated (Fig. 2). The free P-N3 exhibited very weak fluorescence (K = 0.024). However, upon the addition of NaHS (10 equiv.) into the probe solution, a dramatic fluorescence intensity enhancement was observed at around 436 nm with a very large Stokes shift (140 nm). As shown in the inset of Fig. 2, observation of the bright blue emission from “P-N3 + NaHS” sample indicated the formation of fluorescence product P-NH2 (K = 0.412). To further verify this hypothesis, the purified product of the reaction of P-N3 with NaHS was then characterized by 1H NMR, 13C NMR and mass spectra (see Figs. S9–S11), which agreed well with the presynthesized compound 2 (see Figs. S3 and S4) demonstrating that the sensing of H2S with the probe is indeed through an
3. Results and discussion 3.1. Spectral characteristics of P-N3 UV–vis absorption and fluorescence spectra properties of P-N3 were measured in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) at ambient temperature. As shown in Fig. 1, the typical UV–vis absorption peak of P-N3 (10 mM) was observed at 260 nm, which could be attributed to the p–p* transition of the p-conjugated core. When being excited at 300 nm, P-N3 gave a weak emission peak at around 436 nm due to the quenching effect of azido group [38–40].
Fig. 2. Fluorescence spectra of P-N3 (10 mM) before (a) after (b) the incubation with 10 equiv. of NaHS in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) with lex = 300 nm. The insets show the photos of samples illuminated by UV light of 365 nm. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
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fluorescence intensity of P-N3 over a wide pH range (3–10), suggesting that P-N3 was not pH sensitive. Upon treatment with NaHS, the maximal fluorescence signal was observed in the range of pH from 7 to 10. which could be explained by the stronger reducing power of the hydrosulfide ion at neutral to basic environment [33,43]. Therefore, physiological pH (pH 7.4) could be selected as an appropriate working pH in the following experiments. In addition, the influence of pH on the fluorescence intensity of the reaction product P-NH2 was also investigated (Fig. S12). The fluorescence intensity of P-NH2 maintained invariability at neutral to high pH (10). When pH < 6, the emission intensity of P-NH2 gradually increased, this may be due to the protonation of the amine group. The time course study revealed that the reaction between P-N3 and NaHS was completed at about 80 min in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) (Fig. 3B). Scheme 1. Proposed reaction mechanism for H2S.
3.4. The analytical performances of probe azide reduction. Thus, combined with the previously reported conclusion [20,41,42], a possible mechanism was proposed as shown in Scheme 1. 3.3. Optimization of detection conditions Considering the factors affecting the reaction of P-N3 with H2S, pH and incubation time were optimized. We first investigated the effect of pH on fluorescence properties of P-N3 and its response to H2S. As shown in Fig. 3A, there was nearly no change in the
Under the optimal conditions, the fluorescence responses of P-N3 to different concentrations of NaHS were investigated. The fluorescence of the mixture increased as the NaSH concentration increased, and a maximal fluorescence enhancement was obtained when the concentration of NaHS reached 9 equiv. (Fig. 4A). Additionally, there was a good linear relationship between the normalized fluorescence intensity at 436 nm and the concentrations of NaHS in the range of 0–70 mM (Fig. 4B). As shown in inset Fig. 4B, the NaHS with submicromolar level exhibited a linear response. The detection limit was calculated according to previous
Fig. 3. (A) The pH influence on the fluorescence intensity of P-N3 (10 mM) in the absence (&) or presence (*) of NaHS (40 mM). (B) Time-dependent fluorescence spectra of PN3 (10 mM) in the presence of NaHS (70 mM). Data were acquired in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). Time points represent 0, 10, 20, 30, 40, 50, 60, 70, 80 and 100 min.
Fig. 4. (A) Fluorescence spectra of P-N3 (10 mM) in the presence of various concentrations of NaHS (0–90 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). (B) Fluorescence intensity of probe vs. the concentrations of NaHS. (inset B) The fluorescence intensity curve for NaHS at nanomolar concentrations, with error bars that display 3 standard deviations.
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Fig. 5. (A) Fluorescence responses of P-N3 (10 mM) in the present of species (0.5 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v): (1) no addition; (2) H2O2; (3) S2O32; (4) citrate; (5) Cl; (6) CO32; (7) F; (8) HCO3; (9) HPO42; (10) N3; (11) NO2; (12) NO3; (13) OAc; (14) SO42; (15) HSO3; (16) DTT; (17) 2-ME; (18) Cys; (19) Hcy; (20) GSH; (21) HS. (B) Fluorescence responses of P-N3 (10 mM) at 436 nm toward various analytes in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). Black bars represent the addition of a single analyte (1 mM, from left to right: (1) none; (3) DTT; (5) HSO32; (7) 2-ME; (9) Cys; (11) Hcy; (13) GSH). Blue bars represent the mixing corresponding sulfur compounds with HS solution (70 mM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
report [44], which was obtained based on the slope between 0 and 70 mM concentration and the standard deviation of the zero level (i.e., standard approximation 3 SDzero/slope), and limit of quantification was accordingly approximated to 1.9 107 M. These results demonstrated the suitability of P-N3 for the quantitative measurement of H2S. Selectivity is another important factor to evaluate the performance of a new fluorescent probe. Therefore, the fluorescence responses of P-N3 to a series of other biological-related species were investigated. As shown in Fig. 5A, only the addition of HS
caused a large fluorescence enhancement of P-N3. In contrast, the addition of other analytes showed almost no influence on the fluorescence of the probe (the selectivity coefficients toward thiols and sulfite was calculated according to reference [45], which are less than 0.06, see in Table S1). Moreover, considering the relatively high concentration of the possible reducing species in biological system or in the test condition (DTT, 2-ME, Cys, Hcy, GSH and sulfite), we also examined the interference of these species at the level of 1 mM in the detection of HS. As shown in Fig. 5B, distinct fluorescence enhancement can be observed after subsequent
Fig. 6. Fluorescence microscopy images of H2S detection in HeLa cells using P-N3. Bright-field image of live HeLa cells incubated with P-N3 for 40 min and then treated for 80 min in the absence (A) and presence (B) of 60 mM NaHS at 37 C. (C) and (D) represent the fluorescence image images of (A) and (B), respectively.
