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homocysteine, and glutathione
Sensors and Actuators B 235 (2016) 691–697
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Smart probe for rapid and simultaneous detection and discrimination of hydrogen sulfide, cysteine/homocysteine, and glutathione Shuangshuang Ding, 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, PR China
a r t i c l e
i n f o
Article history: Received 12 April 2016 Received in revised form 24 May 2016 Accepted 26 May 2016 Keywords: Biothiols Fluorescent probe Simultaneous detection and discrimination Living cells
a b s t r a c t Development of a simple probe for simultaneous detection and discrimination of the most important four biothiols (H2 S, Cys, Hcy and GSH) is important but a challenging task. In this paper, a very simple but versatile fluorescent probe was reported, which can be used for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This probe not only can be easily prepared, but also shows rapid (within 150 s), selective and sensitive responses for these four biothiols with distinct fluorescent turn-on signal changes at 465 nm. Moreover, this probe is able to show unique absorbance enhancement at 540 nm for H2 S, and additional fluorescence enhancement at 550 nm only for Cys/Hcy, thus providing a rapid, simultaneous detection and discrimination method for H2 S, Cys/Hcy and GSH. Imaging and discrimination of Cys and GSH in HeLa Cells by this probe were also successfully applied. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2 S) are the most important small molecular biothiols that play key but diverse roles in biological systems. In addition, concentration levels of these thiols are very useful in the diagnosis of various closely related disease states. For example, abnormal levels of Cys is closely related to diseases of hair depigmentation, slow growth in children, liver damage, edema, loss of muscle and skin lesions [1,2], whereas abnormal levels of GSH correlate with heart problems, liver damage, leucocyte loss, cancer, and aging [3–6], and abnormal levels of H2 S are implicated in central nervous system diseases such as Down syndrome, Parkinson’s and Alzheimer’s disease [7–9]. Therefore, it is highly important to develop probes for these thiols, and particularly, a single probe that can be used to simultaneously detect and discriminate these thiols would be highly valuable. Optical probes, especially small molecular fluorescent probes which possess advantages of high sensitivity, low cost, convenience and non-invasiveness, are therefore very attractive for molecule sensing. To date, a large number of fluorescent probes have been developed for sensing thiols [10–15]. However, simultaneous detection and differentiation of Cys, Hcy, GSH and H2 S by a
∗ Corresponding author. E-mail address: [email protected] (G. Feng). http://dx.doi.org/10.1016/j.snb.2016.05.146 0925-4005/© 2016 Elsevier B.V. All rights reserved.
single probe remains a challenge [16]. On the one hand, due to the similar structures and reactivities of Cys, Hcy and GSH, many developed probes only can be used to detect these three thiols as a whole without discrimination [10–12]. On the other hand, although great progress have been achieved recently in developing highly selective probes for H2 S [17–23], Cys [24–28], Hcy [29,30], and GSH [31–33], these probes can only sense one of these biothiols at a time. Very recently, chemists started to develop single probes to meet this challenge, and several elegant probes have been found to be able to simultaneously detect and differentiate Cys/Hcy and GSH [34–44]. However, either of these probes are not responsive for the simplest thiol, H2 S, or have not been reported on the response to H2 S. Herein, we report a remarkably simple but versatile probe (probe 1 in Scheme 1) that can be used for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This probe has the following merits: (1) it can be easily prepared from readily available inexpensive reagents. (2) It shows rapid (within 150 s), selective and sensitive fluorescent turn-on signal changes for these four thiols over many other common analytes. (3) In addition, it shows additional characteristic optical changes for H2 S in color and Cys/Hcy in fluorescence, respectively, which can be used to discriminate H2 S, Cys/Hcy and GSH simultaneously. To prove its potential utility, we also demonstrated that this probe can be conveniently used for simultaneous detection and discrimination of Cys and GSH in living cells. Considering its readily available and excellent sensing properties, this probe provided a potentially useful tool for biothiol sensing, detection and discrimination.
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Scheme 1. Structures of H2 S, Cys, Hcy and GSH, and probe 1 for selective detection and simultaneous discrimination of these thiols.
2. Experimental 2.1. Reagents, materials and apparatus All chemicals were purchased from commercial suppliers and used without further purification except HBT, which was synthesized from 2-aminobenzenethiol and 2-hydroxybenzaldehyde by the published procedures [45]. 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. 1 H NMR and 13 C NMR spectra were recorded on a Varian Mercury 400 spectrometer, and resonances (ı) 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, respectively. The fluorescence quantum yields were determined in PBS buffer (10 mM, pH 7.4, 20% DMSO, v:v) at 25 ◦ C, using quinine sulfate (Ф = 0.58 in 1N H2 SO4 ) as standard. Cell imaging was performed in an inverted fluorescence microscopy with a 20× objective lens.
