A ratiometric fluorescent probe with DNBS group for biothiols in aqueous solution

Sensors and Actuators B 223 (2016) 274–279

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A ratiometric fluorescent probe with DNBS group for biothiols in aqueous solution Xi Dai a , Tao Zhang b , Jun-Ying Miao b,∗ , Bao-Xiang Zhao a,∗ a b

Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 14 September 2015 Accepted 20 September 2015 Available online 25 September 2015 Keywords: Ratiometric fluorescence Two-photon Aqueous solution Cell imaging

a b s t r a c t A ratiometric fluorescent probe containing a comarin-pyrazoline fluorophore and a 2,4dinitrobenzenesulfonyl (DNBS) group for thiols has been developed. The probe can fast recognize thiols under both one-photon and two-photon excitations in PBS solution with low detection limits (one-photon 10−8 M and two-photon 10−7 M). The sensing mechanism was confirmed by using ESI-MS and fluorescent spectra. Moreover, the probe was successfully used for the fluorescent imaging of living cells and calf serum. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Low molecular weight biological thiols such as cysteine (Cys), glutathione (GSH) and homocysteine (Hcy) are essential for the physiological processes [1,2]. Cys plays a crucial role in maintaining detoxification, metabolism and biocatalysis [3,4]. GSH is the most abundant non-protein thiol and the main player in either regulating the reducing environment in cells, gene regulation or combating oxidative stress [5]. Furthermore, unexpected changes in the intracellular thiol concentrations in biological fluids usually cause a series of diseases, including liver damage, neurotoxicity, diabetes mellitus, autism spectrum disorders, cytotoxicity and cardiomyopathy [6–8]. Therefore, the qualitative and quantitative assay of individual biothiol is imperative for exploring pathogeny. Although several methods have been developed to detect intracellular thiols [9–13], fluorescence detection has been accepted as the most convenient method due to its low detection limit and simplicity [14–18]. In the past decade, researchers have developed thiol-selective fluorescent probes based on the strong nucleophilicity of the sulfhydryl group, which can readily react with electrophiles such as Michael acceptors and alkylating agents [19–22]. 2,4-Dinitrobenzenesulfonyl (DNBS) group, as a functional

∗ Corresponding author. E-mail addresses: [email protected] (J.-Y. Miao), [email protected] (B.-X. Zhao). http://dx.doi.org/10.1016/j.snb.2015.09.106 0925-4005/© 2015 Elsevier B.V. All rights reserved.

trigger moiety, has been popularly used to detect thiols [23–38] since the first probe reported by Maeda et al. [39]. The outstanding characteristics of those thiol probes include excellent water solubility [40–42], real-time detection [43–45], colorimetric response [45–47] and red fluorescence [48–53]. However, ratiometric fluorescent probes based on DNBS have not been reported because of its strong electron-withdrawing effect. Even though, a strategy concerning dual-reactive or dual-quenching groups was used, the new probe presented a single emission peak [37]. In addition, the excitation wavelengths of reported probes mainly focus on 300–500 nm except two modified BODIPY probes [47,54]. Therefore, ratiometric fluorescent probes based on DNBS with excellent solubility and optical property should be developed. Consequently, we designed a ratiometric and water-soluble fluorescent probe (DPCS) containing a coumarin-pyrazoline moiety as fluorophore and a DNBS group as the reaction site for thiols. Moreover, probe DPCS shows broad prospect for application under both one and two-photon excitations. 2. Experimental 2.1. Materials and methods Thin-layer chromatography (TLC) involved silica gel 60 F254 plates (Merck KGaA) and column chromatography involved silica gel (mesh 200–300). 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were acquired on a Bruker Avance 400 spectrometer, with

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CDCl3 or DMSO used as a solvent and tetramethylsilane (TMS) as an internal standard. Melting points were determined with an XD4 digital micro-melting-point apparatus. IR spectra were recorded with an infra-red (IR) spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). UV–vis spectra were measured by the use of a Hitachi U-4100 spectrophotometer. Fluorescent measurements were performed on a Perkin–Elmer LS-55 luminescence spectrophotometer. Quartz cuvettes with a 1 cm path length and 3 mL volume were used for all measurements. The pH was determined with a model PHS3C pH meter. Unless otherwise stated, all reagents were purchased from J&K or Sinopharm Chemical Reagent Co. and used without further purification. Twice-distilled water was used throughout all experiments. The salts used in stock aqueous solutions of metal ions were KNO3 , Ca(NO3 )2 ·4H2 O, Cu(NO3 )2 ·3H2 O, NaNO3 , Mg(NO3 )2 ·6H2 O, Zn(NO3 )2 ·6H2 O, Fe(NO3 )3 ·9H2 O.

