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A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish Ning Wang a, Wan Xu b, Daqian Song b, Pinyi Ma b, * a b
Department of Medical Science & Education, Jilin Province People’s Hospital, Changchun, 130021, China College of Chemistry, Jilin University, Changchun, 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2019 Received in revised form 7 October 2019 Accepted 22 October 2019 Available online xxx
In this article, a fluorescent probe FCZ with fluorescein-carbazole as the basic skeleton was designed and synthesized. In contrast to the presences of other coexisting anions, active oxygen and organic thiols, the probe could be “turn-on”, exhibiting specific fluorescence performance towards hypochlorite (ClO-). Comprehensive analyses by electrospray ionization mass spectrometry (ESIeMS), thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and density functional theory/time-dependent density functional theory (DFT/TDDFT) revealed that ClO could react with the imine bond of the probe, forming fluorescein, resulting in a significant increase of fluorescence emission intensity. The probe has a detection limit for ClO in water of 0.056 mmol/L. In addition, the probe was successfully applied to detect ClO in water samples, as well as in the imaging of ClO in RAW264.7 cells and zebrafish. © 2019 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Hypochlorite (ClO) Living cells Zebrafish
1. Introduction Reactive oxygen species (ROS) is an important class of reaction intermediates that play critical roles in various biological and pathological processes. They are mainly generated by oxygen during electron transfer process and are used to prevent unnecessary chain reactions caused by alien organisms [1,2]. Excessive amount of ROS is known to increase the oxidative stress, leading to the occurrence of several neurodegenerative diseases, such as Alzheimer’s disease and neuronal degeneration, cancer, kidney disease, and cardiovascular disease [3e5]. In the presence of myeloperoxidase, Cl can react with H2O2 in the immune system and then generate hypochlorite (ClO) [6e8], which is one of the most important active oxygen in the immune system and is an indispensable disinfection in drinking water and household solutions used in our daily life. Therefore, it is important to develop a method can selectively and sensitively detect ClO. Fluorescent probe is a molecule consisting of a suitable recognition group and a fluorophore; it provides convenience for sensitive and selective detection of the targets, especially for in vivo detection [9e13]. Thus far, a variety of fluorescent probes have been developed for detecting ions or molecules [14e18]. Among them are fluorescein and its derivatives, the widely used fluorescent
* Corresponding author. E-mail address: [email protected] (P. Ma).
molecules due to their high fluorescence quantum yield, molar absorption coefficient, chemical and thermal stability, and photostability [19e22]. Another advantage of the probes lies in their structures, which can be easily modified, and the excitation and emission wavelengths can also be adjusted by changing their structures. For this reason, fluorescein and its derivatives have been used widely as fluorescent sensors in various biological/biochemical and immuno-labelling assays. In this study, we designed and synthesized a fluoresceincarbazole-based fluorescent probe, named FCZ, to detect ClO. The sensing ability of the probe in detecting various anions, ROS and organic thiols in 1% DMF aqueous solution were investigated. The mechanism of the reaction between FCZ and ClO was also studied by emission spectra, ESI-MS, and DFT/TDDFT calculations. Finally, the potential use of the probe in fluorescence imaging of ClO in living cells and zebrafish was examined. 2. Experimental section 2.1. Synthesis of FCZ probe The synthetic route of FCZ probe is shown in Fig. 1. Compound 1: 1 was synthesized based on the previously reported method [23]. Compound 2: Fluorescein (1.0 g, 3 mmol) and 30 mL of EtOH were mixed in a 100-mL round bottom flask and stirred until dissolved; and 4 mL of 80% hydrazine hydrate was added thereafter.
https://doi.org/10.1016/j.saa.2019.117692 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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N. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 1. Synthetic route of FCZ probe.
The mixture solution was heated at 100 C and refluxed for 12 h. The resultant precipitate was filtered and then washed twice with 10 mL (each) of cold EtOH, followed by twice with 10 mL (each) of water. After vacuum drying, 0.92 g of pale-yellow powder was obtained (yield: 89%). ESI-MS, m/z: 346.76 [1 þ H]þ (calcd. 347.10 1 for C20H15N2Oþ 4 ) (Fig. S1). H NMR (300 MHz, DMSO-d6) d 7.78 (m, 1H), 7.56e7.42 (m, 2H), 7.04e6.93 (m, 1H), 6.60 (s, 2H), 6.54e6.36 (m, 4H), 4.39 (s, 2H) (Fig. S2). Compound FCZ: Compound 1 (0.13 g, 0.5 mmol) and 0.35 g of compound 2 were mixed with 20 mL of anhydrous EtOH in a 100mL round bottom flask and stirred until dissolved. After that, the mixture was heated at 80 C and refluxed for 12 h. After reflux, the hot mixture was filtered then the residue was washed twice, each with 10 mL of cold MeOH. After being air-dried, 0.32 g of orangeyellow solid was finally obtained (yield: 70%). ESI-MS, m/z: 930.31 [FCZ þ Na]þ (calcd. 930.25 for C56H37N5NaOþ 8 ). HR-ESI-MS, m/z: 908.2705 [FCZ þ Na]þ (calcd. 908.2715 for C56H38N5Oþ 8) (Fig. S3). 1H NMR(300 MHz, DMSO-d6), d 9.95 (s, 4H), 9.15 (s, 2H), 8.14 (d, 2H), 7.98 (d, 2H), 7.73e7.54 (m, 6H), 7.50 (d, 2H), 7.18 (d, 2H), 6.90e6.70 (m, 4H), 6.61 (d, 4H), 6.52 (d, 4H), 4.34e4.25 (m, 2H), 1.21 (t, 3H) (Fig. S4). 13C NMR (75 MHz, DMSO-d6) d 163.47, 158.58, 152.35, 150.52, 141.03, 140.93, 138.46, 133.70, 133.43, 129.30, 129.01, 127.98, 126.15, 125.24, 123.67, 123.11, 122.19, 122.02, 121.28, 121.04, 120.25, 112.31, 110.46, 110.03, 109.33, 102.64, 65.40, 37.35, 13.62 (Fig. S5).