2.2. Synthesis of probe 1 A solution of 4-chloro-7-nitrobenzofurazan (60 mg, 0.3 mmol), HBT (57 mg, 0.25 mmol) and triethylamine (50 L) in anhydrous DMF (3 mL) was stirred at room temperature. A dark green precipitate generated gradually in the solution. After 2 h, the solution was poured into ice-water (10 mL) and the precipitate was collected on a filter, washed with CH3 OH and dried under vacuum to afford probe 1 as a pure yellow solid (83 mg, yield 85%). Mp: 219–220 ◦ C. TLC (silica plate): Rf 0.31 (petroleum ether: ethyl acetate 5:1, v/v). 1 H NMR (400 MHz, CDCl3 ): ı 8.56 (d, J = 7.8 Hz, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 7.1 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H),
7.40 (d, J = 8.0, 1H), 7.36 (d, J = 8.0, 1H), 6.53 (d, J = 8.3 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6 ) ı 161.19, 152.63, 152.46, 150.84, 145.88, 144.88, 135.61, 135.48, 133.66, 131.54, 130.96, 128.28, 127.22, 126.26, 125.82, 123.41, 123.11, 122.65, 110.86. EI-MS: m/z found 390.20 (M+ ). HR-MS: calcd for C19 H11 N4 O4 S+ (M+H+ ), 391.0496; found 391.0521. Elemental analysis calcd (%) for C19 H10 N4 O4 S C 58.46, H 2.58, N 14.35, S 8.21; found C 58.25, H 2.42, N 14.29, S 8.06. 2.3. Optical studies of probe 1 upon addition of various analytes Stock solutions of probe 1 (1 mM) were prepared in HPLC grade DMSO. Stock solutions (1–10 mM) of the analytes including NaHS, Cys, Hcy, GSH, Gln, Phe, Trp, Ala, leu, Thr, Ser, Asp, Ile, Met, Lys, Gly, Glv, Arg, Tyr, Pyr, His, NaF, NaCl, NaBr, NaI, Na2 S2 O7 , Na2 SO3 , Na2 CO3 , Na2 SO4 , NaHSO4 , NaNO3 , NaSCN, NaAcO, H2 NCH2 CH2 NH2 , HOCH2 CH2 NH2 , C6 H5 NH2 , C6 H5 CH2 NH2 , KCl, MnSO4 , FeSO4 , MgCl2 , CaCl2 , HgCl2 , Zn(NO3 )2 , Cd(NO3 )2 , Cu(NO3 )2 , and AgNO3 were prepared in ultrapure water. ROS/RNS were prepared according our published procedure [46,47]. For optical studies, a solution of probe 1 (10 M) was prepared in DMSO-PBS buffer solution (1:4, v/v, 10 mM PBS). Then 3.0 mL of the probe 1 solution was placed in a quartz cuvette. The UV–vis or fluorescent spectra were recorded upon addition of analyte of interest at 25 ◦ C with a temperature controller. 2.4. Cell culture and bioimaging 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 g/mL streptomycin in a 5% CO2 , water saturated incubator at 37 ◦ C, and then were seeded in a 12well culture plate for one night before cell imaging experiments. In the experiment of cell imaging, living cells were incubated with 10 M of probe 1 (with 0.1% DMSO, v/v) at 37 ◦ C for 30 min and washed with PBS for three times, and then imaged immediately. In the experiment of N-ethylmaleimide (NEM) added to the cell
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Scheme 2. The synthesis of probe 1.
culture, HeLa cells were cultured with 500 M NEM for 30 min at 37 ◦ C, washed three times with PBS, and then incubated with 10 M probe 1 for 30 min at 37 ◦ C and imaged immediately. In the imaging of exogenous Cys or GSH, HeLa cells were pre-treated with NEM (500 M) for 30 min at 37 ◦ C, and washed three times with PBS. Then HeLa cells were treated with probe 1 (10 M) for 30 min at 37 ◦ C, washed three times with PBS. Finally, HeLa cells were incubated with Cys (or GSH, 100 M) for 30 min at 37 ◦ C. The imaging of cells was then carried out after washing with PBS buffer.
3. Results and discussion 3.1. Probe design and synthesis Probe 1 was designed to combine two well-known fluorophores, nitrobenzofurazan (NBD) and 2-(2 -hydroxyphenyl)benzothiazole (HBT), which are covalently linked to form an ether. HBT is a typical excited-state intramolecular proton transfer (ESIPT) dye with the merit of large Stokes shifts [25,48,49]. The principle of probe 1 for detection of thiols is illustrated in Scheme 1. First, although NBD and HBT are well-known fluorophores, NBD ether is nonfluorescent and it also can block the ESIPT process and quench the fluorescence of HBT [50–52]. Therefore, probe 1 is expected to be colorless and non-fluorescent, which would provide advantages of low backgrounds. Second, probe 1 can react with thiols by thiolysis of the NBD ether to release the strong fluorescent HBT (em ≈ 465 nm) and different NBD-derivatives. NBD-derivatives have been reported to show different optical properties depending on the thiols [53–56]. NBD-SH and NBD-GSH are S-bound NBD and they are non-fluorescent but the former is purple-red while the later is light yellow in aqueous solution. The S-bound NBD product of Cys/Hcy is not stable and they can change into the corresponding N-bound NBD products quickly via a unique nucleophilic aromatic substitution-intramolecular rearrangement [42,43,53,54]. Thus, NBD-Cys/Hcy is different to NBD-SH and NBDGSH, showing orange color and strong yellow fluorescence at about 550 nm. As a result, probe 1 was expected to show distinct and dif-
ferent optical responses for H2 S, Cys/Hcy and GSH, which would provide a simple and practical method for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. With this expectation, probe 1 was synthesized according to the route outlined in Scheme 2. Overall, probe 1 can be readily synthesized by the readily available NBD-Cl and HBT in good yield (85%), and its structure and purity were well characterized by NMR, Mass and elemental analysis (see experimental section and the Supporting information). It should be noted that the facile synthesis with readily available starting materials is important for the wide use of probes. We noticed that this probe was reported by Yi et al. [51] during we were conducting this work; however, the important ability of this probe to discriminate biothiols is still unkown.