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addition of HS to the probe P-N3 solution in the presence of these species, suggesting the interference of these chemicals was very little, and P-N3 is potentially useful for sensing H2S in the presence of these species. All these results indicate that probe P-N3 has high selectivity for HS detection. This might be due to the fact that the steric effect of thiols in the reaction is greater than HS, which makes the reduction reaction more difficult. 3.5. Fluorescence imaging of H2S in living cells In order to investigate the biological application of P-N3, the fluorescence microscopy experiment was carried out. HeLa cells were incubated with 10 mM P-N3 (in the culture medium containing 1% ethanol) for 40 min and then treated with a blank control or 60 mM NaHS for an additional 80 min at 37 C. This concentration is well within the range that has been used to elicit physiological responses (10–600 mM) [46]. It was found that the control experiments where the cells were not treated with NaHS showed faint fluorescence (Fig. 6C), but those treated with NaHS displayed strong blue fluorescence emission (Fig. 6D). The bright-field images (Fig. 6A and B) confirmed that the cells were viable throughout the imaging experiments. Thus, P-N3 showed excellent cell permeation capability and could efficiently sense H2S in living cells. 4. Conclusion In summary, we have developed an ESIPT-based, off–on fluorescent probe (P-N3) by combining tetraphenylimidazole moiety as the fluorophore and azido group as the recognition unit. The developed probe displayed a large Stokes shift, a wide linear concentration rang and good sensitivity. Meanwhile, this probe showed an excellent selectivity to H2S over other anions, reactive sulfur and oxygen. Fluorescent imaging of intracellular H2S demonstrated its potential for biological applications. Acknowledgments This work was supported by the National Natural Science Foundation of China (21375037, 21275051, 21405043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (20134306110006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.03.017. References [1] L. Li, P. Rose, P.K. Moore, Annu. Rev. Pharmacol. Toxicol. 51 (2011) 169–187. [2] O. Jackson-Weaver, J.M. Osmond, M.A. Riddle, J.S. Naik, L.V.G. Bosc, B.R. Walker, N.L. Kanagy, Am. J. Physiol. Heart Circ. Physiol. 304 (2013) H1446–H1454.
[3] H. Kimura, Mol. Neurobiol. 26 (2002) 13–19. [4] G. Yang, L. Wu, R. Wang, FASEB J. 20 (2006) 553–555. [5] C. Yang, Z. Yang, M. Zhang, Q. Dong, X. Wang, A. Lan, F. Zeng, P. Chen, C. Wang, J. Feng, PLoS One 6 (2011) e21971. [6] L. Li, M. Bhatia, Y.Z. Zhu, Y.C. Zhu, R.D. Ramnath, Z.J. Wang, F.B.M. Anuar, M. Whiteman, M. Salto-Tellez, P.K. Moore, FASEB J. 19 (2005) 1196–1198. [7] K. Abe, H. Kimura, J. Neurosci. 16 (1996) 1066–1071. [8] K. Eto, T. Asada, K. Arima, T. Makifuchi, H. Kimura, Biochem. Biophys. Res. Commun. 293 (2002) 1485–1488. [9] P. Kamoun, M.C. Belardinelli, A. Chabli, K. Lallouchi, B. Chadefaux-Vekemans, Am. J. Med. Genet. Part A 116 (2003) 310–311. [10] W. Yang, G. Yang, X. Jia, L. Wu, R. Wang, J. Physiol. 569 (2005) 519–531. [11] S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah, A. Morelli, Hepatology 42 (2005) 539–548. [12] R.F. Milos, M. Jan, A. Andrea, C. Ricardo, S. Tatyana, H. Martin, I.-B. Ivana, Biochem. J. 441 (2012) 609–621. [13] C.M. Jones, A. Lawrence, P. Wardman, M.J. Burkitt, Free Radic. Biol. Med. 32 (2002) 982–990. [14] X. Gu, C. Liu, Y.-C. Zhu, Y.-Z. Zhu, Tetrahedron Lett. 52 (2011) 5000–5003. [15] D.G. Searcy, M.A. Peterson, Anal. Biochem. 324 (2004) 269–275. [16] C.J. Richardson, E.A.M. Magee, J.H. Cummings, Clin. Chim. Acta 293 (2000) 115–125. [17] M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii, H. Kimura, Antioxid. Redox Signal. 11 (2008) 205–214. [18] Y. Zhao, T.D. Biggs, M. Xian, Chem. Commun. 50 (2014) 11788–11805. [19] A.T. Wright, E.V. Anslyn, Chem. Soc. Rev. 35 (2006) 14–28. [20] J. Wu, W. Liu, J. Ge, H. Zhang, P. Wang, Chem. Soc. Rev. 40 (2011) 3483–3495. [21] M.G. Choi, S. Cha, H. Lee, H.L. Jeon, S.