larvae were anesthetized and subjected to fluorescence imaging using a laser scanning confocal microscope. 3. Results and discussion 3.1. Fluorescence spectra To study the capability of FCZ in identifying anions and ROS, the experiments were carried out in a 10 mmol/L PBS buffer solution at the physiological pH (pH 7.4) containing 1% DMSO. 3.1.1. Selectivity of probe Various ions and organic thiols (each with a concentration of 100 mmol/L) were added to the FCZ solution (10 mmol/L), and the changes in emission spectra were examined. The data showed that among all solutions, only ClO caused significant changes in the fluorescence spectra of FCZ probe (lex ¼ 470 nm, lem ¼ 530 nm); other species did not significant changes (Fig. 2). This indicates that the probe has higher selectivity for ClO. 3.1.2. Emission spectra of FCZ in the presence of ClO To study the effect of ClO on the change of fluorescence spectra of FCZ, fluorescence titration was carried out in 1% DMF aqueous
2.2. Fluorescence measurements The FCZ probe was dissolved in DMSO to prepare a standard stock solution with a concentration of 1 mmol/L. Ten microliters of 1 mmol/L FCZ solution was added to 1 mL of H2O (containing 10 mmol/L PBS buffer solution, pH 7.4) containing the test ions. The ion-free FCZ (or the blank solution) was also prepared using the same procedure. The final solution contained 10 mmol/L FCZ and 1% DMSO. The fluorescence emission spectra were measured from 490 nm to 700 nm using a 1-cm quartz cuvette. The excitation wavelength was 470 nm, and the slit widths of excitation and emission were set at 5.0 nm. 2.3. Pre-treatment of cells and zebrafish RAW264.7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 C for 24 h in an atmosphere saturated with 5% CO2. The cells were inoculated on a Petri dish and subjected to fluorescence imaging using a laser scanning confocal microscope. Adult zebrafish was kept at 28.5 C under 14 h light/10 h darkness, during which the fish was allowed to mate and lay eggs. The embryos were grown in E3 culture medium. Five-day-old zebrafish
Fig. 2. Selectivity of FCZ. The red bars represent the relative emission intensity changes of FCZ (10 mmol/L) in the presence of other compounds (100 mmol/L); the blue bars represent the relative emission intensity changes that occurs upon the subsequent addition of 100 mmol/L of ClO to the above solution. From 0 to 24: none, Kþ, Naþ, Ca2þ, 2 1 Mg2þ, F, Cl, Br, I, NO 3 , AcO , HSO4 , SCN , CN , S , H2O2, $OH, O2 , O2, NO$, ONOO, TBHP, Cys, Hcy, and GSH. lex ¼ 470 nm, lem ¼ 530 nm, 10 mmol/L PBS buffer solution (pH 7.4) containing 1% DMSO.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
N. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 3. Fluorescence emission spectra of FCZ (10 mmol/L) in the presence of ClO (0e200 mmol/L). Inset: calibrations curve in the concentration range of 0e200 mmol/L of ClO. lex ¼ 470 nm, lem ¼ 530 nm, 10 mmol/L PBS buffer solution (pH 7.4) containing 1% DMSO.
solution (containing 10 mmol/L PBS buffer solution, pH 7.4). As shown in Fig. 3, FCZ did not exhibit a distinct fluorescence emission peak. It appears that the spiro ring structure of fluorescein inhibits the intramolecular conjugated chain, resulting in poor coplanarity of the molecule, which hinders the transition of intramolecular electrons. After ClO was added, the peak at around 530 nm
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gradually increased as the concentration of ClO increased, while was accompanied by a weak red shift and a typical emission peak of fluorescein group. Furthermore, in the presence of ClO, the intensity of the absorption band of FCZ at 498 nm was increased, a remarkable color change of solution from colorless to yellow (Fig. S6). These results indicate that FCZ is a promising fluorescent probe for the detection of ClO. By studying the change in fluorescence of the FCZ under 470 nm light condition, we found that the probe has good photostability. Fig. S7 shows the relationship between the time and fluorescence response before and after the addition of ClO. The result revealed that the maximum value can be reached within 1.5 min, indicating that the reaction can be completed in a short time. The responses of FCZ towards ClO at different pH conditions were also investigated (Fig. S8). In the absence of ClO, FCZ kept almost stable over a wide range of pH from 4.0 to 9.0. However, in the presence of ClO, the FCZ gave a remarkable increase fluorescence intensity in the range of 4.0e7.8. Therefore, for practical applications in biological systems, a PBS buffer solution (10 mM) with a pH of 7.4 was selected as the reaction medium. We further found that the change of relative fluorescence emission intensity of FCZ had a linear relationship with ClO concentration at a range of 0e200 mmol/L. The linear regression equation was (IeI0)/I0 ¼ 0.08112[ClO] 0.1378 (the unit of [ClO] is mmol/L) with a correlation coefficient of 0.9981. The limit of detection (LOD) of the probe in detecting ClO was 0.056 mmol/L (3s/k), which is lower than those of other fluorescent probes previously reported (Table 1). The FCZ probe was further applied to detect ClO in water samples, and the results are shown in Table 2. The concentration of sodium hypochlorite disinfectant in tap water and lake water were found to be 2.12 mmol/L and 0.05 mmol/L, respectively, while that in
Table 1 Comparison of the present method with other reported ClO selective fluorescence probe. Probe structure
Reaction medium
lex/lem (nm)
LOD (mmol/L)
Ref.