3.2. Simultaneous detection and discrimination of thiols by probe 1 The sensing property of probe 1 was investigated in PBS buffer (10 mM, pH 7.4, containing 20% DMSO, v/v) at 25 ◦ C. As expected, probe 1 is colorless (maximum abs = 375 nm) and non-fluorescent (Ф = 0.003), and upon addition of various analytes including thiols (H2 S, Cys, Hcy and GSH. NaHS was used as a standard source for H2 S), amino acids such as Gln, Phe, Trp, Ala, leu, Thr, Ser, Asp, Ile, Met, Lys, Gly, Glv, Arg, Tyr, Pyr and His, common anions such as F− , Cl− , Br− , I− , S2 O7 2− , SO3 2− , CO3 2− , SO4 2− , NO3 − , SCN− and AcO− , other nucleophilic agents such as H2 NCH2 CH2 NH2 , HOCH2 CH2 NH2 , C6 H5 NH2 and C6 H5 CH2 NH2 , and reactive oxygen/nitrogen species (ROS/RNS) such as ClO− , H2 O2 , NO2 − , t BuOO• and • OH, we observed that the probe 1 solution showed selective changes for NaSH, Cys, Hcy and GSH over the other analytes. As shown in Fig. 1a, only the addition of NaHS, Cys, Hcy and GSH induced significant changes to the UV–vis spectrum of probe 1. Among them, the addition of NaHS induced the most remarkable red-shift ( = 165 nm, from 375 nm to 540 nm) to the maximum absorption of probe 1, while that for Cys/Hcy and GSH is only 105 nm and 45 nm, respectively. As a result, the color of the probe 1 solution changed from colorless to purple-red, orange and pale-
Fig. 1. (a) UV–vis spectra changes of probe 1 (10 M) upon addition of various analytes (100 M of each, including: (1) none, (2) Cys, (3) Hcy, (4) GSH, (5) NaHS, (6) SO3 2− , (7) CO3 2− , (8) SO4 2− , (9) S2 O7 2− , (10) Cl− , (11) Br− , (12) F− , (13) I− , (14) S2 O3 2− , (15) NO3 − , (16) SCN− , (17) AcO− , (18) H2 NCH2 CH2 NH2 , (19) HOCH2 CH2 NH2 , (20) C6 H5 NH2 , (21) C6 H5 CH2 NH2 , (22) Gln, (23) Phe, (24) Trp, (25) Ala, (26) Leu, (27) Thr, (28) Ser, (29) Asp, 30. Ile, (31) Met, (32) Lys, (33) Gly, (34) Glv, (35) Arg, (36) Tyr, (37) Pyr, (38) His, (39) ClO− , (40) H2 O2 , (41) NO2 − , (42) t BuOO• , (43) • OH). (b) The corresponding absorbance change of probe 1 at 540 nm for each analyte. Insert: A photo of color changes of probe 1 for thiols. All spectra were measured in PBS buffer (10 mM, pH 7.4) with 20% DMSO at 25 ◦ C and each spectrum was obtained 150 s after addition.
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yellow for NaHS, Cys/Hcy and GSH, respectively (insert in Fig. 1b). In contrast, other analytes did not show any noticeable changes in the UV–vis spectra or the color of probe 1 (Fig. S1). It should be noted that the color change induced by NaHS is most distinct to the “naked eyes”, which can be attributed to the production of NBDSH (max ∼540 nm) [52,55,56]. The production of NBD-SH and HBT from probe 1 upon addition of NaHS was confirmed by HPLC, NMR and Mass analysis (Fig. S2). Notably, the distinct absorbance change at 540 nm is unique for H2 S (Fig. 1b). Therefore, these results not only indicate that probe 1 can be used as a selective colorimetric sensor for H2 S, Cys, Hcy and GSH, but also indicate that probe 1 can be used to discriminate H2 S from Cys, Hcy and GSH by the characteristic absorption changes at 540 nm. Fig. 2a shows the fluorescence spectra changes of the probe 1 solution to various analytes at an excitation wavelength of 330 nm. One can see that only the addition of NaHS, Cys, Hcy and GSH induced significant fluorescence turn-on changes at 465 nm. This fluorescence enhancement can be ascribed to the formation of HBT (em ∼ 465 nm with Ф = 0.052) via the thiolysis reaction induced by thiols, which was confirmed by HPLC, TLC and Mass analysis (Fig. S2). In contrast, other analytes induced almost no changes to the fluorescence of the probe 1 solution. In addition, probe 1 is not responsive to common metal ions such as Na+ , K + , Mn2+ , Zn2+ , Mg2+ , Cd2+ , Ca2+ , Hg2+ , Cu2+ , Fe2+ , and Ag+ (Fig. S3). All these results indicate that probe 1 can be also used as a selective fluorescent turn-on sensor for H2 S, Cys, Hcy and GSH. Notably, if we select a longer excitation wavelength that is efficient to light-up the fluorescence of NBD, the probe 1 solution was found to show high selectivity for Cys/Hcy. As shown in Fig. 2c, when the excitation wavelength was set at 475 nm, only Cys/Hcy induced the probe 1 solution significant fluorescence enhancement at 550 nm, while both H2 S and GSH and other analytes showed no effect. This clearly indicates that probe 1 can be used to discriminate Cys/Hcy from H2 S and GSH. It is worth noting that when the excitation wavelength was used at 415 nm, a wavelength between 330 nm and 475 nm, we observed fluorescence enhancement of
the probe 1 solution at both 465 nm and 550 nm upon addition of Cys/Hcy, while addition of H2 S and GSH still only induced fluorescence enhancement at 465 nm (Fig. S4). These results indicate that besides the formation of HBT (emission at 465 nm), the reaction of probe 1 with Cys generated another fluorescent product, NBDCys, which was confirmed by Mass analyses (Fig. S5). Similarly, the product of NBD-GSH was also confirmed by Mass analyses from the mixture of probe 1 and GSH (Fig. S6). Therefore, the sensing mechanism of probe 1 for H2 S, Cys/Hcy, and GSH is most likely the process as illustrated in Scheme 1, which is consistent with the literature [53,54]. Overall, probe 1 can be used for simultaneous detection of these four thiols by fluorescence changes at 465 nm, or selective detection of H2 S from Cys, Hcy and GSH by absorbance changes at 540 nm, or selective detection of Cys/Hcy from H2 S and GSH by fluorescence changes at 550 nm, which provides a convenient method for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. 3.3. The sensitivity of probe 1 for thiols The sensitivity of probe 1 for H2 S, Cys/Hcy and GSH was also investigated. Kinetics studies showed that probe 1 itself is quite stable but responses rapidly to these thiols. As shown in Fig. 3a, probe 1 showed the fastest response for NaHS (completed almost immediately) and similar reactivities for other thiols (completed within 150 s). These rapid responses indicate that probe 1 could be very sensitive to H2 S, Cys/Hcy and GSH, which is highly favourable for a rapid detection. It should be noted that although Hcy exhibits a similar fluorescence response to Cys, the concentration of Hcy is generally at least ten times lower than that of Cys in living systems [54]. Thus, we focused on the sensitivity of probe 1 for H2 S, Cys and GSH. As shown in Fig. S7, the fluorescence intensity at 465 nm gradually increased with increasing amounts of each thiol, and saturation all occurred at addition of about 5 equiv. of thiols. In addition, as shown in Fig. 3b–d (also see Fig. S8–10 with ex = 330 nm), good linear relationship between the fluorescence
Fig. 2. Fluorescent spectra changes of probe 1 (10 M) in PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v) upon addition of 100 M various analytes at 25 ◦ C. (a) ex = 330 nm. (b) The corresponding fluorescence change of probe 1 at 465 nm for each analyte. (c) ex = 475 nm. (d) The corresponding fluorescence change of probe 1 at 550 nm for each analyte. Slit width: dex = dem = 5 nm. Emission color changes of probe 1 for NaHS, Cys, Hcy and GSH under a light of 365 nm and 460 nm were inserted in (a) and (c), respectively.
