Article pubs.acs.org/ac
Near-Infrared Fluorescent Probe for Detection of Thiophenols in Water Samples and Living Cells Dehuan Yu, Feihu Huang, Shuangshuang Ding, and Guoqiang Feng* Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China S Supporting Information *
ABSTRACT: The development of probes for rapid, selective, and sensitive detection of the highly toxic thiophenols is of great importance in both environmental and biological science. Despite the appealing advantages of near-infrared (NIR) fluorescent detection, no NIR fluorescent probes have been reported for thiophenols to date. Using the chemical properties of thiophenols that are able to cleave sulfonamide selectively and efficiently under mild conditions, we herein report a dicyanomethylene-benzopyran (DCMB)-based NIR fluorescent probe for thiophenols. This probe features remarkable large Stokes shift and shows a rapid, highly selective, and sensitive detection process for thiophenols with significant NIR fluorescent turn-on responses. The potential applications of this new NIR fluorescent probe were demonstrated by the quantitative detection of thiophenol in real water samples and by fluorescent imaging of thiophenol in living cells. sensitivity. Since then, several more fluorescent probes have been developed for thiophenols.11−23 However, available fluorescent probes for thiophenols are still very limited, and all of them show fluorescent responses only in the visible region. So far, no near-infrared (NIR, 650−900 nm) fluorescent probe for thiophenols has been reported, despite the fact that NIR fluorescent detection has unique advantages for tracing molecular in vitro and in vivo, such as deep tissue penetration, minimum photodamage to biological samples, and minimum interference from background autofluorescence by biomolecules in the living systems, which were well stated in several recent reviews.24,25 Herein, we report a NIR fluorescent probe (probe 1 in Scheme 1) for thiophenols. This probe uses a known NIR fluorescent dye (compound 2 in Scheme 1) as the fluorophore and the strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl (DNBS) group as a recognition unit. Notably, this probe was found to show high selectivity and sensitivity for thiophenols, and also importantly, it is able to detect thiophenols rapidly (within a few minutes) with significant NIR fluorescent turn-on responses around 670 nm. In addition, it shows a remarkable large Stokes shift (>180 nm), which is highly desirable for a fluorescent probe to achieve reliable and sensitive fluorescent detection. Moreover, the quantitative detection of thiophenol in water samples and fluorescent thiophenol imaging in living cells by this probe were also successfully applied.
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hiophenols are a class of highly toxic and pollutant compounds. Their median lethal dose (LC50) values for fish were reported in a low range of 0.01−0.4 mM.1 Generally, thiophenols are more toxic than aliphatic thiols, and exposure to thiophenol liquid and vapor was reported to cause a series of serious health problems, including central nervous system damage, increased respiration, muscle weakness, hind limb paralysis, coma, and even death.2 However, highly toxic thiophenols are useful chemicals, and they are widely used in preparation of agrochemicals, pharmaceuticals, and various industrial products.3−5 Considering their highly toxic property and the continuing environmental concerns, simple, rapid, sensitive, and selective detection of thiophenols is therefore of considerable interest in both environmental and biological science. Recently, fluorescent detection especially using small molecular fluorescent probes has attracted much attention due to these probes being able to provide simple, highly sensitive, and low cost detection by fluorescent signal responses. Accordingly, much effort has been given to the development of fluorescent probes for thiols, and as a result, many thiols reactive and selective fluorescent probes have been developed in the past few decades.6−9 However, these probes are designed mainly for discrimination of aliphatic thiols such as cysteine and glutathione from other amino acids, and in general, they cannot clearly discriminate thiophenols over aliphatic thiols.6−9 In fact, the first fluorescent probe capable of selective detection of thiophenols over aliphatic thiols was reported very recently by Wang et al.,10 who innovatively developed a reaction-based fluorescent probe for selective detection of thiophenols, in spite of the drawbacks of this probe showing relatively weak fluorescence intensity and low © XXXX American Chemical Society
Received: June 17, 2014 Accepted: August 7, 2014
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Scheme 1. Diagram of the Probe Design for Thiophenol Detection
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EXPERIMENTAL SECTION Materials and Chemicals. All chemicals were purchased from commercial suppliers and used without further purification. All solvents were purified prior to use. Distilled water was used after passing through a water ultrapurification system. TLC analysis was performed using precoated silica plates. IR spectra were recorded on a FT-IR spectrophotometer as KBr pellets and were reported in cm−1. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury 600 or 400 spectrometer, and resonances (δ) are given in parts per million relative to tetramethylsilane (TMS). Coupling constants (J values) are reported in hertz. The low-resolution MS spectra were performed on an electron ionization mass spectrometer. HR-MS data were obtained with an LC/Q-TOF MS spectrometer. UV−vis and fluorescence spectra were recorded at 25 °C on a UV−vis spectrophotometer and a fluorescence spectrophotometer with a temperature controller, respectively. Standard quartz cuvettes with a 10 mm lightpath were used for all optical spectra measurements. Cell imaging was performed in an inverted fluorescence microscope with a 20× objective lens. Synthesis of Compound 2. Compound 426 (208 mg, 1.0 mmol) and N-(4-formyl-phenyl) acetamide (150 mg, 0.92 mmol) were dissolved in 30 mL of toluene, and then, 0.5 mL of piperidine and 0.5 mL of acetic acid were added. A dean-stark head was fitted, and the reaction mixture was heated under reflux for 12 h. After the completion of the reaction, the mixture was allowed to cool to room temperature and then condensed under reduced pressure. A red solid was obtained (262.6 mg, yield 74.4%). This solid product was used in the next step without further purification (TLC and 1H NMR analysis showed this solid is pure enough). Mp: > 300 °C. TLC (silica plate): Rf 0.22 (hexane/ethyl acetate 1:1, v/v). 1H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H, NH), 8.73 (d, J = 8.3 Hz, 1H), 7.93 (t, J = 8.0 Hz,1H), 7.80 (d, J = 8.5 Hz, 1H), 7.21−7.67 (m, 5H), 7.62 (t, J = 8.0 Hz,1H), 7.39 (d, J = 16.1 Hz, 1H), 6.99 (s, 1H), 2.09 (s, 3H, CH3). EI-MS: m/z found 353.26 (M+, 72%), 311.27 (M+ − CH3CO, 100%). This red solid (65 mg) was then refluxed in a solution of conc. HCl and ethanol (2:1, v/v, 30 mL) for 16 h before the pH of the solution was adjusted to neutral. The aqueous solution was extracted with ethyl acetate three times, and then, the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the crude product which was purified by silica column chromatography to yield a deep purple solid (45.3 mg, Yield 76%). Mp: 259.5−261 °C. TLC (silica plate): Rf 0.73 (hexane/ethyl acetate 1:1, v/v). 1H NMR (600 MHz, DMSO-d6) δ 8.71 (d, J = 8.3 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 15.8 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 15.6 Hz, 1H), 6.86 (s, 1H), 6.62 (d, J = 8.2 Hz, 2H), 6.11 (br s, 2H, NH2). 13C NMR (150 MHz, DMSO-d6) 159.6, 152.4, 152.0,