H2O (1%DMSO, PBS 10 mmol/L, pH 7.4)
470/530
0.056
This work
PBS solution (pH 7.4, 10 mmol/L).
49/530
0.068
[24]
EtOH/H2O (1:1, v/v)
560/652
0.080
[25]
20% DMF solution (10 mmol/L Tris buffer, pH 7.4)
420/500
0.15
[26]
EtOH/PBS solution (v/v ¼ 3/7, pH 7.4)
380/(455/632)
0.182
[27]
20% DMF solution (10 mmol/L PBS, pH 7.4)
488/660
0.29
[28]
DMF/PBS solution (v/v ¼ 5/5, pH 7.4, 10 mmol/L)
425/554
0.59
[29]
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Table 2 Analytical results of ClO in water samples (n ¼ 5). Sample
Spiked (mmol/L)
Found (mmol/L)
Recovery (%)
Tap water
0.00 10.00 20.00 0.00 5.00 10.00 0.00 20.00 50.00
2.12 ± 0.07 12.28 ± 0.13 21.85 ± 0.25 0.05 ± 0.02 5.38 ± 0.18 11.45 ± 0.45 Non-detected 19.07 ± 0.24 48.87 ± 0.38
e 101.60 98.65 e 106.60 114.00 e 95.35 97.74
Lake water
Bottled water
bottled water was not detected; and the recovery rates were between 95.35% and 114%. The data indicate that the probe can successfully detect ClO in water samples.
3.2. Reaction mechanism To understand the reaction mechanism, the progress of reaction between FCZ and ClO was monitored by electrospray ionization mass spectrometry (ESI-MS). When excess amount of NaClO was added to the solution, positive ion mode became disordered (Fig. 4a); by contrast, a peak at m/z ¼ 331.061 was observed in negative ion mode. After calculation and analysis, the peak was found to correspond to fluorescein anion (Fig. 4b). We also employed thin-layer chromatography (TLC) to analyze the reaction progress. The results showed that FCZ reacted with ClO to generate two major new products, as indicated by the green fluorescence spots (Fig. S9). Based on its Rf value, the new product was speculated to be sodium fluorescein. Considering the structural changes, we speculate that ClO- can break C]N of FCZ, resulting in the opening of lactam’s spiro ring, causing the conversion to carboxyl anion. To further confirm the structure of the new product, the product was separated by column chromatography (silica gel, CH2Cl2:MeOH ¼ 20:1), after which its structure was characterized by 1H NMR (Fig. S10). The results showed that the product had the structure of fluorescein, which further confirms the reaction
between FCZ and ClO- occurred. Based on the results shown above, the reaction mechanism between FCZ and ClO was elucidated, as depicted Fig. 5. The mechanism is similar to those papers previously reported [30,31]. 3.3. Theoretical calculation To understand the relationship between the structure and spectral changes of FZC after the addition of ClO, the structures, electrons and optical properties of FCZ and Flu were calculated by DFT and TDDFT using the Gaussian 16 software [32]. Solvent was calibrated by solvation model based on density (SMD) solvent model using B3LYP/6-31G (d) as the base group and water as the solvent. Fig. 6 shows the optimal geometric structure and electronic transitions of FCZ and Flu. Considering the energy, the HOMO and LUMO energy levels of FCZ and Flu were different by 3.579 eV and 3.249 eV, respectively. The main electronic transition of FCZ was HOMO/LUMO (f ¼ 0.7293, CI ¼ 0.6958, 96.8%), and the overlap of electron cloud were located mainly in the carbazole region. The calculated first vertical excitation energy was 3.086 eV, which is correspondent to the excitation wavelength of 401.78 nm. This excitation wavelength is different from that used in the experiment (470 nm); therefore, the fluorescence signal of FCZ was not observed at this wavelength. When ClO reacted with FCZ to generate Flu, the main electronic transition was HOMO/LUMO (f ¼ 0.5143, CI ¼ 0.6821, 93.1%), and the electron cloud is distributed mainly in the p-conjugated system of oxonium. Its calculated fluorescence emission wavelength was 515.31 nm, which is slightly smaller than that used in the experiment (530 nm). Considering both the effects of solvent model and intermolecular interaction, the difference is considered acceptable. Overall, the DFT/TDDFT calculation could reveal the optical properties of FCZ and Flu. 3.4. Application of FCZ in bio-imaging In order to study the possible biological application of the FCZ probe, firstly, the cytotoxicity of FCZ probe was evaluated using MCF-7 cells by MTT assay; the results are shown in Fig. S11. The cell
Fig. 4. ESI-MS spectra of FCZ upon addition of NaClO (excess) in CH3OH/H2O mixture (1:1, v/v). (a) Positive ion mode. (b) Negative ion mode.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Fig. 5. The proposed mechanism of the reaction between FCZ and ClO.
viability remained above 80% after being treated with 50 mmol/L FCZ for 24 h, which indicates that FCZ has no cytotoxicity at low concentration and after long incubation time; thus, it should be safe for using in bio-imaging. To examine whether FCZ can be used for in situ detection of ClO in living cells, fluorescence imaging experiments were carried out to detect exogenous and endogenous ClO.