-K. Chang, Chem. Commun. 47 (2009) 7390–7392. [22] 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. [23] 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. [24] L.A. Montoya, T.F. Pearce, R.J. Hansen, L.N. Zakharov, M.D. Pluth, J. Org. Chem. 78 (2013) 6550–6557. [25] X. Chen, S. Wu, J. Han, S. Han, Bioorg. Med. Chem. Lett. 23 (2013) 5295–5299. [26] C. Wei, Q. Zhu, W. Liu, W. Chen, Z. Xi, L. Yi, Org. Biomol. Chem. 12 (2014) 479–485. [27] Z. Wu, Z. Li, L. Yang, J. Han, S. Han, Chem. Commun. 48 (2012) 10120–10122. [28] K. Zheng, W. Lin, L. Tan, Org. Biomol. Chem. 10 (2012) 9683–9688. [29] H. Peng, Y. Cheng, C. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, Angew. Chem. Int. Ed. 50 (2011) 9672–9675. [30] 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. [31] C. Liu, B. Peng, S. Li, C.-M. Park, A.R. Whorton, M. Xian, Org. Lett. 14 (2012) 2184–2187. [32] C. Liu, J. Pan, S. Li, Y. Zhao, L.Y. Wu, C.E. Berkman, A.R. Whorton, M. Xian, Angew. Chem. Int. Ed. 50 (2011) 10327–10329. [33] W. Xuan, R. Pan, Y. Cao, K. Liu, W. Wang, Chem. Commun. 48 (2012) 10669–10671. [34] L.A. Montoya, M.D. Pluth, Anal. Chem. 86 (12) (2014) 6032–6039. [35] T. Mutai, H. Tomoda, T. Ohkawa, Y. Yabe, K. Araki, Angew. Chem. Int. Ed. 47 (2008) 9522–9524. [36] B. Liu, J. Wang, G. Zhang, R. Bai, Y. Pang, ACS Appl. Mater. Interfaces 6 (2014) 4402–4407. [37] K. Skonieczny, A.I. Ciuciu, E.M. Nichols, V. Hugues, M. Blanchard-Desce, L. Flamigni, D.T. Gryko, J. Mater. Chem. 22 (2012) 20649–20664. [38] F. Xie, K. Sivakumar, Q. Zeng, M.A. Bruckman, B. Hodges, Q. Wang, Tetrahedron 64 (2008) 2906–2914. [39] T. Saha, D. Kand, P. Talukdar, Org. Biomol. Chem. 11 (2013) 8166–8170. [40] Y. Cai, L. Li, Z. Wang, J.Z. Sun, A. Qin, B.Z. Tang, Chem. Commun. 50 (2014) 8892–8895. [41] Y. Liu, G. Feng, Org. Biomol. Chem. 12 (2014) 438–445. [42] J. Zhao, S. Ji, Y. Chen, H. Guo, P. Yang, Phys. Chem. Chem. Phys. 14 (2012) 8803–8817. [43] Z. Wu, Z. Li, L. Yang, J. Han, S. Han, Chem. Commun. 48 (2012) 10120–10122. [44] A. Hakonen, Anal. Chem. 81 (2009) 4555–4559. [45] X. Pei, H. Tian, W. Zhang, A.M. Brouwer, J. Qian, Analyst 139 (2014) 5290–5296. [46] A.R. Lippert, E.J. New, C.J. Chang, J. Am. Chem. Soc. 133 (2011) 10078–10080.
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Analytica Chimica Acta xxx (2015) xxx–xxx
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A tetraphenylimidazole-based fluorescent probe for the detection of hydrogen sulfide and its application in living cells Biao Gu, Naxiu Mi, Youyu Zhang, Pen Yin, Haitao Li * , Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A sensitive and selective fluorescent probe for sensing H2S was developed. The sensing mechanism of the synthesized probe was based on ESIPT. Upon treatment with H2S, fluorescence of the probe shows a large Stokes shift. The probe can be used for fluorescence imaging of H2S in living cells.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2014 Received in revised form 16 February 2015 Accepted 10 March 2015 Available online xxx
A novel probe based on the fluorescence off–on strategy was prepared to optically detect hydrogen sulfide (H2S) via an excited state intramolecular proton transfer (ESIPT) mechanism. The probe shows high sensitivity and excellent selectivity to H2S. It also displays a large Stokes shift (140 nm) and a remarkable quantum yield enhancement (K = 0.412) after interaction with H2S. Moreover, the cellular imaging experiment demonstrated that it has potential utility for H2S sensing in biological sciences. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Probe Hydrogen sulfide ESIPT Cellular imaging
1. Introduction As one of the most important gaseous signaling molecule [1], hydrogen sulfide (H2S) plays an important role in many biological
* Corresponding author. Tel.: +86 731 88865515; fax: +86 731 88865515. E-mail address: [email protected] (H. Li).
