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A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells
Talanta 198 (2019) 185–192
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A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells
T
Ting Caoa, Zhidong Tengb, Deyan Gonga, Jing Qiana, Wei Liua, Kanwal Iqbala, Wenwu Qina, , ⁎ Huichen Guob, ⁎
a
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu Province 730046, PR China
ARTICLE INFO
ABSTRACT
Keywords: H2S Fluorescent sensor Ratiometric Endogenous and exogenous Cells imaging
A ratiometric visualized fluorescent probe of H2S of intramolecular charge transfer (ICT) and excited intramolecular proton transfer (ESIPT) mechanisms due to solvation effects has been designed and synthesized. This chemosensor shows the distinct signal changes with dual-emission in blue and green fluorescence spectral channel (from 495 nm to 525 nm) upon the addition of H2S in a single wavelength excitation. This chemosensor exhibits the low detection limit (91 nM) and high sensitivity and selectivity. Based on this, this chemosensor was successfully used not only to monitor H2S exogenously but also used to detect and image the endogenously generated H2S in HeLa cells with excellent performance.
1. Introduction It is generally accepted that hydrogen sulfide (H2S), as the third endogenous toxic gas signaling compound after NO and CO [1,2] (gas transmitter), is known for having the rotten egg like odour. H2S belongs to the active sulfur substance (RSS), containing thiols [3], S-nitrosomercaptan [4], sulfonic acid [5], and sulfite [6]. The mammalian cells having H2S consists mainly of two endogenous enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [7,8] by disassembling cysteine and cysteine substrates derivatives of the enzyme reaction. Paradoxically, our body produces H2S in a small amount and it plays a crucial role in regulating of redox status [9] in cells and other essential signals at normal concentrations [10]. For example, it has been confirmed that H2S can not only relax the vascular smooth muscle, but also can induce vasodilation and reduce the blood pressure. Besides, in vascular inflammation of rats, leukocyte adherence in mesenteric microcirculation can be inhibited by H2S, which indicates that H2S is an effective anti-inflammatory particle. In addition, H2S is an effective antioxidant that can increase the defense ability of antioxidants in chronic conditions. In spite of the increasing interest in H2S research, the underlying problems of its production and effects the mechanism at the normal level, as well as the basic problems of its destruction, still exist. Such as, owing to the unabiding essence of H2S and less content in
⁎
most tissues [11], and how to detect the endogenous H2S with highly sensitivity - selectivity and not affected by other biological thiols [12], many approaches, such as colorimetric, electrochemical analysis and gas chromatography, have been applied to measure and trace the H2S [13,14]. Among the various available detection methods, fluorescence detection is a very sensitive detection method, especially fluorescence technology has become the most attractive biomolecule in vivo detection because of its high sensitivity, selectivity, non-destructive and affinity towards the living cells and tissues, molecular imaging technology has been recognized as the efficient molecular tool [15,16]. During the last several decades, many kinds of fluorescent H2S probes have emerged as a hot topic in the scientific literature, most of which involve development of reactivity and reducing properties of H2S with specific chemical reactions [17,18]. Whereas, the many probes display turn-off or turn-on response with H2S, this may affects the accuracy of quantitative detection because of many reasons [19,20]. Thus, compared with the single emission probes of H2S, the ratio type fluorescence probes can reduce some errors by self- calibration by dualemission spectra bands. Up to now, there have been few ratiometric fluorescence sensors for endogenous H2S [21,22]. Based on the above deficiencies, in order to better monitor and image the dual roles of H2S in cells and even in vivo, our main research program is to select a fluorescent group which can
Corresponding authors. E-mail addresses: [email protected] (W. Qin), [email protected] (H. Guo).
https://doi.org/10.1016/j.talanta.2019.02.017 Received 28 August 2018; Received in revised form 28 January 2019; Accepted 3 February 2019 Available online 05 February 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. The synthetic procedure of probe 1. (a) o-hydroxyacetophenone, CH3CH2OH, NaOH, 50 °C reflux 4 h, HOAc (neutralized pH = 7), 82%; (b) MeOH, NaOH, H2O2, rt 7 h, 79%; (c) CHCl3, PySSPy, rt 12 h, 95%; (d) EDC, DMAP, DCM, rt 12 h, 59%, DMAP = 4dimethylaminopyridine; DCM = dichloromethane, EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
provide high quantum yield, long emission spectrum wavelength, and pH-insensitive and a simple mature reaction group can alter the fluorescent properties of the probe by reacting with H2S. In this report, we designed and synthesized a new type of ratio fluorescent probe 1, in which 4′-(diethylamino)-3-hydroxyflavone [23] was modified with 2,2′-dipyridyl disulfide benzoic acid [24,25] to produce an intramolecular charge transfer (ICT) process ( Scheme 1). Therefore, we can monitor and image endogenous and exogenous H2S with the change of ratio signal.
3. Results and discussion It was confirmed that the NMR and MS spectrum of the product was indeed for probe 1 (Figs. S11–S12) and probe 1-H2S (Figs. S13–S14) as presented in Scheme 1. The normalized absorption spectrum and fluorescence emission spectrum of probe 1 in a few solvents of varying polarity are shown in Fig. S1, and their photophysical properties are listed in Table S1. The absorption spectrum has one main band at ~385 nm in all solvents and which shows that there is a negligible effect by the polarity of solvent. However, the maximum red shift (~70 nm) of the fluorescence emission spectrum of probe 1 is appeared. The Φf value was higher in the non-polar solvent, such as cyclohexane (Φf = 0.16), while in the polar solvents, the fluorescence intensity gradually decreased (Φf = 0.15 in chloroform and Φf = 0.067 in acetonitrile), and the fluorescence quantum yield Φf also depends on the polarity of the solvent. The low Φf value of the probe 1 in the polar solvent is because of effective quenching of the electron acceptor of the pyridine nitrogen atom through the ICT effect.
2. Experimental section 2.1. Instruments and reagents All experiment reagents and chemicals were purchased from commercially available providers. The source of hydrogen sulfide used in all experiments were sodium sulfide nonahydrate hydrate, purchased from Guangfu. Double distilled water was used. Coumarine 102 in ethanol (λex = 389 nm, Φf = 0.783) was used as standard of fluorescence for the quantum yield test [26]. All measurements were carried out in CTAB/MeCN/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) at room temperature.