151.9, 140.5, 134.9, 130.6, 125.8, 124.4, 122.2, 118.8, 117.7, 117.1, 116.3, 113.7, 112.3, 104.6, 57.3. IR (KBr, cm−1) 3344, 2974, 2900, 2262, 2208, 1716, 1623, 1552, 1589, 1516, 1499, 1479, 1457, 1399, 1330, 1229, 1171, 1140, 1089, 1050, 976, 834, 769, 664. EI-MS: m/z found 311.24 (M+, 100%). HRMS calcd for C20H14N3O+ [M + H +], 312.11314; found, 312.11288. Synthesis of Probe 1. Compound 2 (100 mg, 0.32 mmol) was dissolved in dry pyridine (10 mL). The mixture was then cooled to 0 °C, and a solution of 2,4-dinitrobenzenesulfonyl chloride (260 mg) in dry CH2Cl2 (5 mL) was slowly added. After being stirred at 0 °C for 30 min, the mixture was then stirred at room temperature overnight. After the completion of the reaction, the mixture was washed in turn by 50 mL of water and saturated brine and dried with anhydrous Na2SO4. After removing the solvent under reduced pressure, the residue was purified by flash column chromatography to give the title compound as a red solid (78 mg, yield 45%). Mp: >300 °C. TLC (silica plate): Rf 0.47 (hexane/ethyl acetate 1:1, v/v). 1H NMR (400 MHz, CDCl3) δ 11.44 (br s, 1H, NH), 8.92 (s, 1H), 8.72 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 8.7 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 7.91 (t, J = 7.3 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 16.3 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 16.3 Hz, 1H), 7.22 (d, J = 8.1 Hz, 2H), 6.99 (s, 1H). 13C NMR (150 MHz, DMSO-d6) 158.0, 152.8, 151.9, 149.9, 147.7, 137.7, 137.1, 136.4, 135.3, 131.4, 131.0, 129.4, 127.1, 126.1, 124.5, 120.4, 120.2, 118.9, 118.7, 117.1, 117.0, 115.8, 106.5, 60.0. IR (KBr, cm−1) 3445, 3108, 2213, 2026, 1716, 1631, 1602, 1552, 1479, 1456, 1407, 1385, 1361, 1225, 1163, 1124, 1076, 1035, 979, 838, 756, 664. EI-MS: m/z found 541.35 (M+). HRMS calcd for C26H14N5O7S− (M − H)−, 540.0619; found, 540.0615. Preparation of Solutions of Probe 1 and Analytes. Stock solution of probe 1 (1 mM) was prepared in HPLC grade DMSO. Stock solutions of C 6 H 5 SH, C 6 H 5 OH, C6H5NH2, OHCH2CH2SH, (CH3)3CSH, p-CH3−C6H4SH, pNH2−C6H4SH, p-CH3O−C6H4SH, and p-NO2−C6H4SH were prepared in DMSO (10 mM, respectively). Analytes cysteine (Cys), homocysteine (Hcy), glutathione (GSH), alanine (Ala), NaN3, NaF, NaCl, NaBr, NaI, NaNO3, NaClO4, AcONa, Na2C2O4, Na2SO4, NaHCO3, NaH2PO4, Na3PO4, Na2S2O7, KI, KCN, LiCl, MgCl2, CaCl2, Al2(SO4)3, MnSO4, Ce(NO3)3, Co(NO3)3, Ni(NO3)2, and Zn(NO3)2 were dissolved in distilled water (10 mL) to afford 2.5 mM (for Cys) or 10 mM (for others) aqueous solution. The stock solutions were used freshly and were diluted to desired concentrations with water when needed. The preparation of reactive nitrogen and oxygen species such as hypochlorite, hydrogen peroxide, superoxide, nitrite, nitroxyl, nitric oxide, peroxynitrite, etc. can be found in the Supporting Information, and they were used immediately after being generated. B
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incubator at 37 °C and then were seeded in a 12-well culture plate for one night before the cell imaging experiments. For living cell imaging experiments, some of the cells were incubated with 20 μM probe 1 (with 0.4% DMSO, v/v) for 30 min at 37 °C, washed three times with prewarmed PBS buffer, and then imaged. Meanwhile, another portion of the cells were pretreated with thiophenol (20 μM 0.4% DMSO) for 30 min at 37 °C, washed three times with prewarmed PBS buffer, and then incubated with probe (20 μM in 0.4% DMSO) for 30 min. Cell imaging was then carried out after washing cells with prewarmed PBS buffer.
Measurements of Fluorescence Changes of Probe 1 upon Addition of Various Analytes. A solution of probe 1 (10 μM) was prepared in DMSO-H2O solution (3:7, v/v, 10 mM PBS buffer). Then, 3.0 mL of the probe 1 solution was placed in a quartz cell (10.0 mm width) until the temperature reached 25 °C over a few minutes. The fluorescent spectra were recorded upon addition of various analytes. Measurements of Thiophenol in Water Samples. The crude water samples from the Yangtse River, East Lake, and South Lake in Wuhan City were passed through a microfiltration membrane before use. The pH values of the water samples (50 mL) were adjusted using a sodium phosphate buffer (10 mM, pH 7.4), and aliquots of the water samples were then spiked with different concentrations of thiophenol (1, 5, 10, 20, 50 μM) that had been accurately prepared. The resulting samples were then treated with probe 1 in a phosphate buffer (10 mM, pH 7.4) to give the final mixtures (3.0 mL) containing probe 1 (10 μM) and thiophenol (1, 5, 10, 20, or 50 μM). The solutions were incubated for 10 min at 25 °C, and the fluorescence was measured at 670 nm. The results shown in Table 1 were reported as the mean ± standard deviation of triplicate experiments for thiophenol spiked from 0 to 20 μM.
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RESULTS AND DISCUSSION Probe Design and Synthesis. To develop a NIR fluorescent probe for thiophenols, a NIR fluorescent dye as the fluorophore is needed. It was reported that conjugated dicyanomethylene-benzopyran (DCMB) derivatives not only can show controllable emission wavelength in the NIR region but also have high photostability.27 The attractive features of this kind of dye have been applied recently to develop NIR fluorescent probes for several important analytes.26,28−33 Particularly, compound 2 shows attractive spectroscopic properties.31−33 Besides emission wavelength in NIR region (∼670 nm), it shows large Stokes shift (>100 nm in different solvents) with good fluorescence quantum yield and good photostability.31−33 In addition, it can be easily prepared. Therefore, compound 2 was selected as the fluorophore to construct the NIR fluorescent probe for thiophenols in this work. Using the strategy developed by Wang et al.,10 protection of the amino group by the strongly electron-withdrawing 2,4dinitrobenzenesulfonyl (DNBS) group is expected to quench the fluorescence of compound 2. Moreover, the resulting sulfonamide should be readily cleaved by thiophenols (pKa ≈ 6.5) under physiological pH through an SNAr process, but not by aliphatic thiols (pKa > 8.0), due to the big differences in their pKa values. As a result, compound 2 will be released, leading to the recovery of its NIR fluorescence and thus showing a NIR fluorescent turn-on detection process for thiophenols (Scheme 1). To investigate the feasibility of our design concept, probe 1 was synthesized. The synthesis route for probe 1 is outlined in Scheme 2, in which probe 1 can be readily prepared via reaction of compound 2 with 2,4-dinitrobenzene-1-sulfonyl chloride. Both compound 2 and the intermediate compound 4 are known compounds, and they were prepared according to the previously published procedures.26,31 Structural identification of probe 1 and compound 2 was confirmed by conventional NMR, IR, and HR-MS spectroscopy. Detailed synthetic procedures and structure characterizations are given in the Experimental Section and in the Supporting Information.
Table 1. Determination of Thiophenol Concentrations in Water Samples sample Yangtse River water
East Lake water
South Lake water
thiophenol spiked (μM)
thiophenol recovered (μM)
0
not detected
1 5 10 20 0 1 5 10 20 0 1 5 10 20
0.97 ± 0.01 4.53 ± 0.32 9.05 ± 0.11 20.32 ± 0.94 not detected 1.07 ± 0.07 5.01 ± 0.22 10.79 ± 0.27 19.34 ± 0.65 not detected 0.91 ± 0.02 4.55 ± 0.27 10.32 ± 0.48 20.69 ± 0.62
recovery (%)
97 91 90 102 107 101 108 95 91 91 103 104
Cell Culture and Imaging. HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (fetal bovine serum), 100 mg/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2, water saturated Scheme 2. Synthesis of Probe 1a
a
Reagent and conditions: (a) Na, CH3COOC2H5, 4 h, 53%; (b) AcOH, H2SO4, 30 min, 80%; (c) malononitrile, AcOH, H2SO4, 14 h, 34%; (d) N(4-formyl-phenyl) acetamide, toluene, piperidine, AcOH, 12 h, 74% and then conc. HCl, ethanol, 76%; (e) 2,4-dinitrobenzene-1-sulfonyl chloride, Et3N, CH2Cl2, r.t., 45%. C
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Figure 1. (a) Fluorescent spectral changes of probe 1 (10 μM) upon addition of PhSH (100 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The spectra were collected at 1 min intervals after mixing thiophenol with probe 1. (b) Kinetic curve of probe 1 (10 μM) at 670 nm with PhSH (100 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The data were reported as the mean ± standard deviation of triplicate experiments and fitted (red line) by a first-order reaction scheme as shown in the figure.
