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A coumarin-based fluorescent probe for differential identification of sulfide and sulfite in CTAB micelle solution† Haiyu Tian,a Junhong Qian,*a Qian Sun,a Chenjia Jiang,a Runsheng Zhangb and Weibing Zhang*a Sulfite and sulfide share several similarities in terms of chemical properties, such as nucleophilic and reducing reactivities. Therefore, they may disturb the detection of each other. In order to discriminate between these two kinds of sulfur-containing species, a new probe TSSP-N3 was developed, in which para-azidobenzenyl ketone was covalently incorporated to a coumarin fluorophore linked by a C]C double bond. Sulfite and sulfide can respectively react with the C]C double bond and the azido group
Received 13th March 2014 Accepted 5th April 2014
to give different products, consequently, they can be differentially identified by UV-vis and fluorescence
DOI: 10.1039/c4an00478g
good candidate for the detection of sulfide and sulfite. The bioimaging experiment demonstrates the
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potential of the TSSP-N3 probe for the differential imaging of sulfide and sulfite in living cells.
spectroscopy as well as by the naked eye. Selectivity and competition results reveal that TSSP-N3 is a
Introduction Hydrogen sulde (H2S) and sulte (SO32), two important members of sulfur-containing species, play vital roles in environmental and physiological processes. In cells and tissues, H2S comes from the decomposition of L-cysteine catalyzed by several live enzymes and has been conrmed to be a gaseous signaling molecule.1 Sulfur dioxide (SO2), a gaseous form of sulte, can also be produced in vivo from sulfur-containing amino acids.2 The endogenous and/or environmental levels of these sulfurcontaining species are believed to be related to some diseases: abnormal concentrations of H2S may be associated with liver cirrhosis3 and Down's syndrome,4 while excessive amounts of SO32 are a cause of difficulty in breathing, gastrointestinal distress, wheezing, etc.5 The functions of H2S and SO32 are quite different, it is of great interest to differentially detect H2S and SO32 with high sensitivity and selectivity to study their biological functions. Several techniques such as colorimetric/electrochemical assays, gas chromatography and metal complex precipitation have been developed for the detection of H2S and sulte.6 Among these, the uorescence method has received much attention due to the advantages of convenience, high sensitivity
a
Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, China. E-mail: [email protected]; [email protected]
b
Shanghai Key Laboratory of Scene Material Evidence, Center of Material Evidence Identication, Shanghai Public Security Bureau, Shanghai, 200083, China † Electronic supplementary 10.1039/c4an00478g
information
(ESI)
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available.
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DOI:
and selectivity, fast response and ease of live-cell imaging.7 H2S and SO32 share some common features such as nucleophilic and reducing properties, and many colorimetric/uorescent probes for sulde or sulte have been designed based on these properties,8 consequently, they may interfere with the detection of each other. For example, some reported probes8e–g,9a for sulte are based on the nucleophilic addition of sulte to C]C or C]O double bonds, and sulde showed interference to some extent; a number of uorescent probes for sulde utilize the reduction of azide to amine8a,9b or the removal of a 2,4-dinitrobenzenesulfonyl group8h triggered by the sulde anion, however, the selectivity for sulde over sulte was not so satisfactory. Therefore, construction of a molecular probe that can discriminate multiple analytes with similar properties has attracted our attention. In our previous work,9 we designed a couple of uorescent probes with a,b-unsaturated ketone and azide as the receptors for sulte and sulde, respectively. During the detection of sulte, we found that the selectivity over sulde is relatively poor. Considering the much faster reaction rate of the reduction of azide than that of the nucleophilic addition of a,b-unsaturated ketone with sulde, we designed a new uorescent probe TSSP-N3 to diminish the effect of sulde on the detection of sulte. TSSP-N3 was constructed by covalently incorporating para-azidobenzenyl ketone to a coumarin uorophore linked by a C]C double bond (Scheme 1). Michael addition between sulte and the a,b-unsaturated ketone shortens the p-conjugation of the probe, which will result in signicant blue shis in the absorption and emission spectra. Sulde can react with both azido and a,b-unsaturated ketone at different reaction rates, and the reduction product TSSP-NH2 is dominant under
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min 5% B, and 7–10 min 95% B. Injection volume: 50 mL; ow rate: 1.0 mL min1; detection wavelength: 410 nm.
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Live cell culture and uorescence imaging
Scheme 1
The chemical structures of the three dyes studied.
the experimental conditions. Compared to the azide compound TSSP-N3, the electronic push–pull effect of the amine product TSSP-NH2 is weakened, and slight blue shis in the absorption and emission peaks could be expected. Therefore, sulde and sulte will induce different spectral changes of TSSP-N3, which makes it possible to differentially identify H2S and SO32. In addition, two compounds TSSP-NH2 and TSSP-H were employed as the references to investigate the substitution effect on the spectral properties of the probe.