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In exogenous ClO experiment, after RAW264.7 cells were incubated with 10 mmol/L FCZ for 30 min, the fluorescence signal was not obviously observed in the cells, which is consistent with the fluorescent properties of FCZ; however, this can indicate that FCZ could penetrate, to a certain extent, into the cells (Fig. 7a). After the cells were incubated with 20 mmol/L ClO for 5 min, green fluorescence was observed in the cells, which is consistent with the results from the in vitro experiments (Fig. 7b). These results indicate that the sensitive fluorescent probe FCZ can specifically recognize ClO and is suitable for in vivo fluorescence imaging. Lipopolysaccharide (LPS) and polyacid lipopolysaccharide (PMA) are known to promote the production of ClO in RAW264.7 cells, and N-acetyl-L-cysteine (NAC) is known to consume ROS in the cells [33,34]. In endogenous ClO experiments, cells were incubated with 1 mg/L LPS for 12 h, followed by 1 mg/L PMA and 10 mmol/L FCZ, each for 30 min. Obvious green fluorescence in the cells was observed, which shows that endogenous ClO was produced inside the cells upon the stimulation of LPS and
Fig. 6. Optimized structures and the molecular orbitals of FCZ and Flu.
Fig. 7. Fluorescence microscopy images in RAW264.7. (a) Cells treated with FCZ (10 mmol/L) for 30 min. (b) Cells treated with FCZ (10 mmol/L) for 30 min. Cells incubated with NaClO (20 mmol/L) for 5 min. (c) Cells incubated with LPS (1 mg/L) for 12 h and PMA (1 mg/L) for 30 min. Then the cells were loaded with FCZ (10 mmol/L) for 30 min. (d) Cells incubated with LPS (1 mg/L) for 12 h, PMA (1 mg/L) for 30 min, and NAC (0.5 mmol/L) for 10 min. Then the cells were loaded with FCZ (10 mmol/L). (e) Normalized fluorescence intensities of cells. lex ¼ 480 nm, lem ¼ 500e560 nm.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Fig. 8. In vivo fluorescence imaging of endogenous ClO in zebrafish larvae. (a) Zebrafish was incubated with FCZ (10 mmol/L) for 30 min. (b) Zebrafish was fed with LPS (2 mg/L) for 4 h, and then incubated with PMA (2 mg/L) for 30 min. Then the zebrafish was loaded with FCZ (10 mmol/L) for 30 min. (c) Zebrafish was fed with LPS (2 mg/L) for 4 h, PMA (2 mg/L) for 30 min, and NAC (1 mmol/L) for 10 min. Then the zebrafish was loaded with FCZ (10 mmol/L). (e) Normalized fluorescence intensities of zebrafish. lex ¼ 480 nm, lem ¼ 500e560 nm.
PMA (Fig. 7c). As a control experiment, the cells stimulated by LPS and PMA were subsequently incubated with 0.5 mmol/L NAC for 10 min, followed by 10 mmol/L FCZ for 30 min. As shown in Fig. 7d, only weak fluorescence signal was observed inside the cells. These results indicate that the change of fluorescence signal intensity of FCZ can be used as an indicator to track and/or to quantify endogenous ClO. In the experiment using zebrafish, fluorescence signal was not obviously observed in zebrafish larvae cultured in the presence of 10 mmol/L FCZ for 30 min (Fig. 8a). After being treated with LPS and PMA (2 mg/L each), followed by FCZ (10 mmol/L), the zebrafish larvae produce green fluorescent (Fig. 8b). Similarly to the results from cell experiment, the green fluorescence inside the zebrafish was weakened when it was treated with an active oxygen scavenger, N-acetylcysteine (NAC) (Fig. 8c). These results indicate that FCZ can be used to monitor ClO in vivo. 4. Conclusion In summary, a novel fluorescent probe FCZ was designed and synthesized to detect ClO in aqueous solution under physiological pH. The ClO sensing mechanism and the luminescence properties of the probe were investigated via ESI-MS, TLC, NMR, and DFT/ TDDFT calculations. The fluorescence titration results showed that the probe had a good linear relationship (r ¼ 0.9981) and a low LOD of 0.056 mmol/L (3s/k). In addition, the probe was successfully used in fluorescence imaging of exogenous and endogenous ClO in RAW264.7 cells. The probe also had low cytotoxicity and good cell permeability, thus can enter into the cells. In an experiment using zebrafish, the FCZ probe exhibited good biocompatibility; it may be developed as a tool for monitoring ClO in vivo. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Science and Technology Developing Foundation of Jilin Province of China (Nos. 20180201050YY and
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Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish Ning Wang a, Wan Xu b, Daqian Song b, Pinyi Ma b, * a b
Department of Medical Science & Education, Jilin Province People’s Hospital, Changchun, 130021, China College of Chemistry, Jilin University, Changchun, 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2019 Received in revised form 7 October 2019 Accepted 22 October 2019 Available online xxx