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Fig. 3. (a) Fluorescent kinetics of probe 1 (10 M) with 100 M of NaHS, Cys, Hcy and GSH. (b–d) Linear relationship of fluorescence change at 465 nm as a function of thiol concentration. All spectra were measured in PBS buffer (10 mM, pH 7.4, 20% DMSO, v/v) at 25 ◦ C after an incubation time of 150 s ex = 415 nm, dex = 5 nm, dem = 10 nm.
change and a certain concentration of thiols (NaHS: 0–30 M, Cys and GSH: 0–10 M) can be observed, respectively. Thus, the detection limit (S/N = 3) of probe 1 for NaHS, Cys and GSH was calculated to be about 0.10 M, 0.08 M and 0.06 M, respectively. These results indicated that probe 1 can be used to detect these thiols quantitatively with high sensitivity. Moreover, good linearity can be also observed between the absorption change at 540 nm (the characteristic optical change for NaHS) and the concentration of NaHS in the range of 0–30 M (Fig. S11). Similar result can be obtained for Cys, if we monitor the fluorescence change at 550 nm (the characteristic optical change for Cys) (Fig. S12). It should be
noted that these results are important, because we can apply the additivity of absorption and fluorescence to determine the concentration of each thiol if they coexist. To prove this, different concentrations of Cys and GSH were added to a solution of probe 1 including a certain concentration of NaHS (all of their concentrations are in the linear range), and the fluorescence intensity at 465 nm of the mixed system was tested. As shown in Fig. S13, the fluorescence intensity at 465 nm obtained from the mixture samples accords well with the additive fluorescence intensity obtained from each sample with only one component (error < 5%). Therefore, if NaHS, Cys and GSH coexist, the concentration of NaHS can
Fig. 4. Imaging and discrimination of Cys and GSH in HeLa Cells by probe 1. Row A: bright field images. Row B: fluorescent images from the blue channel with excitation wavelength at 395–420 nm. Row C: fluorescent images from the green channel with excitation wavelength at 420–485 nm. Column 1: HeLa cells were incubated with probe 1 (10 M) for 30 min. Column 2: HeLa cells were incubated with NEM (500 M) for 30 min and then treated with probe 1 (10 M) for 30 min. Column 3: HeLa cells were pre-incubated with NEM (500 M) for 30 min and then treated with Cys (100 M) for 30 min, and followed with probe 1 (10 M) for 30 min. Columns 4 is similar to Column 3, but with GSH (100 M) instead of Cys. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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be measured through the absorption at 540 nm at first, and then the concentration of Cys can be determined through the fluorescence intensity at 550 nm. Finally, the concentration of GSH can be calculated by subtracting the fluorescence intensity contributed by NaHS and Cys at 465 nm.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.05.146. References
3.4. The effect of pH The effect of pH for sensing thiols was also investigated. As shown in Fig. S14–15, probe 1 can be used for simultaneous detection and differentiation of H2 S, Cys/Hcy and GSH over a wide pH range including a physiologically relevant pH, which is favorable for its biological applications. 3.5. The potential applications of probe 1 Since Cys and GSH are the well-known two main thiols in biosystems that play separate, critically important roles in human physiology and pathology, great attention has been given in recently to the simultaneous detection and discrimination of these two thiols in living cells [34–39,41–44]. Encouraged by our aforementioned results, we evaluated the capability of probe 1 to selectively image intracellular Cys and GSH. The cytotoxicity of probe 1 was first investigated by MTT assays, and the result showed that probe 1 is of low cytotoxicity to living cells (Fig. S16). Cell imaging was then explored. As shown in Fig. 4, when HeLa cells were incubated with probe 1 (10 M) for 30 min, blue and green fluorescence was observed at different channels simultaneously (column 1). However, when the cell culture was incubated with a thiol trapping reagent, N-ethylmaleimide (NEM) [28] before incubation of probe 1, the cells showed almost no fluorescence at both channels (column 2). These results clearly indicate that probe 1 can be applied to image intracellular thiols in living cells. In another set of experiments, after HeLa cells were incubated with NEM, we added Cys and GSH to the cell culture, respectively, and then incubated the cells with probe 1. In this case, different responses were observed. As shown in Fig. 4 (column 3–4), addition of Cys generated blue and green fluorescence (column 3), while addition of GSH only induced blue fluorescence (columns 4). These results are consistent with the results shown in Fig. 2, indicating that probe 1 can be conveniently used for simultaneous detection and differentiation of Cys and GSH in living cells. 4. Conclusions In summary, we reported a rather simple but versatile fluorescent probe for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This smart probe not only can be easily prepared, but also shows a rapid, simultaneous detection process for these four important thiols with high selectivity and sensitivity in aqueous solution under mild conditions. Moreover, this probe is able to show unique absorbance enhancement at 540 nm for H2 S, and additional fluorescence enhancement at 550 nm only for Cys/Hcy, thus providing a simultaneous discrimination method for H2 S, Cys/Hcy and GSH. In addition, we also demonstrated that this probe can be used to simultaneously detect and discriminate of Cys and GSH in living cells with low cytotoxicity, which indicated that this probe has potential application prospects. Acknowledgments We thank the National Natural Science Foundation of China (Grant Nos. 21472066and 21172086) and the Natural Science Foundation of Hubei Province (No. 2014CFA042) for financial support.