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The fluorescence intensity quantitation was carried out using the ImageJ image analysis tool. 2.5. Cytotoxicity assay The cytotoxicity in vitro of the probe to A549 cells was obtained through a standard sulforhodamine B (SRB) assay. Briefly, A549 cells were loaded in 96-well culture plates at 4 × 104 cells per well. After cultured for 12 h, cells were incubated with fresh 1640 containing 10 ␮M the probe for 12 h. Then cells were fixed with 4% TCA for 1 h at 4 ◦ C, and washed 5 times with deionized water. 50 ␮L SRB was added to each well for 10 min, and then removed by washing each well with 1% acetic acid solution and 100 ␮L Tris–HCl. Absorbance at 540 nm was measured in a 96-well multiwell-plate reader (TECAN). 3. Results and discussion

2.2. Absorption and fluorescence spectroscopy A 1.0 × 10−3 M stock solution of probe DPCS was prepared in DMSO. The amino acids (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, sarcosine, serine, threonine, tryptophan, valine), cationic (K+ , Ca2+ , Na+ , Mg2+ , Zn2+ , Fe3+ , Cu2+ ), H2 O2 and glucose stocks were all in deionized water at 10−2 M for UV–vis absorption and fluorescence analysis. Test solutions were prepared by displacing 100 ␮L of the stock solution and an appropriate aliquot of each testing species solution into a 10 mL volumetric flask, and the solution was diluted to 10 mL in an aqueous solution at pH 7.4 (PBS buffer, 1 mM CTAB). The resulting solution was shaken well and incubated for 4.5 h at room temperature before recording the spectra. Fluorescence quantum yield was determined at room temperature with fluorescein (˚s = 0.95 in 0.1 M NaOH) as standard, and it was calculated by Eq. (1), ˚ = ˚s (IAs /Is A)(2 /s2 )

(1)

in which, A is the absorbance, I is the integrated fluorescence intensity, and  is the refractive index of the solvent [55,56]. 2.3. Quantification of thiols in calf serum Calf serum (CS, from Hyclone) was divided into 8 groups, each group diluted with different multiples of PBS: no calf serum (PBS); dilution 100 times (0.01 × CS), 50 times (0.02 × CS), 20 times (0.05 × CS), 10 times (0.1 × CS), 5 times (0.2 × CS), 2 times (0.5 × CS) and not diluted (1 × CS). Then CS was incubated with probe DPCS (stock solution: 0.1 M in DMSO), and the reaction was blended in an oscillator to a required concentration of DPCS (5 ␮M). After incubation for 60 min at 37 ◦ C in the incubator, the reaction liquid underwent photoluminescence imaging measurement by luminescence microscopy (Nikon TE2000-E). Each reaction liquid was moved to black 96-well plates, and the photoluminescence image was recorded by a fluorescence microplate reader (Victor 3TM). 2.4. Cell culture and cell imaging A549 cells were cultured in DMEM. After adherent-cultured in 24-well culture plates for 12 h, A549 cells were washed from the culture medium, and then incubated with 1, 5, 10 ␮M probe solution at 37 ◦ C, respectively. Finally, the cells were washed 3 times with phosphate buffered saline (PBS) and imaged with a Nikon TE2000-E fluorescent microscope. In the control experiments, cells were pretreated with a thiol scavenger N-ethylmaleimide before the addition of the probe to remove intracellular thiols.