Experimental
HeLa cells were cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 C and under 5% CO2 in a CO2 incubator. The cells were washed with phosphate buffered solution (PBS) and preincubated with 100 mM Na2S or Na2SO3, then incubated with TSSP-N3 (5 mM) and CTAB (1 mM) in DMEM for 30 min at 37 C and washed 3 times with PBS. For the control experiment, the cells were only incubated with 5 mM of TSSP-N3 and CTAB (1 mM) for 30 min. Cell imaging was carried out aer washing the cells with PBS. Emission was collected at 460–480 nm for the blue channel, at 550–560 nm for the green channel and at 590– 600 nm for the red channel. The excitation wavelength was set at 404 nm for the blue channel or 488 nm for green and red channels. Determination of the detection limit The detection limit (LOD) was obtained by 3Sb/k, where Sb is the standard deviation of 10 blank measurements, and k is the slope of the tted line.
Materials and reagents All the chemicals were purchased from Adamas-beta Corporation and were used without further purication. Ultra-pure water was prepared through the Sartorius Arium611DI system. Phosphate salts were used to maintain a stable pH and ion strength in detection systems. Instruments Absorption spectra were measured using an Evolution 220 UVvisible spectrophotometer (Thermo Scientic). Fluorescence spectra were recorded on a Lumina uorescence spectrometer (Thermo Scientic). 1H and 13C spectra were recorded using a BrukerAV-400 spectrometer (400 MHz). Mass spectra were recorded on a MA 1212 Instrument under standard conditions (ESI, 70 eV).
Results and discussion Photophysical properties of three dyes in CTAB–PBS First, the absorption and emission spectra of different dyes in CTAB–PBS were investigated to understand the substitution effect. Compared with TSSP-H, both the absorption and emission wavelengths of TSSP-NH2 were a bit shorter, while those of TSSP-N3 were slightly longer (Fig. 1). It can be seen from Scheme 1 that all three dyes possess electronic push–pull characteristics with N,N-diethyl amino group as the electron-donor and the carbonyl group as the electron-acceptor. The strong electrondonating amino group at the benzenyl ring weakened the push– pull effect, which results in blue-shis in both absorption and emission peaks. In the case of TSSP-N3, the electron withdrawing azido group strengthens the push–pull effect and leads to slight red-shis in the absorbance and emission maxima.
Sulde and sulte titration A stock solution of 3 102 M TSSP-N3 in DMF, 10 mM Na2S– PBS, 10 mM Na2SO3–PBS and CTAB (1 mM) phosphate buffer solutions (20 mM, pH 7.4) were prepared in advance. The stock solution of TSSP-N3 was added to 3 mL of the above CTAB–PBS solution to make [TSSP-N3] ¼ 2 mM. 0–50 mL of sulte or sulde stock solution were added to the dye–PBS solution to obtain appropriate concentrations of sulte or sulde.
Photophysical responses of probe TSSP-N3 to sulde and sulte in CTAB–PBS The sensing behavior of TSSP-N3 towards sulde and sulte was studied using UV-vis and uorescence spectroscopy. In the
HPLC traces HPLC spectra were recorded using an Elliot 1203 system and a Zobax C18 reversed-phase column (4.6 mm 10 cm). The mobile phases were degassed using an ultrasonic apparatus for 10 min. Mobile phase: A: water, B: acetonitrile; gradient elution: 1–7 min 5–95% B, 10–11 min 95–5% B; isocratic elution: 0–1
3374 | Analyst, 2014, 139, 3373–3377
Fig. 1 The normalized absorption (a) and emission (b) spectra of the three dyes.
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CTAB–PBS system, the absorption and emission peaks of TSSPN3 were centered at 475 nm and 590 nm, respectively. Upon the addition of 10 equiv. sulde (Na2S as the sulde donor, the reaction was too fast for us to monitor the wavelength changes in the presence of 50 equiv. sulde), the absorption and emission peaks respectively shied to 462 nm and 564 nm within 10 min (Fig. 2a and b). The spectral properties of the nal product is in line with those of TSSP-NH2 (Fig. 1), indicating that sulde reduced the azido group in the probe to give TSSP-NH2. Fig. 2c and d demonstrate the time-dependent absorption and emission spectra of TSSP-N3 in the presence of 50 equiv. sulte. As the Michael addition between sulte and TSSP-N3 progressed, the original absorption band at 475 nm decreased, accompanied by the appearance and development of a new band at 386 nm (Fig. 2c), and an isosbestic point at 410 nm was clearly observed. The uorescence spectrum of TSSP-N3 displayed a similar behavior towards sulte: the emission band dramatically shied from 590 nm to 460 nm (Fig. 2d) with an isoemission point at 543 nm. Fig. 3 shows that sulde and sulte induce different color changes of the TSSP-N3 solution, which could easily be distinguished by the naked eye. Sulte triggered the change of the solution's color from orange to colorless, and that of the uorescence color from red to blue; while sulde changed the solution's color to yellow. Therefore, the probe TSSP-N3 can be used to differentiate between sulte and sulde. It is clear from Fig. 2 that the reaction between TSSP-N3 and sulde was completed aer 10 min, and about 95% of the probe TSSP-N3 was transformed to the addition product with sulte aer 1 h (t1/2 z 7 min, Fig. S3a†). Therefore, the assay time of 1 h is used in the following photophysical experiments. HPLC traces of the reaction processes of TSSP-N3 with sulde and sulte To get more insight into the sensing mechanism of TSSP-N3, the HPLC traces of the reaction processes were recorded as shown
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Fig. 3 The proposed recognition mechanisms for sulfide and sulfite, [TSSP-N3] ¼ 10 mM, [sulfite] ¼ 500 mM, [sulfide] ¼ 100 mM.