In this article, a fluorescent probe FCZ with fluorescein-carbazole as the basic skeleton was designed and synthesized. In contrast to the presences of other coexisting anions, active oxygen and organic thiols, the probe could be “turn-on”, exhibiting specific fluorescence performance towards hypochlorite (ClO-). Comprehensive analyses by electrospray ionization mass spectrometry (ESIeMS), thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and density functional theory/time-dependent density functional theory (DFT/TDDFT) revealed that ClO could react with the imine bond of the probe, forming fluorescein, resulting in a significant increase of fluorescence emission intensity. The probe has a detection limit for ClO in water of 0.056 mmol/L. In addition, the probe was successfully applied to detect ClO in water samples, as well as in the imaging of ClO in RAW264.7 cells and zebrafish. © 2019 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Hypochlorite (ClO) Living cells Zebrafish
1. Introduction Reactive oxygen species (ROS) is an important class of reaction intermediates that play critical roles in various biological and pathological processes. They are mainly generated by oxygen during electron transfer process and are used to prevent unnecessary chain reactions caused by alien organisms [1,2]. Excessive amount of ROS is known to increase the oxidative stress, leading to the occurrence of several neurodegenerative diseases, such as Alzheimer’s disease and neuronal degeneration, cancer, kidney disease, and cardiovascular disease [3e5]. In the presence of myeloperoxidase, Cl can react with H2O2 in the immune system and then generate hypochlorite (ClO) [6e8], which is one of the most important active oxygen in the immune system and is an indispensable disinfection in drinking water and household solutions used in our daily life. Therefore, it is important to develop a method can selectively and sensitively detect ClO. Fluorescent probe is a molecule consisting of a suitable recognition group and a fluorophore; it provides convenience for sensitive and selective detection of the targets, especially for in vivo detection [9e13]. Thus far, a variety of fluorescent probes have been developed for detecting ions or molecules [14e18]. Among them are fluorescein and its derivatives, the widely used fluorescent
* Corresponding author. E-mail address: [email protected] (P. Ma).
molecules due to their high fluorescence quantum yield, molar absorption coefficient, chemical and thermal stability, and photostability [19e22]. Another advantage of the probes lies in their structures, which can be easily modified, and the excitation and emission wavelengths can also be adjusted by changing their structures. For this reason, fluorescein and its derivatives have been used widely as fluorescent sensors in various biological/biochemical and immuno-labelling assays. In this study, we designed and synthesized a fluoresceincarbazole-based fluorescent probe, named FCZ, to detect ClO. The sensing ability of the probe in detecting various anions, ROS and organic thiols in 1% DMF aqueous solution were investigated. The mechanism of the reaction between FCZ and ClO was also studied by emission spectra, ESI-MS, and DFT/TDDFT calculations. Finally, the potential use of the probe in fluorescence imaging of ClO in living cells and zebrafish was examined. 2. Experimental section 2.1. Synthesis of FCZ probe The synthetic route of FCZ probe is shown in Fig. 1. Compound 1: 1 was synthesized based on the previously reported method [23]. Compound 2: Fluorescein (1.0 g, 3 mmol) and 30 mL of EtOH were mixed in a 100-mL round bottom flask and stirred until dissolved; and 4 mL of 80% hydrazine hydrate was added thereafter.
https://doi.org/10.1016/j.saa.2019.117692 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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N. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 1. Synthetic route of FCZ probe.
The mixture solution was heated at 100 C and refluxed for 12 h. The resultant precipitate was filtered and then washed twice with 10 mL (each) of cold EtOH, followed by twice with 10 mL (each) of water. After vacuum drying, 0.92 g of pale-yellow powder was obtained (yield: 89%). ESI-MS, m/z: 346.76 [1 þ H]þ (calcd. 347.10 1 for C20H15N2Oþ 4 ) (Fig. S1). H NMR (300 MHz, DMSO-d6) d 7.78 (m, 1H), 7.56e7.42 (m, 2H), 7.04e6.93 (m, 1H), 6.60 (s, 2H), 6.54e6.36 (m, 4H), 4.39 (s, 2H) (Fig. S2). Compound FCZ: Compound 1 (0.13 g, 0.5 mmol) and 0.35 g of compound 2 were mixed with 20 mL of anhydrous EtOH in a 100mL round bottom flask and stirred until dissolved. After that, the mixture was heated at 80 C and refluxed for 12 h. After reflux, the hot mixture was filtered then the residue was washed twice, each with 10 mL of cold MeOH. After being air-dried, 0.32 g of orangeyellow solid was finally obtained (yield: 70%). ESI-MS, m/z: 930.31 [FCZ þ Na]þ (calcd. 930.25 for C56H37N5NaOþ 8 ). HR-ESI-MS, m/z: 908.2705 [FCZ þ Na]þ (calcd. 908.2715 for C56H38N5Oþ 8) (Fig. S3). 1H NMR(300 MHz, DMSO-d6), d 9.95 (s, 4H), 9.15 (s, 2H), 8.14 (d, 2H), 7.98 (d, 2H), 7.73e7.54 (m, 6H), 7.50 (d, 2H), 7.18 (d, 2H), 6.90e6.70 (m, 4H), 6.61 (d, 4H), 6.52 (d, 4H), 4.34e4.25 (m, 2H), 1.21 (t, 3H) (Fig. S4). 13C NMR (75 MHz, DMSO-d6) d 163.47, 158.58, 152.35, 150.52, 141.03, 140.93, 138.46, 133.70, 133.43, 129.30, 129.01, 127.98, 126.15, 125.24, 123.67, 123.11, 122.19, 122.02, 121.28, 121.04, 120.25, 112.31, 110.46, 110.03, 109.33, 102.64, 65.40, 37.35, 13.62 (Fig. S5).