Figure 2. (a) Fluorescent spectral changes of probe 1 (10 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C upon addition of different concentrations of PhSH (0−100 μM). Each spectrum was collected 10 min after PhSH addition. (b) A plot of fluorescence intensity changes of probe 1 at 670 nm against [PhSH] from 0 to 30 μM, which can be linearly fitted (red line).
Sensing Property of Probe 1 for Thiophenols. Since compound 2 is the expected product of probe 1 upon treatment of thiophenols, we first examined the differences of the absorption and fluorescent spectra between probe 1 and the reference sample of 2. As shown in Figure S1 (Supporting Information), although the absorption spectra difference between probe 1 (λmax = 470 nm, ε ≈ 2.53 × 104 M−1 cm−1) and 2 (λmax = 478 nm, ε ≈ 3.16 × 104 M−1 cm−1) is not that noticeable, their fluorescent spectra showed a significant difference. As expected, probe 1 shows almost no fluorescence while 2 shows strong NIR fluorescence at 670 nm when excited at 490 nm. This low background fluorescence of the probe itself and the strong fluorescence of the expected product upon thiophenol treatment should be highly desirable for a sensitive detection of thiophenols. The sensing ability of probe 1 for thiophenols was then investigated in 10 mM PBS buffer (pH 7.4) with 30% DMSO (v/v) at 25 °C.34 The fluorescence response of probe 1 toward thiophenol (PhSH) was first investigated. As shown in Figure 1, as anticipated, the fluorescence of probe 1 (10 μM) showed a large enhancement around 670 nm (∼25-fold, excited at 490 nm) within a few minutes upon addition of thiophenol (100 μM). Kinetic analysis showed that the fluorescence enhancement at 670 nm reached a plateau at about 6 min (Figure 1b), and the reaction obeys a typical pseudo-first-order with rate constant kobs determined to be about 0.45 min−1 (t1/2 ≈ 1.56 min). This fast and distinct fluorescence signal change in the NIR region indicates that probe 1 can be used as a rapid and sensitive NIR fluorescent turn-on sensor for thiophenol. In
addition, it is also worth mentioning that probe 1 showed a remarkable large Stokes shift, which is highly desirable for a fluorescent probe, because the large gap between the excitation wavelength and the emission wavelength can effectively diminish the measurement error caused by the excitation light and scattered light. Thus, we have established a NIR fluorescent turn-on probe for rapid detection of thiophenol with remarkable large Stokes shift and significant NIR fluorescent enhancement. To shed light on the sensitivity of probe 1, the kinetics of probe 1 (10 μM) with addition of different concentrations of thiophenol were also measured by monitoring the fluorescence changes at 670 nm (Figure S3, Supporting Information). We can see that, in the absence of thiophenol, the fluorescence signal of probe 1 is quite stable under the test conditions; however, in the presence of thiophenols, the fluorescence signal of probe 1 showed changes and the change speeds are dependent on the concentrations of thiophenol added. Since the reaction can be completed within 10 min when more than 5 equiv of thiophenol is added, the full fluorescence spectra changes of probe 1 (10 μM) were then investigated after 10 min upon addition of increasing concentrations of thiophenol. As shown in Figure 2a, upon progressive addition of thiophenol, the fluorescence of the solution gradually increases until it reaches a plateau after the addition of more than 5 equiv of thiophenol. Notably, significant fluorescence signal changes can be still observed even when thiophenol was added at or below stoichiometric conditions (1−10 μM). A linear calibration graph of the responses of the fluorescent intensity D
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Figure 3. (a) Fluorescence spectra changes of probe 1 (10 μM) toward various analytes (100 μM). (b) Fluorescence responses of probe 1 (10 μM) at 670 nm toward 100 μM of various analytes. All experiments were performed in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C, and each spectrum was obtained 10 min after addition of an analyte. λex = 490 nm; slit width: (5, 10).
at 670 nm to the thiophenol concentrations from 0 to 30 μM can be observed (Figure 2b), which indicates that probe 1 can be potentially employed to detect thiophenol quantitatively. From this linear calibration graph, the detection limit of probe 1 for thiophenol is found to be about 0.15 μM based on signalto-noise ratio (S/N) = 3 under the test conditions. This result further proved that probe 1 is highly sensitive to thiophenol. The effect of pH on the fluorescence signal responses of probe 1 toward thiophenol was also investigated. As shown in Figure S4 (Supporting Information), the probe 1 itself is inert to pH change in a wide range (from 4 to 10); however, in the presence of thiophenol, the fluorescence intensity of probe 1 showed distinct changes over the pH range from 5 to 10. Notably, the fluorescence changes of probe 1 to thiophenol reached a maximum and became almost constant in the pH range of 7−10, which is consistent with the pKa value of thiophenol (pKa ≈ 6.5), because strong ionization of thiophenol at pH over 6.5 is anticipated. This result suggests that probe 1 is able to work over a relatively wide pH range. To evaluate the selectivity of probe 1 for thiophenols, various analytes including thiophenol derivatives (C6H5SH, p-CH3− C6H4SH, p-NH2−C6H4SH, p-CH3O−C6H4SH, and p-NO2− C6H4SH), aliphatic thiols Cys, Hcy, GSH, (CH3)3CSH, and OHCH2CH2SH, and other potential interfering substances such as some nucleophilic species (Ala, NaN3, KI, KCN, C6H5OH, C6H5NH2, and NaSH) and various anions and metal ions (NaF, NaCl, NaBr, NaI, NaNO3, NaClO4, AcONa, Na2C2O4, Na2SO4, NaHCO3, NaH2PO4, Na3PO4, Na2S2O7, LiCl, MgCl2, CaCl2, Al2(SO4)3, MnSO4, Ce(NO3)3, Co(NO3)3, Ni(NO3)2, and Zn(NO3)2) were tested. As shown in Figure 3, only the introduction of thiophenols such as C6H5SH, p-CH3− C6H4SH, p-NH2−C6H4SH, and p-CH3O−C6H4SH to the probe 1 solution induced a significant enhancement in the fluorescent intensity at 670 nm. One exception is that the fluorescent detection of p-NO2−C6H4SH with a strong electron-withdrawing NO2 group by probe 1 is difficult, which is consistent with reports in the literature,10,19,22 probably because of the low activity of p-NO2−C6H4S− in the SNAr reaction toward sulfonamide owing to its relatively large local softness (ssulfur−) value.19 Except thiophenols, other tested species mentioned above including aliphatic thiols such as Cys and Hcy, thiophenol analogues such as C6H5OH and C6H5NH2, common anions, and metal ions did not induce any obvious fluorescence enhancement to the probe 1 solution (Figure 3 and Figure S5, Supporting Information). In addition, probe 1 shows high selectivity for thiophenol against reactive
nitrogen and oxygen species such as hypochlorite, hydrogen peroxide, superoxide, nitrite, nitroxyl, nitric oxide, peroxynitrite, etc., and detection of thiophenol using probe 1 in the presence of various analytes including these reactive species is still effective (Figure 4 and Figure S5, Supporting Information).
Figure 4. Fluorescence responses of probe 1 (10 μM) at 670 nm toward PhSH in the presence of some representative analytes. Black bars represent the addition of a single analyte (100 μM) including: none, Cys, Hcy, GSH, Ala, NaN3, KI, KCN, C6H5OH, C6H5NH2, OHCH2CH2SH, (CH3)3CSH, NaHS. Red bars represent the subsequent addition of C6H5SH (100 μM) to the mixture. All experiments were performed in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C, and data was obtained 10 min after addition of an analyte. λex = 490 nm; slit width: (5, 10).
Therefore, all these results clearly indicate that probe 1 has high selectivity to thiophenols (except p-NO2−C6H4SH) over other analytes mentioned above. The fluorescence changes of probe 1 in the presence of thiophenol suggest that compound 2 was produced. To confirm that the fluorescence sensing response of probe 1 to thiophenol is indeed due to the conversion of probe 1 to compound 2, the reaction products of probe 1 with thiophenol were isolated and subjected to 1H NMR and MS analysis. Two products were isolated, and one of them showed an essentially identical Rf (in the TLC plate) and 1H NMR spectrum with that of the standard compound 2 (Figures S6 and S7, Supporting Information). Moreover, a peak of 311.27 found in the MS spectrum further proved the formation of compound 2 (Figure S8, Supporting Information). In addition, 1H NMR, 13C NMR, and MS analysis proved another isolated product to be compound 3 (Figures S9−S11, Supporting Information). E
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Figure 5. (a) Fluorescent detection of different concentrations of PhSH in “distilled water”, “Yangtse River water”, “East Lake water”, and “South Lake water” by probe 1 (10 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The reaction is monitored at 670 nm. Each condition was measured three times, and the average values are shown. PhSH is spiked in 0, 1, 5, 10, 20, and 50 μM, respectively. λex = 490 nm; slit width: (5, 10). (b−d) Linear plot of fluorescence intensity changes of probe 1 at 670 nm against the spiked concentrations of PhSH from 0 to 20 μM for each real water sample.