Fig. 4 Time-dependent HPLC spectra of the reaction between TSSPN3 and sulfide (a) or sulfite (b) in the PBS–CTAB (1 mM) system. [TSSPN3] ¼ 15 mM, [sulfide] ¼ [sulfite] ¼ 750 mM, pH ¼ 7.4, 20 mM PBS, 25 C, detection wavelength ¼ 410 nm.
in Fig. 4. It is clear from Fig. 4a that the peaks of TSSP-N3 and its reduction product (TSSP-NH2) are at about 7.5 min and 6.5 min, respectively. With the reaction between sulde and the probe progressing, the peak of TSSP-N3 decreased dramatically and disappeared aer 1 h accompanied by the emergence and development of a new peak at about 6.5 min, which further supports the reaction mechanism between the probe and sulde. The reaction between sulte and the probe is more complex but interesting (Fig. 4b). With the decrement of the peak at 7.5 min, two new peaks respectively at 3.8 min and 4.1 min emerged. The mass spectral analysis (Fig. S4†) indicates that the peak at 3.8 min is from the sulte addition product of TSSP-NH2 (cal. for M 1 ¼ 443.1277, found 443.1282), while that at 4.1 min is supposed to be from the sulte addition product of TSSP-N3. The ratio of the peak intensities at 3.8 min and 4.1 min increases with time, which suggests that sulte can reduce the azido group to amino, but the reaction rate of reduction is lower than that of the addition (Fig. 4b). The selectivity and competition of TSSP-N3 towards sulde and sulte over various analytes in the CTAB–PBS system
Time-dependent absorption and emission spectra of TSSP-N3 (2 mM) in the presence of 10 equiv. sulfide (a and b) and 50 equiv. sulfite (c and d) in the PBS–CTAB (1 mM) system. Data in (a) and (b) are normalized. [TSSP-N3] ¼ 2 mM, 20 mM PBS, pH 7.4, 25 C, excited at the isosbestic point 410 nm. Fig. 2
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We further investigated the selectivity prole of TSSP-N3 in response to some other anions and biological thiols in the CTAB–PBS system (20 mM, pH 7.4, 25 C). No evident uorescence intensity change of the emission band was observed in the presence of 50 equiv. of F, Cl, Br, I, AcO, HCO3, CN, NO2, NO3, PO43, SCN, HS, SO42, S2O32, Cys and Hcy (Fig. 5). GSH induced a uorescence enhancement at 460 nm but to a much smaller extent (I460/I590 z 3), which revealed that the nucleophilic addition between GSH and TSSP-N3 happened at a much lower rate. It can also be seen from Fig. 5a that the selectivity of TSSP-N3 towards sulde over all the anions and thiols detected is satisfactory. The results in Fig. 5 indicated
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Fig. 5 The fluorescence spectra (a) and the ratio of fluorescence intensities at 460 nm and 590 nm (I460/I590) (b) in the presence of 50 equiv. of various additives with (red) or without (black) sulfite in CTAB– PBS for 1 h (in the competition experiment of sulfite towards sulfide the reaction time was 4 h), (1) blank; (2) F; (3) Cl; (4) Br; (5) I; (6) AcO; (7) HCO3; (8) CN; (9) NO2; (10) NO3; (11) PO43; (12) HS; (13) SCN; (14) SO42; (15) S2O32; (16) Cys; (17) Hcy; (18) GSH. 20 mM PBS, pH 7.4, 25 C, [CTAB] ¼ 1 mM, [TSSP-N3] ¼ 2 mM, [sulfite] ¼ 100 mM, excited at the isosbestic point 410 nm.
that the probe TSSP-N3 was highly specic for sulte and H2S. The uorescence responses of TSSP-N3 towards sulte in the presence of other anions or thiols were explored in order to determine the possible interference from other analytes. Fig. 5b demonstrates that the other analytes except for sulde did not exhibit notable interference with the detection of sulte. The presence of sulde decreased the uorescence intensity of the sulte addition product within 1 hour, however, the interference can be eliminated by prolonging the detection time to 3 hours (Fig. S5†). The selectivity and the competition results reveal that TSSP-N3 is a good candidate for the detection of sulte.