processes such as the relaxation of blood vessels [2], neural signal transmission [3], cell growth regulation [4] and inflammation [5]. Scientific evidence has demonstrated that H2S is present at levels of about 10–100 mM in blood [6] and may reach 600 mM in the brains of human [7]. However, the abnormal H2S level in cells could be related to many health issues such as Alzheimer’s disease [8], congenital down low intelligence [9], diabetes [10] and liver cirrhosis [11], as H2S is involved in the chemical regulation of many
http://dx.doi.org/10.1016/j.aca.2015.03.017 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
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biological processes. It has also been reported that H2S can act as a neuromodulator in the brain [7] and a scavenger for endogenous oxidants such as peroxynitrite, superoxide and hydrogen peroxide [12,13]. Therefore, the development of efficient methods for H2S detection and direct indication of its contribution in living systems is of considerable significance for the related biomedical research and disease diagnosis. Up to now, a number of analytical methods for the detection of H2S have been developed, including colorimetric method [14], electrochemical analysis [15], chromatography [16], metal-induced precipitation [17] and fluorescence spectroscopy [18]. Among these methods, fluorescence probes are more favorable due to their high selectivity, simplicity, real-time spatial imaging and non-destructive advantage [19,20]. In the past few years, there have been several kinds of probes reported for detecting H2S. In general, these probes were designed by taking advantage of chemical properties of H2S, such as binding affinity with copper ion [21–23], nucleophilic reaction [24–26] and good reducing property [27–29]. The probes based on copper sulfide precipitation for H2S showed good sensitivity and selectivity, but the poor live cell permeability of the probes and toxicity of Cu2+ limited their biological applications [30]. The probe based on nucleophilic substitution suffered the possible interference from biological thiols [31,32], such as cysteine, glutathione and homocysteine, because of their similar reactivity of thiolcontaining molecules and the biological thiols are typically higher in cellular levels than H2S in living cells [33,34]. Recently, the probe based on H2S reductive property have been developed through either intramolecular charge transfer (ICT) or photoinduced electron transfer (PET) processes. Nevertheless, some of them exhibited relatively weak fluorescence intensity and small Stokes shift, which might decrease their sensing performance. In comparison with ICT and PET processes, excited state intramolecular proton transfer (ESIPT) process generally has large Stokes shift [35,36], which offers an advantage that minimizes the selfabsorption and reduces the interference from auto-fluorescence for in vivo application. Therefore, it is very meaningful to develop a novel probe based on ESIPT process for detecting H2S in living cells. Recently, Skonieczny et al. reported the synthesis and optical properties of imidazole derivatives and their analogues, an interesting new class of fluorophores [37]. A large Stokes shift, strong fluorescence and high fluorescence quantum yield of 2-(1(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2-yl)aniline prompted us to select it as the active fluorophore. We envisioned that the conversion of the amino group into an azido, which could occlude the ESIPT process, would result in quenching of fluorescence. Due to the reducing ability of H2S, the azido group could be converted into an amino group and consequently turn on the ESIPT, which could restore the fluorescence properties of the fluorophore. Therefore, the strategy should be meaningful to the new design of tetraphenylimidazole-based ESIPT molecular probe (P-N3). Indeed, P-N3 as a new probe for H2S has many advantages including the wide concentration range for linear response, the larger Stokes shift and the high selectivity and sensitivity for H2S. Further more, cell imaging experiments suggested that the biological application of the probe is prospective. 2. Experimental 2.1. Materials and instruments Phenanthrenequinone, p-toluidine, and o-nitrobbenzaldehyde were obtained from Sigma–Aldrich Company. Ethanol, dichloromethane and sodium hydrosulfide were purchased from Sinopharm Chemical Reagent Company. All other chemicals used in this work were of analytical grade. The detection buffer was PBS buffer
(0.01 M, pH 7.4). Milli-Q ultrapure water (Millipore, 18 MV cm) was used throughout. Except the specific statement, solvents were purified by distillation. Silica gel 300–400 mesh (37–54 mm) was used for column chromatography. The thin-layer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. Bruker AVB-500 spectrometer, ZQ2000 mass spectrometer (Manchester, UK), Nexus 670 Fourier Transform Infrared Spectrometer (Nicolet Co., USA) and F-4500 FL spectrophotometer (Hitachi Co., Japan) were used to characterize the probe P-N3. Fluorescence images of HeLa cells were performed by fluorescence microscopy (Nikon, Eclipse Ti-S). 