3.1. UV absorption and emission fluorescence spectra The absorption and emission spectra of probe 1 towards H2S are shown in Fig. S2, and the photophysical corresponding data are listed in Table S2. As shown in Fig. 1a, the absorption band at 392 nm dropped off, meanwhile, a new peak arised at about 425 nm and the intensity enhanced with the increased concentration of H2S. The two distinct isoelectric points (375 and 405 nm) given by the visible absorption spectra signal changes at 425 and 392 nm with different concentrations of H2S versus probe 1 both indicates the appearance of new species. This change is in accordance with the fluorescence lifetime measurement and numerical fitting (Table S3). As shown in Fig. 1b, the maximum emission wavelength of the probe 1 is 495 nm when H2S is not exist. But when H2S was added to the PBS within 10 μM probe 1 in buffer, a new fluorescence peak was observed in 525 nm, and with the gradually increased amount of H2S, the fluorescence peak at 495 nm was descend, meanwhile, accompanied by a strong green fluorescence appeared at 525 nm. As shown in Table S2, this change is consistent with the measured and calculated quantum yield of fluorescence. The Φf = 0.078 ± 0.001 in the blank sample with only the probe, and the Φf = 0.258 ± 0.003 at 525 nm after the addition of H2S.
2.2. Synthesis of Probe 1 As shown in Scheme 1, compound A (with a minor revision) and compound B were synthesized respectively by reported method [23,27]. Compound B (131 mg, 0.5 mmol), compound A (15.6 mg, 0.05 mmol), EDC (96 mg, 0.5 mmol), and DMAP (6.1 mg, 0.05 mmol) were dissolved in dry DCM (25 mL) at 25 °C and stirred for 12 h. After the reaction finished, removed the solvent and then purified by using silica gel column to get probe 1 as a pale yellow solid (130 mg, 59%). 1 H NMR (400 MHz, CDCl3, TMS) 8.83–8.85 (3H, m), 8.35 (1H, t, H-l, J = 8.0 Hz), 7.88 (2H, d, H-k, J = 8.0 Hz), 7.80 (1H, d, H-j, J = 8.0 Hz), 7.67 (1H, d, H-e, J = 8.0 Hz), 7.55 (2H, d, H-d, H-f, J = 8.0 Hz), 7.37–7.42 (4H, m), 6.68 (2H, d, H-a, J = 8.0 Hz), 3.41 (4H, q, 2 × CH2, J = 8.0 Hz), 1.19 (6H, t, 2 × CH3, J = 8.0 Hz). MS calcd for C31H26N2O4S2, 554.13, found 555.11 (M + 1). 2.3. Synthesis of Probe 1-H2S (compound A)
3.2. The limit of detection calculation
27.7 mg (0.05 mmol) of probe 1 was dissolved in 15 mL of dry DCM, and then 7.8 mg H2S (1.0 mmol) was added at 25 °C and stirred for 0.5 h. The solvent was removed and then separated by silica gel column to give product probe 1-H2S as a deep yellow solid (15 mg) in 97% yield. 1H NMR (400 MHz, CDCl3, TMS) 8.22 (1H, d, H-c, J = 8.0 Hz), 8.16 (2H, d, H-b, J = 8.0 Hz), 7.63 (1H, t, H-e, J = 8.0 Hz), 7.55 (1H, d, H-f, J = 8.0 Hz), 7.38 ( 1H, t, H-d, J = 8.0 Hz), 6.75 (2H, d, H-a, J = 8.0 Hz), 5.29 (-OH, s), 3.44 (4H, q, 2 × CH2, J = 8.0 Hz), 1.22 (6H, t, 2 × CH3, J = 8.0 Hz). MS calcd for C19H19NO3, 309.14, found 310.14 (M + 1).
Fig. 2a shows titration curve of the probe 1 for H2S. It can be seen from the titration diagram that the value of the fluorescence signal changes obviously when the low concentration of H2S is added to the PBS, after the treatment of high concentration the changes slowly and steadily. Fig. 2b shows the correction curve of probe 1 (R = F525 nm/F495 nm) with different concentrations of H2S. Because the concentration of probe 1 in the titration experiment is 10 μM, as the reaction progresses, the concentration of H2S in the analyte is increased. Due to the 186
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Fig. 2. a. Extremum titration curve of the probe 1 to H2S; b. Linear calibration curve of fluorescence Intensity ratio R = F525 nm/F495 nm of probe 1 with H2S, probe 1 = 10 μM, λex = 380 nm.
Fig. 1. (a) UV absorption spectrum; (b) Fluorescence spectrum of probe 1 towards H2S in PBS buffer, probe 1 = 10 μM.
consumption of the probe, the reaction proceeds smoothly and the fluorescence intensity reaches the maximum. Therefore, in the initial concentration range of 0–10 μM, the linear increase value is more obvious, while in the concentration range of 10–100 μM, the ratio value change is relatively small. After calculation, the DL of probe 1 towards H2S was 0.091 μM (1–10 μM) and 0.703 μM (10–100 μM). To ours pleasure, this detection limit have an ideal effect compared with others reported literature (Table S4). 3.3. Time response and kinetic study of probe 1 toward H2S As shown in Fig. 3, the time-dependent fluorescence curve of probe 1 towards H2S was conducted in PBS solution at room temperature. The fluorescence intensity is stable over time only when the probe 1 is present. When addition of several (10, 50, and 100 μM) concentration of H2S in this system, the emission band from 495 nm moved to 525 nm apparently within the initial 5 min and reached a maximum at about 10–15 min and as well as it maintained its strong green fluorescence. Therefore, the detection of H2S was obtained through the detection of probe 1 within ~15 min. This response time is relatively short compared to most of the previously reported literature (Table S4). For a chemodosimeter, the response time of a signal depends on the rate of reaction between the object species and the target substance. Therefore, the kinetics of the time dependent dosimetric response was assessed between the probe 1 and the H2S. According to the calculation method in literature reported [28], kinetic studies were observed to calculate the apparent rate constant k for probe 1 towards H2S at indoor temperature. The pseudo-first-order reaction rate constant kobs was calculated to be 0.17 min−1 (Fig. S3) When probe 1 was responded to 10 μM of H2S.