Effects of sulde and sulte concentrations on the uorescence intensity of TSSP-N3 The effect of sulte on the emission spectrum of the probe TSSP-N3 in CTAB–PBS was studied as well. The titration experiments were carried out by gradually adding sulte to the probe and collecting the absorption and emission spectra aer 1 hour (Fig. 6 and S6†). As shown in Fig. 6b, a linear relationship was found between the uorescence intensity ratio (I460/I590) and sulte concentration in the range of 0–100 mM. The detection limit for sulte was 0.1 mM, which was comparable to those of the reported sensors.8f,9a However, sulde-induced changes in the absorbance and the uorescence intensity of TSSP-N3 were relatively small (Fig. S7†), TSSP-N3 cannot be employed to
Fig. 6 The emission spectra (a) of TSSP-N3 with different concentrations of sulfite after 1 h, and ratio of fluorescence intensities at 460 nm and 590 nm (b, I460/I590) as a function of sulfite concentration. 20 mM PBS, pH 7.4, 25 C, [CTAB] ¼ 1 mM, [TSSP-N3] ¼ 2 mM, excited at 410 nm.
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Fig. 7 The merged fluorescence images of blue, green and red channels for HeLa cells incubated with TSSP-N3 and CTAB (1 mM). (a and c) nothing else, (b) preincubated with 100 mM sulfite and (d) pretreated with 100 mM sulfide. (a and b) excited at 404 nm; (c and d) excited at 488 nm.
quantify sulde, but it can sense the existence of small amounts of H2S (as low as 5 mM) by the wavelength change (Fig. S8†). Bioimaging of sulde and sulte in living cells Finally, the possibility of using TSSP-N3 for uorescence imaging of sulde and sulte in living cells was evaluated. Aer incubating with 5 mM of TSSP-N3 for 30 min, remarkable intracellular green and red uorescence could be observed (Fig. S9†). Pretreating the cells with 100 mM sulte followed by incubation with TSSP-N3 led to a marked increase in blue emission and slight decreases in both green and red emissions (Fig. S10b–d†). When HeLa cells were preincubated with 100 mM sulde and then with TSSP-N3, a clear increment in green emission and decrement in red uorescence were observed (Fig. S11†). These results verify the potential application of TSSP-N3 for the differential sensing of intracellular sulte and sulde from different emission channels. Fig. 7 shows the overlapped uorescence images of the three channels, from which sulde and sulte could be clearly distinguished by the naked eye.
Conclusions In conclusion, a new colorimetric and ratiometric uorescent probe TSSP-N3 was developed to differentiate between sulde and sulte in CTAB micellar solution. The signicant blueshis in the absorption and emission peaks induced by sulte are based on the 1,4-addition of the a,b-unsaturated ketone in the probe by sulte, which results in a color change from orange to colorless, and thus can serve as a ‘‘naked-eye’’ probe for sulte. The slight blue-shis triggered by sulde are ascribed to the reduction of the azido group by sulde, which changes the electronic effect of the substitution at the benzene ring. A clear uorescence color change from red to yellow was observed upon the addition of sulde. Furthermore, we have demonstrated that the probe can be used for the uorescence imaging of cellular sulte and sulde. We hope that this probe may inspire
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the exploitation of new uorescent sensors for multiple analytes.
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Acknowledgements This work was nancially supported by the National 973 Program (no. 2011CB910403), NSFC (21235005) and the Science and Technology Commission of Shanghai Municipality (no. 12JG0500200). Dr J. Qian thanks the Scientic Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We appreciate Prof. A. M. Brouwer (University of Amsterdam) for his kind help.
Notes and references 1 (a) C. Szab´ o, Nat. Rev. Drug Discovery, 2007, 6, 917; (b) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51, 169; (c) H. Kimura, Amino Acids, 2011, 41, 113; (d) C. W. Leffler, H. Parfenova, J. H. Jaggar and R. Wang, J. Appl. Physiol., 2006, 100, 1065. 2 (a) M. H. Stipanuk and I. Ueki, J. Inherited Metab. Dis., 2011, 34, 17; (b) M. Whiteman and P. K. Moore, J. Cell. Mol. Med., 2009, 13, 488. 3 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005, 42, 539. 4 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-Vekemans, Am. J. Med. Genet., Part A, 2003, 116, 310. 5 (a) H. Vally, N. L. Misso and V. Madan, Clin. Exp. Allergy, 2009, 39, 1643; (b) R. C. Claudia and J. C. Francisco, Food Chem., 2009, 112, 487; (c) S. L. Taylor, R. K. Bush, J. C. Selner, J. A. Nordlee, M. B. Wiener, K. Holden, J. W. Koepke and W. W. Busse, J. Allergy Clin. Immunol., 1988, 81, 1159. 6 (a) W. Lei and P. K. Dasgupta, Anal. Chim. Acta, 1989, 226, 165; (b) A. Sen, J. D. Albarella, J. R. Carey, P. Kim and