3.1. Design and synthesis of the probe 2,4-Dinitrobenzenesulfonyl (DNBS) group was utilized as the trigger group for thiols, because of its strong electron-withdrawing and water-soluble properties [39,55]. Importantly, DNBS can be used to design turn-on fluorescent probes since the group could totally quench fluorescence to reduce background interference. Inspired by these features, we wanted to bring in a fluorophore with self-correction fluorescence [56,57], for example 7-hydroxy coumarin pyrazoline (H-DPC), which shows two emission peaks with special excitation wavelength (Fig. S1) [56]. The attractive features of H-DPC were applied recently to develop a ratiometric fluorescent probe (DPCA) for Cys [56]. As well known, the two-photon properties of coumarin dyes have been popular in biological application since the first report in 1995 [58–60]. Coumarin derivatives exhibit high two-photon absorption coefficients and their photosensitivity and photostability in the NIR region could be improved by modifying their structures [61]. Therefore, as a continuation of our study, a turn-on fluorescence probe for biothiols, 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3yl)-2-oxo-2H-chromen-7-yl 2,4-dinitrobenzenesulfonate (DPCS), was simply synthesized and confirmed by conventional IR, NMR, and HRMS spectroscopy (Scheme 1). 3.2. Sensing property of probe DPCS for thiols All the samples were studied in PBS-CTAB buffer solution without toxic organic solvents. Cationic surfactant CTAB could build a micellar system in water solution to improve the solubility and sensitivity of the probe [52,62]. Because of CTAB has cellular toxicity, it was not used in cell imaging. Biothiols (Cys, GSH and Hcy) induced an UV–vis absorption band enhancement around 458 nm with a deepened yellow solution (Fig. S2). Absorption titrated spectra showed that the absorbance at 458 nm peaked in the presence of 2 equivalent thiols (Fig. S3). In view of the character of H-DPC, fluorescence spectra of probe DPCS with different excitation wavelengths were exemplified by its reaction with GSH (Fig. 1, Fig. S4). Probe DPCS had no fluorescence no matter at any excitation wavelengths, however, GSH can induce DPCS to show two emission peaks at 470 and 540 nm under 410 nm excitation. Interestingly, with the increase of excitation wavelength, the emission intensity at 470 nm decreased and that at 540 nm increased, until at 450 nm excitation wavelength, the intensity at 470 nm disappeared and that at 540 nm peaked. Based on the ratiometric responses (I540 /I470 ) and enhancement, we selected 410 and 450 nm as the optimized excitation wavelength (Fig. S4f).

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Scheme 1. Synthesis of probe DPCS and proposed reaction mechanism of probe DPCS with thiols.

Fig. 1. Fluorescence spectra of DPCS (1 × 10−5 M) with or without GSH (10 equiv.) at different excitation wavelengths in PBS buffer solution (CTAB 1 mM, pH 7.4, slit: 10.0 nm/4.0 nm).

Firstly, we selected the wavelength of maximum absorbance as excitation wavelength, and the fluorescence spectra of DPCS with various analytes (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, sarcosine, serine, threonine,

Fig. 2. Fluorescence spectra of DPCS (1 × 10−5 M) with various analytes (10 equiv.) in PBS buffer solution (CTAB 1 mM, pH 7.4, ex = 450 nm, slit: 10.0 nm/4.0 nm). Inset photograph is DPCS with or without thiols in UV light.

tryptophan, valine, K+ , Ca2+ , Na+ , Mg2+ , Zn2+ , Fe3+ , Cu2+ , H2 O2 and glucose) were recorded. Only biothiols triggered a marked single peak at 540 nm with a color change from colorless to bright yellow (Fig. 2). Other analytes were still no response. Competitive

Fig. 3. Fluorescence spectra of DPCS (1 × 10−5 M) with or without biothiols (10 equiv.) in PBS buffer solution (CTAB 1 mM, pH 7.4, (a) ex = 900 nm; (b) ex = 410 nm, slit: 10.0 nm/4.0 nm).