Fig. 3. Time course of the response at F525 nm/F495 nm of probe 1 with several equiv. of H2S, probe 1 = 10 μM.
3.4. Study on the optical stability and pH of the probe 1 The probe 1 has good light resistance within 10 min, the fluorescence intensity is very stable, and shows good light stability (Fig. S5), and the influence of illumination on the detection process can be excluded during the experiment. At the same time, we also measured the effect of pH on the blank probe 1 and the addition of H2S. It can be seen from Fig. S6 that the fluorescence ratio of the probe 1 is very stable within a certain pH range. When H2S is added for 15 min, the fluorescence ratio increases significantly in the range of pH 4–7, and tends to 187
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Fig. 5. Time-resolved curve of probe 1 (10 μM) towards H2S, λex = 380 nm.
Fig. 4. The ratio fluorescence intensity (F525 nm/F495 nm) of probe 1 (10 μM) towards active sulfur (100 μM) in PBS solution.
experimental conditions and fluorescence spectrum were obtained after 15 min. As shown in Fig. 4 and Fig. S4, about 30 nm bathochromic-shift clearly shows that the Probe 1 has an excellent response for H2S due to the efficiently ester cyclization reaction, while other typical active sulfur substance do not shows a clear change in fluorescence ratio under the same reaction conditions. This result forcefully shows that Probe 1 was superior for H2S in selectivity compared with other sulfur-containing active substances. The competition experiments were also used to carried out, which also indicates that Probe 1 still detects the H2S with high selectivity although other sulfur-containing species were still existed.
be smooth in the range of 7–10 This experimental result is superior for the imaging of probes for further hydrogen sulfide in the cellular environment. 3.5. The specificity of probe 1 towards H2S To investigate the selectivity in aqueous solution, the probe 1 was added in various species of sulfur, including common interference such as Cys, GSH, Hcy, SO32−, S2O32− (all at 100 μM) under the same
Scheme 2. The response mechanism of the probe 1 towards H2S and other thiols. 188
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Fig. 6. a) HeLa cells were cultured with probe 1 for 1 h; b), c) Cells were cultured with probe 1 for 1 h after further cultured with H2S (10 µM and 50 µM) for another 30 min, respectively; d) The ratio of mean fluorescence intensity of blue and green channels of HeLa cells, R=F525 nm/F495 nm. probe 1 = 10 µM. Scale bar = 20 µm.
3.6. The exploration of the mechanism
tautomer formation. On the contrary, the emission spectra of compound A in ethanol or PBS (polar proton solvent) both displays a broad peak at 525 nm (Fig. S7c). Because in aqueous media where the ESIPT process could be inhibited by intermolecular H-bonding in solvent, the emission peak at 525 nm could be due to the formation of species which are formed due to the presence of intermolecular hydrogen-bonding in solvent molecules. The reaction of probe 1 with H2S confirmed this mechanism. Meanwhile, after the other biological thiols reacted with the probe 1 under the same experimental conditions, the compound E was obtained, however, since compound E has no SH functional group, the intramolecular nucleophilic attack process could not be further performed. Therefore, other biological thiols could not change the fluorescence and only H2S could cause the fluorescence spectrum to produce a change in the ratio signal.
As shown in Scheme 2, owing to the strong electron-giving ability of hydroxyl group, compound B acts as a H2S acceptor, the probe 1 can form sulfhydryl-containing intermediate with H2S, the by-product compound C was formed by spontaneous cyclization and the fluorophore (Compound A) was released at the same time. The process and mechanism of this reaction are similar to those already reported for the detection of H2S [23,24,29–32] Fig. S7 shows the fluorescence emission spectra of Compound A in solvents having different polarities, which proves that Compound A shows dual fluorescence in the reduced-polarity solvent. Compound A with a higher energy band around ~420 nm is due to the emission of enol Compound A tautomer while a large stokes shift emission spectrum at ~550 nm in cyclohexane can be due to the emission at ESIPT in the keto-tautomers formed (Scheme 2). The fluorescence quantum yield Φf of compound A is strongly solvent dependent. The Φf values are higher in cyclohexane (0.083), somewhat lower in acetonitrile (0.021) (Table S1). The emission spectrum in other solvents also exhibits two bands, respectively. One is ranges from 430 nm to 515 nm and another at ~565 nm. If the solvent polarity was increased from cyclohexane (430 nm) to acetonitrile (510 nm), it can be seen that the band of the spectrum bathochromic-shifts (~90 nm) at higher energy level. Consequently, the emission spectra at 500 nm band is almost not affected by the polarity of the solvents, the emission peak within 430–510 nm are owe to the emission of enol tautomer, the large stokes shift at 550 nm probably could be due to the enol tautomer excited to keto
3.7. Fluorescence decay traces of probe 1 toward different concentrations of H2 S A global fitting of fluorescence decays was done by using FAST software supplied by Edinburgh Instruments Company. It can be expected that global analysis with three decay traces would estimate the {τi αi} values with higher accuracy than single-curve analysis. All global curve fittings presented here had χ2 values < 1.20. As shown in Fig. 5, the fluorescence decay of probe 1 shows two main components (Table S3, Fig. S8 and Fig. S10), with attenuation constants of 0.36 ns and 0.83 ns, respectively. The contributions from the slow decay time 0.83 ns increased at longer wavelengths (from 24% at 485 nm to 34% at 189
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Fig. 7. a) Cells were cultured with probe 1 for 1 h; b) cells were cultured with Cys (1000 µM) for 1 h followed by probe 1 for another 1 h; c); d); e) cells were handled with PAG (10 µM), AOAA (10 µM), PAG and AOAA (10 µM + 10 µM) for 1 h; then cultured with Cys (1000 µM) for 1 h; finally, cultured with probe 1 for further 1 h; f) The ratio of mean fluorescence intensity of blue and green channels of HeLa cells, R=F525 nm/F495 nm. probe 1 = 10 µM. Scale bar = 20 µm. λex = 405 nm; λem = 405–495 nm (blue channel), λem = 495–530 nm (green channel).