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Biographies Shuangshuang Ding is studying for MS degree at Central China Normal University. Her research focuses on the development of new fluorescent probes. Guoqiang Feng is a Professor of the College of Chemistry, Central China Normal University (CCNU). He received his PhD in 2003 from Institute of Chemistry, Chinese Academy of Sciences. After completing postdoctoral research at the University of Sheffield and the University of Cambridge, he joined the faculty at Central China Normal University in 2009. His recent research focuses on molecular recognition and chemosensors.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Smart probe for rapid and simultaneous detection and discrimination of hydrogen sulfide, cysteine/homocysteine, and glutathione Shuangshuang Ding, 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, PR China
a r t i c l e
i n f o
Article history: Received 12 April 2016 Received in revised form 24 May 2016 Accepted 26 May 2016 Keywords: Biothiols Fluorescent probe Simultaneous detection and discrimination Living cells
a b s t r a c t Development of a simple probe for simultaneous detection and discrimination of the most important four biothiols (H2 S, Cys, Hcy and GSH) is important but a challenging task. In this paper, a very simple but versatile fluorescent probe was reported, which can be used for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This probe not only can be easily prepared, but also shows rapid (within 150 s), selective and sensitive responses for these four biothiols with distinct fluorescent turn-on signal changes at 465 nm. Moreover, this probe is able to show unique absorbance enhancement at 540 nm for H2 S, and additional fluorescence enhancement at 550 nm only for Cys/Hcy, thus providing a rapid, simultaneous detection and discrimination method for H2 S, Cys/Hcy and GSH. Imaging and discrimination of Cys and GSH in HeLa Cells by this probe were also successfully applied. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2 S) are the most important small molecular biothiols that play key but diverse roles in biological systems. In addition, concentration levels of these thiols are very useful in the diagnosis of various closely related disease states. For example, abnormal levels of Cys is closely related to diseases of hair depigmentation, slow growth in children, liver damage, edema, loss of muscle and skin lesions [1,2], whereas abnormal levels of GSH correlate with heart problems, liver damage, leucocyte loss, cancer, and aging [3–6], and abnormal levels of H2 S are implicated in central nervous system diseases such as Down syndrome, Parkinson’s and Alzheimer’s disease [7–9]. Therefore, it is highly important to develop probes for these thiols, and particularly, a single probe that can be used to simultaneously detect and discriminate these thiols would be highly valuable. Optical probes, especially small molecular fluorescent probes which possess advantages of high sensitivity, low cost, convenience and non-invasiveness, are therefore very attractive for molecule sensing. To date, a large number of fluorescent probes have been developed for sensing thiols [10–15]. However, simultaneous detection and differentiation of Cys, Hcy, GSH and H2 S by a
∗ Corresponding author. E-mail address: [email protected] (G. Feng). http://dx.doi.org/10.1016/j.snb.2016.05.146 0925-4005/© 2016 Elsevier B.V. All rights reserved.
single probe remains a challenge [16]. On the one hand, due to the similar structures and reactivities of Cys, Hcy and GSH, many developed probes only can be used to detect these three thiols as a whole without discrimination [10–12]. On the other hand, although great progress have been achieved recently in developing highly selective probes for H2 S [17–23], Cys [24–28], Hcy [29,30], and GSH [31–33], these probes can only sense one of these biothiols at a time. Very recently, chemists started to develop single probes to meet this challenge, and several elegant probes have been found to be able to simultaneously detect and differentiate Cys/Hcy and GSH [34–44]. However, either of these probes are not responsive for the simplest thiol, H2 S, or have not been reported on the response to H2 S. Herein, we report a remarkably simple but versatile probe (probe 1 in Scheme 1) that can be used for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This probe has the following merits: (1) it can be easily prepared from readily available inexpensive reagents. (2) It shows rapid (within 150 s), selective and sensitive fluorescent turn-on signal changes for these four thiols over many other common analytes. (3) In addition, it shows additional characteristic optical changes for H2 S in color and Cys/Hcy in fluorescence, respectively, which can be used to discriminate H2 S, Cys/Hcy and GSH simultaneously. To prove its potential utility, we also demonstrated that this probe can be conveniently used for simultaneous detection and discrimination of Cys and GSH in living cells. Considering its readily available and excellent sensing properties, this probe provided a potentially useful tool for biothiol sensing, detection and discrimination.
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S. Ding, G. Feng / Sensors and Actuators B 235 (2016) 691–697
Scheme 1. Structures of H2 S, Cys, Hcy and GSH, and probe 1 for selective detection and simultaneous discrimination of these thiols.
2. Experimental 2.1. Reagents, materials and apparatus All chemicals were purchased from commercial suppliers and used without further purification except HBT, which was synthesized from 2-aminobenzenethiol and 2-hydroxybenzaldehyde by the published procedures [45]. 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. 1 H NMR and 13 C NMR spectra were recorded on a Varian Mercury 400 spectrometer, and resonances (ı) 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, respectively. The fluorescence quantum yields were determined in PBS buffer (10 mM, pH 7.4, 20% DMSO, v:v) at 25 ◦ C, using quinine sulfate (Ф = 0.58 in 1N H2 SO4 ) as standard. Cell imaging was performed in an inverted fluorescence microscopy with a 20× objective lens.