larvae were anesthetized and subjected to fluorescence imaging using a laser scanning confocal microscope. 3. Results and discussion 3.1. Fluorescence spectra To study the capability of FCZ in identifying anions and ROS, the experiments were carried out in a 10 mmol/L PBS buffer solution at the physiological pH (pH 7.4) containing 1% DMSO. 3.1.1. Selectivity of probe Various ions and organic thiols (each with a concentration of 100 mmol/L) were added to the FCZ solution (10 mmol/L), and the changes in emission spectra were examined. The data showed that among all solutions, only ClO caused significant changes in the fluorescence spectra of FCZ probe (lex ¼ 470 nm, lem ¼ 530 nm); other species did not significant changes (Fig. 2). This indicates that the probe has higher selectivity for ClO. 3.1.2. Emission spectra of FCZ in the presence of ClO To study the effect of ClO on the change of fluorescence spectra of FCZ, fluorescence titration was carried out in 1% DMF aqueous
2.2. Fluorescence measurements The FCZ probe was dissolved in DMSO to prepare a standard stock solution with a concentration of 1 mmol/L. Ten microliters of 1 mmol/L FCZ solution was added to 1 mL of H2O (containing 10 mmol/L PBS buffer solution, pH 7.4) containing the test ions. The ion-free FCZ (or the blank solution) was also prepared using the same procedure. The final solution contained 10 mmol/L FCZ and 1% DMSO. The fluorescence emission spectra were measured from 490 nm to 700 nm using a 1-cm quartz cuvette. The excitation wavelength was 470 nm, and the slit widths of excitation and emission were set at 5.0 nm. 2.3. Pre-treatment of cells and zebrafish RAW264.7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 C for 24 h in an atmosphere saturated with 5% CO2. The cells were inoculated on a Petri dish and subjected to fluorescence imaging using a laser scanning confocal microscope. Adult zebrafish was kept at 28.5 C under 14 h light/10 h darkness, during which the fish was allowed to mate and lay eggs. The embryos were grown in E3 culture medium. Five-day-old zebrafish
Fig. 2. Selectivity of FCZ. The red bars represent the relative emission intensity changes of FCZ (10 mmol/L) in the presence of other compounds (100 mmol/L); the blue bars represent the relative emission intensity changes that occurs upon the subsequent addition of 100 mmol/L of ClO to the above solution. From 0 to 24: none, Kþ, Naþ, Ca2þ, 2 1 Mg2þ, F, Cl, Br, I, NO 3 , AcO , HSO4 , SCN , CN , S , H2O2, $OH, O2 , O2, NO$, ONOO, TBHP, Cys, Hcy, and GSH. lex ¼ 470 nm, lem ¼ 530 nm, 10 mmol/L PBS buffer solution (pH 7.4) containing 1% DMSO.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
N. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 3. Fluorescence emission spectra of FCZ (10 mmol/L) in the presence of ClO (0e200 mmol/L). Inset: calibrations curve in the concentration range of 0e200 mmol/L of ClO. lex ¼ 470 nm, lem ¼ 530 nm, 10 mmol/L PBS buffer solution (pH 7.4) containing 1% DMSO.
solution (containing 10 mmol/L PBS buffer solution, pH 7.4). As shown in Fig. 3, FCZ did not exhibit a distinct fluorescence emission peak. It appears that the spiro ring structure of fluorescein inhibits the intramolecular conjugated chain, resulting in poor coplanarity of the molecule, which hinders the transition of intramolecular electrons. After ClO was added, the peak at around 530 nm
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gradually increased as the concentration of ClO increased, while was accompanied by a weak red shift and a typical emission peak of fluorescein group. Furthermore, in the presence of ClO, the intensity of the absorption band of FCZ at 498 nm was increased, a remarkable color change of solution from colorless to yellow (Fig. S6). These results indicate that FCZ is a promising fluorescent probe for the detection of ClO. By studying the change in fluorescence of the FCZ under 470 nm light condition, we found that the probe has good photostability. Fig. S7 shows the relationship between the time and fluorescence response before and after the addition of ClO. The result revealed that the maximum value can be reached within 1.5 min, indicating that the reaction can be completed in a short time. The responses of FCZ towards ClO at different pH conditions were also investigated (Fig. S8). In the absence of ClO, FCZ kept almost stable over a wide range of pH from 4.0 to 9.0. However, in the presence of ClO, the FCZ gave a remarkable increase fluorescence intensity in the range of 4.0e7.8. Therefore, for practical applications in biological systems, a PBS buffer solution (10 mM) with a pH of 7.4 was selected as the reaction medium. We further found that the change of relative fluorescence emission intensity of FCZ had a linear relationship with ClO concentration at a range of 0e200 mmol/L. The linear regression equation was (IeI0)/I0 ¼ 0.08112[ClO] 0.1378 (the unit of [ClO] is mmol/L) with a correlation coefficient of 0.9981. The limit of detection (LOD) of the probe in detecting ClO was 0.056 mmol/L (3s/k), which is lower than those of other fluorescent probes previously reported (Table 1). The FCZ probe was further applied to detect ClO in water samples, and the results are shown in Table 2. The concentration of sodium hypochlorite disinfectant in tap water and lake water were found to be 2.12 mmol/L and 0.05 mmol/L, respectively, while that in
Table 1 Comparison of the present method with other reported ClO selective fluorescence probe. Probe structure
Reaction medium
lex/lem (nm)
LOD (mmol/L)
Ref.