505 nm), indicating that the excited state fast decay compound (τ1 ≈ 0.36 ns) in PBS is a local excitation state, while the slow decay time τ2 can be attributed to the lifetime of the ICT state. Upon the addition of different concentration of H2S (20 and 100 μM) in PBS buffer, it can be seen that the fluorescence decays to a single index, and the average fluorescence decay lifetime at the emission wavelengths of 515 nm, 525 nm, and 535 nm increases from 1.20 ns to 1.23 ns. The above experimental data shows that the fluorescence emission peak at 525 nm after the addition of H2S is mainly the formation of a substance derived from intermolecular hydrogen bonding in a solvent molecule, and the newly formed product (compound A) content is almost 100%. The
rationality of the reaction mechanism of H2S and probe 1 in Scheme 2 was further proved by analyzing the experimental data. 3.8. Imaging of exogenous H2S in living cells The probe 1 was used to determine the real-time tracking and detection of H2S in HeLa cells. Before cell imaging, an MTS assay was carried out to evaluate the cytotoxicity effect and compatibility of the probe 1. The MTS test concentration of probe 1 in HeLa cells varies from 5 μM to 100 μM (Fig. S9), which clearly indicates that the probe 1 exhibits low cytotoxicity and excellent biocompatibility for cultured 190
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cell lines under the experimental conditions keeping the concentration 10 μM. In Fig. 6a, cells were only treated with probe 1 initially showed blue fluorescence and a relatively weaker green fluorescence. However, when probe 1-stained cells cultured with H2S (10 μM and 50 μM), showed the relatively strong green fluorescence in the green channel, while the blue channel showed relatively weak blue fluorescence (Figs. 6b and 6c). Taking together, this increasing proportion of the signal clearly shows probe 1 have better cell membrane permeability which confirming that probe 1 is a possible tool used to detect the content of H2S in living cells in real time.
of China (2014DFA31890). The authors would like to thank the Natural Science Foundation of China (No. 21771092). This work was also supported by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China, [email protected].
3.9. Imaging of endogenous H2S in living cells
References
The probes for detecting H2S is shown in Table S4, most are turn-off or turn-on type, and the ratio type is relatively small. The relatively novel probes that are both ratios and endogenous for the detection of H2S are rare [33–35]. On the one hand, the ratio probe has dual emission compared to the conventional turn-off or turn-on type probe. The probe 1 reacts with the substance to be tested, and the emission wavelength changes before and after. The emission wavelength are two, one high and one low, compared with the subsequent Ratio-related, such probes can reduce background interference and have better sensitivity; on the other hand, since the biological system generates responsive substances with probes through certain metabolic processes, endogenous detection have little damage to the cells themselves, It is more widely used in biological systems. Thus, based on the support of these experimental data, we attempted to test the practicability of probe 1 with endogenic H2S in HeLa cells. It is known that H2S can be produced by the catalytic activity of Cys in living cells through the catalysis of two enzymes of CBS and CSE. As shown in Fig. 7, as we expected, only in the presence of probe 1 (a), the strong blue fluorescence and the weaker green fluorescence were collected. On the contrary, the fluorescence emission of the blue channel was basically unchanged after the cell was incubated with Cys, Meanwhile, the fluorescence of the green channel is obviously stronger (b). To demonstrate that Cys indeed produce hydrogen sulfide due to the catalysis of the two enzymes mentioned above, we also performed experiment for the inhibition of CBS and CSE by enzyme inhibitors (propargylglycine (PAG) or aminooxyacetic acid (AOAA)) using reports from known literature [34]. As Fig. 7(c–e) shows, cells were handled with PAG or AOAA or PAG and AOAA, and then incubated with Cys for 1 h, and followed by addition of probe 1 for further 1 h. There was almost negligible ratiometric signal of the fluorescence intensity changes was observed compared to the control group. These results verified that the production of endogenous H2S was inhibited resulted from the inactivation of the enzyme.
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2019.02.017.
4. Conclusion In short, an efficient synthetic method is successfully development for the preparation of a ratio fluorescent dual-emission probe for tracing not only the exogenous but also the endogenous H2S in living cells. The probe 1 showed the excellent selectivity-sensitive fluorescence response in the emission ratio of H2S for other biothiol (F525 nm/F495 nm) having low limit of detection of 91 nM (0–10 μM) in aqueous solution for ~15 min. In addition, the mechanism of probe 1 for H2S was also analyzed and determined by ICT and ESIPT due to solvation effects. Based on the excellent ratio of probe 1 and the endogenous detection of the properties of H2S, the probe 1 could be an useful tool which can be effectively used for the detection and imaging analysis of H2S in biological cells. Acknowledgments This work was supported by the Ministry of Science and Technology 191
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T. Cao et al. [27] H. Peng, Y. Cheng, C. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood, Angew. Chem. Int. Ed. 50 (41) (2011) 9672–9675. [28] Z. Huang, S. Ding, D. Yu, F. Huang, G. Feng, Aldehyde group assisted thiolysis of dinitrophenyl ether: a new promising approach for efficient hydrogen sulfide probes, Chem. Commun. 50 (65) (2014) 9185–9187. [29] C. Liu, J. Pan, S. Li, Y. Zhao, L.Y. Wu, C.E. Berkman, A.R. Whorton, M. Xian, Capture and visualization of hydrogen sulfide by a fluorescent probe, Angew. Chem. Int. Ed. 50 (44) (2011) 10327–10329. [30] F. Wang, J. Wu, X. Zhuang, W. Zhang, W. Liu, P. Wang, S. Wu, A highly selective fluorescent sensor for fluoride in aqueous solution based on the inhibition of excited-state intramolecular proton transfer, Sens. Actuators B Chem. 146 (1) (2010) 260–265. [31] C. Zhang, G. Zhang, L. Feng, J. Li, A ratiometric fluorescent probe for sensitive and selective detection of hydrogen sulfide and its application for bioimaging, Sens. Actuators B Chem. 216 (2015) 412–417.