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Fig. 4. Fluorescence titration spectra of DPCS (1 × 10−5 M) with GSH at various equivalents (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 4.0 and 5.0 equiv.) at (a) ex = 450 nm, (b) ex = 900 nm in PBS buffer solution (CTAB 1 mM, pH 7.4). Data are mean ± SE (bars) (n = 3).

experiments were investigated by treating DPCS with biothiols in the presence of other related amino acids and metal ions, respectively, which showed scarce interference (Fig. S5). Furthermore, two-photon fluorescence spectra and ratiometric fluorescence spectra of DPCS with biothiols were measured under the same conditions, which showed a single emission peak (em = 580 nm, ex = 900 nm) and two emission peaks (em = 540 and 470 nm, ex = 410 nm) respectively (Fig. 3). The data demonstrated that biothiols could induce strong fluorescence under one-photon or two-photon excitation. 3.3. Fluorescence spectra of probe DPCS titrated with GSH Upon addition of GSH to the solution of DPCS, the fluorescence intensity at 540 nm increased until GSH reached approximately 3.0 equivalents at 450 nm excitation (Fig. 4a, Fig. S6b). Based on the linearly proportional relation, the limit of detection (LOD) was 1.5 × 10−8 M (Fig. S7). Correspondingly, two-photon fluorescence titrated spectra were measured (ex 900 nm), which showed a gradual increase of the intensity at 580 nm (Fig. 4b, Fig. S8b). Under the optimized conditions, the LOD was calculated to be 2.8 × 10−7 M (Fig. S9). Besides, we examined the concentration-dependent fluorescence ratio changes of DPCS upon addition of GSH at ex 410 nm (Fig. S10c). It was observed that free probe was essentially nonfluorescent; however, the introduction of GSH caused a dramatic fluorescence increase at 540 and 470 nm. The data shed light on that probe DPCS could detect sensitively and quantitatively biothiols in PBS buffer solution at three different excitation wavelengths. It is significance progress that the probe has ratiometric fluorescence response. Therefore, H-DPC could be potential fluorophore to develop new fluorescent probes.

with increasing pH value from 6.0 to 10.0, the response of DPCS toward biothiols at 540 nm increased and peaked at pH 8.0. Because sulfydryl group of thiols had less nucleophilicity under weak acid condition (pH 5.0–6.0), probe DPCS did not respond to biothiols. Probe DPCS could function over a wide range of pH from 6.5 to 10.0. Finally, physiological pH 7.4 was chosen as the experimental conditions. 3.5. Mechanism of the probe response toward thiols Probe DPCS, with the electron-withdrawing group DNBS connecting to comarin fluorophores, was cleaved by sulfydryl of thiols to form compound H-DPC and new compound 5. H-DPC is responsible for the final fluorescence (Scheme 1, and Fig. S1). The reaction products were subjected to electrospray ionization mass spectral analyses (Figs. S14–S16). Every solution had always two peaks corresponding to compound H-DPC and product 5, which verified the proposed sensing mechanism. In addition, the fluorescence sensing behaviors of H-DPC, DPCS and DPCS with thiols were measured in PBS buffer solution (Fig. S17). Upon addition of thiols to non-fluorescent DPCS (Fluorescence quantum yield ˚DPCS = 0.00188), turn-on fluorescence was observed and the final intensity almost reached the intensity of precursor compound H-DPC (˚H-DPC = 0.0321). Fluorescent quantum yields

3.4. Reaction time and pH effect For better testing probe DPCS effect toward thiols, timedependent fluorescent spectra were exemplified by one of 450 nm excitation (Fig. S12). The fluorescence intensity change (I540 nm) of DPCS with GSH was the fastest, which almost peaked within 100 min. On the other hand, Cys and Hcy had the same reaction rate with DPCS, and the intensity stabilized after 250 min. Probe DPCS without any hydrolysis in 300 min exhibited stronger stabilization than reported probes including DBNS group. Follow on, the application range of pH for probe DPCS was investigated (Fig. S13). Probe DPCS was stable in a wide range of pH from 5.0 to 8.5, and appeared low level of hydrolysis at strong alkali condition (pH > 9). Obviously,

Fig. 5. Fluorescence images of DPCS (5 ␮M) with different concentrations of calf serum. Calf serum was diluted 100 times (1%), 50 times (2%), 20 times (5%), 10 times (10%), 5 times (20%), 2 times (50%) with PBS and not diluted (100%). (n > 3; *p < 0.05, **p < 0.01 vs. control).