2.2. Synthetic procedure 2.2.1. Synthesis of 2-(2-nitrophenyl)-1-(p-tolyl)-1H-phenanthro[9,10d]imidazole (1) Phenanthrenequinone (416.4 mg, 2.00 mmol) and ammonium acetate (770.8 mg, 10.00 mmol) were added to a solution of o-nitrobbenzaldehyde (302.2 mg, 2.00 mmol) and p-toluidine (321.5 mg, 3.00 mmol) in glacial acetic acid. The reaction mixture was refluxed for 12 h and then cooled to room temperature. Subsequently, the reaction mixture was poured into 80 mL water, and then extracted with ethyl acetate. The organic layer was washed with brine, dried with Na2SO4, filtered and concentrated. An orange crystalline solid (790.2 mg, 92%) was obtained from crystallization and characterized by NMR. 1H NMR (500 MHz, CDCl3): d 8.78 (d, J = 8.4 Hz, 1H), 8.75 (dd, J = 7.9, 0.8 Hz, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.71 (dd, J = 11.0, 3.9 Hz, 1H), 7.68–7.60 (m, 3H), 7.54 (tt, J = 8.4, 2.5 Hz, 2H), 7.32–7.27 (m, 3H), 7.24 (d, J = 8.0 Hz, 3H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3): d 148.87, 147.70, 139.81, 134.46, 133.51, 132.99, 130.51, 130.47, 129.35, 128.27, 127.36, 127.11, 126.66, 126.34, 125.68, 125.17, 124.66, 124.10, 123.15, 122.89, 122.71, 120.96, 22.40. 2.2.2. Synthesis of 2-(1-(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2yl)aniline (2) Compound 1 (584.1 mg, 1.36 mmol) was dissolved in 40 mL ethyl acetate and reduced with hydrogen (1 atm) on 10% Pd-C (40 mg) as a catalyst. After the reaction completed, the catalyst was filtered off. Then, the crude product was purified by crystallization from ethyl acetate to afford a dark green crystalline solid (523.0 mg, 96%) and the solid was measured with NMR. 1H NMR (500 MHz, CDCl3): d 8.80 (dd, J = 7.9, 0.9 Hz, 1H), 8.77 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 7.78–7.70 (m, 1H), 7.68–7.62 (m, 1H), 7.51 (ddd, J = 8.3, 6.7, 1.6 Hz, 1H), 7.40–7.32 (m, 4H), 7.31–7.26 (m, 2H), 7.15–6.98 (m, 1H), 6.88 (dd, J = 7.8, 1.3 Hz, 1H), 6.78 (dd, J = 8.1, 0.7 Hz, 1H), 6.45 (td, J = 7.9, 1.1 Hz, 1H), 5.32 (d, J = 28.1 Hz, 2H), 2.51 (s, 3H). 13C NMR (126 MHz, CDCl3): d 149.57, 147.29, 139.64, 136.59, 135.95, 130.62, 130.36, 129.89, 129.18, 128.73, 128.25, 127.24, 126.98, 126.31, 125.57, 124.86, 124.07, 123.17, 123.07, 122.63, 121.13, 116.77, 116.40, 113.92, 77.34, 77.09, 76.83, 21.47. 2.2.3. Synthesis of 2-(2-azidophenyl)-1-(p-tolyl)-1H-phenanthro [9,10-d]imidazole (P-N3) Compound 2 (259.7 mg, 0.65 mmol) was dissolved slowly in HCl aq. (37.5%, 3 mL) at room temperature. The solution was cooled in an ice bath and 3 mL solution of NaNO2 (67.6 mg, 0.98 mmol) was added. The reaction mixture was stirred for 1 h in an ice bath. This was followed by the addition of potassium acetate to adjust the pH of the resulting solution to 4. Then, NaN3 (126.7 mg, 1.95 mmol) was added in portions. Subsequently, the mixture was stirred at 0–5 C for another 4 h, and it was extracted with ethyl acetate. The organic layer was washed with brine, dried with Na2SO4, filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (15:1, petrol ether:ethyl acetate, v/v) to afford the desired product as an orange solid
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(141.1 mg, 51%). 1H NMR (500 MHz, CDCl3): d 8.94–8.80 (m, 1H), 8.77 (d, J = 8.5 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 7.72 (dd, J = 11.0, 4.0 Hz, 1H), 7.68–7.62 (m, 1H), 7.53 (ddd, J = 8.3, 6.0, 2.3 Hz, 1H), 7.47 (dd, J = 7.6, 1.4 Hz, 1H), 7.40 (td, J = 8.0, 1.5 Hz, 1H), 7.34–7.28 (m, 4H), 7.28–7.20 (m, 4H), 2.44 (s, 3H). 13C NMR (126 MHz, CDCl3): d 148.74, 139.89, 139.40, 137.29, 135.14, 132.84, 130.93, 130.04, 129.30, 128.26, 127.40, 126.23, 125.53, 124.99, 124.42, 124.05, 123.06, 122.79, 121.03, 118.45, 21.36. MS (ESI+) [M + H]: 425.64. IR (KBr, cm1): 2132 (N3). The synthesis processes and structure characterization of intermediates and probe are provided in the Supplementary information (see Scheme S1 and Figs. S1–S8). 2.3. Determination of the fluorescence quantum yield The fluorescence quantum yields of P-N3 and P-NH2 were determined in ethanol at 25 C, using quinine sulfate (F = 0.546 in 1 N H2SO4) as a reference. The quantum yield was calculated using the following equation: 2 AF ns Fs ¼ Fr r s As F r nr 2 where s and r denote sample and reference, respectively, A is the absorbance, F is the relative integrated fluorescence intensity, and n is the refractive index of the solvent. 2.4. Fluorescent detection of H2S For all tests, the experiments were repeated at least three times to ensure the accuracy of the measurements. The fluorescent measurements for H2S were carried out as follows: P-N3 was dissolved in ethanol to prepare a stock solution (1 mM). The fluorescence of P-N3 (10 mM) was detected after incubating 80 min at 37 C in phosphate buffer (0.01 M, pH 7.4) in the presence and absence of H2S (NaHS was used as the hydrogen sulfide source in all experiments). Then, fluorescence spectra were recorded with excitation wavelength at 300 nm. The fluorescence emission intensity at 436 nm was used for quantitative analysis of H2S. 2.5. Fluorescence imaging The HeLa cells were incubated on 96-well plate in the culture medium and allowed to adhere for 24 h at 37 C. Immediately prior to the imaging experiments, the cells were washed with phosphate buffered saline (PBS), incubated with 10 mM P-N3 (in the culture medium containing 1% ethanol) for 40 min at 37 C, then washed with PBS for three times, and imaged. After incubating with 60 mM NaHS for another 80 min at 37 C, the HeLa cells were washed with PBS three times and imaged again. The fluorescence imaging of living HeLa cells was observed and imaged on fluorescence microscopy with a 40 objective lens.