[32] X. Li, H. Ma, J. Qian, T. Cao, Z. Teng, K. Iqbal, W. Qin, H. Guo, Ratiometric fluorescent probe based on ESIPT for the highly selective detection of cysteine in living cells, Talanta 194 (2019) 717–722. [33] C. Zhao, X. Zhang, K. Li, S. Zhu, Z. Guo, L. Zhang, F. Wang, Q. Fei, S. Luo, P. Shi, H. Tian, W.H. Zhu, Forster resonance energy transfer switchable self-assembled micellar nanoprobe: ratiometric fluorescent trapping of endogenous H2S generation via fluvastatin-stimulated upregulation, J. Am. Chem. Soc. 137 (26) (2015) 8490–8498. [34] X. Feng, T. Zhang, J.T. Liu, J.Y. Miao, B.X. Zhao, A new ratiometric fluorescent probe for rapid, sensitive and selective detection of endogenous hydrogen sulfide in mitochondria, Chem. Commun. 52 (15) (2016) 3131–3134. [35] L. Zhang, S. Li, M. Hong, Y. Xu, S. Wang, Y. Liu, Y. Qian, J. Zhao, A colorimetric and ratiometric fluorescent probe for the imaging of endogenous hydrogen sulphide in living cells and sulphide determination in mouse hippocampus, Org. Biomol. Chem. 12 (28) (2014) 5115–5125.
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A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells
T
Ting Caoa, Zhidong Tengb, Deyan Gonga, Jing Qiana, Wei Liua, Kanwal Iqbala, Wenwu Qina, , ⁎ Huichen Guob, ⁎
a
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu Province 730046, PR China
ARTICLE INFO
ABSTRACT
Keywords: H2S Fluorescent sensor Ratiometric Endogenous and exogenous Cells imaging
A ratiometric visualized fluorescent probe of H2S of intramolecular charge transfer (ICT) and excited intramolecular proton transfer (ESIPT) mechanisms due to solvation effects has been designed and synthesized. This chemosensor shows the distinct signal changes with dual-emission in blue and green fluorescence spectral channel (from 495 nm to 525 nm) upon the addition of H2S in a single wavelength excitation. This chemosensor exhibits the low detection limit (91 nM) and high sensitivity and selectivity. Based on this, this chemosensor was successfully used not only to monitor H2S exogenously but also used to detect and image the endogenously generated H2S in HeLa cells with excellent performance.
1. Introduction It is generally accepted that hydrogen sulfide (H2S), as the third endogenous toxic gas signaling compound after NO and CO [1,2] (gas transmitter), is known for having the rotten egg like odour. H2S belongs to the active sulfur substance (RSS), containing thiols [3], S-nitrosomercaptan [4], sulfonic acid [5], and sulfite [6]. The mammalian cells having H2S consists mainly of two endogenous enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [7,8] by disassembling cysteine and cysteine substrates derivatives of the enzyme reaction. Paradoxically, our body produces H2S in a small amount and it plays a crucial role in regulating of redox status [9] in cells and other essential signals at normal concentrations [10]. For example, it has been confirmed that H2S can not only relax the vascular smooth muscle, but also can induce vasodilation and reduce the blood pressure. Besides, in vascular inflammation of rats, leukocyte adherence in mesenteric microcirculation can be inhibited by H2S, which indicates that H2S is an effective anti-inflammatory particle. In addition, H2S is an effective antioxidant that can increase the defense ability of antioxidants in chronic conditions. In spite of the increasing interest in H2S research, the underlying problems of its production and effects the mechanism at the normal level, as well as the basic problems of its destruction, still exist. Such as, owing to the unabiding essence of H2S and less content in
⁎
most tissues [11], and how to detect the endogenous H2S with highly sensitivity - selectivity and not affected by other biological thiols [12], many approaches, such as colorimetric, electrochemical analysis and gas chromatography, have been applied to measure and trace the H2S [13,14]. Among the various available detection methods, fluorescence detection is a very sensitive detection method, especially fluorescence technology has become the most attractive biomolecule in vivo detection because of its high sensitivity, selectivity, non-destructive and affinity towards the living cells and tissues, molecular imaging technology has been recognized as the efficient molecular tool [15,16]. During the last several decades, many kinds of fluorescent H2S probes have emerged as a hot topic in the scientific literature, most of which involve development of reactivity and reducing properties of H2S with specific chemical reactions [17,18]. Whereas, the many probes display turn-off or turn-on response with H2S, this may affects the accuracy of quantitative detection because of many reasons [19,20]. Thus, compared with the single emission probes of H2S, the ratio type fluorescence probes can reduce some errors by self- calibration by dualemission spectra bands. Up to now, there have been few ratiometric fluorescence sensors for endogenous H2S [21,22]. Based on the above deficiencies, in order to better monitor and image the dual roles of H2S in cells and even in vivo, our main research program is to select a fluorescent group which can
Corresponding authors. E-mail addresses: [email protected] (W. Qin), [email protected] (H. Guo).
https://doi.org/10.1016/j.talanta.2019.02.017 Received 28 August 2018; Received in revised form 28 January 2019; Accepted 3 February 2019 Available online 05 February 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. The synthetic procedure of probe 1. (a) o-hydroxyacetophenone, CH3CH2OH, NaOH, 50 °C reflux 4 h, HOAc (neutralized pH = 7), 82%; (b) MeOH, NaOH, H2O2, rt 7 h, 79%; (c) CHCl3, PySSPy, rt 12 h, 95%; (d) EDC, DMAP, DCM, rt 12 h, 59%, DMAP = 4dimethylaminopyridine; DCM = dichloromethane, EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
provide high quantum yield, long emission spectrum wavelength, and pH-insensitive and a simple mature reaction group can alter the fluorescent properties of the probe by reacting with H2S. In this report, we designed and synthesized a new type of ratio fluorescent probe 1, in which 4′-(diethylamino)-3-hydroxyflavone [23] was modified with 2,2′-dipyridyl disulfide benzoic acid [24,25] to produce an intramolecular charge transfer (ICT) process ( Scheme 1). Therefore, we can monitor and image endogenous and exogenous H2S with the change of ratio signal.