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W. B. McNamara III, Sens. Actuators, B, 2008, 134, 234; (c) G. J. Mohr, Chem. Commun., 2002, 2646; (d) N. S. Lawrence, R. P. Deo and J. Wang, Anal. Chim. Acta, 2004, 517, 131; (e) A. Isaac, A. J. Wain, R. G. Compton, C. Livingstone and J. Davis, Analyst, 2005, 130, 1343; (f) T. Ubuka, T. Abe, R. Kajikawa and K. Morino, J. Chromatogr. B: Biomed. Sci. Appl., 2001, 757, 31; (g) M. Balazy, I. A. Abu-Yousef, D. N. Harpp and J. Park, Biochem. Biophys. Res. Commun., 2003, 311, 728; (h) S. Virji, J. D. Fowler, C. O. Baker, J. Huang, R. B. Kaner and B. H. Weiller, Small, 2005, 1, 624; (i) M. Albrecht, R. A. Gossage, M. Lutz, A. L. Spek and G. V. Koten, Chem.–Eur. J., 2000, 6, 1431. 7 (a) J. S. Kim and D. T. Quang, Chem. Rev., 2007, 107, 3780; (b) T. Q. Duong and J. S. Kim, Chem. Rev., 2010, 110, 6280; (c) M. S. T. Gonçalves, Chem. Rev., 2009, 109, 190; (d) M. Fern´ andez-Su´ arez and A. Y. Ting, Nat. Rev. Mol. Cell Biol., 2008, 9, 929. 8 (a) C. Yu, X. Li, F. Zeng, F. Zheng and S. Wu, Chem. Commun., 2013, 49, 403; (b) Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011, 11, 495; (c) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 50, 10327; (d) Y.-Q. Sun, J. Liu, J. Zhang, T. Yang and W. Guo, Chem. Commun., 2013, 49, 2637; (e) L. E. Santos-Figueroa, C. Gim´ enez, A. Agostini, E. Aznar, ˇ ez and M. D. Marcos, F. Sancen´ on, R. Mart´ınez-M´ an P. Amor´ os, Angew. Chem., Int. Ed., 2013, 52, 13712; (f) M.-Y. Wu, T. He, K. Li, M.-B. Wu, Z. Huang and X.-Q. Yu, Analyst, 2013, 138, 3018; (g) Y. Sun, S. Fan, S. Zhang, D. Zhao, L. Duan and R. Li, Sens. Actuators, B, 2014, 193, 173; (h) T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013, 15, 2310. 9 (a) H. Tian, J. Qian, Q. Sun, H. Bai and W. Zhang, Anal. Chim. Acta, 2013, 788, 165; (b) H. Tian, J. Qian, H. Bai, Q. Sun, L. Zhang and W. Zhang, Anal. Chim. Acta, 2013, 768, 136.
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A coumarin-based fluorescent probe for differential identification of sulfide and sulfite in CTAB micelle solution† Haiyu Tian,a Junhong Qian,*a Qian Sun,a Chenjia Jiang,a Runsheng Zhangb and Weibing Zhang*a Sulfite and sulfide share several similarities in terms of chemical properties, such as nucleophilic and reducing reactivities. Therefore, they may disturb the detection of each other. In order to discriminate between these two kinds of sulfur-containing species, a new probe TSSP-N3 was developed, in which para-azidobenzenyl ketone was covalently incorporated to a coumarin fluorophore linked by a C]C double bond. Sulfite and sulfide can respectively react with the C]C double bond and the azido group
Received 13th March 2014 Accepted 5th April 2014
to give different products, consequently, they can be differentially identified by UV-vis and fluorescence
DOI: 10.1039/c4an00478g
good candidate for the detection of sulfide and sulfite. The bioimaging experiment demonstrates the
www.rsc.org/analyst
potential of the TSSP-N3 probe for the differential imaging of sulfide and sulfite in living cells.
spectroscopy as well as by the naked eye. Selectivity and competition results reveal that TSSP-N3 is a
Introduction Hydrogen sulde (H2S) and sulte (SO32), two important members of sulfur-containing species, play vital roles in environmental and physiological processes. In cells and tissues, H2S comes from the decomposition of L-cysteine catalyzed by several live enzymes and has been conrmed to be a gaseous signaling molecule.1 Sulfur dioxide (SO2), a gaseous form of sulte, can also be produced in vivo from sulfur-containing amino acids.2 The endogenous and/or environmental levels of these sulfurcontaining species are believed to be related to some diseases: abnormal concentrations of H2S may be associated with liver cirrhosis3 and Down's syndrome,4 while excessive amounts of SO32 are a cause of difficulty in breathing, gastrointestinal distress, wheezing, etc.5 The functions of H2S and SO32 are quite different, it is of great interest to differentially detect H2S and SO32 with high sensitivity and selectivity to study their biological functions. Several techniques such as colorimetric/electrochemical assays, gas chromatography and metal complex precipitation have been developed for the detection of H2S and sulte.6 Among these, the uorescence method has received much attention due to the advantages of convenience, high sensitivity
a
Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, China. E-mail: [email protected]; [email protected]
b
Shanghai Key Laboratory of Scene Material Evidence, Center of Material Evidence Identication, Shanghai Public Security Bureau, Shanghai, 200083, China † Electronic supplementary 10.1039/c4an00478g
information
(ESI)
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available.
