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sensitive chemodosimeter for detection on bisulfate and its living cell imaging
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 220 (2019) 117148
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A dual electron-withdrawing enhanced selective/sensitive chemodosimeter for detection on bisulfate and its living cell imaging Xixi Xie a, Fangjun Huo b, Jianbin Chao b, Yongbin Zhang b, Caixia Yin a,⁎ a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
a r t i c l e
i n f o
Article history: Received 15 March 2019 Received in revised form 20 May 2019 Accepted 21 May 2019 Available online 22 May 2019 Keywords: Selective/sensitive Bisulfate Dual electron-withdrawing Imaging
a b s t r a c t Fluorescence detection of sulfur dioxide has attracted great interest from researchers in recent years. Usually double bonds and aldehyde group were employed as reaction sites for sulfur dioxide. In this work, the double bond was linked with cyano and carboxyl group as dual electron-withdrawing to enhance the reaction reactivity between the probe and sulfite. Meanwhile, coumarin with good biocompatibility was introduced as fluorophore. Thus D-π-A form constructs intramolecular charge transfer (ICT), the probe has weak yellow fluorescence emission (565 nm), after addition reaction taking place between the probe and bisulfate, conjugated double bond is broken, the system showed a short-wavelength fluorescence emission (483 nm). All these realized a ratiometric fluorescence detection for bisulfate. The study found that dual electron-withdrawing groups enhanced the specificity and sensibility (with a low detection limit 82 nM) of the probe recognizing bisulfate. These excellent properties led directly to the use of probes to image sulfur dioxide in living cells. Further applications are still being on the way. © 2019 Elsevier B.V. All rights reserved.
1. Introduction SO2 in vivo, one metabolite of amino acids containing sulfhydryl groups, is catalyzed by cysteine (Cys) under aerobic conditions via cysteine dioxygenase (CDO) and aspartate aminotransferase (AAT) in the cell [1–3]. Sulfur dioxide (SO2), as the 4th signal gas, is not only present in living organisms (as a metabolism product of thiol), but also existed far and wide in air (as one of air pollutants), foods and living goods (as an additive or bleach), and have received considerable attention in recent years [4–8]. When the pH of the environment is 7.4, such as the physiological conditions, SO2 exists in the form of hydrolyzed products, − liking SO2− 3 and HSO3 [9,10]. Studies found that SO2 or its derivatives 2− − SO3 and HSO3 in cells can change of the content of thiols in protein, cause respiratory diseases, lung cancer [11], damage the nervous system [12,13] and hinder the REDOX balance [14,15], however, in some subcellular structures, for example, in the cytosol or mitochondria, can adjust the cardiovascular function, such as lowering blood pressure, relaxing blood vessels and the negative inotropic action of the heart [2,16–18]. It is still a challenge to determine the action mechanism of sulfite derivatives during the pathology, due to the lack of bio− monitoring methods for SO2 or its derivatives SO2− 3 and HSO3 .
⁎ Corresponding author. E-mail address: [email protected] (C. Yin).
https://doi.org/10.1016/j.saa.2019.117148 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Although plenty of methods such as photo colorimetric method, titration, gas or ion chromatography [19–21], and chemiluminescent immunoassay (CLIA), electrochemical methods [22,23], and sensors, and enzyme photometry have been demonstrated, fluorescent probes are more popular than the above methods because of the simple synthesis method, short detection time, and the advantages of visually distinguishable and in vivo imaging [24–26]. A tremendous amount of fluorescent probes have been reported in recent years. For example, in 2016, a colorimetric and ratiometric fluorescent probe was developed by Zhang's group for the detection of sulfite in sugar，because of its high selectivity [27]. Feng and companions designed and synthesized a NIR fluorescent probe in 2014, a conjugated coumarin–indolium system, based ICT mechanism for detection of bisulfite/sulfite in real food samples, serum samples and living cells [7]. In 2016, Yin's group reported a fluorescent probe for the visualization of Cys metabolismbased on the dual-recognition sites for Cys and its metabolite SO2 [25]. Nevertheless, these probes have some drawbacks more or less, such as poor water solubility, nonspecific recognition and unsatisfactory detection limit. On the basis of careful reading of a large number of literatures, we rational designed using coumarin as the fluorophore of the fluorescence probe of sulfite, because of its high fluorescence quantum yield, low toxicity, great water solubility, wide spectral range and function [28–30]. In this regard, we sought to construct a new sulfite high selectivity fluorescent probe in which coumarin was conjugated with cyanoacetic acid.
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− Scheme 1. Design of probe 1 for SO2− 3 /HSO3 .