3. Results and discussion It was confirmed that the NMR and MS spectrum of the product was indeed for probe 1 (Figs. S11–S12) and probe 1-H2S (Figs. S13–S14) as presented in Scheme 1. The normalized absorption spectrum and fluorescence emission spectrum of probe 1 in a few solvents of varying polarity are shown in Fig. S1, and their photophysical properties are listed in Table S1. The absorption spectrum has one main band at ~385 nm in all solvents and which shows that there is a negligible effect by the polarity of solvent. However, the maximum red shift (~70 nm) of the fluorescence emission spectrum of probe 1 is appeared. The Φf value was higher in the non-polar solvent, such as cyclohexane (Φf = 0.16), while in the polar solvents, the fluorescence intensity gradually decreased (Φf = 0.15 in chloroform and Φf = 0.067 in acetonitrile), and the fluorescence quantum yield Φf also depends on the polarity of the solvent. The low Φf value of the probe 1 in the polar solvent is because of effective quenching of the electron acceptor of the pyridine nitrogen atom through the ICT effect.
2. Experimental section 2.1. Instruments and reagents All experiment reagents and chemicals were purchased from commercially available providers. The source of hydrogen sulfide used in all experiments were sodium sulfide nonahydrate hydrate, purchased from Guangfu. Double distilled water was used. Coumarine 102 in ethanol (λex = 389 nm, Φf = 0.783) was used as standard of fluorescence for the quantum yield test [26]. All measurements were carried out in CTAB/MeCN/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) at room temperature.
3.1. UV absorption and emission fluorescence spectra The absorption and emission spectra of probe 1 towards H2S are shown in Fig. S2, and the photophysical corresponding data are listed in Table S2. As shown in Fig. 1a, the absorption band at 392 nm dropped off, meanwhile, a new peak arised at about 425 nm and the intensity enhanced with the increased concentration of H2S. The two distinct isoelectric points (375 and 405 nm) given by the visible absorption spectra signal changes at 425 and 392 nm with different concentrations of H2S versus probe 1 both indicates the appearance of new species. This change is in accordance with the fluorescence lifetime measurement and numerical fitting (Table S3). As shown in Fig. 1b, the maximum emission wavelength of the probe 1 is 495 nm when H2S is not exist. But when H2S was added to the PBS within 10 μM probe 1 in buffer, a new fluorescence peak was observed in 525 nm, and with the gradually increased amount of H2S, the fluorescence peak at 495 nm was descend, meanwhile, accompanied by a strong green fluorescence appeared at 525 nm. As shown in Table S2, this change is consistent with the measured and calculated quantum yield of fluorescence. The Φf = 0.078 ± 0.001 in the blank sample with only the probe, and the Φf = 0.258 ± 0.003 at 525 nm after the addition of H2S.
2.2. Synthesis of Probe 1 As shown in Scheme 1, compound A (with a minor revision) and compound B were synthesized respectively by reported method [23,27]. Compound B (131 mg, 0.5 mmol), compound A (15.6 mg, 0.05 mmol), EDC (96 mg, 0.5 mmol), and DMAP (6.1 mg, 0.05 mmol) were dissolved in dry DCM (25 mL) at 25 °C and stirred for 12 h. After the reaction finished, removed the solvent and then purified by using silica gel column to get probe 1 as a pale yellow solid (130 mg, 59%). 1 H NMR (400 MHz, CDCl3, TMS) 8.83–8.85 (3H, m), 8.35 (1H, t, H-l, J = 8.0 Hz), 7.88 (2H, d, H-k, J = 8.0 Hz), 7.80 (1H, d, H-j, J = 8.0 Hz), 7.67 (1H, d, H-e, J = 8.0 Hz), 7.55 (2H, d, H-d, H-f, J = 8.0 Hz), 7.37–7.42 (4H, m), 6.68 (2H, d, H-a, J = 8.0 Hz), 3.41 (4H, q, 2 × CH2, J = 8.0 Hz), 1.19 (6H, t, 2 × CH3, J = 8.0 Hz). MS calcd for C31H26N2O4S2, 554.13, found 555.11 (M + 1). 2.3. Synthesis of Probe 1-H2S (compound A)
3.2. The limit of detection calculation
27.7 mg (0.05 mmol) of probe 1 was dissolved in 15 mL of dry DCM, and then 7.8 mg H2S (1.0 mmol) was added at 25 °C and stirred for 0.5 h. The solvent was removed and then separated by silica gel column to give product probe 1-H2S as a deep yellow solid (15 mg) in 97% yield. 1H NMR (400 MHz, CDCl3, TMS) 8.22 (1H, d, H-c, J = 8.0 Hz), 8.16 (2H, d, H-b, J = 8.0 Hz), 7.63 (1H, t, H-e, J = 8.0 Hz), 7.55 (1H, d, H-f, J = 8.0 Hz), 7.38 ( 1H, t, H-d, J = 8.0 Hz), 6.75 (2H, d, H-a, J = 8.0 Hz), 5.29 (-OH, s), 3.44 (4H, q, 2 × CH2, J = 8.0 Hz), 1.22 (6H, t, 2 × CH3, J = 8.0 Hz). MS calcd for C19H19NO3, 309.14, found 310.14 (M + 1).
Fig. 2a shows titration curve of the probe 1 for H2S. It can be seen from the titration diagram that the value of the fluorescence signal changes obviously when the low concentration of H2S is added to the PBS, after the treatment of high concentration the changes slowly and steadily. Fig. 2b shows the correction curve of probe 1 (R = F525 nm/F495 nm) with different concentrations of H2S. Because the concentration of probe 1 in the titration experiment is 10 μM, as the reaction progresses, the concentration of H2S in the analyte is increased. Due to the 186
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Fig. 2. a. Extremum titration curve of the probe 1 to H2S; b. Linear calibration curve of fluorescence Intensity ratio R = F525 nm/F495 nm of probe 1 with H2S, probe 1 = 10 μM, λex = 380 nm.