Figure 6. Imaging of thiophenol in HeLa Cells by probe 1. (A) Bright field images of HeLa cells after being treated with probe 1 (20 μM) for 30 min. (B) Bright field images of HeLa cells preincubated with thiophenol (2 μM) and then incubated with probe 1 (20 μM). A1 and B1 are fluorescence images of A and B, respectively.
Thus, the mechanism of probe 1 for sensing of thiophenol is most likely the cleavage of the sulfonamide process mediated by thiophenol as shown in Scheme 1. Detection of Thiophenol in Water Samples. Considering the toxicity of thiophenols and their potential as pollutants, we employed probe 1 to determine thiophenol concentrations in water samples to validate its practical utility in environmental science. The water samples were collected from the Yangtse River, East Lake, and South Lake in Wuhan City. These water samples were directly analyzed first and then spiked with thiophenol at different levels (1, 5, 10, 20, and 50 μM), and the fluorescence responses of probe 1 at 670 nm toward all these samples were examined, respectively. The results are shown in Figure 5a, and they are compared with those determined in distilled water. We can see that they have a good consistency between each other (Figure 5a). For each real water sample, a linear relationship between the responses of the fluorescent intensity at 670 nm to the spiked thiophenol concentrations from 0 to 20 μM was observed (Figure 5b−d), which is in good agreement with the result obtained in distilled water (Figure
2b). In addition, we can see that the recoveries of thiophenol ranged from 90% to 108% (Table 1), which indicates that the thiophenol in the water samples could be accurately measured with good recovery when probe 1 was applied as the probe. This result showed that probe 1 has potential application for quantitative detection of thiophenols in water samples. Detection of Thiophenol in Living Cells. We also investigated the practical utilities of probe 1 for fluorescent cell imaging with HeLa cells. As shown in Figure 6, when HeLa cells were incubated directly with probe 1 (20 μM) only at 37 °C, no fluorescence was observed. However, when HeLa cells were preincubated with thiophenol (2 μM) and then incubated with probe 1 (20 μM), we observed strong red fluorescence. These results indicate that probe 1 can be applied to detect thiophenols in living cells.
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CONCLUSION In summary, we developed a NIR fluorescent probe (probe 1) for rapid, selective, and sensitive detection of thiophenols. This new probe is reaction-based, which uses a conjugated F
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(17) Kand, D.; Mishra, P. K.; Saha, T.; Lahiri, M.; Talukdar, P. Analyst 2012, 137, 3921−3924. (18) Liu, X. L.; Duan, X. Y.; Du, X. J.; Song, Q. H. Chem.Asian J. 2012, 7, 2696−2702. (19) Shiraishi, Y.; Yamamoto, K.; Sumiya, S.; Hirai, T. Chem. Commun. 2013, 49, 11680−11682. (20) Zhao, C.; Wang, X.; Cao, J.; Feng, P.; Zhang, J.; Zhang, Y.; Yang, Y.; Yang, Z. Dyes Pigm. 2013, 96, 328−332. (21) Kand, D.; Mandal, P. S.; Datar, A.; Talukdar, P. Dyes Pigm. 2014, 106, 25−31. (22) Li, J.; Zhang, C.-F.; Yang, S.-H.; Yang, W.-C.; Yang, G.-F. Anal. Chem. 2014, 86, 3037−3042. (23) Balkrishna, S. J.; Hodage, A. S.; Kumar, S.; Panini, P.; Kumar, S. RSC Adv. 2014, 4, 11535−11538. (24) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev. 2014, 43, 16−29. (25) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (26) Huang, X.; Guo, Z.; Zhu, W.; Xie, Y.; Tian, H. Chem. Commun. 2008, 5143−5145. (27) Guo, Z.; Zhu, W.; Tian, H. Chem. Commun. 2012, 48, 6073− 6084. (28) Zhu, W.; Huang, X.; Guo, Z.; Wu, X.; Yu, H.; Tian, H. Chem. Commun. 2012, 48, 1784−1786. (29) Cao, J.; Zhao, C.; Zhu, W. Tetrahedron Lett. 2012, 53, 2107− 2110. (30) Li, M.; Wu, X.; Wang, Y.; Li, Y.; Zhu, W.; James, T. D. Chem. Commun. 2014, 50, 1751−1753. (31) Sun, W.; Fan, J.; Hu, C.; Cao, J.; Zhang, H.; Xiong, X.; Wang, J.; Shuang, C.; Sun, S.; Peng, X. Chem. Commun. 2013, 49, 3890−3892. (32) Zheng, Y.; Zhao, M.; Qiao, Q.; Liu, H.; Lang, H.; Xu, Z. Dyes Pigm. 2013, 98, 367−371. (33) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. J. Am. Chem. Soc. 2014, 136, 3579−3588. (34) Note: We have investigated the effect of different amounts of DMSO as cosolvent to the sensing property of probe 1 for thiophenol; considering the fluorescent intensity changes and the reaction speed, 30% DMSO was chosen in this study (Figure S2, Supporting Information). The working temperature was set at 25 °C without further optimization, although a higher temperature may be optimal for more rapid thiophenol detection.
dicyanomethylene-benzopyran molecule (compound 2) as the fluorophore and 2,4-dinitrobenzene-1-sulfonamide as the reaction site. The merits of this probe for detection of thiophenols (except p-NO2−C6H4SH) include rapid and significant NIR fluorescence turn-on responses, high selectivity and sensitivity, working under very mild conditions, and having a remarkably large Stokes shift. In addition, this probe can be successfully applied to detect thiophenol in real water samples and in living cells. Therefore, this work provides a promising NIR probe for the rapid detection and quantification of the highly toxic thiophenols found in environmental and biological samples.