Herein, we utilized a large π-conjugated coumarin–cyanoacetic acid system, double-bonds as a sulfite binding site. Thus, cyano and carboxyl group was introduced as dual electron-withdrawing to enhance reactivity, also carboxyl group as an effective factor of water solubility, which could improve the solubility of the probe (Scheme 1). As designed, the probe displayed good water solubility and highly selectivity to sulfite with colorimetric and ratiometric fluorescent response upon which peaked with 72-fold fluorescent enhancement at 483 nm, and a detection limit as low as ∼82 nM. Therefore, this probe 1 could be used for de2− tecting HSO− in living Hela cells, which promoted the probe to 3 /SO3 detect bisulfite in the living Hela cells. 2. Material and methods 2.1. Synthesis of probe 1 Compound D was synthesized according to the previous references [31]. Compound D (0.049 g, 0.20 mmol), and 2-cyanoacetic acid (0.025 g, 0.30 mmol) were mixed and dissolved in EtOH (15 mL). 40 μL piperidine was added and the system was stirred at 78 °C refluxed for 4 h (TLC monitoring process). After cooling to 20 °C, reducing pressure distillation, the reddish brown solid was purified by column chromatography using dichloromethane/methanol/glacial acetic acid as eluent to gain 0.0576 g (0.17 mmol) probe 1 as a dark red powder with 85% yields (Scheme 2). 1H NMR (600 MHz, DMSO d6) δ (ppm): 8.76 (s, 1H), 8.25 (s, 1H), 7.61 (d, J = 9.1 Hz, 1H), 6.84 (d, J = 6.8 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 3.54 (q, J = 7.0 Hz, 5H), 1.16 (t, J = 7.0 Hz, 7H) (Fig. S1). 13C NMR (151 MHz, DMSO d6) δ (ppm): 164.1, 160.7, 158.0, 153.9, 147.4, 144.4, 132.8, 117.2, 111.2, 110.1, 108.5, 97.1, 45.1, 12.9 (Fig. S2). HR-MS [probe 1 + Na] +: m/z Calcd 335.1002, Found 335.1002 (Fig. S3). 2.2. Measurement procedure Amino acid, metal chloride salts and anionic sodium salt, which including Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val, K+, Na+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, − 2− − − 2− Zn2+, F−, Cl−, SO2− were dis4 , CO3 , H2PO4 , NO3 , HS , and S2O3 solved in distilled water to prepare the stock solution (0.2 M).
Fig. 1. (a) UV–vis absorption spectra of 10 μM probe 1 upon addition SO2− 3 (0–350 μM) in PBS-DMSO (pH 7.4, v/v, 4:1) system; (b) fluorescent response of probe 1 (10 μM) upon 2− addition of SO3 (0–300 μM) in PBS-DMSO (pH 7.4, v/v, 4:1) system. Each spectrum was obtained 15 min after addition (λex = 405 nm, slit: 2.5 nm/5 nm).
Scheme 2. The synthesis of probe 1.
X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 220 (2019) 117148
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3. Results and discussion 3.1. Spectral characteristics
Fig. 2. Working curve of probe 1 to detect Cys obtained by addition of various concentrations of SO2− 3 (0–250 μM) to probe 1 (10 μM).
Fluorescence spectrum was carried out at excitation of 415 nm with slit 2.5/5 nm.
2.3. Cell culture and imaging The HeLa cells were fostered in Dulbecco's modified Eagle's medium (DMEM) added with fetal calf serum (10%) and penicillin–streptomycin (1%) [32]. And part of cells were incubated with 10 μM of probe 1 and then imaged some minutes later. Meanwhile, part of cells were preincubated with Na2SO3, then incubated with 10 μM of probe 1 and imaged. PBS buffer solution (10 mM) was rinsed three times before each step. In addition, in order to verify the process of detecting SO2− by 3 probe 1 in cells, the Na2SO3 was pre-incubated, and the confocal fluorescence imaging intensity was implemented immediately after with 10 μM of probe 1 was incubated.