Fig. 1. (a) UV absorption spectrum; (b) Fluorescence spectrum of probe 1 towards H2S in PBS buffer, probe 1 = 10 μM.
consumption of the probe, the reaction proceeds smoothly and the fluorescence intensity reaches the maximum. Therefore, in the initial concentration range of 0–10 μM, the linear increase value is more obvious, while in the concentration range of 10–100 μM, the ratio value change is relatively small. After calculation, the DL of probe 1 towards H2S was 0.091 μM (1–10 μM) and 0.703 μM (10–100 μM). To ours pleasure, this detection limit have an ideal effect compared with others reported literature (Table S4). 3.3. Time response and kinetic study of probe 1 toward H2S As shown in Fig. 3, the time-dependent fluorescence curve of probe 1 towards H2S was conducted in PBS solution at room temperature. The fluorescence intensity is stable over time only when the probe 1 is present. When addition of several (10, 50, and 100 μM) concentration of H2S in this system, the emission band from 495 nm moved to 525 nm apparently within the initial 5 min and reached a maximum at about 10–15 min and as well as it maintained its strong green fluorescence. Therefore, the detection of H2S was obtained through the detection of probe 1 within ~15 min. This response time is relatively short compared to most of the previously reported literature (Table S4). For a chemodosimeter, the response time of a signal depends on the rate of reaction between the object species and the target substance. Therefore, the kinetics of the time dependent dosimetric response was assessed between the probe 1 and the H2S. According to the calculation method in literature reported [28], kinetic studies were observed to calculate the apparent rate constant k for probe 1 towards H2S at indoor temperature. The pseudo-first-order reaction rate constant kobs was calculated to be 0.17 min−1 (Fig. S3) When probe 1 was responded to 10 μM of H2S.
Fig. 3. Time course of the response at F525 nm/F495 nm of probe 1 with several equiv. of H2S, probe 1 = 10 μM.
3.4. Study on the optical stability and pH of the probe 1 The probe 1 has good light resistance within 10 min, the fluorescence intensity is very stable, and shows good light stability (Fig. S5), and the influence of illumination on the detection process can be excluded during the experiment. At the same time, we also measured the effect of pH on the blank probe 1 and the addition of H2S. It can be seen from Fig. S6 that the fluorescence ratio of the probe 1 is very stable within a certain pH range. When H2S is added for 15 min, the fluorescence ratio increases significantly in the range of pH 4–7, and tends to 187
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Fig. 5. Time-resolved curve of probe 1 (10 μM) towards H2S, λex = 380 nm.
Fig. 4. The ratio fluorescence intensity (F525 nm/F495 nm) of probe 1 (10 μM) towards active sulfur (100 μM) in PBS solution.
experimental conditions and fluorescence spectrum were obtained after 15 min. As shown in Fig. 4 and Fig. S4, about 30 nm bathochromic-shift clearly shows that the Probe 1 has an excellent response for H2S due to the efficiently ester cyclization reaction, while other typical active sulfur substance do not shows a clear change in fluorescence ratio under the same reaction conditions. This result forcefully shows that Probe 1 was superior for H2S in selectivity compared with other sulfur-containing active substances. The competition experiments were also used to carried out, which also indicates that Probe 1 still detects the H2S with high selectivity although other sulfur-containing species were still existed.
be smooth in the range of 7–10 This experimental result is superior for the imaging of probes for further hydrogen sulfide in the cellular environment. 3.5. The specificity of probe 1 towards H2S To investigate the selectivity in aqueous solution, the probe 1 was added in various species of sulfur, including common interference such as Cys, GSH, Hcy, SO32−, S2O32− (all at 100 μM) under the same
Scheme 2. The response mechanism of the probe 1 towards H2S and other thiols. 188
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Fig. 6. a) HeLa cells were cultured with probe 1 for 1 h; b), c) Cells were cultured with probe 1 for 1 h after further cultured with H2S (10 µM and 50 µM) for another 30 min, respectively; d) The ratio of mean fluorescence intensity of blue and green channels of HeLa cells, R=F525 nm/F495 nm. probe 1 = 10 µM. Scale bar = 20 µm.
3.6. The exploration of the mechanism
tautomer formation. On the contrary, the emission spectra of compound A in ethanol or PBS (polar proton solvent) both displays a broad peak at 525 nm (Fig. S7c). Because in aqueous media where the ESIPT process could be inhibited by intermolecular H-bonding in solvent, the emission peak at 525 nm could be due to the formation of species which are formed due to the presence of intermolecular hydrogen-bonding in solvent molecules. The reaction of probe 1 with H2S confirmed this mechanism. Meanwhile, after the other biological thiols reacted with the probe 1 under the same experimental conditions, the compound E was obtained, however, since compound E has no SH functional group, the intramolecular nucleophilic attack process could not be further performed. Therefore, other biological thiols could not change the fluorescence and only H2S could cause the fluorescence spectrum to produce a change in the ratio signal.