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ASSOCIATED CONTENT
S Supporting Information *
Structure characterizations for compound 2 and probe 1, additional fluorescence data, and data for investigation of the sensing mechanism. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grant Nos. 21172086 and 21032001) for financial support. This work was also sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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REFERENCES
(1) Heil, T. P.; Lindsay, R. C. J. Environ. Sci. Health B 1989, 24, 349− 360. (2) Amrolia, P.; Sullivan, S. G.; Stern, A.; Munday, R. J. Appl. Toxicol. 1989, 9, 113−118. (3) Roy, K.-M. In Ullmann’s Encyclopedia of Industrial Chemistry, 7th ed.; Wiley: New York, 2007. (4) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103−1170. (5) EychmKller, A.; Rogach, A. L. Pure Appl. Chem. 2000, 72, 179− 188. (6) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52−67. (7) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019−6031. (8) Yin, C.; Huo, F.; Zhang, J.; Martínez-Máñez, R.; Yang, Y.; Lv, H.; Li, S. Chem. Soc. Rev. 2013, 42, 6032−6059. (9) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351−5358. (10) Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8445−8448. (11) Lin, W.; Long, L.; Tan, W. Chem. Commun. 2010, 46, 1503− 1505. (12) Jiang, W.; Cao, Y.; Liu, Y.; Wang, W. Chem. Commun. 2010, 46, 1944−1946. (13) Zhao, W. W.; Liu, W. M.; Ge, J. C.; Wu, J. S.; Zhang, W. J.; Meng, X. M.; Wang, P. F. J. Mater. Chem. 2011, 21, 13561−13568. (14) Zhao, C.; Zhou, Y.; Lin, Q.; Zhu, L.; Feng, P.; Zhang, Y.; Cao, J. J. Phys. Chem. B 2011, 115, 642−647. (15) Wang, X.; Cao, J.; Zhao, C. Org. Biomol. Chem. 2012, 10, 4689− 4691. (16) Wang, Z.; Han, D. M.; Jia, W. P.; Zhou, Q. Z.; Deng, W. P. Anal. Chem. 2012, 84, 4915−4920. G
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Near-Infrared Fluorescent Probe for Detection of Thiophenols in Water Samples and Living Cells Dehuan Yu, Feihu Huang, Shuangshuang Ding, and Guoqiang Feng* Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China S Supporting Information *
ABSTRACT: The development of probes for rapid, selective, and sensitive detection of the highly toxic thiophenols is of great importance in both environmental and biological science. Despite the appealing advantages of near-infrared (NIR) fluorescent detection, no NIR fluorescent probes have been reported for thiophenols to date. Using the chemical properties of thiophenols that are able to cleave sulfonamide selectively and efficiently under mild conditions, we herein report a dicyanomethylene-benzopyran (DCMB)-based NIR fluorescent probe for thiophenols. This probe features remarkable large Stokes shift and shows a rapid, highly selective, and sensitive detection process for thiophenols with significant NIR fluorescent turn-on responses. The potential applications of this new NIR fluorescent probe were demonstrated by the quantitative detection of thiophenol in real water samples and by fluorescent imaging of thiophenol in living cells. sensitivity. Since then, several more fluorescent probes have been developed for thiophenols.11−23 However, available fluorescent probes for thiophenols are still very limited, and all of them show fluorescent responses only in the visible region. So far, no near-infrared (NIR, 650−900 nm) fluorescent probe for thiophenols has been reported, despite the fact that NIR fluorescent detection has unique advantages for tracing molecular in vitro and in vivo, such as deep tissue penetration, minimum photodamage to biological samples, and minimum interference from background autofluorescence by biomolecules in the living systems, which were well stated in several recent reviews.24,25 Herein, we report a NIR fluorescent probe (probe 1 in Scheme 1) for thiophenols. This probe uses a known NIR fluorescent dye (compound 2 in Scheme 1) as the fluorophore and the strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl (DNBS) group as a recognition unit. Notably, this probe was found to show high selectivity and sensitivity for thiophenols, and also importantly, it is able to detect thiophenols rapidly (within a few minutes) with significant NIR fluorescent turn-on responses around 670 nm. In addition, it shows a remarkable large Stokes shift (>180 nm), which is highly desirable for a fluorescent probe to achieve reliable and sensitive fluorescent detection. Moreover, the quantitative detection of thiophenol in water samples and fluorescent thiophenol imaging in living cells by this probe were also successfully applied.
T
hiophenols are a class of highly toxic and pollutant compounds. Their median lethal dose (LC50) values for fish were reported in a low range of 0.01−0.4 mM.1 Generally, thiophenols are more toxic than aliphatic thiols, and exposure to thiophenol liquid and vapor was reported to cause a series of serious health problems, including central nervous system damage, increased respiration, muscle weakness, hind limb paralysis, coma, and even death.2 However, highly toxic thiophenols are useful chemicals, and they are widely used in preparation of agrochemicals, pharmaceuticals, and various industrial products.3−5 Considering their highly toxic property and the continuing environmental concerns, simple, rapid, sensitive, and selective detection of thiophenols is therefore of considerable interest in both environmental and biological science. Recently, fluorescent detection especially using small molecular fluorescent probes has attracted much attention due to these probes being able to provide simple, highly sensitive, and low cost detection by fluorescent signal responses. Accordingly, much effort has been given to the development of fluorescent probes for thiols, and as a result, many thiols reactive and selective fluorescent probes have been developed in the past few decades.6−9 However, these probes are designed mainly for discrimination of aliphatic thiols such as cysteine and glutathione from other amino acids, and in general, they cannot clearly discriminate thiophenols over aliphatic thiols.6−9 In fact, the first fluorescent probe capable of selective detection of thiophenols over aliphatic thiols was reported very recently by Wang et al.,10 who innovatively developed a reaction-based fluorescent probe for selective detection of thiophenols, in spite of the drawbacks of this probe showing relatively weak fluorescence intensity and low © XXXX American Chemical Society
Received: June 17, 2014 Accepted: August 7, 2014
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Scheme 1. Diagram of the Probe Design for Thiophenol Detection
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EXPERIMENTAL SECTION Materials and Chemicals. All chemicals were purchased from commercial suppliers and used without further purification. All solvents were purified prior to use. Distilled water was used after passing through a water ultrapurification system. TLC analysis was performed using precoated silica plates. IR spectra were recorded on a FT-IR spectrophotometer as KBr pellets and were reported in cm−1. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury 600 or 400 spectrometer, and resonances (δ) are given in parts per million relative to tetramethylsilane (TMS). Coupling constants (J values) are reported in hertz. The low-resolution MS spectra were performed on an electron ionization mass spectrometer. HR-MS data were obtained with an LC/Q-TOF MS spectrometer. UV−vis and fluorescence spectra were recorded at 25 °C on a UV−vis spectrophotometer and a fluorescence spectrophotometer with a temperature controller, respectively. Standard quartz cuvettes with a 10 mm lightpath were used for all optical spectra measurements. Cell imaging was performed in an inverted fluorescence microscope with a 20× objective lens. Synthesis of Compound 2. Compound 426 (208 mg, 1.0 mmol) and N-(4-formyl-phenyl) acetamide (150 mg, 0.92 mmol) were dissolved in 30 mL of toluene, and then, 0.5 mL of piperidine and 0.5 mL of acetic acid were added. A dean-stark head was fitted, and the reaction mixture was heated under reflux for 12 h. After the completion of the reaction, the mixture was allowed to cool to room temperature and then condensed under reduced pressure. A red solid was obtained (262.6 mg, yield 74.4%). This solid product was used in the next step without further purification (TLC and 1H NMR analysis showed this solid is pure enough). Mp: > 300 °C. TLC (silica plate): Rf 0.22 (hexane/ethyl acetate 1:1, v/v). 1H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H, NH), 8.73 (d, J = 8.3 Hz, 1H), 7.93 (t, J = 8.0 Hz,1H), 7.80 (d, J = 8.5 Hz, 1H), 7.21−7.67 (m, 5H), 7.62 (t, J = 8.0 Hz,1H), 7.39 (d, J = 16.1 Hz, 1H), 6.99 (s, 1H), 2.09 (s, 3H, CH3). EI-MS: m/z found 353.26 (M+, 72%), 311.27 (M+ − CH3CO, 100%). This red solid (65 mg) was then refluxed in a solution of conc. HCl and ethanol (2:1, v/v, 30 mL) for 16 h before the pH of the solution was adjusted to neutral. The aqueous solution was extracted with ethyl acetate three times, and then, the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the crude product which was purified by silica column chromatography to yield a deep purple solid (45.3 mg, Yield 76%). Mp: 259.5−261 °C. TLC (silica plate): Rf 0.73 (hexane/ethyl acetate 1:1, v/v). 1H NMR (600 MHz, DMSO-d6) δ 8.71 (d, J = 8.3 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 15.8 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 15.6 Hz, 1H), 6.86 (s, 1H), 6.62 (d, J = 8.2 Hz, 2H), 6.11 (br s, 2H, NH2). 13C NMR (150 MHz, DMSO-d6) 159.6, 152.4, 152.0,