Spectra of the UV–Vis and fluorescent responses of probe 1 towards SO2− 3 were monitored systematically in PBS-DMSO (pH 7.4, 10 mM, v/v, 4:1) buffer system. As the Fig. 1a showed, the absorption peak of probe 1 (10 μM) itself in the above system was at 310 nm and 480 nm, which decreased with upon addition of Na2SO3 (350 μM) and a distinct new peak at 400 nm appeared with two isosbestic points at 335/425 nm, with a color change from red to yellow. As shown in the Fig. 1b, probe 1 presented yellow fluorescent emission in the system, however, the addition of Na2SO3 induced a ratiometric fluorescence change with fluorescent emission decreasing at 565 nm and increasing (72-fold) at 483 nm. The fluorescence intensity was linear correlative with the SO2− 3 concentration in the range of 0–250 μM, implying probe 1 as a potential quantitative detector for SO2− 3 . Linear regression equation of calibration curve of the fluorescence intensity is F483/F565 = 0.3474 c − −5 0.1907 (c: [SO2− M) with the correlation coefficient of 0.9952. 3 ]/10 The corresponding detection limit (CDL) of probe 1 for SO2− 3 was reckoned to be 82 nM based on 3 Sb/m from 10 blank solutions (Fig. 2) [33–35]. The bonding constant K is obtained as 1.42 × 104 M−1 (R2 = 0.979) between the probe and SO2− by using GraphPad Prism 7 3 (Fig. S8). The constant K for probe and SO2− 3 calculated from the plots of A0/(A − A0) versus probe 1-SO2− based on the standard Benesi3 Hildebrand method is 1.37 × 104 M−1 (R2 = 0.987) (Fig. S9) [36]. It is notable that a 1:1 complexation stoichiometry for probe 1-SO2− was 3 established through a Job's plot analysis, where the products between molar fractions and the intensity of the fluorescence intensity ratio were plotted against molar fractions of probe 1 under the condition of a constant total concentration. When the molar fraction of [probe 1]/ [probe 1 + SO2− 3 ] was 0.50, the emission intensity ratio of probe 1SO2− 3 reached maximum (Fig. S10) [37,38]. Time-dependent fluorescent response of probe 1 towards SO2− 3 (1 eq. probe 1: 30 eq. SO2− 3 ) at 483 nm showed that the determination process balanced within 15 min and the subsequent fluorescent data were all measured 15 min after the addition of analytes (Fig. S4). After doubling the volume, the reaction equilibrium should be moved backwards, and the absorption of 1-SO2− 3 should be less than 1/2 of the absorption of the undiluted system (Table S1, Fig. S13), the system is considered to be recyclable. The fluorescence response of probe 1 to
Fig. 3. (a) The fluorescence emission spectra of probe 1 towards 0.2 M various analytes (e.g. Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val, K+, Na+, Ca2 − 2− − − 2− , Mg2+, Mn2+, Ba2+, Cu2+, Zn2+, F−, Cl−, SO2− 4 , CO3 , H2PO4 , NO3 , HS , S2O3 ). All data were obtained 15 min after addition of each analyte in PBS-DMSO (4/1, v/v, pH 7.4), λex = 405 nm, slit: 2.5 nm/5 nm.
+
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sodium salt (K+, Na+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, Zn2+, F−, Cl−, − 2− − − 2− SO2− 4 , CO3 , H2PO4 , NO3 , HS , S2O3 ) were involved to study. Each of them was added into PBS-DMSO (pH 7.4, 10 mM, v/v, 4:1) buffer system containing probe. Negligible fluorescent changes took place on the spectrum both at 483 nm and at 565 of ensemble with slit 2.5/5 nm (Fig. 3). Thus, high selectivity provides an advantage that the probe can detect hydrogen sulfide in complex biological systems. 3.3. pH dependence Further, the pH effect and its applicable pH range of probe 1 were investigated. Upon addition of 300 μM SO2− 3 into a mixture of PB-DMSO (v/v, 4:1) containing 10 μM probe 1 with broad pH change from 2 to 12 (Fig. 4). The F483/F565 of probe 1 itself remains substantially during the pH changing. Meanwhile, the results showed that the probe could response well on SO2− 3 in the range of pH 4–10, manifesting its gentle pH range for the detection of SO2− 3 in vivo assay. (See Fig. 5.) 3.4. Proposed mechanism of probe 1 to SO2− 3 Fig. 4. pH effect of probe 1 towards SO2− 3 detection process, each spectrum was obtained 15 min after addition, λex = 405 nm, slit: 2.5 nm/5 nm.
sulfite is reversible. 2− 1 þ SO2− 3 ⇄1−SO3
ð1 : probe 1Þ
3.2. Selectivity of probe 1 To assess the selective effect of probe for diverse analytes, including amino acids (0.2 M Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val), some metal chloride salts and anionic
1 To investigate the proposed mechanism of probe 1 towards SO2− 3 , H NMR titration experiment and HR-MS analysis were performed. 1H NMR comparison spectra of probe 1 in DMSO d6 revealed that the original signal at 8.76 disappeared which belongs to the activated vinyl proton, and new signals appeared at 4.52 and 3.42 (Fig. S5). HR-MS (Fig. S6) data of SO2− added into containing probe 1 system displayed signal 3 peaked at 393.0761. These results can be explained that the fluorescent responses of probe 1 towards SO2− 3 were induced by the nucleophilic addition reaction of SO2− with the activated vinyl group of the 3 coumarin-cyanoacetic acid conjugated system of probe 1. To further investigate the sensing mechanism, we performed theoretical calculations by the Gaussian 09 program. The optimized structures of Probe 1 and Probe 1-SO2− in ground state are shown in 3
Fig. 5. Frontier molecular orbital plots of probe 1 and probe 1-SO2− 3 in water (CPCM model). Green and red shapes are corresponding to the different phases of the molecular wave functions for HOMO and LUMO orbitals.