2.2. Synthesis of probe 1 A solution of 4-chloro-7-nitrobenzofurazan (60 mg, 0.3 mmol), HBT (57 mg, 0.25 mmol) and triethylamine (50 L) in anhydrous DMF (3 mL) was stirred at room temperature. A dark green precipitate generated gradually in the solution. After 2 h, the solution was poured into ice-water (10 mL) and the precipitate was collected on a filter, washed with CH3 OH and dried under vacuum to afford probe 1 as a pure yellow solid (83 mg, yield 85%). Mp: 219–220 ◦ C. TLC (silica plate): Rf 0.31 (petroleum ether: ethyl acetate 5:1, v/v). 1 H NMR (400 MHz, CDCl3 ): ı 8.56 (d, J = 7.8 Hz, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 7.1 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H),
7.40 (d, J = 8.0, 1H), 7.36 (d, J = 8.0, 1H), 6.53 (d, J = 8.3 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6 ) ı 161.19, 152.63, 152.46, 150.84, 145.88, 144.88, 135.61, 135.48, 133.66, 131.54, 130.96, 128.28, 127.22, 126.26, 125.82, 123.41, 123.11, 122.65, 110.86. EI-MS: m/z found 390.20 (M+ ). HR-MS: calcd for C19 H11 N4 O4 S+ (M+H+ ), 391.0496; found 391.0521. Elemental analysis calcd (%) for C19 H10 N4 O4 S C 58.46, H 2.58, N 14.35, S 8.21; found C 58.25, H 2.42, N 14.29, S 8.06. 2.3. Optical studies of probe 1 upon addition of various analytes Stock solutions of probe 1 (1 mM) were prepared in HPLC grade DMSO. Stock solutions (1–10 mM) of the analytes including NaHS, Cys, Hcy, GSH, Gln, Phe, Trp, Ala, leu, Thr, Ser, Asp, Ile, Met, Lys, Gly, Glv, Arg, Tyr, Pyr, His, NaF, NaCl, NaBr, NaI, Na2 S2 O7 , Na2 SO3 , Na2 CO3 , Na2 SO4 , NaHSO4 , NaNO3 , NaSCN, NaAcO, H2 NCH2 CH2 NH2 , HOCH2 CH2 NH2 , C6 H5 NH2 , C6 H5 CH2 NH2 , KCl, MnSO4 , FeSO4 , MgCl2 , CaCl2 , HgCl2 , Zn(NO3 )2 , Cd(NO3 )2 , Cu(NO3 )2 , and AgNO3 were prepared in ultrapure water. ROS/RNS were prepared according our published procedure [46,47]. For optical studies, a solution of probe 1 (10 M) was prepared in DMSO-PBS buffer solution (1:4, v/v, 10 mM PBS). Then 3.0 mL of the probe 1 solution was placed in a quartz cuvette. The UV–vis or fluorescent spectra were recorded upon addition of analyte of interest at 25 ◦ C with a temperature controller. 2.4. Cell culture and bioimaging 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 g/mL streptomycin in a 5% CO2 , water saturated incubator at 37 ◦ C, and then were seeded in a 12well culture plate for one night before cell imaging experiments. In the experiment of cell imaging, living cells were incubated with 10 M of probe 1 (with 0.1% DMSO, v/v) at 37 ◦ C for 30 min and washed with PBS for three times, and then imaged immediately. In the experiment of N-ethylmaleimide (NEM) added to the cell
S. Ding, G. Feng / Sensors and Actuators B 235 (2016) 691–697
693
Scheme 2. The synthesis of probe 1.
culture, HeLa cells were cultured with 500 M NEM for 30 min at 37 ◦ C, washed three times with PBS, and then incubated with 10 M probe 1 for 30 min at 37 ◦ C and imaged immediately. In the imaging of exogenous Cys or GSH, HeLa cells were pre-treated with NEM (500 M) for 30 min at 37 ◦ C, and washed three times with PBS. Then HeLa cells were treated with probe 1 (10 M) for 30 min at 37 ◦ C, washed three times with PBS. Finally, HeLa cells were incubated with Cys (or GSH, 100 M) for 30 min at 37 ◦ C. The imaging of cells was then carried out after washing with PBS buffer.
3. Results and discussion 3.1. Probe design and synthesis Probe 1 was designed to combine two well-known fluorophores, nitrobenzofurazan (NBD) and 2-(2 -hydroxyphenyl)benzothiazole (HBT), which are covalently linked to form an ether. HBT is a typical excited-state intramolecular proton transfer (ESIPT) dye with the merit of large Stokes shifts [25,48,49]. The principle of probe 1 for detection of thiols is illustrated in Scheme 1. First, although NBD and HBT are well-known fluorophores, NBD ether is nonfluorescent and it also can block the ESIPT process and quench the fluorescence of HBT [50–52]. Therefore, probe 1 is expected to be colorless and non-fluorescent, which would provide advantages of low backgrounds. Second, probe 1 can react with thiols by thiolysis of the NBD ether to release the strong fluorescent HBT (em ≈ 465 nm) and different NBD-derivatives. NBD-derivatives have been reported to show different optical properties depending on the thiols [53–56]. NBD-SH and NBD-GSH are S-bound NBD and they are non-fluorescent but the former is purple-red while the later is light yellow in aqueous solution. The S-bound NBD product of Cys/Hcy is not stable and they can change into the corresponding N-bound NBD products quickly via a unique nucleophilic aromatic substitution-intramolecular rearrangement [42,43,53,54]. Thus, NBD-Cys/Hcy is different to NBD-SH and NBDGSH, showing orange color and strong yellow fluorescence at about 550 nm. As a result, probe 1 was expected to show distinct and dif-
ferent optical responses for H2 S, Cys/Hcy and GSH, which would provide a simple and practical method for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. With this expectation, probe 1 was synthesized according to the route outlined in Scheme 2. Overall, probe 1 can be readily synthesized by the readily available NBD-Cl and HBT in good yield (85%), and its structure and purity were well characterized by NMR, Mass and elemental analysis (see experimental section and the Supporting information). It should be noted that the facile synthesis with readily available starting materials is important for the wide use of probes. We noticed that this probe was reported by Yi et al. [51] during we were conducting this work; however, the important ability of this probe to discriminate biothiols is still unkown.