See
DOI:
and selectivity, fast response and ease of live-cell imaging.7 H2S and SO32 share some common features such as nucleophilic and reducing properties, and many colorimetric/uorescent probes for sulde or sulte have been designed based on these properties,8 consequently, they may interfere with the detection of each other. For example, some reported probes8e–g,9a for sulte are based on the nucleophilic addition of sulte to C]C or C]O double bonds, and sulde showed interference to some extent; a number of uorescent probes for sulde utilize the reduction of azide to amine8a,9b or the removal of a 2,4-dinitrobenzenesulfonyl group8h triggered by the sulde anion, however, the selectivity for sulde over sulte was not so satisfactory. Therefore, construction of a molecular probe that can discriminate multiple analytes with similar properties has attracted our attention. In our previous work,9 we designed a couple of uorescent probes with a,b-unsaturated ketone and azide as the receptors for sulte and sulde, respectively. During the detection of sulte, we found that the selectivity over sulde is relatively poor. Considering the much faster reaction rate of the reduction of azide than that of the nucleophilic addition of a,b-unsaturated ketone with sulde, we designed a new uorescent probe TSSP-N3 to diminish the effect of sulde on the detection of sulte. TSSP-N3 was constructed by covalently incorporating para-azidobenzenyl ketone to a coumarin uorophore linked by a C]C double bond (Scheme 1). Michael addition between sulte and the a,b-unsaturated ketone shortens the p-conjugation of the probe, which will result in signicant blue shis in the absorption and emission spectra. Sulde can react with both azido and a,b-unsaturated ketone at different reaction rates, and the reduction product TSSP-NH2 is dominant under
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min 5% B, and 7–10 min 95% B. Injection volume: 50 mL; ow rate: 1.0 mL min1; detection wavelength: 410 nm.
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Live cell culture and uorescence imaging
Scheme 1
The chemical structures of the three dyes studied.
the experimental conditions. Compared to the azide compound TSSP-N3, the electronic push–pull effect of the amine product TSSP-NH2 is weakened, and slight blue shis in the absorption and emission peaks could be expected. Therefore, sulde and sulte will induce different spectral changes of TSSP-N3, which makes it possible to differentially identify H2S and SO32. In addition, two compounds TSSP-NH2 and TSSP-H were employed as the references to investigate the substitution effect on the spectral properties of the probe.
Experimental
HeLa cells were cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 C and under 5% CO2 in a CO2 incubator. The cells were washed with phosphate buffered solution (PBS) and preincubated with 100 mM Na2S or Na2SO3, then incubated with TSSP-N3 (5 mM) and CTAB (1 mM) in DMEM for 30 min at 37 C and washed 3 times with PBS. For the control experiment, the cells were only incubated with 5 mM of TSSP-N3 and CTAB (1 mM) for 30 min. Cell imaging was carried out aer washing the cells with PBS. Emission was collected at 460–480 nm for the blue channel, at 550–560 nm for the green channel and at 590– 600 nm for the red channel. The excitation wavelength was set at 404 nm for the blue channel or 488 nm for green and red channels. Determination of the detection limit The detection limit (LOD) was obtained by 3Sb/k, where Sb is the standard deviation of 10 blank measurements, and k is the slope of the tted line.
Materials and reagents All the chemicals were purchased from Adamas-beta Corporation and were used without further purication. Ultra-pure water was prepared through the Sartorius Arium611DI system. Phosphate salts were used to maintain a stable pH and ion strength in detection systems. Instruments Absorption spectra were measured using an Evolution 220 UVvisible spectrophotometer (Thermo Scientic). Fluorescence spectra were recorded on a Lumina uorescence spectrometer (Thermo Scientic). 1H and 13C spectra were recorded using a BrukerAV-400 spectrometer (400 MHz). Mass spectra were recorded on a MA 1212 Instrument under standard conditions (ESI, 70 eV).
Results and discussion Photophysical properties of three dyes in CTAB–PBS First, the absorption and emission spectra of different dyes in CTAB–PBS were investigated to understand the substitution effect. Compared with TSSP-H, both the absorption and emission wavelengths of TSSP-NH2 were a bit shorter, while those of TSSP-N3 were slightly longer (Fig. 1). It can be seen from Scheme 1 that all three dyes possess electronic push–pull characteristics with N,N-diethyl amino group as the electron-donor and the carbonyl group as the electron-acceptor. The strong electrondonating amino group at the benzenyl ring weakened the push– pull effect, which results in blue-shis in both absorption and emission peaks. In the case of TSSP-N3, the electron withdrawing azido group strengthens the push–pull effect and leads to slight red-shis in the absorbance and emission maxima.
Sulde and sulte titration A stock solution of 3 102 M TSSP-N3 in DMF, 10 mM Na2S– PBS, 10 mM Na2SO3–PBS and CTAB (1 mM) phosphate buffer solutions (20 mM, pH 7.4) were prepared in advance. The stock solution of TSSP-N3 was added to 3 mL of the above CTAB–PBS solution to make [TSSP-N3] ¼ 2 mM. 0–50 mL of sulte or sulde stock solution were added to the dye–PBS solution to obtain appropriate concentrations of sulte or sulde.
Photophysical responses of probe TSSP-N3 to sulde and sulte in CTAB–PBS The sensing behavior of TSSP-N3 towards sulde and sulte was studied using UV-vis and uorescence spectroscopy. In the
HPLC traces HPLC spectra were recorded using an Elliot 1203 system and a Zobax C18 reversed-phase column (4.6 mm 10 cm). The mobile phases were degassed using an ultrasonic apparatus for 10 min. Mobile phase: A: water, B: acetonitrile; gradient elution: 1–7 min 5–95% B, 10–11 min 95–5% B; isocratic elution: 0–1
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Fig. 1 The normalized absorption (a) and emission (b) spectra of the three dyes.