X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 220 (2019) 117148
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inhibited. This could lead to a blue-shift in the absorption and emission spectra.
Table 1 Determination of sulfite in real water samples with the probe 1. Determined/μM
Add/μM
Found/μM
Recovery (%)
Tap water
1.21
Pool water
4.51
3.00 6.00 3.00 6.00
4.19 7.23 7.53 10.48
100.47 99.72 99.73 100.29
Fig. S12. The optimized structure of Probe 1 is essentially planar between the coumarin ring and the cyanoacetic acid with a dihedral angle about 7.5° via a conjugated bridge (–C_C–), whereas Probe 12− SO2− 3 has a dihedral angle about 80.5° (73° for Probe 1-SO3 ) between the coumarin ring and cyanoacetic acid. This structural difference of probe 1 and probe 1-SO2− exhibits their significant variation in π3 conjunction. We then further compared the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of compounds probe 1, probe 1-SO2− 3 . In the case of free probe 1, the electronic transition of free probe 1 is mainly contributed by HOMO-LUMO transition. The π electrons on the LUMO of probe 1 are located around the whole π-conjunction structure, however, the π electrons on the HOMO is mostly positioned at the coumarin unit. Thus, an ICT takes place through the conjugated bridge between the coumarin and cyanoacetic acid. However, the π-conjunction between coumarin ring and cyanoacetic acid moieties is destroyed. Thus, the ICT process between the coumarin ring and cyanoacetic acid of Probe 1-SO2− is 3
3.5. The probe 1 detecting SO2− 3 from various water Tap water and pool water were used for detecting the content of SO2− 3 . For optical measurements, probe 1 was diluted in water-DMSO (v/v, 4:1), and 2.0 mL of the resulting solution was placed in a cuvette each time (Table 1). The fluorescence spectra were recorded upon the addition of the analytes. 3.6. Cellular imaging Cell Counting Kit-8 (CCK-8) was used for investigating the cytotoxicity of probe 1 against HeLa cells. The experiments results displayed negligible cytotoxicity of probe 1 to cells at a concentration of 30 μM (Fig. S7). The application of probe 1 for cells imaging was then carried out. As showed in Fig. 6, comparing to the blank, probe 1 directly incubated HeLa cells showed turquoise fluorescent and distinct yellow fluorescent signals in cells [39,40]. As expected, SO2− 3 pre-incubated cells further cultured with probe 1 induced enhanced turquoise fluorescent emission while the yellow fluorescent signal changed weakly in the cytoplasm. The above results declared that probe 1 could react to endogenous and exogenous SO2− in living cells. Subsequently, after treated 3 with 200 μM SO2− 3 , and 10 μM probe 1 successively, the fluorescent emission in the turquoise channel enhanced gradually while yellow
Fig. 6. (a) Free; (b) Hela cells incubated with probe 1 (10 μM) for 20 min; (c) Hela cells pre-treated with 200 μM SO2− 3 for 15 min, then incubated with probe 1 (10 μM) for 15 min. Scale bar: 20 μm.
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Fig. 7. Time-dependent confocal images of exogenous SO2− 3 in Hela cells. Hela cells pretreated with 200 μM Na2SO3 for 15 min, then incubated with probe 1 (10 μM). Turquoise channel, λem = 440–530 nm (λex = 405 nm); yellow channel, λem = 540–600 nm (λex = 405 nm). Scale bar: 20 μm.