3.2. Simultaneous detection and discrimination of thiols by probe 1 The sensing property of probe 1 was investigated in PBS buffer (10 mM, pH 7.4, containing 20% DMSO, v/v) at 25 ◦ C. As expected, probe 1 is colorless (maximum abs = 375 nm) and non-fluorescent (Ф = 0.003), and upon addition of various analytes including thiols (H2 S, Cys, Hcy and GSH. NaHS was used as a standard source for H2 S), amino acids such as Gln, Phe, Trp, Ala, leu, Thr, Ser, Asp, Ile, Met, Lys, Gly, Glv, Arg, Tyr, Pyr and His, common anions such as F− , Cl− , Br− , I− , S2 O7 2− , SO3 2− , CO3 2− , SO4 2− , NO3 − , SCN− and AcO− , other nucleophilic agents such as H2 NCH2 CH2 NH2 , HOCH2 CH2 NH2 , C6 H5 NH2 and C6 H5 CH2 NH2 , and reactive oxygen/nitrogen species (ROS/RNS) such as ClO− , H2 O2 , NO2 − , t BuOO• and • OH, we observed that the probe 1 solution showed selective changes for NaSH, Cys, Hcy and GSH over the other analytes. As shown in Fig. 1a, only the addition of NaHS, Cys, Hcy and GSH induced significant changes to the UV–vis spectrum of probe 1. Among them, the addition of NaHS induced the most remarkable red-shift ( = 165 nm, from 375 nm to 540 nm) to the maximum absorption of probe 1, while that for Cys/Hcy and GSH is only 105 nm and 45 nm, respectively. As a result, the color of the probe 1 solution changed from colorless to purple-red, orange and pale-
Fig. 1. (a) UV–vis spectra changes of probe 1 (10 M) upon addition of various analytes (100 M of each, including: (1) none, (2) Cys, (3) Hcy, (4) GSH, (5) NaHS, (6) SO3 2− , (7) CO3 2− , (8) SO4 2− , (9) S2 O7 2− , (10) Cl− , (11) Br− , (12) F− , (13) I− , (14) S2 O3 2− , (15) NO3 − , (16) SCN− , (17) AcO− , (18) H2 NCH2 CH2 NH2 , (19) HOCH2 CH2 NH2 , (20) C6 H5 NH2 , (21) C6 H5 CH2 NH2 , (22) Gln, (23) Phe, (24) Trp, (25) Ala, (26) Leu, (27) Thr, (28) Ser, (29) Asp, 30. Ile, (31) Met, (32) Lys, (33) Gly, (34) Glv, (35) Arg, (36) Tyr, (37) Pyr, (38) His, (39) ClO− , (40) H2 O2 , (41) NO2 − , (42) t BuOO• , (43) • OH). (b) The corresponding absorbance change of probe 1 at 540 nm for each analyte. Insert: A photo of color changes of probe 1 for thiols. All spectra were measured in PBS buffer (10 mM, pH 7.4) with 20% DMSO at 25 ◦ C and each spectrum was obtained 150 s after addition.
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yellow for NaHS, Cys/Hcy and GSH, respectively (insert in Fig. 1b). In contrast, other analytes did not show any noticeable changes in the UV–vis spectra or the color of probe 1 (Fig. S1). It should be noted that the color change induced by NaHS is most distinct to the “naked eyes”, which can be attributed to the production of NBDSH (max ∼540 nm) [52,55,56]. The production of NBD-SH and HBT from probe 1 upon addition of NaHS was confirmed by HPLC, NMR and Mass analysis (Fig. S2). Notably, the distinct absorbance change at 540 nm is unique for H2 S (Fig. 1b). Therefore, these results not only indicate that probe 1 can be used as a selective colorimetric sensor for H2 S, Cys, Hcy and GSH, but also indicate that probe 1 can be used to discriminate H2 S from Cys, Hcy and GSH by the characteristic absorption changes at 540 nm. Fig. 2a shows the fluorescence spectra changes of the probe 1 solution to various analytes at an excitation wavelength of 330 nm. One can see that only the addition of NaHS, Cys, Hcy and GSH induced significant fluorescence turn-on changes at 465 nm. This fluorescence enhancement can be ascribed to the formation of HBT (em ∼ 465 nm with Ф = 0.052) via the thiolysis reaction induced by thiols, which was confirmed by HPLC, TLC and Mass analysis (Fig. S2). In contrast, other analytes induced almost no changes to the fluorescence of the probe 1 solution. In addition, probe 1 is not responsive to common metal ions such as Na+ , K + , Mn2+ , Zn2+ , Mg2+ , Cd2+ , Ca2+ , Hg2+ , Cu2+ , Fe2+ , and Ag+ (Fig. S3). All these results indicate that probe 1 can be also used as a selective fluorescent turn-on sensor for H2 S, Cys, Hcy and GSH. Notably, if we select a longer excitation wavelength that is efficient to light-up the fluorescence of NBD, the probe 1 solution was found to show high selectivity for Cys/Hcy. As shown in Fig. 2c, when the excitation wavelength was set at 475 nm, only Cys/Hcy induced the probe 1 solution significant fluorescence enhancement at 550 nm, while both H2 S and GSH and other analytes showed no effect. This clearly indicates that probe 1 can be used to discriminate Cys/Hcy from H2 S and GSH. It is worth noting that when the excitation wavelength was used at 415 nm, a wavelength between 330 nm and 475 nm, we observed fluorescence enhancement of
the probe 1 solution at both 465 nm and 550 nm upon addition of Cys/Hcy, while addition of H2 S and GSH still only induced fluorescence enhancement at 465 nm (Fig. S4). These results indicate that besides the formation of HBT (emission at 465 nm), the reaction of probe 1 with Cys generated another fluorescent product, NBDCys, which was confirmed by Mass analyses (Fig. S5). Similarly, the product of NBD-GSH was also confirmed by Mass analyses from the mixture of probe 1 and GSH (Fig. S6). Therefore, the sensing mechanism of probe 1 for H2 S, Cys/Hcy, and GSH is most likely the process as illustrated in Scheme 1, which is consistent with the literature [53,54]. Overall, probe 1 can be used for simultaneous detection of these four thiols by fluorescence changes at 465 nm, or selective detection of H2 S from Cys, Hcy and GSH by absorbance changes at 540 nm, or selective detection of Cys/Hcy from H2 S and GSH by fluorescence changes at 550 nm, which provides a convenient method for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. 3.3. The sensitivity of probe 1 for thiols The sensitivity of probe 1 for H2 S, Cys/Hcy and GSH was also investigated. Kinetics studies showed that probe 1 itself is quite stable but responses rapidly to these thiols. As shown in Fig. 3a, probe 1 showed the fastest response for NaHS (completed almost immediately) and similar reactivities for other thiols (completed within 150 s). These rapid responses indicate that probe 1 could be very sensitive to H2 S, Cys/Hcy and GSH, which is highly favourable for a rapid detection. It should be noted that although Hcy exhibits a similar fluorescence response to Cys, the concentration of Hcy is generally at least ten times lower than that of Cys in living systems [54]. Thus, we focused on the sensitivity of probe 1 for H2 S, Cys and GSH. As shown in Fig. S7, the fluorescence intensity at 465 nm gradually increased with increasing amounts of each thiol, and saturation all occurred at addition of about 5 equiv. of thiols. In addition, as shown in Fig. 3b–d (also see Fig. S8–10 with ex = 330 nm), good linear relationship between the fluorescence
Fig. 2. Fluorescent spectra changes of probe 1 (10 M) in PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v) upon addition of 100 M various analytes at 25 ◦ C. (a) ex = 330 nm. (b) The corresponding fluorescence change of probe 1 at 465 nm for each analyte. (c) ex = 475 nm. (d) The corresponding fluorescence change of probe 1 at 550 nm for each analyte. Slit width: dex = dem = 5 nm. Emission color changes of probe 1 for NaHS, Cys, Hcy and GSH under a light of 365 nm and 460 nm were inserted in (a) and (c), respectively.