H2O (1%DMSO, PBS 10 mmol/L, pH 7.4)
470/530
0.056
This work
PBS solution (pH 7.4, 10 mmol/L).
49/530
0.068
[24]
EtOH/H2O (1:1, v/v)
560/652
0.080
[25]
20% DMF solution (10 mmol/L Tris buffer, pH 7.4)
420/500
0.15
[26]
EtOH/PBS solution (v/v ¼ 3/7, pH 7.4)
380/(455/632)
0.182
[27]
20% DMF solution (10 mmol/L PBS, pH 7.4)
488/660
0.29
[28]
DMF/PBS solution (v/v ¼ 5/5, pH 7.4, 10 mmol/L)
425/554
0.59
[29]
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Table 2 Analytical results of ClO in water samples (n ¼ 5). Sample
Spiked (mmol/L)
Found (mmol/L)
Recovery (%)
Tap water
0.00 10.00 20.00 0.00 5.00 10.00 0.00 20.00 50.00
2.12 ± 0.07 12.28 ± 0.13 21.85 ± 0.25 0.05 ± 0.02 5.38 ± 0.18 11.45 ± 0.45 Non-detected 19.07 ± 0.24 48.87 ± 0.38
e 101.60 98.65 e 106.60 114.00 e 95.35 97.74
Lake water
Bottled water
bottled water was not detected; and the recovery rates were between 95.35% and 114%. The data indicate that the probe can successfully detect ClO in water samples.
3.2. Reaction mechanism To understand the reaction mechanism, the progress of reaction between FCZ and ClO was monitored by electrospray ionization mass spectrometry (ESI-MS). When excess amount of NaClO was added to the solution, positive ion mode became disordered (Fig. 4a); by contrast, a peak at m/z ¼ 331.061 was observed in negative ion mode. After calculation and analysis, the peak was found to correspond to fluorescein anion (Fig. 4b). We also employed thin-layer chromatography (TLC) to analyze the reaction progress. The results showed that FCZ reacted with ClO to generate two major new products, as indicated by the green fluorescence spots (Fig. S9). Based on its Rf value, the new product was speculated to be sodium fluorescein. Considering the structural changes, we speculate that ClO- can break C]N of FCZ, resulting in the opening of lactam’s spiro ring, causing the conversion to carboxyl anion. To further confirm the structure of the new product, the product was separated by column chromatography (silica gel, CH2Cl2:MeOH ¼ 20:1), after which its structure was characterized by 1H NMR (Fig. S10). The results showed that the product had the structure of fluorescein, which further confirms the reaction
between FCZ and ClO- occurred. Based on the results shown above, the reaction mechanism between FCZ and ClO was elucidated, as depicted Fig. 5. The mechanism is similar to those papers previously reported [30,31]. 3.3. Theoretical calculation To understand the relationship between the structure and spectral changes of FZC after the addition of ClO, the structures, electrons and optical properties of FCZ and Flu were calculated by DFT and TDDFT using the Gaussian 16 software [32]. Solvent was calibrated by solvation model based on density (SMD) solvent model using B3LYP/6-31G (d) as the base group and water as the solvent. Fig. 6 shows the optimal geometric structure and electronic transitions of FCZ and Flu. Considering the energy, the HOMO and LUMO energy levels of FCZ and Flu were different by 3.579 eV and 3.249 eV, respectively. The main electronic transition of FCZ was HOMO/LUMO (f ¼ 0.7293, CI ¼ 0.6958, 96.8%), and the overlap of electron cloud were located mainly in the carbazole region. The calculated first vertical excitation energy was 3.086 eV, which is correspondent to the excitation wavelength of 401.78 nm. This excitation wavelength is different from that used in the experiment (470 nm); therefore, the fluorescence signal of FCZ was not observed at this wavelength. When ClO reacted with FCZ to generate Flu, the main electronic transition was HOMO/LUMO (f ¼ 0.5143, CI ¼ 0.6821, 93.1%), and the electron cloud is distributed mainly in the p-conjugated system of oxonium. Its calculated fluorescence emission wavelength was 515.31 nm, which is slightly smaller than that used in the experiment (530 nm). Considering both the effects of solvent model and intermolecular interaction, the difference is considered acceptable. Overall, the DFT/TDDFT calculation could reveal the optical properties of FCZ and Flu. 3.4. Application of FCZ in bio-imaging In order to study the possible biological application of the FCZ probe, firstly, the cytotoxicity of FCZ probe was evaluated using MCF-7 cells by MTT assay; the results are shown in Fig. S11. The cell
Fig. 4. ESI-MS spectra of FCZ upon addition of NaClO (excess) in CH3OH/H2O mixture (1:1, v/v). (a) Positive ion mode. (b) Negative ion mode.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Fig. 5. The proposed mechanism of the reaction between FCZ and ClO.
viability remained above 80% after being treated with 50 mmol/L FCZ for 24 h, which indicates that FCZ has no cytotoxicity at low concentration and after long incubation time; thus, it should be safe for using in bio-imaging. To examine whether FCZ can be used for in situ detection of ClO in living cells, fluorescence imaging experiments were carried out to detect exogenous and endogenous ClO.