channel changed weakly for within 10 min (Fig. 7), which reflected the efficient response time of probe 1 towards SO2− 3 in the cells. 4. Conclusions In general, we developed a ratiometric fluorescent probe based on D-π-A form constructs intramolecular charge transfer (ICT), after addition reaction taking place between the probe and sulfite, conjugated double bond was broken. The study found that dual electronwithdrawing groups enhanced the specificity and sensibility (with a low detection limit 82 nM) of the probe recognizing bisulfate. These excellent properties led directly to the use of probes to image sulfur dioxide in living cells. Acknowledgments We thank the National Natural Science Foundation of China (Nos. 21775096, 21672131, 21705102), One Hundred People Plan of Shanxi Province, Shanxi Province “1331 Project” Key Innovation Team Construction Plan Cultivation Team (2018-CT-1), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi Collaborative Innovation Center of High Value-added Utilization of Coal-related Wastes, China Institute for Radiation Production and Scientific Instrument Center of Shanxi University (201512). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117148. References [1] W. Xu, C.L. Teoh, J.J. Peng, D.D. Su, L. Yuan, Y.T. Chang, A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells, Biomaterials 56 (2015) 1–9.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A dual electron-withdrawing enhanced selective/sensitive chemodosimeter for detection on bisulfate and its living cell imaging Xixi Xie a, Fangjun Huo b, Jianbin Chao b, Yongbin Zhang b, Caixia Yin a,⁎ a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
a r t i c l e
i n f o
Article history: Received 15 March 2019 Received in revised form 20 May 2019 Accepted 21 May 2019 Available online 22 May 2019 Keywords: Selective/sensitive Bisulfate Dual electron-withdrawing Imaging
a b s t r a c t Fluorescence detection of sulfur dioxide has attracted great interest from researchers in recent years. Usually double bonds and aldehyde group were employed as reaction sites for sulfur dioxide. In this work, the double bond was linked with cyano and carboxyl group as dual electron-withdrawing to enhance the reaction reactivity between the probe and sulfite. Meanwhile, coumarin with good biocompatibility was introduced as fluorophore. Thus D-π-A form constructs intramolecular charge transfer (ICT), the probe has weak yellow fluorescence emission (565 nm), after addition reaction taking place between the probe and bisulfate, conjugated double bond is broken, the system showed a short-wavelength fluorescence emission (483 nm). All these realized a ratiometric fluorescence detection for bisulfate. The study found that dual electron-withdrawing groups enhanced the specificity and sensibility (with a low detection limit 82 nM) of the probe recognizing bisulfate. These excellent properties led directly to the use of probes to image sulfur dioxide in living cells. Further applications are still being on the way. © 2019 Elsevier B.V. All rights reserved.
1. Introduction SO2 in vivo, one metabolite of amino acids containing sulfhydryl groups, is catalyzed by cysteine (Cys) under aerobic conditions via cysteine dioxygenase (CDO) and aspartate aminotransferase (AAT) in the cell [1–3]. Sulfur dioxide (SO2), as the 4th signal gas, is not only present in living organisms (as a metabolism product of thiol), but also existed far and wide in air (as one of air pollutants), foods and living goods (as an additive or bleach), and have received considerable attention in recent years [4–8]. When the pH of the environment is 7.4, such as the physiological conditions, SO2 exists in the form of hydrolyzed products, − liking SO2− 3 and HSO3 [9,10]. Studies found that SO2 or its derivatives 2− − SO3 and HSO3 in cells can change of the content of thiols in protein, cause respiratory diseases, lung cancer [11], damage the nervous system [12,13] and hinder the REDOX balance [14,15], however, in some subcellular structures, for example, in the cytosol or mitochondria, can adjust the cardiovascular function, such as lowering blood pressure, relaxing blood vessels and the negative inotropic action of the heart [2,16–18]. It is still a challenge to determine the action mechanism of sulfite derivatives during the pathology, due to the lack of bio− monitoring methods for SO2 or its derivatives SO2− 3 and HSO3 .
⁎ Corresponding author. E-mail address: [email protected] (C. Yin).
https://doi.org/10.1016/j.saa.2019.117148 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Although plenty of methods such as photo colorimetric method, titration, gas or ion chromatography [19–21], and chemiluminescent immunoassay (CLIA), electrochemical methods [22,23], and sensors, and enzyme photometry have been demonstrated, fluorescent probes are more popular than the above methods because of the simple synthesis method, short detection time, and the advantages of visually distinguishable and in vivo imaging [24–26]. A tremendous amount of fluorescent probes have been reported in recent years. For example, in 2016, a colorimetric and ratiometric fluorescent probe was developed by Zhang's group for the detection of sulfite in sugar，because of its high selectivity [27]. Feng and companions designed and synthesized a NIR fluorescent probe in 2014, a conjugated coumarin–indolium system, based ICT mechanism for detection of bisulfite/sulfite in real food samples, serum samples and living cells [7]. In 2016, Yin's group reported a fluorescent probe for the visualization of Cys metabolismbased on the dual-recognition sites for Cys and its metabolite SO2 [25]. Nevertheless, these probes have some drawbacks more or less, such as poor water solubility, nonspecific recognition and unsatisfactory detection limit. On the basis of careful reading of a large number of literatures, we rational designed using coumarin as the fluorophore of the fluorescence probe of sulfite, because of its high fluorescence quantum yield, low toxicity, great water solubility, wide spectral range and function [28–30]. In this regard, we sought to construct a new sulfite high selectivity fluorescent probe in which coumarin was conjugated with cyanoacetic acid.
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− Scheme 1. Design of probe 1 for SO2− 3 /HSO3 .