As shown in Scheme 2, owing to the strong electron-giving ability of hydroxyl group, compound B acts as a H2S acceptor, the probe 1 can form sulfhydryl-containing intermediate with H2S, the by-product compound C was formed by spontaneous cyclization and the fluorophore (Compound A) was released at the same time. The process and mechanism of this reaction are similar to those already reported for the detection of H2S [23,24,29–32] Fig. S7 shows the fluorescence emission spectra of Compound A in solvents having different polarities, which proves that Compound A shows dual fluorescence in the reduced-polarity solvent. Compound A with a higher energy band around ~420 nm is due to the emission of enol Compound A tautomer while a large stokes shift emission spectrum at ~550 nm in cyclohexane can be due to the emission at ESIPT in the keto-tautomers formed (Scheme 2). The fluorescence quantum yield Φf of compound A is strongly solvent dependent. The Φf values are higher in cyclohexane (0.083), somewhat lower in acetonitrile (0.021) (Table S1). The emission spectrum in other solvents also exhibits two bands, respectively. One is ranges from 430 nm to 515 nm and another at ~565 nm. If the solvent polarity was increased from cyclohexane (430 nm) to acetonitrile (510 nm), it can be seen that the band of the spectrum bathochromic-shifts (~90 nm) at higher energy level. Consequently, the emission spectra at 500 nm band is almost not affected by the polarity of the solvents, the emission peak within 430–510 nm are owe to the emission of enol tautomer, the large stokes shift at 550 nm probably could be due to the enol tautomer excited to keto
3.7. Fluorescence decay traces of probe 1 toward different concentrations of H2 S A global fitting of fluorescence decays was done by using FAST software supplied by Edinburgh Instruments Company. It can be expected that global analysis with three decay traces would estimate the {τi αi} values with higher accuracy than single-curve analysis. All global curve fittings presented here had χ2 values < 1.20. As shown in Fig. 5, the fluorescence decay of probe 1 shows two main components (Table S3, Fig. S8 and Fig. S10), with attenuation constants of 0.36 ns and 0.83 ns, respectively. The contributions from the slow decay time 0.83 ns increased at longer wavelengths (from 24% at 485 nm to 34% at 189
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Fig. 7. a) Cells were cultured with probe 1 for 1 h; b) cells were cultured with Cys (1000 µM) for 1 h followed by probe 1 for another 1 h; c); d); e) cells were handled with PAG (10 µM), AOAA (10 µM), PAG and AOAA (10 µM + 10 µM) for 1 h; then cultured with Cys (1000 µM) for 1 h; finally, cultured with probe 1 for further 1 h; f) The ratio of mean fluorescence intensity of blue and green channels of HeLa cells, R=F525 nm/F495 nm. probe 1 = 10 µM. Scale bar = 20 µm. λex = 405 nm; λem = 405–495 nm (blue channel), λem = 495–530 nm (green channel).
505 nm), indicating that the excited state fast decay compound (τ1 ≈ 0.36 ns) in PBS is a local excitation state, while the slow decay time τ2 can be attributed to the lifetime of the ICT state. Upon the addition of different concentration of H2S (20 and 100 μM) in PBS buffer, it can be seen that the fluorescence decays to a single index, and the average fluorescence decay lifetime at the emission wavelengths of 515 nm, 525 nm, and 535 nm increases from 1.20 ns to 1.23 ns. The above experimental data shows that the fluorescence emission peak at 525 nm after the addition of H2S is mainly the formation of a substance derived from intermolecular hydrogen bonding in a solvent molecule, and the newly formed product (compound A) content is almost 100%. The
rationality of the reaction mechanism of H2S and probe 1 in Scheme 2 was further proved by analyzing the experimental data. 3.8. Imaging of exogenous H2S in living cells The probe 1 was used to determine the real-time tracking and detection of H2S in HeLa cells. Before cell imaging, an MTS assay was carried out to evaluate the cytotoxicity effect and compatibility of the probe 1. The MTS test concentration of probe 1 in HeLa cells varies from 5 μM to 100 μM (Fig. S9), which clearly indicates that the probe 1 exhibits low cytotoxicity and excellent biocompatibility for cultured 190
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cell lines under the experimental conditions keeping the concentration 10 μM. In Fig. 6a, cells were only treated with probe 1 initially showed blue fluorescence and a relatively weaker green fluorescence. However, when probe 1-stained cells cultured with H2S (10 μM and 50 μM), showed the relatively strong green fluorescence in the green channel, while the blue channel showed relatively weak blue fluorescence (Figs. 6b and 6c). Taking together, this increasing proportion of the signal clearly shows probe 1 have better cell membrane permeability which confirming that probe 1 is a possible tool used to detect the content of H2S in living cells in real time.
of China (2014DFA31890). The authors would like to thank the Natural Science Foundation of China (No. 21771092). This work was also supported by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China, [email protected].
3.9. Imaging of endogenous H2S in living cells
References
The probes for detecting H2S is shown in Table S4, most are turn-off or turn-on type, and the ratio type is relatively small. The relatively novel probes that are both ratios and endogenous for the detection of H2S are rare [33–35]. On the one hand, the ratio probe has dual emission compared to the conventional turn-off or turn-on type probe. The probe 1 reacts with the substance to be tested, and the emission wavelength changes before and after. The emission wavelength are two, one high and one low, compared with the subsequent Ratio-related, such probes can reduce background interference and have better sensitivity; on the other hand, since the biological system generates responsive substances with probes through certain metabolic processes, endogenous detection have little damage to the cells themselves, It is more widely used in biological systems. Thus, based on the support of these experimental data, we attempted to test the practicability of probe 1 with endogenic H2S in HeLa cells. It is known that H2S can be produced by the catalytic activity of Cys in living cells through the catalysis of two enzymes of CBS and CSE. As shown in Fig. 7, as we expected, only in the presence of probe 1 (a), the strong blue fluorescence and the weaker green fluorescence were collected. On the contrary, the fluorescence emission of the blue channel was basically unchanged after the cell was incubated with Cys, Meanwhile, the fluorescence of the green channel is obviously stronger (b). To demonstrate that Cys indeed produce hydrogen sulfide due to the catalysis of the two enzymes mentioned above, we also performed experiment for the inhibition of CBS and CSE by enzyme inhibitors (propargylglycine (PAG) or aminooxyacetic acid (AOAA)) using reports from known literature [34]. As Fig. 7(c–e) shows, cells were handled with PAG or AOAA or PAG and AOAA, and then incubated with Cys for 1 h, and followed by addition of probe 1 for further 1 h. There was almost negligible ratiometric signal of the fluorescence intensity changes was observed compared to the control group. These results verified that the production of endogenous H2S was inhibited resulted from the inactivation of the enzyme.
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2019.02.017.
4. Conclusion In short, an efficient synthetic method is successfully development for the preparation of a ratio fluorescent dual-emission probe for tracing not only the exogenous but also the endogenous H2S in living cells. The probe 1 showed the excellent selectivity-sensitive fluorescence response in the emission ratio of H2S for other biothiol (F525 nm/F495 nm) having low limit of detection of 91 nM (0–10 μM) in aqueous solution for ~15 min. In addition, the mechanism of probe 1 for H2S was also analyzed and determined by ICT and ESIPT due to solvation effects. Based on the excellent ratio of probe 1 and the endogenous detection of the properties of H2S, the probe 1 could be an useful tool which can be effectively used for the detection and imaging analysis of H2S in biological cells. Acknowledgments This work was supported by the Ministry of Science and Technology 191
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