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In exogenous ClO experiment, after RAW264.7 cells were incubated with 10 mmol/L FCZ for 30 min, the fluorescence signal was not obviously observed in the cells, which is consistent with the fluorescent properties of FCZ; however, this can indicate that FCZ could penetrate, to a certain extent, into the cells (Fig. 7a). After the cells were incubated with 20 mmol/L ClO for 5 min, green fluorescence was observed in the cells, which is consistent with the results from the in vitro experiments (Fig. 7b). These results indicate that the sensitive fluorescent probe FCZ can specifically recognize ClO and is suitable for in vivo fluorescence imaging. Lipopolysaccharide (LPS) and polyacid lipopolysaccharide (PMA) are known to promote the production of ClO in RAW264.7 cells, and N-acetyl-L-cysteine (NAC) is known to consume ROS in the cells [33,34]. In endogenous ClO experiments, cells were incubated with 1 mg/L LPS for 12 h, followed by 1 mg/L PMA and 10 mmol/L FCZ, each for 30 min. Obvious green fluorescence in the cells was observed, which shows that endogenous ClO was produced inside the cells upon the stimulation of LPS and
Fig. 6. Optimized structures and the molecular orbitals of FCZ and Flu.
Fig. 7. Fluorescence microscopy images in RAW264.7. (a) Cells treated with FCZ (10 mmol/L) for 30 min. (b) Cells treated with FCZ (10 mmol/L) for 30 min. Cells incubated with NaClO (20 mmol/L) for 5 min. (c) Cells incubated with LPS (1 mg/L) for 12 h and PMA (1 mg/L) for 30 min. Then the cells were loaded with FCZ (10 mmol/L) for 30 min. (d) Cells incubated with LPS (1 mg/L) for 12 h, PMA (1 mg/L) for 30 min, and NAC (0.5 mmol/L) for 10 min. Then the cells were loaded with FCZ (10 mmol/L). (e) Normalized fluorescence intensities of cells. lex ¼ 480 nm, lem ¼ 500e560 nm.
Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692
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Fig. 8. In vivo fluorescence imaging of endogenous ClO in zebrafish larvae. (a) Zebrafish was incubated with FCZ (10 mmol/L) for 30 min. (b) Zebrafish was fed with LPS (2 mg/L) for 4 h, and then incubated with PMA (2 mg/L) for 30 min. Then the zebrafish was loaded with FCZ (10 mmol/L) for 30 min. (c) Zebrafish was fed with LPS (2 mg/L) for 4 h, PMA (2 mg/L) for 30 min, and NAC (1 mmol/L) for 10 min. Then the zebrafish was loaded with FCZ (10 mmol/L). (e) Normalized fluorescence intensities of zebrafish. lex ¼ 480 nm, lem ¼ 500e560 nm.
PMA (Fig. 7c). As a control experiment, the cells stimulated by LPS and PMA were subsequently incubated with 0.5 mmol/L NAC for 10 min, followed by 10 mmol/L FCZ for 30 min. As shown in Fig. 7d, only weak fluorescence signal was observed inside the cells. These results indicate that the change of fluorescence signal intensity of FCZ can be used as an indicator to track and/or to quantify endogenous ClO. In the experiment using zebrafish, fluorescence signal was not obviously observed in zebrafish larvae cultured in the presence of 10 mmol/L FCZ for 30 min (Fig. 8a). After being treated with LPS and PMA (2 mg/L each), followed by FCZ (10 mmol/L), the zebrafish larvae produce green fluorescent (Fig. 8b). Similarly to the results from cell experiment, the green fluorescence inside the zebrafish was weakened when it was treated with an active oxygen scavenger, N-acetylcysteine (NAC) (Fig. 8c). These results indicate that FCZ can be used to monitor ClO in vivo. 4. Conclusion In summary, a novel fluorescent probe FCZ was designed and synthesized to detect ClO in aqueous solution under physiological pH. The ClO sensing mechanism and the luminescence properties of the probe were investigated via ESI-MS, TLC, NMR, and DFT/ TDDFT calculations. The fluorescence titration results showed that the probe had a good linear relationship (r ¼ 0.9981) and a low LOD of 0.056 mmol/L (3s/k). In addition, the probe was successfully used in fluorescence imaging of exogenous and endogenous ClO in RAW264.7 cells. The probe also had low cytotoxicity and good cell permeability, thus can enter into the cells. In an experiment using zebrafish, the FCZ probe exhibited good biocompatibility; it may be developed as a tool for monitoring ClO in vivo. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Science and Technology Developing Foundation of Jilin Province of China (Nos. 20180201050YY and
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Please cite this article as: N. Wang et al., A fluorescein-carbazole-based fluorescent probe for imaging of endogenous hypochlorite in living cells and zebrafish, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117692