Herein, we utilized a large π-conjugated coumarin–cyanoacetic acid system, double-bonds as a sulfite binding site. Thus, cyano and carboxyl group was introduced as dual electron-withdrawing to enhance reactivity, also carboxyl group as an effective factor of water solubility, which could improve the solubility of the probe (Scheme 1). As designed, the probe displayed good water solubility and highly selectivity to sulfite with colorimetric and ratiometric fluorescent response upon which peaked with 72-fold fluorescent enhancement at 483 nm, and a detection limit as low as ∼82 nM. Therefore, this probe 1 could be used for de2− tecting HSO− in living Hela cells, which promoted the probe to 3 /SO3 detect bisulfite in the living Hela cells. 2. Material and methods 2.1. Synthesis of probe 1 Compound D was synthesized according to the previous references [31]. Compound D (0.049 g, 0.20 mmol), and 2-cyanoacetic acid (0.025 g, 0.30 mmol) were mixed and dissolved in EtOH (15 mL). 40 μL piperidine was added and the system was stirred at 78 °C refluxed for 4 h (TLC monitoring process). After cooling to 20 °C, reducing pressure distillation, the reddish brown solid was purified by column chromatography using dichloromethane/methanol/glacial acetic acid as eluent to gain 0.0576 g (0.17 mmol) probe 1 as a dark red powder with 85% yields (Scheme 2). 1H NMR (600 MHz, DMSO d6) δ (ppm): 8.76 (s, 1H), 8.25 (s, 1H), 7.61 (d, J = 9.1 Hz, 1H), 6.84 (d, J = 6.8 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 3.54 (q, J = 7.0 Hz, 5H), 1.16 (t, J = 7.0 Hz, 7H) (Fig. S1). 13C NMR (151 MHz, DMSO d6) δ (ppm): 164.1, 160.7, 158.0, 153.9, 147.4, 144.4, 132.8, 117.2, 111.2, 110.1, 108.5, 97.1, 45.1, 12.9 (Fig. S2). HR-MS [probe 1 + Na] +: m/z Calcd 335.1002, Found 335.1002 (Fig. S3). 2.2. Measurement procedure Amino acid, metal chloride salts and anionic sodium salt, which including Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val, K+, Na+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, − 2− − − 2− Zn2+, F−, Cl−, SO2− were dis4 , CO3 , H2PO4 , NO3 , HS , and S2O3 solved in distilled water to prepare the stock solution (0.2 M).
Fig. 1. (a) UV–vis absorption spectra of 10 μM probe 1 upon addition SO2− 3 (0–350 μM) in PBS-DMSO (pH 7.4, v/v, 4:1) system; (b) fluorescent response of probe 1 (10 μM) upon 2− addition of SO3 (0–300 μM) in PBS-DMSO (pH 7.4, v/v, 4:1) system. Each spectrum was obtained 15 min after addition (λex = 405 nm, slit: 2.5 nm/5 nm).
Scheme 2. The synthesis of probe 1.
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3
3. Results and discussion 3.1. Spectral characteristics
Fig. 2. Working curve of probe 1 to detect Cys obtained by addition of various concentrations of SO2− 3 (0–250 μM) to probe 1 (10 μM).
Fluorescence spectrum was carried out at excitation of 415 nm with slit 2.5/5 nm.
2.3. Cell culture and imaging The HeLa cells were fostered in Dulbecco's modified Eagle's medium (DMEM) added with fetal calf serum (10%) and penicillin–streptomycin (1%) [32]. And part of cells were incubated with 10 μM of probe 1 and then imaged some minutes later. Meanwhile, part of cells were preincubated with Na2SO3, then incubated with 10 μM of probe 1 and imaged. PBS buffer solution (10 mM) was rinsed three times before each step. In addition, in order to verify the process of detecting SO2− by 3 probe 1 in cells, the Na2SO3 was pre-incubated, and the confocal fluorescence imaging intensity was implemented immediately after with 10 μM of probe 1 was incubated.