Fig. 1. (a) Absorption spectra, (b) excitation spectra, and (c) emission spectra of probe P-N3 (10 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v).
3.2. Mechanism of the detection of H2S Initially, we proposed the optical probe P-N3 for the detection of H2S based on the tetraphenylimidazole dye. Briefly, the probe P-N3 was designed by combining tetraphenylimidazole moiety as the fluorophore and azido group as the recognition unit. The fluorescence of the fluorophore could be quenched by the azido group due to the hampered ESIPT process. Once reduction of azide to amine was triggered by H2S, the tetraphenylimidazole moiety would shed high fluorescence because of the resulting ESIPT modulated fluorescence off–on response. To verify the proposed mechanism, the fluorescence of P-N3 in the absence and presence of H2S (NaHS was used as a hydrogen sulfide source in all experiments) was investigated (Fig. 2). The free P-N3 exhibited very weak fluorescence (K = 0.024). However, upon the addition of NaHS (10 equiv.) into the probe solution, a dramatic fluorescence intensity enhancement was observed at around 436 nm with a very large Stokes shift (140 nm). As shown in the inset of Fig. 2, observation of the bright blue emission from “P-N3 + NaHS” sample indicated the formation of fluorescence product P-NH2 (K = 0.412). To further verify this hypothesis, the purified product of the reaction of P-N3 with NaHS was then characterized by 1H NMR, 13C NMR and mass spectra (see Figs. S9–S11), which agreed well with the presynthesized compound 2 (see Figs. S3 and S4) demonstrating that the sensing of H2S with the probe is indeed through an
3. Results and discussion 3.1. Spectral characteristics of P-N3 UV–vis absorption and fluorescence spectra properties of P-N3 were measured in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) at ambient temperature. As shown in Fig. 1, the typical UV–vis absorption peak of P-N3 (10 mM) was observed at 260 nm, which could be attributed to the p–p* transition of the p-conjugated core. When being excited at 300 nm, P-N3 gave a weak emission peak at around 436 nm due to the quenching effect of azido group [38–40].
Fig. 2. Fluorescence spectra of P-N3 (10 mM) before (a) after (b) the incubation with 10 equiv. of NaHS in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) with lex = 300 nm. The insets show the photos of samples illuminated by UV light of 365 nm. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
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fluorescence intensity of P-N3 over a wide pH range (3–10), suggesting that P-N3 was not pH sensitive. Upon treatment with NaHS, the maximal fluorescence signal was observed in the range of pH from 7 to 10. which could be explained by the stronger reducing power of the hydrosulfide ion at neutral to basic environment [33,43]. Therefore, physiological pH (pH 7.4) could be selected as an appropriate working pH in the following experiments. In addition, the influence of pH on the fluorescence intensity of the reaction product P-NH2 was also investigated (Fig. S12). The fluorescence intensity of P-NH2 maintained invariability at neutral to high pH (10). When pH < 6, the emission intensity of P-NH2 gradually increased, this may be due to the protonation of the amine group. The time course study revealed that the reaction between P-N3 and NaHS was completed at about 80 min in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v) (Fig. 3B). Scheme 1. Proposed reaction mechanism for H2S.
3.4. The analytical performances of probe azide reduction. Thus, combined with the previously reported conclusion [20,41,42], a possible mechanism was proposed as shown in Scheme 1. 3.3. Optimization of detection conditions Considering the factors affecting the reaction of P-N3 with H2S, pH and incubation time were optimized. We first investigated the effect of pH on fluorescence properties of P-N3 and its response to H2S. As shown in Fig. 3A, there was nearly no change in the
Under the optimal conditions, the fluorescence responses of P-N3 to different concentrations of NaHS were investigated. The fluorescence of the mixture increased as the NaSH concentration increased, and a maximal fluorescence enhancement was obtained when the concentration of NaHS reached 9 equiv. (Fig. 4A). Additionally, there was a good linear relationship between the normalized fluorescence intensity at 436 nm and the concentrations of NaHS in the range of 0–70 mM (Fig. 4B). As shown in inset Fig. 4B, the NaHS with submicromolar level exhibited a linear response. The detection limit was calculated according to previous
Fig. 3. (A) The pH influence on the fluorescence intensity of P-N3 (10 mM) in the absence (&) or presence (*) of NaHS (40 mM). (B) Time-dependent fluorescence spectra of PN3 (10 mM) in the presence of NaHS (70 mM). Data were acquired in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). Time points represent 0, 10, 20, 30, 40, 50, 60, 70, 80 and 100 min.
Fig. 4. (A) Fluorescence spectra of P-N3 (10 mM) in the presence of various concentrations of NaHS (0–90 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). (B) Fluorescence intensity of probe vs. the concentrations of NaHS. (inset B) The fluorescence intensity curve for NaHS at nanomolar concentrations, with error bars that display 3 standard deviations.