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Fig. 3. (a) Fluorescent kinetics of probe 1 (10 M) with 100 M of NaHS, Cys, Hcy and GSH. (b–d) Linear relationship of fluorescence change at 465 nm as a function of thiol concentration. All spectra were measured in PBS buffer (10 mM, pH 7.4, 20% DMSO, v/v) at 25 ◦ C after an incubation time of 150 s ex = 415 nm, dex = 5 nm, dem = 10 nm.
change and a certain concentration of thiols (NaHS: 0–30 M, Cys and GSH: 0–10 M) can be observed, respectively. Thus, the detection limit (S/N = 3) of probe 1 for NaHS, Cys and GSH was calculated to be about 0.10 M, 0.08 M and 0.06 M, respectively. These results indicated that probe 1 can be used to detect these thiols quantitatively with high sensitivity. Moreover, good linearity can be also observed between the absorption change at 540 nm (the characteristic optical change for NaHS) and the concentration of NaHS in the range of 0–30 M (Fig. S11). Similar result can be obtained for Cys, if we monitor the fluorescence change at 550 nm (the characteristic optical change for Cys) (Fig. S12). It should be
noted that these results are important, because we can apply the additivity of absorption and fluorescence to determine the concentration of each thiol if they coexist. To prove this, different concentrations of Cys and GSH were added to a solution of probe 1 including a certain concentration of NaHS (all of their concentrations are in the linear range), and the fluorescence intensity at 465 nm of the mixed system was tested. As shown in Fig. S13, the fluorescence intensity at 465 nm obtained from the mixture samples accords well with the additive fluorescence intensity obtained from each sample with only one component (error < 5%). Therefore, if NaHS, Cys and GSH coexist, the concentration of NaHS can
Fig. 4. Imaging and discrimination of Cys and GSH in HeLa Cells by probe 1. Row A: bright field images. Row B: fluorescent images from the blue channel with excitation wavelength at 395–420 nm. Row C: fluorescent images from the green channel with excitation wavelength at 420–485 nm. Column 1: HeLa cells were incubated with probe 1 (10 M) for 30 min. Column 2: HeLa cells were incubated with NEM (500 M) for 30 min and then treated with probe 1 (10 M) for 30 min. Column 3: HeLa cells were pre-incubated with NEM (500 M) for 30 min and then treated with Cys (100 M) for 30 min, and followed with probe 1 (10 M) for 30 min. Columns 4 is similar to Column 3, but with GSH (100 M) instead of Cys. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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be measured through the absorption at 540 nm at first, and then the concentration of Cys can be determined through the fluorescence intensity at 550 nm. Finally, the concentration of GSH can be calculated by subtracting the fluorescence intensity contributed by NaHS and Cys at 465 nm.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.05.146. References
3.4. The effect of pH The effect of pH for sensing thiols was also investigated. As shown in Fig. S14–15, probe 1 can be used for simultaneous detection and differentiation of H2 S, Cys/Hcy and GSH over a wide pH range including a physiologically relevant pH, which is favorable for its biological applications. 3.5. The potential applications of probe 1 Since Cys and GSH are the well-known two main thiols in biosystems that play separate, critically important roles in human physiology and pathology, great attention has been given in recently to the simultaneous detection and discrimination of these two thiols in living cells [34–39,41–44]. Encouraged by our aforementioned results, we evaluated the capability of probe 1 to selectively image intracellular Cys and GSH. The cytotoxicity of probe 1 was first investigated by MTT assays, and the result showed that probe 1 is of low cytotoxicity to living cells (Fig. S16). Cell imaging was then explored. As shown in Fig. 4, when HeLa cells were incubated with probe 1 (10 M) for 30 min, blue and green fluorescence was observed at different channels simultaneously (column 1). However, when the cell culture was incubated with a thiol trapping reagent, N-ethylmaleimide (NEM) [28] before incubation of probe 1, the cells showed almost no fluorescence at both channels (column 2). These results clearly indicate that probe 1 can be applied to image intracellular thiols in living cells. In another set of experiments, after HeLa cells were incubated with NEM, we added Cys and GSH to the cell culture, respectively, and then incubated the cells with probe 1. In this case, different responses were observed. As shown in Fig. 4 (column 3–4), addition of Cys generated blue and green fluorescence (column 3), while addition of GSH only induced blue fluorescence (columns 4). These results are consistent with the results shown in Fig. 2, indicating that probe 1 can be conveniently used for simultaneous detection and differentiation of Cys and GSH in living cells. 4. Conclusions In summary, we reported a rather simple but versatile fluorescent probe for simultaneous detection and discrimination of H2 S, Cys/Hcy and GSH. This smart probe not only can be easily prepared, but also shows a rapid, simultaneous detection process for these four important thiols with high selectivity and sensitivity in aqueous solution under mild conditions. Moreover, this probe is able to show unique absorbance enhancement at 540 nm for H2 S, and additional fluorescence enhancement at 550 nm only for Cys/Hcy, thus providing a simultaneous discrimination method for H2 S, Cys/Hcy and GSH. In addition, we also demonstrated that this probe can be used to simultaneously detect and discriminate of Cys and GSH in living cells with low cytotoxicity, which indicated that this probe has potential application prospects. Acknowledgments We thank the National Natural Science Foundation of China (Grant Nos. 21472066and 21172086) and the Natural Science Foundation of Hubei Province (No. 2014CFA042) for financial support.
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Biographies Shuangshuang Ding is studying for MS degree at Central China Normal University. Her research focuses on the development of new fluorescent probes. Guoqiang Feng is a Professor of the College of Chemistry, Central China Normal University (CCNU). He received his PhD in 2003 from Institute of Chemistry, Chinese Academy of Sciences. After completing postdoctoral research at the University of Sheffield and the University of Cambridge, he joined the faculty at Central China Normal University in 2009. His recent research focuses on molecular recognition and chemosensors.