151.9, 140.5, 134.9, 130.6, 125.8, 124.4, 122.2, 118.8, 117.7, 117.1, 116.3, 113.7, 112.3, 104.6, 57.3. IR (KBr, cm−1) 3344, 2974, 2900, 2262, 2208, 1716, 1623, 1552, 1589, 1516, 1499, 1479, 1457, 1399, 1330, 1229, 1171, 1140, 1089, 1050, 976, 834, 769, 664. EI-MS: m/z found 311.24 (M+, 100%). HRMS calcd for C20H14N3O+ [M + H +], 312.11314; found, 312.11288. Synthesis of Probe 1. Compound 2 (100 mg, 0.32 mmol) was dissolved in dry pyridine (10 mL). The mixture was then cooled to 0 °C, and a solution of 2,4-dinitrobenzenesulfonyl chloride (260 mg) in dry CH2Cl2 (5 mL) was slowly added. After being stirred at 0 °C for 30 min, the mixture was then stirred at room temperature overnight. After the completion of the reaction, the mixture was washed in turn by 50 mL of water and saturated brine and dried with anhydrous Na2SO4. After removing the solvent under reduced pressure, the residue was purified by flash column chromatography to give the title compound as a red solid (78 mg, yield 45%). Mp: >300 °C. TLC (silica plate): Rf 0.47 (hexane/ethyl acetate 1:1, v/v). 1H NMR (400 MHz, CDCl3) δ 11.44 (br s, 1H, NH), 8.92 (s, 1H), 8.72 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 8.7 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 7.91 (t, J = 7.3 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 16.3 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 16.3 Hz, 1H), 7.22 (d, J = 8.1 Hz, 2H), 6.99 (s, 1H). 13C NMR (150 MHz, DMSO-d6) 158.0, 152.8, 151.9, 149.9, 147.7, 137.7, 137.1, 136.4, 135.3, 131.4, 131.0, 129.4, 127.1, 126.1, 124.5, 120.4, 120.2, 118.9, 118.7, 117.1, 117.0, 115.8, 106.5, 60.0. IR (KBr, cm−1) 3445, 3108, 2213, 2026, 1716, 1631, 1602, 1552, 1479, 1456, 1407, 1385, 1361, 1225, 1163, 1124, 1076, 1035, 979, 838, 756, 664. EI-MS: m/z found 541.35 (M+). HRMS calcd for C26H14N5O7S− (M − H)−, 540.0619; found, 540.0615. Preparation of Solutions of Probe 1 and Analytes. Stock solution of probe 1 (1 mM) was prepared in HPLC grade DMSO. Stock solutions of C 6 H 5 SH, C 6 H 5 OH, C6H5NH2, OHCH2CH2SH, (CH3)3CSH, p-CH3−C6H4SH, pNH2−C6H4SH, p-CH3O−C6H4SH, and p-NO2−C6H4SH were prepared in DMSO (10 mM, respectively). Analytes cysteine (Cys), homocysteine (Hcy), glutathione (GSH), alanine (Ala), NaN3, NaF, NaCl, NaBr, NaI, NaNO3, NaClO4, AcONa, Na2C2O4, Na2SO4, NaHCO3, NaH2PO4, Na3PO4, Na2S2O7, KI, KCN, LiCl, MgCl2, CaCl2, Al2(SO4)3, MnSO4, Ce(NO3)3, Co(NO3)3, Ni(NO3)2, and Zn(NO3)2 were dissolved in distilled water (10 mL) to afford 2.5 mM (for Cys) or 10 mM (for others) aqueous solution. The stock solutions were used freshly and were diluted to desired concentrations with water when needed. The preparation of reactive nitrogen and oxygen species such as hypochlorite, hydrogen peroxide, superoxide, nitrite, nitroxyl, nitric oxide, peroxynitrite, etc. can be found in the Supporting Information, and they were used immediately after being generated. B
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incubator at 37 °C and then were seeded in a 12-well culture plate for one night before the cell imaging experiments. For living cell imaging experiments, some of the cells were incubated with 20 μM probe 1 (with 0.4% DMSO, v/v) for 30 min at 37 °C, washed three times with prewarmed PBS buffer, and then imaged. Meanwhile, another portion of the cells were pretreated with thiophenol (20 μM 0.4% DMSO) for 30 min at 37 °C, washed three times with prewarmed PBS buffer, and then incubated with probe (20 μM in 0.4% DMSO) for 30 min. Cell imaging was then carried out after washing cells with prewarmed PBS buffer.
Measurements of Fluorescence Changes of Probe 1 upon Addition of Various Analytes. A solution of probe 1 (10 μM) was prepared in DMSO-H2O solution (3:7, v/v, 10 mM PBS buffer). Then, 3.0 mL of the probe 1 solution was placed in a quartz cell (10.0 mm width) until the temperature reached 25 °C over a few minutes. The fluorescent spectra were recorded upon addition of various analytes. Measurements of Thiophenol in Water Samples. The crude water samples from the Yangtse River, East Lake, and South Lake in Wuhan City were passed through a microfiltration membrane before use. The pH values of the water samples (50 mL) were adjusted using a sodium phosphate buffer (10 mM, pH 7.4), and aliquots of the water samples were then spiked with different concentrations of thiophenol (1, 5, 10, 20, 50 μM) that had been accurately prepared. The resulting samples were then treated with probe 1 in a phosphate buffer (10 mM, pH 7.4) to give the final mixtures (3.0 mL) containing probe 1 (10 μM) and thiophenol (1, 5, 10, 20, or 50 μM). The solutions were incubated for 10 min at 25 °C, and the fluorescence was measured at 670 nm. The results shown in Table 1 were reported as the mean ± standard deviation of triplicate experiments for thiophenol spiked from 0 to 20 μM.
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RESULTS AND DISCUSSION Probe Design and Synthesis. To develop a NIR fluorescent probe for thiophenols, a NIR fluorescent dye as the fluorophore is needed. It was reported that conjugated dicyanomethylene-benzopyran (DCMB) derivatives not only can show controllable emission wavelength in the NIR region but also have high photostability.27 The attractive features of this kind of dye have been applied recently to develop NIR fluorescent probes for several important analytes.26,28−33 Particularly, compound 2 shows attractive spectroscopic properties.31−33 Besides emission wavelength in NIR region (∼670 nm), it shows large Stokes shift (>100 nm in different solvents) with good fluorescence quantum yield and good photostability.31−33 In addition, it can be easily prepared. Therefore, compound 2 was selected as the fluorophore to construct the NIR fluorescent probe for thiophenols in this work. Using the strategy developed by Wang et al.,10 protection of the amino group by the strongly electron-withdrawing 2,4dinitrobenzenesulfonyl (DNBS) group is expected to quench the fluorescence of compound 2. Moreover, the resulting sulfonamide should be readily cleaved by thiophenols (pKa ≈ 6.5) under physiological pH through an SNAr process, but not by aliphatic thiols (pKa > 8.0), due to the big differences in their pKa values. As a result, compound 2 will be released, leading to the recovery of its NIR fluorescence and thus showing a NIR fluorescent turn-on detection process for thiophenols (Scheme 1). To investigate the feasibility of our design concept, probe 1 was synthesized. The synthesis route for probe 1 is outlined in Scheme 2, in which probe 1 can be readily prepared via reaction of compound 2 with 2,4-dinitrobenzene-1-sulfonyl chloride. Both compound 2 and the intermediate compound 4 are known compounds, and they were prepared according to the previously published procedures.26,31 Structural identification of probe 1 and compound 2 was confirmed by conventional NMR, IR, and HR-MS spectroscopy. Detailed synthetic procedures and structure characterizations are given in the Experimental Section and in the Supporting Information.
Table 1. Determination of Thiophenol Concentrations in Water Samples sample Yangtse River water
East Lake water
South Lake water
thiophenol spiked (μM)
thiophenol recovered (μM)
0
not detected
1 5 10 20 0 1 5 10 20 0 1 5 10 20
0.97 ± 0.01 4.53 ± 0.32 9.05 ± 0.11 20.32 ± 0.94 not detected 1.07 ± 0.07 5.01 ± 0.22 10.79 ± 0.27 19.34 ± 0.65 not detected 0.91 ± 0.02 4.55 ± 0.27 10.32 ± 0.48 20.69 ± 0.62
recovery (%)
97 91 90 102 107 101 108 95 91 91 103 104
Cell Culture and Imaging. HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (fetal bovine serum), 100 mg/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2, water saturated Scheme 2. Synthesis of Probe 1a
a
Reagent and conditions: (a) Na, CH3COOC2H5, 4 h, 53%; (b) AcOH, H2SO4, 30 min, 80%; (c) malononitrile, AcOH, H2SO4, 14 h, 34%; (d) N(4-formyl-phenyl) acetamide, toluene, piperidine, AcOH, 12 h, 74% and then conc. HCl, ethanol, 76%; (e) 2,4-dinitrobenzene-1-sulfonyl chloride, Et3N, CH2Cl2, r.t., 45%. C
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Figure 1. (a) Fluorescent spectral changes of probe 1 (10 μM) upon addition of PhSH (100 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The spectra were collected at 1 min intervals after mixing thiophenol with probe 1. (b) Kinetic curve of probe 1 (10 μM) at 670 nm with PhSH (100 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The data were reported as the mean ± standard deviation of triplicate experiments and fitted (red line) by a first-order reaction scheme as shown in the figure.
Figure 2. (a) Fluorescent spectral changes of probe 1 (10 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C upon addition of different concentrations of PhSH (0−100 μM). Each spectrum was collected 10 min after PhSH addition. (b) A plot of fluorescence intensity changes of probe 1 at 670 nm against [PhSH] from 0 to 30 μM, which can be linearly fitted (red line).