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Fig. 5. (A) Fluorescence responses of P-N3 (10 mM) in the present of species (0.5 mM) in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v): (1) no addition; (2) H2O2; (3) S2O32; (4) citrate; (5) Cl; (6) CO32; (7) F; (8) HCO3; (9) HPO42; (10) N3; (11) NO2; (12) NO3; (13) OAc; (14) SO42; (15) HSO3; (16) DTT; (17) 2-ME; (18) Cys; (19) Hcy; (20) GSH; (21) HS. (B) Fluorescence responses of P-N3 (10 mM) at 436 nm toward various analytes in PBS buffer (0.01 M, pH 7.4, containing 10% ethanol, v/v). Black bars represent the addition of a single analyte (1 mM, from left to right: (1) none; (3) DTT; (5) HSO32; (7) 2-ME; (9) Cys; (11) Hcy; (13) GSH). Blue bars represent the mixing corresponding sulfur compounds with HS solution (70 mM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
report [44], which was obtained based on the slope between 0 and 70 mM concentration and the standard deviation of the zero level (i.e., standard approximation 3 SDzero/slope), and limit of quantification was accordingly approximated to 1.9 107 M. These results demonstrated the suitability of P-N3 for the quantitative measurement of H2S. Selectivity is another important factor to evaluate the performance of a new fluorescent probe. Therefore, the fluorescence responses of P-N3 to a series of other biological-related species were investigated. As shown in Fig. 5A, only the addition of HS
caused a large fluorescence enhancement of P-N3. In contrast, the addition of other analytes showed almost no influence on the fluorescence of the probe (the selectivity coefficients toward thiols and sulfite was calculated according to reference [45], which are less than 0.06, see in Table S1). Moreover, considering the relatively high concentration of the possible reducing species in biological system or in the test condition (DTT, 2-ME, Cys, Hcy, GSH and sulfite), we also examined the interference of these species at the level of 1 mM in the detection of HS. As shown in Fig. 5B, distinct fluorescence enhancement can be observed after subsequent
Fig. 6. Fluorescence microscopy images of H2S detection in HeLa cells using P-N3. Bright-field image of live HeLa cells incubated with P-N3 for 40 min and then treated for 80 min in the absence (A) and presence (B) of 60 mM NaHS at 37 C. (C) and (D) represent the fluorescence image images of (A) and (B), respectively.
Please cite this article in press as: B. Gu, et al., A tetraphenylimidazole-based fluorescent probe for the detection of hydrogen sulfide and its application in living cells, Anal. Chim. Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.03.017
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addition of HS to the probe P-N3 solution in the presence of these species, suggesting the interference of these chemicals was very little, and P-N3 is potentially useful for sensing H2S in the presence of these species. All these results indicate that probe P-N3 has high selectivity for HS detection. This might be due to the fact that the steric effect of thiols in the reaction is greater than HS, which makes the reduction reaction more difficult. 3.5. Fluorescence imaging of H2S in living cells In order to investigate the biological application of P-N3, the fluorescence microscopy experiment was carried out. HeLa cells were incubated with 10 mM P-N3 (in the culture medium containing 1% ethanol) for 40 min and then treated with a blank control or 60 mM NaHS for an additional 80 min at 37 C. This concentration is well within the range that has been used to elicit physiological responses (10–600 mM) [46]. It was found that the control experiments where the cells were not treated with NaHS showed faint fluorescence (Fig. 6C), but those treated with NaHS displayed strong blue fluorescence emission (Fig. 6D). The bright-field images (Fig. 6A and B) confirmed that the cells were viable throughout the imaging experiments. Thus, P-N3 showed excellent cell permeation capability and could efficiently sense H2S in living cells. 4. Conclusion In summary, we have developed an ESIPT-based, off–on fluorescent probe (P-N3) by combining tetraphenylimidazole moiety as the fluorophore and azido group as the recognition unit. The developed probe displayed a large Stokes shift, a wide linear concentration rang and good sensitivity. Meanwhile, this probe showed an excellent selectivity to H2S over other anions, reactive sulfur and oxygen. Fluorescent imaging of intracellular H2S demonstrated its potential for biological applications. Acknowledgments This work was supported by the National Natural Science Foundation of China (21375037, 21275051, 21405043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (20134306110006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.03.017. References [1] L. Li, P. Rose, P.K. Moore, Annu. Rev. Pharmacol. Toxicol. 51 (2011) 169–187. [2] O. Jackson-Weaver, J.M. Osmond, M.A. Riddle, J.S. Naik, L.V.G. Bosc, B.R. Walker, N.L. Kanagy, Am. J. Physiol. Heart Circ. Physiol. 304 (2013) H1446–H1454.