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CTAB–PBS system, the absorption and emission peaks of TSSPN3 were centered at 475 nm and 590 nm, respectively. Upon the addition of 10 equiv. sulde (Na2S as the sulde donor, the reaction was too fast for us to monitor the wavelength changes in the presence of 50 equiv. sulde), the absorption and emission peaks respectively shied to 462 nm and 564 nm within 10 min (Fig. 2a and b). The spectral properties of the nal product is in line with those of TSSP-NH2 (Fig. 1), indicating that sulde reduced the azido group in the probe to give TSSP-NH2. Fig. 2c and d demonstrate the time-dependent absorption and emission spectra of TSSP-N3 in the presence of 50 equiv. sulte. As the Michael addition between sulte and TSSP-N3 progressed, the original absorption band at 475 nm decreased, accompanied by the appearance and development of a new band at 386 nm (Fig. 2c), and an isosbestic point at 410 nm was clearly observed. The uorescence spectrum of TSSP-N3 displayed a similar behavior towards sulte: the emission band dramatically shied from 590 nm to 460 nm (Fig. 2d) with an isoemission point at 543 nm. Fig. 3 shows that sulde and sulte induce different color changes of the TSSP-N3 solution, which could easily be distinguished by the naked eye. Sulte triggered the change of the solution's color from orange to colorless, and that of the uorescence color from red to blue; while sulde changed the solution's color to yellow. Therefore, the probe TSSP-N3 can be used to differentiate between sulte and sulde. It is clear from Fig. 2 that the reaction between TSSP-N3 and sulde was completed aer 10 min, and about 95% of the probe TSSP-N3 was transformed to the addition product with sulte aer 1 h (t1/2 z 7 min, Fig. S3a†). Therefore, the assay time of 1 h is used in the following photophysical experiments. HPLC traces of the reaction processes of TSSP-N3 with sulde and sulte To get more insight into the sensing mechanism of TSSP-N3, the HPLC traces of the reaction processes were recorded as shown
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Fig. 3 The proposed recognition mechanisms for sulfide and sulfite, [TSSP-N3] ¼ 10 mM, [sulfite] ¼ 500 mM, [sulfide] ¼ 100 mM.
Fig. 4 Time-dependent HPLC spectra of the reaction between TSSPN3 and sulfide (a) or sulfite (b) in the PBS–CTAB (1 mM) system. [TSSPN3] ¼ 15 mM, [sulfide] ¼ [sulfite] ¼ 750 mM, pH ¼ 7.4, 20 mM PBS, 25 C, detection wavelength ¼ 410 nm.
in Fig. 4. It is clear from Fig. 4a that the peaks of TSSP-N3 and its reduction product (TSSP-NH2) are at about 7.5 min and 6.5 min, respectively. With the reaction between sulde and the probe progressing, the peak of TSSP-N3 decreased dramatically and disappeared aer 1 h accompanied by the emergence and development of a new peak at about 6.5 min, which further supports the reaction mechanism between the probe and sulde. The reaction between sulte and the probe is more complex but interesting (Fig. 4b). With the decrement of the peak at 7.5 min, two new peaks respectively at 3.8 min and 4.1 min emerged. The mass spectral analysis (Fig. S4†) indicates that the peak at 3.8 min is from the sulte addition product of TSSP-NH2 (cal. for M 1 ¼ 443.1277, found 443.1282), while that at 4.1 min is supposed to be from the sulte addition product of TSSP-N3. The ratio of the peak intensities at 3.8 min and 4.1 min increases with time, which suggests that sulte can reduce the azido group to amino, but the reaction rate of reduction is lower than that of the addition (Fig. 4b). The selectivity and competition of TSSP-N3 towards sulde and sulte over various analytes in the CTAB–PBS system
Time-dependent absorption and emission spectra of TSSP-N3 (2 mM) in the presence of 10 equiv. sulfide (a and b) and 50 equiv. sulfite (c and d) in the PBS–CTAB (1 mM) system. Data in (a) and (b) are normalized. [TSSP-N3] ¼ 2 mM, 20 mM PBS, pH 7.4, 25 C, excited at the isosbestic point 410 nm. Fig. 2
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We further investigated the selectivity prole of TSSP-N3 in response to some other anions and biological thiols in the CTAB–PBS system (20 mM, pH 7.4, 25 C). No evident uorescence intensity change of the emission band was observed in the presence of 50 equiv. of F, Cl, Br, I, AcO, HCO3, CN, NO2, NO3, PO43, SCN, HS, SO42, S2O32, Cys and Hcy (Fig. 5). GSH induced a uorescence enhancement at 460 nm but to a much smaller extent (I460/I590 z 3), which revealed that the nucleophilic addition between GSH and TSSP-N3 happened at a much lower rate. It can also be seen from Fig. 5a that the selectivity of TSSP-N3 towards sulde over all the anions and thiols detected is satisfactory. The results in Fig. 5 indicated
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Fig. 5 The fluorescence spectra (a) and the ratio of fluorescence intensities at 460 nm and 590 nm (I460/I590) (b) in the presence of 50 equiv. of various additives with (red) or without (black) sulfite in CTAB– PBS for 1 h (in the competition experiment of sulfite towards sulfide the reaction time was 4 h), (1) blank; (2) F; (3) Cl; (4) Br; (5) I; (6) AcO; (7) HCO3; (8) CN; (9) NO2; (10) NO3; (11) PO43; (12) HS; (13) SCN; (14) SO42; (15) S2O32; (16) Cys; (17) Hcy; (18) GSH. 20 mM PBS, pH 7.4, 25 C, [CTAB] ¼ 1 mM, [TSSP-N3] ¼ 2 mM, [sulfite] ¼ 100 mM, excited at the isosbestic point 410 nm.