Spectra of the UV–Vis and fluorescent responses of probe 1 towards SO2− 3 were monitored systematically in PBS-DMSO (pH 7.4, 10 mM, v/v, 4:1) buffer system. As the Fig. 1a showed, the absorption peak of probe 1 (10 μM) itself in the above system was at 310 nm and 480 nm, which decreased with upon addition of Na2SO3 (350 μM) and a distinct new peak at 400 nm appeared with two isosbestic points at 335/425 nm, with a color change from red to yellow. As shown in the Fig. 1b, probe 1 presented yellow fluorescent emission in the system, however, the addition of Na2SO3 induced a ratiometric fluorescence change with fluorescent emission decreasing at 565 nm and increasing (72-fold) at 483 nm. The fluorescence intensity was linear correlative with the SO2− 3 concentration in the range of 0–250 μM, implying probe 1 as a potential quantitative detector for SO2− 3 . Linear regression equation of calibration curve of the fluorescence intensity is F483/F565 = 0.3474 c − −5 0.1907 (c: [SO2− M) with the correlation coefficient of 0.9952. 3 ]/10 The corresponding detection limit (CDL) of probe 1 for SO2− 3 was reckoned to be 82 nM based on 3 Sb/m from 10 blank solutions (Fig. 2) [33–35]. The bonding constant K is obtained as 1.42 × 104 M−1 (R2 = 0.979) between the probe and SO2− by using GraphPad Prism 7 3 (Fig. S8). The constant K for probe and SO2− 3 calculated from the plots of A0/(A − A0) versus probe 1-SO2− based on the standard Benesi3 Hildebrand method is 1.37 × 104 M−1 (R2 = 0.987) (Fig. S9) [36]. It is notable that a 1:1 complexation stoichiometry for probe 1-SO2− was 3 established through a Job's plot analysis, where the products between molar fractions and the intensity of the fluorescence intensity ratio were plotted against molar fractions of probe 1 under the condition of a constant total concentration. When the molar fraction of [probe 1]/ [probe 1 + SO2− 3 ] was 0.50, the emission intensity ratio of probe 1SO2− 3 reached maximum (Fig. S10) [37,38]. Time-dependent fluorescent response of probe 1 towards SO2− 3 (1 eq. probe 1: 30 eq. SO2− 3 ) at 483 nm showed that the determination process balanced within 15 min and the subsequent fluorescent data were all measured 15 min after the addition of analytes (Fig. S4). After doubling the volume, the reaction equilibrium should be moved backwards, and the absorption of 1-SO2− 3 should be less than 1/2 of the absorption of the undiluted system (Table S1, Fig. S13), the system is considered to be recyclable. The fluorescence response of probe 1 to
Fig. 3. (a) The fluorescence emission spectra of probe 1 towards 0.2 M various analytes (e.g. Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val, K+, Na+, Ca2 − 2− − − 2− , Mg2+, Mn2+, Ba2+, Cu2+, Zn2+, F−, Cl−, SO2− 4 , CO3 , H2PO4 , NO3 , HS , S2O3 ). All data were obtained 15 min after addition of each analyte in PBS-DMSO (4/1, v/v, pH 7.4), λex = 405 nm, slit: 2.5 nm/5 nm.
+
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sodium salt (K+, Na+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, Zn2+, F−, Cl−, − 2− − − 2− SO2− 4 , CO3 , H2PO4 , NO3 , HS , S2O3 ) were involved to study. Each of them was added into PBS-DMSO (pH 7.4, 10 mM, v/v, 4:1) buffer system containing probe. Negligible fluorescent changes took place on the spectrum both at 483 nm and at 565 of ensemble with slit 2.5/5 nm (Fig. 3). Thus, high selectivity provides an advantage that the probe can detect hydrogen sulfide in complex biological systems. 3.3. pH dependence Further, the pH effect and its applicable pH range of probe 1 were investigated. Upon addition of 300 μM SO2− 3 into a mixture of PB-DMSO (v/v, 4:1) containing 10 μM probe 1 with broad pH change from 2 to 12 (Fig. 4). The F483/F565 of probe 1 itself remains substantially during the pH changing. Meanwhile, the results showed that the probe could response well on SO2− 3 in the range of pH 4–10, manifesting its gentle pH range for the detection of SO2− 3 in vivo assay. (See Fig. 5.) 3.4. Proposed mechanism of probe 1 to SO2− 3 Fig. 4. pH effect of probe 1 towards SO2− 3 detection process, each spectrum was obtained 15 min after addition, λex = 405 nm, slit: 2.5 nm/5 nm.
sulfite is reversible. 2− 1 þ SO2− 3 ⇄1−SO3
ð1 : probe 1Þ
3.2. Selectivity of probe 1 To assess the selective effect of probe for diverse analytes, including amino acids (0.2 M Cys, GSH, Hcy, Arg, Asp, Cystine, Glu, Gly, Leu, Lys, Met, Phe, Pro, Thr, Try, Tyr, Val), some metal chloride salts and anionic
1 To investigate the proposed mechanism of probe 1 towards SO2− 3 , H NMR titration experiment and HR-MS analysis were performed. 1H NMR comparison spectra of probe 1 in DMSO d6 revealed that the original signal at 8.76 disappeared which belongs to the activated vinyl proton, and new signals appeared at 4.52 and 3.42 (Fig. S5). HR-MS (Fig. S6) data of SO2− added into containing probe 1 system displayed signal 3 peaked at 393.0761. These results can be explained that the fluorescent responses of probe 1 towards SO2− 3 were induced by the nucleophilic addition reaction of SO2− with the activated vinyl group of the 3 coumarin-cyanoacetic acid conjugated system of probe 1. To further investigate the sensing mechanism, we performed theoretical calculations by the Gaussian 09 program. The optimized structures of Probe 1 and Probe 1-SO2− in ground state are shown in 3
Fig. 5. Frontier molecular orbital plots of probe 1 and probe 1-SO2− 3 in water (CPCM model). Green and red shapes are corresponding to the different phases of the molecular wave functions for HOMO and LUMO orbitals.