[3] H. Kimura, Mol. Neurobiol. 26 (2002) 13–19. [4] G. Yang, L. Wu, R. Wang, FASEB J. 20 (2006) 553–555. [5] C. Yang, Z. Yang, M. Zhang, Q. Dong, X. Wang, A. Lan, F. Zeng, P. Chen, C. Wang, J. Feng, PLoS One 6 (2011) e21971. [6] L. Li, M. Bhatia, Y.Z. Zhu, Y.C. Zhu, R.D. Ramnath, Z.J. Wang, F.B.M. Anuar, M. Whiteman, M. Salto-Tellez, P.K. Moore, FASEB J. 19 (2005) 1196–1198. [7] K. Abe, H. Kimura, J. Neurosci. 16 (1996) 1066–1071. [8] K. Eto, T. Asada, K. Arima, T. Makifuchi, H. Kimura, Biochem. Biophys. Res. Commun. 293 (2002) 1485–1488. [9] P. Kamoun, M.C. Belardinelli, A. Chabli, K. Lallouchi, B. Chadefaux-Vekemans, Am. J. Med. Genet. Part A 116 (2003) 310–311. [10] W. Yang, G. Yang, X. Jia, L. Wu, R. Wang, J. Physiol. 569 (2005) 519–531. [11] S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah, A. Morelli, Hepatology 42 (2005) 539–548. [12] R.F. Milos, M. Jan, A. Andrea, C. Ricardo, S. Tatyana, H. Martin, I.-B. Ivana, Biochem. J. 441 (2012) 609–621. [13] C.M. Jones, A. Lawrence, P. Wardman, M.J. Burkitt, Free Radic. Biol. Med. 32 (2002) 982–990. [14] X. Gu, C. Liu, Y.-C. Zhu, Y.-Z. Zhu, Tetrahedron Lett. 52 (2011) 5000–5003. [15] D.G. Searcy, M.A. Peterson, Anal. Biochem. 324 (2004) 269–275. [16] C.J. Richardson, E.A.M. Magee, J.H. Cummings, Clin. Chim. Acta 293 (2000) 115–125. [17] M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii, H. Kimura, Antioxid. Redox Signal. 11 (2008) 205–214. [18] Y. Zhao, T.D. Biggs, M. Xian, Chem. Commun. 50 (2014) 11788–11805. [19] A.T. Wright, E.V. Anslyn, Chem. Soc. Rev. 35 (2006) 14–28. [20] J. Wu, W. Liu, J. Ge, H. Zhang, P. Wang, Chem. Soc. Rev. 40 (2011) 3483–3495. [21] M.G. Choi, S. Cha, H. Lee, H.L. Jeon, S.-K. Chang, Chem. Commun. 47 (2009) 7390–7392. [22] 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. [23] 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. [24] L.A. Montoya, T.F. Pearce, R.J. Hansen, L.N. Zakharov, M.D. Pluth, J. Org. Chem. 78 (2013) 6550–6557. [25] X. Chen, S. Wu, J. Han, S. Han, Bioorg. Med. Chem. Lett. 23 (2013) 5295–5299. [26] C. Wei, Q. Zhu, W. Liu, W. Chen, Z. Xi, L. Yi, Org. Biomol. Chem. 12 (2014) 479–485. [27] Z. Wu, Z. Li, L. Yang, J. Han, S. Han, Chem. Commun. 48 (2012) 10120–10122. [28] K. Zheng, W. Lin, L. Tan, Org. Biomol. Chem. 10 (2012) 9683–9688. [29] H. Peng, Y. Cheng, C. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, Angew. Chem. Int. Ed. 50 (2011) 9672–9675. [30] 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. [31] C. Liu, B. Peng, S. Li, C.-M. Park, A.R. Whorton, M. Xian, Org. Lett. 14 (2012) 2184–2187. [32] C. Liu, J. Pan, S. Li, Y. Zhao, L.Y. Wu, C.E. Berkman, A.R. Whorton, M. Xian, Angew. Chem. Int. Ed. 50 (2011) 10327–10329. [33] W. Xuan, R. Pan, Y. Cao, K. Liu, W. Wang, Chem. Commun. 48 (2012) 10669–10671. [34] L.A. Montoya, M.D. Pluth, Anal. Chem. 86 (12) (2014) 6032–6039. [35] T. Mutai, H. Tomoda, T. Ohkawa, Y. Yabe, K. Araki, Angew. Chem. Int. Ed. 47 (2008) 9522–9524. [36] B. Liu, J. Wang, G. Zhang, R. Bai, Y. Pang, ACS Appl. Mater. Interfaces 6 (2014) 4402–4407. [37] K. Skonieczny, A.I. Ciuciu, E.M. Nichols, V. Hugues, M. Blanchard-Desce, L. Flamigni, D.T. Gryko, J. Mater. Chem. 22 (2012) 20649–20664. [38] F. Xie, K. Sivakumar, Q. Zeng, M.A. Bruckman, B. Hodges, Q. Wang, Tetrahedron 64 (2008) 2906–2914. [39] T. Saha, D. Kand, P. Talukdar, Org. Biomol. Chem. 11 (2013) 8166–8170. [40] Y. Cai, L. Li, Z. Wang, J.Z. Sun, A. Qin, B.Z. Tang, Chem. Commun. 50 (2014) 8892–8895. [41] Y. Liu, G. Feng, Org. Biomol. Chem. 12 (2014) 438–445. [42] J. Zhao, S. Ji, Y. Chen, H. Guo, P. Yang, Phys. Chem. Chem. Phys. 14 (2012) 8803–8817. [43] Z. Wu, Z. Li, L. Yang, J. Han, S. Han, Chem. Commun. 48 (2012) 10120–10122. [44] A. Hakonen, Anal. Chem. 81 (2009) 4555–4559. [45] X. Pei, H. Tian, W. Zhang, A.M. Brouwer, J. Qian, Analyst 139 (2014) 5290–5296. [46] A.R. Lippert, E.J. New, C.J. Chang, J. Am. Chem. Soc. 133 (2011) 10078–10080.
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