that the probe TSSP-N3 was highly specic for sulte and H2S. The uorescence responses of TSSP-N3 towards sulte in the presence of other anions or thiols were explored in order to determine the possible interference from other analytes. Fig. 5b demonstrates that the other analytes except for sulde did not exhibit notable interference with the detection of sulte. The presence of sulde decreased the uorescence intensity of the sulte addition product within 1 hour, however, the interference can be eliminated by prolonging the detection time to 3 hours (Fig. S5†). The selectivity and the competition results reveal that TSSP-N3 is a good candidate for the detection of sulte.
Effects of sulde and sulte concentrations on the uorescence intensity of TSSP-N3 The effect of sulte on the emission spectrum of the probe TSSP-N3 in CTAB–PBS was studied as well. The titration experiments were carried out by gradually adding sulte to the probe and collecting the absorption and emission spectra aer 1 hour (Fig. 6 and S6†). As shown in Fig. 6b, a linear relationship was found between the uorescence intensity ratio (I460/I590) and sulte concentration in the range of 0–100 mM. The detection limit for sulte was 0.1 mM, which was comparable to those of the reported sensors.8f,9a However, sulde-induced changes in the absorbance and the uorescence intensity of TSSP-N3 were relatively small (Fig. S7†), TSSP-N3 cannot be employed to
Fig. 6 The emission spectra (a) of TSSP-N3 with different concentrations of sulfite after 1 h, and ratio of fluorescence intensities at 460 nm and 590 nm (b, I460/I590) as a function of sulfite concentration. 20 mM PBS, pH 7.4, 25 C, [CTAB] ¼ 1 mM, [TSSP-N3] ¼ 2 mM, excited at 410 nm.
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Fig. 7 The merged fluorescence images of blue, green and red channels for HeLa cells incubated with TSSP-N3 and CTAB (1 mM). (a and c) nothing else, (b) preincubated with 100 mM sulfite and (d) pretreated with 100 mM sulfide. (a and b) excited at 404 nm; (c and d) excited at 488 nm.
quantify sulde, but it can sense the existence of small amounts of H2S (as low as 5 mM) by the wavelength change (Fig. S8†). Bioimaging of sulde and sulte in living cells Finally, the possibility of using TSSP-N3 for uorescence imaging of sulde and sulte in living cells was evaluated. Aer incubating with 5 mM of TSSP-N3 for 30 min, remarkable intracellular green and red uorescence could be observed (Fig. S9†). Pretreating the cells with 100 mM sulte followed by incubation with TSSP-N3 led to a marked increase in blue emission and slight decreases in both green and red emissions (Fig. S10b–d†). When HeLa cells were preincubated with 100 mM sulde and then with TSSP-N3, a clear increment in green emission and decrement in red uorescence were observed (Fig. S11†). These results verify the potential application of TSSP-N3 for the differential sensing of intracellular sulte and sulde from different emission channels. Fig. 7 shows the overlapped uorescence images of the three channels, from which sulde and sulte could be clearly distinguished by the naked eye.
Conclusions In conclusion, a new colorimetric and ratiometric uorescent probe TSSP-N3 was developed to differentiate between sulde and sulte in CTAB micellar solution. The signicant blueshis in the absorption and emission peaks induced by sulte are based on the 1,4-addition of the a,b-unsaturated ketone in the probe by sulte, which results in a color change from orange to colorless, and thus can serve as a ‘‘naked-eye’’ probe for sulte. The slight blue-shis triggered by sulde are ascribed to the reduction of the azido group by sulde, which changes the electronic effect of the substitution at the benzene ring. A clear uorescence color change from red to yellow was observed upon the addition of sulde. Furthermore, we have demonstrated that the probe can be used for the uorescence imaging of cellular sulte and sulde. We hope that this probe may inspire
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the exploitation of new uorescent sensors for multiple analytes.
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Acknowledgements This work was nancially supported by the National 973 Program (no. 2011CB910403), NSFC (21235005) and the Science and Technology Commission of Shanghai Municipality (no. 12JG0500200). Dr J. Qian thanks the Scientic Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We appreciate Prof. A. M. Brouwer (University of Amsterdam) for his kind help.
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