X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 220 (2019) 117148
5
inhibited. This could lead to a blue-shift in the absorption and emission spectra.
Table 1 Determination of sulfite in real water samples with the probe 1. Determined/μM
Add/μM
Found/μM
Recovery (%)
Tap water
1.21
Pool water
4.51
3.00 6.00 3.00 6.00
4.19 7.23 7.53 10.48
100.47 99.72 99.73 100.29
Fig. S12. The optimized structure of Probe 1 is essentially planar between the coumarin ring and the cyanoacetic acid with a dihedral angle about 7.5° via a conjugated bridge (–C_C–), whereas Probe 12− SO2− 3 has a dihedral angle about 80.5° (73° for Probe 1-SO3 ) between the coumarin ring and cyanoacetic acid. This structural difference of probe 1 and probe 1-SO2− exhibits their significant variation in π3 conjunction. We then further compared the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of compounds probe 1, probe 1-SO2− 3 . In the case of free probe 1, the electronic transition of free probe 1 is mainly contributed by HOMO-LUMO transition. The π electrons on the LUMO of probe 1 are located around the whole π-conjunction structure, however, the π electrons on the HOMO is mostly positioned at the coumarin unit. Thus, an ICT takes place through the conjugated bridge between the coumarin and cyanoacetic acid. However, the π-conjunction between coumarin ring and cyanoacetic acid moieties is destroyed. Thus, the ICT process between the coumarin ring and cyanoacetic acid of Probe 1-SO2− is 3
3.5. The probe 1 detecting SO2− 3 from various water Tap water and pool water were used for detecting the content of SO2− 3 . For optical measurements, probe 1 was diluted in water-DMSO (v/v, 4:1), and 2.0 mL of the resulting solution was placed in a cuvette each time (Table 1). The fluorescence spectra were recorded upon the addition of the analytes. 3.6. Cellular imaging Cell Counting Kit-8 (CCK-8) was used for investigating the cytotoxicity of probe 1 against HeLa cells. The experiments results displayed negligible cytotoxicity of probe 1 to cells at a concentration of 30 μM (Fig. S7). The application of probe 1 for cells imaging was then carried out. As showed in Fig. 6, comparing to the blank, probe 1 directly incubated HeLa cells showed turquoise fluorescent and distinct yellow fluorescent signals in cells [39,40]. As expected, SO2− 3 pre-incubated cells further cultured with probe 1 induced enhanced turquoise fluorescent emission while the yellow fluorescent signal changed weakly in the cytoplasm. The above results declared that probe 1 could react to endogenous and exogenous SO2− in living cells. Subsequently, after treated 3 with 200 μM SO2− 3 , and 10 μM probe 1 successively, the fluorescent emission in the turquoise channel enhanced gradually while yellow
Fig. 6. (a) Free; (b) Hela cells incubated with probe 1 (10 μM) for 20 min; (c) Hela cells pre-treated with 200 μM SO2− 3 for 15 min, then incubated with probe 1 (10 μM) for 15 min. Scale bar: 20 μm.
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Fig. 7. Time-dependent confocal images of exogenous SO2− 3 in Hela cells. Hela cells pretreated with 200 μM Na2SO3 for 15 min, then incubated with probe 1 (10 μM). Turquoise channel, λem = 440–530 nm (λex = 405 nm); yellow channel, λem = 540–600 nm (λex = 405 nm). Scale bar: 20 μm.
channel changed weakly for within 10 min (Fig. 7), which reflected the efficient response time of probe 1 towards SO2− 3 in the cells. 4. Conclusions In general, we developed a ratiometric fluorescent probe based on D-π-A form constructs intramolecular charge transfer (ICT), after addition reaction taking place between the probe and sulfite, conjugated double bond was broken. The study found that dual electronwithdrawing groups enhanced the specificity and sensibility (with a low detection limit 82 nM) of the probe recognizing bisulfate. These excellent properties led directly to the use of probes to image sulfur dioxide in living cells. Acknowledgments We thank the National Natural Science Foundation of China (Nos. 21775096, 21672131, 21705102), One Hundred People Plan of Shanxi Province, Shanxi Province “1331 Project” Key Innovation Team Construction Plan Cultivation Team (2018-CT-1), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi Collaborative Innovation Center of High Value-added Utilization of Coal-related Wastes, China Institute for Radiation Production and Scientific Instrument Center of Shanxi University (201512). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117148. References [1] W. Xu, C.L. Teoh, J.J. Peng, D.D. Su, L. Yuan, Y.T. Chang, A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells, Biomaterials 56 (2015) 1–9.
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