Sensing Property of Probe 1 for Thiophenols. Since compound 2 is the expected product of probe 1 upon treatment of thiophenols, we first examined the differences of the absorption and fluorescent spectra between probe 1 and the reference sample of 2. As shown in Figure S1 (Supporting Information), although the absorption spectra difference between probe 1 (λmax = 470 nm, ε ≈ 2.53 × 104 M−1 cm−1) and 2 (λmax = 478 nm, ε ≈ 3.16 × 104 M−1 cm−1) is not that noticeable, their fluorescent spectra showed a significant difference. As expected, probe 1 shows almost no fluorescence while 2 shows strong NIR fluorescence at 670 nm when excited at 490 nm. This low background fluorescence of the probe itself and the strong fluorescence of the expected product upon thiophenol treatment should be highly desirable for a sensitive detection of thiophenols. The sensing ability of probe 1 for thiophenols was then investigated in 10 mM PBS buffer (pH 7.4) with 30% DMSO (v/v) at 25 °C.34 The fluorescence response of probe 1 toward thiophenol (PhSH) was first investigated. As shown in Figure 1, as anticipated, the fluorescence of probe 1 (10 μM) showed a large enhancement around 670 nm (∼25-fold, excited at 490 nm) within a few minutes upon addition of thiophenol (100 μM). Kinetic analysis showed that the fluorescence enhancement at 670 nm reached a plateau at about 6 min (Figure 1b), and the reaction obeys a typical pseudo-first-order with rate constant kobs determined to be about 0.45 min−1 (t1/2 ≈ 1.56 min). This fast and distinct fluorescence signal change in the NIR region indicates that probe 1 can be used as a rapid and sensitive NIR fluorescent turn-on sensor for thiophenol. In
addition, it is also worth mentioning that probe 1 showed a remarkable large Stokes shift, which is highly desirable for a fluorescent probe, because the large gap between the excitation wavelength and the emission wavelength can effectively diminish the measurement error caused by the excitation light and scattered light. Thus, we have established a NIR fluorescent turn-on probe for rapid detection of thiophenol with remarkable large Stokes shift and significant NIR fluorescent enhancement. To shed light on the sensitivity of probe 1, the kinetics of probe 1 (10 μM) with addition of different concentrations of thiophenol were also measured by monitoring the fluorescence changes at 670 nm (Figure S3, Supporting Information). We can see that, in the absence of thiophenol, the fluorescence signal of probe 1 is quite stable under the test conditions; however, in the presence of thiophenols, the fluorescence signal of probe 1 showed changes and the change speeds are dependent on the concentrations of thiophenol added. Since the reaction can be completed within 10 min when more than 5 equiv of thiophenol is added, the full fluorescence spectra changes of probe 1 (10 μM) were then investigated after 10 min upon addition of increasing concentrations of thiophenol. As shown in Figure 2a, upon progressive addition of thiophenol, the fluorescence of the solution gradually increases until it reaches a plateau after the addition of more than 5 equiv of thiophenol. Notably, significant fluorescence signal changes can be still observed even when thiophenol was added at or below stoichiometric conditions (1−10 μM). A linear calibration graph of the responses of the fluorescent intensity D
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Figure 3. (a) Fluorescence spectra changes of probe 1 (10 μM) toward various analytes (100 μM). (b) Fluorescence responses of probe 1 (10 μM) at 670 nm toward 100 μM of various analytes. All experiments were performed in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C, and each spectrum was obtained 10 min after addition of an analyte. λex = 490 nm; slit width: (5, 10).
at 670 nm to the thiophenol concentrations from 0 to 30 μM can be observed (Figure 2b), which indicates that probe 1 can be potentially employed to detect thiophenol quantitatively. From this linear calibration graph, the detection limit of probe 1 for thiophenol is found to be about 0.15 μM based on signalto-noise ratio (S/N) = 3 under the test conditions. This result further proved that probe 1 is highly sensitive to thiophenol. The effect of pH on the fluorescence signal responses of probe 1 toward thiophenol was also investigated. As shown in Figure S4 (Supporting Information), the probe 1 itself is inert to pH change in a wide range (from 4 to 10); however, in the presence of thiophenol, the fluorescence intensity of probe 1 showed distinct changes over the pH range from 5 to 10. Notably, the fluorescence changes of probe 1 to thiophenol reached a maximum and became almost constant in the pH range of 7−10, which is consistent with the pKa value of thiophenol (pKa ≈ 6.5), because strong ionization of thiophenol at pH over 6.5 is anticipated. This result suggests that probe 1 is able to work over a relatively wide pH range. To evaluate the selectivity of probe 1 for thiophenols, various analytes including thiophenol derivatives (C6H5SH, p-CH3− C6H4SH, p-NH2−C6H4SH, p-CH3O−C6H4SH, and p-NO2− C6H4SH), aliphatic thiols Cys, Hcy, GSH, (CH3)3CSH, and OHCH2CH2SH, and other potential interfering substances such as some nucleophilic species (Ala, NaN3, KI, KCN, C6H5OH, C6H5NH2, and NaSH) and various anions and metal ions (NaF, NaCl, NaBr, NaI, NaNO3, NaClO4, AcONa, Na2C2O4, Na2SO4, NaHCO3, NaH2PO4, Na3PO4, Na2S2O7, LiCl, MgCl2, CaCl2, Al2(SO4)3, MnSO4, Ce(NO3)3, Co(NO3)3, Ni(NO3)2, and Zn(NO3)2) were tested. As shown in Figure 3, only the introduction of thiophenols such as C6H5SH, p-CH3− C6H4SH, p-NH2−C6H4SH, and p-CH3O−C6H4SH to the probe 1 solution induced a significant enhancement in the fluorescent intensity at 670 nm. One exception is that the fluorescent detection of p-NO2−C6H4SH with a strong electron-withdrawing NO2 group by probe 1 is difficult, which is consistent with reports in the literature,10,19,22 probably because of the low activity of p-NO2−C6H4S− in the SNAr reaction toward sulfonamide owing to its relatively large local softness (ssulfur−) value.19 Except thiophenols, other tested species mentioned above including aliphatic thiols such as Cys and Hcy, thiophenol analogues such as C6H5OH and C6H5NH2, common anions, and metal ions did not induce any obvious fluorescence enhancement to the probe 1 solution (Figure 3 and Figure S5, Supporting Information). In addition, probe 1 shows high selectivity for thiophenol against reactive
nitrogen and oxygen species such as hypochlorite, hydrogen peroxide, superoxide, nitrite, nitroxyl, nitric oxide, peroxynitrite, etc., and detection of thiophenol using probe 1 in the presence of various analytes including these reactive species is still effective (Figure 4 and Figure S5, Supporting Information).
Figure 4. Fluorescence responses of probe 1 (10 μM) at 670 nm toward PhSH in the presence of some representative analytes. Black bars represent the addition of a single analyte (100 μM) including: none, Cys, Hcy, GSH, Ala, NaN3, KI, KCN, C6H5OH, C6H5NH2, OHCH2CH2SH, (CH3)3CSH, NaHS. Red bars represent the subsequent addition of C6H5SH (100 μM) to the mixture. All experiments were performed in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C, and data was obtained 10 min after addition of an analyte. λex = 490 nm; slit width: (5, 10).
Therefore, all these results clearly indicate that probe 1 has high selectivity to thiophenols (except p-NO2−C6H4SH) over other analytes mentioned above. The fluorescence changes of probe 1 in the presence of thiophenol suggest that compound 2 was produced. To confirm that the fluorescence sensing response of probe 1 to thiophenol is indeed due to the conversion of probe 1 to compound 2, the reaction products of probe 1 with thiophenol were isolated and subjected to 1H NMR and MS analysis. Two products were isolated, and one of them showed an essentially identical Rf (in the TLC plate) and 1H NMR spectrum with that of the standard compound 2 (Figures S6 and S7, Supporting Information). Moreover, a peak of 311.27 found in the MS spectrum further proved the formation of compound 2 (Figure S8, Supporting Information). In addition, 1H NMR, 13C NMR, and MS analysis proved another isolated product to be compound 3 (Figures S9−S11, Supporting Information). E
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Figure 5. (a) Fluorescent detection of different concentrations of PhSH in “distilled water”, “Yangtse River water”, “East Lake water”, and “South Lake water” by probe 1 (10 μM) in PBS buffer (10 mM, pH 7.4) with 30% DMSO at 25 °C. The reaction is monitored at 670 nm. Each condition was measured three times, and the average values are shown. PhSH is spiked in 0, 1, 5, 10, 20, and 50 μM, respectively. λex = 490 nm; slit width: (5, 10). (b−d) Linear plot of fluorescence intensity changes of probe 1 at 670 nm against the spiked concentrations of PhSH from 0 to 20 μM for each real water sample.