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Fig. 6. (a) Fluorescence images of DPCS in A549 cells at different concentrations and times. (b) Fluorescence intensity quantification. Data are mean ± SE (n > 3; *p < 0.05, **p < 0.01 vs. control).

correspond to the experiment results (˚Cys = 0.0228, ˚GSH = 0.0234, ˚Hcy = 0.0237). The results conformed to the reported cleavage mechanism of sulfonate ester by thiols [33–35,39]. Moreover, if thiols were pre-incubated with N-methylmaleimide (NEM, a thiolblocking agent), there were not fluorescent emission upon the addition of probe DPCS. The phenomenon proved the specificity of probe DPCS for biothiols.

calf serum. Moreover, the sensing mechanism of probe DPCS was confirmed by ESI-MS and fluorescence spectra. Acknowledgements This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004) and the National Natural Science Foundation of China (91313303).

3.6. Application of probe DPCS Appendix A. Supplementary data To explore the practical application of probe DPCS, we incubated DPCS (5 ␮M) with different concentrations of calf serum (CS) at 37 ◦ C. We obtained an increasing bright yellow fluorescence images after 60 min (Fig. 5). Moreover, we got fluorescence images of various concentrations of CS without probe DPCS (Fig. S19). Although CS has weak autofluorescence at higher concentration, DPCS can monitor the increase of CS (Fig. 5). The images showed excellent fluorescence intensity and were superior to our previous work and that of other research groups [32,56,63,64]. Sulforhodamine B assays of probe DPCS (10 ␮M) in living A549 cells for 12 h were carried out to research the cytotoxicity (Fig. S20). The cell viability showed that probe DPCS is low cytotoxic and could be available in biological systems. Next, living A549 cells were incubated with DPCS to demonstrate its cell imaging capability to detect biothiols in vivo. Obviously, the fluorescence intensity depended on the concentrations of the probe and response time. Strong fluorescence was observed after 1 h with 10 ␮M DPCS (Fig. 6). The intensity was quantified using imageJ that gave us a clear dosedependent relationship. To confirm that fluorescence signals from cells was attributed to the reaction of the probe with biothiols, the control experiment was performed. Cells were pretreated with different concentrations of a thiol scavenger (N-ethylmaleimide) to remove the endogenous intracellular thiols, and then incubating with 10 ␮M DPCS (Fig. S22). The weak yellow fluorescence confirmed that probe DPCS was specific to biothiols in living cells. 4. Conclusions In summary, the ratiometric fluorescent probe DPCS including a coumarin-pyrazoline fluorophore and a 2,4dinitrobenzenesulfonyl group has been developed, which exhibits a special fluorescence character over other reported probes based on bond-cleaving reaction. The merits of DPCS include high sensitivity in a wide range of pH, ratio and two-photon signal variation. DPCS has been applied for the detection of thiols in living cells and

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Biographies Xi Dai received her B.Sc. in chemistry from Qufu Normal University in 2011. At present, she is a PhD student in Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University. Her current research interest involves the synthesis and application of fluorescent sensors. Tao Zhang is studying assiduously for his M.Sc. at present in Institute of Developmental Biology, School of Life Science, Shandong University. His current research interest involves the chemosensor applied in living cells. Bao-Xiang Zhao received his PhD (1998) in chemistry from the Nagoya University, Japan. Then, he became Associate Professor at Shandong University. After postdoctoral fellowship with Professor S. Blechert at Technology University Berlin, he spent his career at the Shandong University where he has been Professor of Organic Chemistry since 2000. His main scientific interests are the design and synthesis of small molecule with structural diversity for chemical biology research. One of currently his research interests is synthesis of fluorescent probe for detecting bithiols in water and in living cells. Jun-Ying Miao received her PhD (1997) in Cell Biology from the Nagoya University, Japan. She spent her career at the Shandong University where she has been Professor of Cell Biology since 1999. Her main scientific interests are the research on differentiation, autophagy, apoptosis and modulation of cell fate by small molecules with structural diversity.