Figure 6. Imaging of thiophenol in HeLa Cells by probe 1. (A) Bright field images of HeLa cells after being treated with probe 1 (20 μM) for 30 min. (B) Bright field images of HeLa cells preincubated with thiophenol (2 μM) and then incubated with probe 1 (20 μM). A1 and B1 are fluorescence images of A and B, respectively.
Thus, the mechanism of probe 1 for sensing of thiophenol is most likely the cleavage of the sulfonamide process mediated by thiophenol as shown in Scheme 1. Detection of Thiophenol in Water Samples. Considering the toxicity of thiophenols and their potential as pollutants, we employed probe 1 to determine thiophenol concentrations in water samples to validate its practical utility in environmental science. The water samples were collected from the Yangtse River, East Lake, and South Lake in Wuhan City. These water samples were directly analyzed first and then spiked with thiophenol at different levels (1, 5, 10, 20, and 50 μM), and the fluorescence responses of probe 1 at 670 nm toward all these samples were examined, respectively. The results are shown in Figure 5a, and they are compared with those determined in distilled water. We can see that they have a good consistency between each other (Figure 5a). For each real water sample, a linear relationship between the responses of the fluorescent intensity at 670 nm to the spiked thiophenol concentrations from 0 to 20 μM was observed (Figure 5b−d), which is in good agreement with the result obtained in distilled water (Figure
2b). In addition, we can see that the recoveries of thiophenol ranged from 90% to 108% (Table 1), which indicates that the thiophenol in the water samples could be accurately measured with good recovery when probe 1 was applied as the probe. This result showed that probe 1 has potential application for quantitative detection of thiophenols in water samples. Detection of Thiophenol in Living Cells. We also investigated the practical utilities of probe 1 for fluorescent cell imaging with HeLa cells. As shown in Figure 6, when HeLa cells were incubated directly with probe 1 (20 μM) only at 37 °C, no fluorescence was observed. However, when HeLa cells were preincubated with thiophenol (2 μM) and then incubated with probe 1 (20 μM), we observed strong red fluorescence. These results indicate that probe 1 can be applied to detect thiophenols in living cells.
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CONCLUSION In summary, we developed a NIR fluorescent probe (probe 1) for rapid, selective, and sensitive detection of thiophenols. This new probe is reaction-based, which uses a conjugated F
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(17) Kand, D.; Mishra, P. K.; Saha, T.; Lahiri, M.; Talukdar, P. Analyst 2012, 137, 3921−3924. (18) Liu, X. L.; Duan, X. Y.; Du, X. J.; Song, Q. H. Chem.Asian J. 2012, 7, 2696−2702. (19) Shiraishi, Y.; Yamamoto, K.; Sumiya, S.; Hirai, T. Chem. Commun. 2013, 49, 11680−11682. (20) Zhao, C.; Wang, X.; Cao, J.; Feng, P.; Zhang, J.; Zhang, Y.; Yang, Y.; Yang, Z. Dyes Pigm. 2013, 96, 328−332. (21) Kand, D.; Mandal, P. S.; Datar, A.; Talukdar, P. Dyes Pigm. 2014, 106, 25−31. (22) Li, J.; Zhang, C.-F.; Yang, S.-H.; Yang, W.-C.; Yang, G.-F. Anal. Chem. 2014, 86, 3037−3042. (23) Balkrishna, S. J.; Hodage, A. S.; Kumar, S.; Panini, P.; Kumar, S. RSC Adv. 2014, 4, 11535−11538. (24) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev. 2014, 43, 16−29. (25) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (26) Huang, X.; Guo, Z.; Zhu, W.; Xie, Y.; Tian, H. Chem. Commun. 2008, 5143−5145. (27) Guo, Z.; Zhu, W.; Tian, H. Chem. Commun. 2012, 48, 6073− 6084. (28) Zhu, W.; Huang, X.; Guo, Z.; Wu, X.; Yu, H.; Tian, H. Chem. Commun. 2012, 48, 1784−1786. (29) Cao, J.; Zhao, C.; Zhu, W. Tetrahedron Lett. 2012, 53, 2107− 2110. (30) Li, M.; Wu, X.; Wang, Y.; Li, Y.; Zhu, W.; James, T. D. Chem. Commun. 2014, 50, 1751−1753. (31) Sun, W.; Fan, J.; Hu, C.; Cao, J.; Zhang, H.; Xiong, X.; Wang, J.; Shuang, C.; Sun, S.; Peng, X. Chem. Commun. 2013, 49, 3890−3892. (32) Zheng, Y.; Zhao, M.; Qiao, Q.; Liu, H.; Lang, H.; Xu, Z. Dyes Pigm. 2013, 98, 367−371. (33) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. J. Am. Chem. Soc. 2014, 136, 3579−3588. (34) Note: We have investigated the effect of different amounts of DMSO as cosolvent to the sensing property of probe 1 for thiophenol; considering the fluorescent intensity changes and the reaction speed, 30% DMSO was chosen in this study (Figure S2, Supporting Information). The working temperature was set at 25 °C without further optimization, although a higher temperature may be optimal for more rapid thiophenol detection.
dicyanomethylene-benzopyran molecule (compound 2) as the fluorophore and 2,4-dinitrobenzene-1-sulfonamide as the reaction site. The merits of this probe for detection of thiophenols (except p-NO2−C6H4SH) include rapid and significant NIR fluorescence turn-on responses, high selectivity and sensitivity, working under very mild conditions, and having a remarkably large Stokes shift. In addition, this probe can be successfully applied to detect thiophenol in real water samples and in living cells. Therefore, this work provides a promising NIR probe for the rapid detection and quantification of the highly toxic thiophenols found in environmental and biological samples.
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ASSOCIATED CONTENT
S Supporting Information *
Structure characterizations for compound 2 and probe 1, additional fluorescence data, and data for investigation of the sensing mechanism. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grant Nos. 21172086 and 21032001) for financial support. This work was also sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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REFERENCES
(1) Heil, T. P.; Lindsay, R. C. J. Environ. Sci. Health B 1989, 24, 349− 360. (2) Amrolia, P.; Sullivan, S. G.; Stern, A.; Munday, R. J. Appl. Toxicol. 1989, 9, 113−118. (3) Roy, K.-M. In Ullmann’s Encyclopedia of Industrial Chemistry, 7th ed.; Wiley: New York, 2007. (4) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103−1170. (5) EychmKller, A.; Rogach, A. L. Pure Appl. Chem. 2000, 72, 179− 188. (6) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52−67. (7) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019−6031. (8) Yin, C.; Huo, F.; Zhang, J.; Martínez-Máñez, R.; Yang, Y.; Lv, H.; Li, S. Chem. Soc. Rev. 2013, 42, 6032−6059. (9) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351−5358. (10) Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8445−8448. (11) Lin, W.; Long, L.; Tan, W. Chem. Commun. 2010, 46, 1503− 1505. (12) Jiang, W.; Cao, Y.; Liu, Y.; Wang, W. Chem. Commun. 2010, 46, 1944−1946. (13) Zhao, W. W.; Liu, W. M.; Ge, J. C.; Wu, J. S.; Zhang, W. J.; Meng, X. M.; Wang, P. F. J. Mater. Chem. 2011, 21, 13561−13568. (14) Zhao, C.; Zhou, Y.; Lin, Q.; Zhu, L.; Feng, P.; Zhang, Y.; Cao, J. J. Phys. Chem. B 2011, 115, 642−647. (15) Wang, X.; Cao, J.; Zhao, C. Org. Biomol. Chem. 2012, 10, 4689− 4691. (16) Wang, Z.; Han, D. M.; Jia, W. P.; Zhou, Q. Z.; Deng, W. P. Anal. Chem. 2012, 84, 4915−4920. G
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