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Reversible ratiometric fluorescent probe for sensing bisulphate/H2O2 and its application in zebrafish weijie zhang, tao liu, Fangjun Huo, Peng Ning, Xiangming Meng, and Caixia Yin Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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Analytical Chemistry

Reversible ratiometric fluorescent probe for bisulphate/H2O2 and its application in zebrafish

sensing

§Weijie Zhang,† §Tao Liu,† Fangjun Huo,‡ Peng Ning, ǁ Xiangming Meng,ǁ Caixia Yin †,* †

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 030-006, China. ‡ Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China ǁ

Department of Chemistry, Anhui University, Hefei, 230601, China

*Corresponding author. * E-mail: [email protected]. Tel/Fax: +86-351-7011022. ABSTRACT: Herein, a novel near-infrared fluorescent probe for ratiometric detection of bisulphate was designed and developed based on a conjugation of naphthopyran-benzothiazolium system. The sensor showed excellent selectivity, high sensitivity and a rapid response toward bisulfite in aqueous solution. Upon the addition of HSO3-, the sensor displayed 37-fold (I520/I630) fluorescence intensity enhancement, accompanied with an apparent color change from violet to colorless, suggesting that the sensor can be used to detect HSO3- with “naked-eye”. Notably, the addition product can be applied to the design of regenerative chemodosimeters based on the H2O2 promoted elimination of bisulfite and recovery of probe 1. Further cell and zebrafish imaging experiment demonstrated that the sensor could image the bisulfite/H2O2 redox cycle in biological system with ratiometric manners.

Recently, studies have suggested that bisulfite could be endogenously generated during the decomposition of sulfurcontaining amino acids by reactive oxygen species (ROS), which could induce oxidative stress, and diseases caused by aging 1-4. H2O2, as one type of reactive oxygen species (ROS) is an inevitable byproduct of cell metabolism and a common marker and signal molecule of oxidative stress.5-7 Sulfur dioxide (SO2), as a toxic environment pollutant,8 is usually associated with the symptoms of neurological disorders, cardiovascular diseases, and lung cancer.9 The toxicity of SO2 is mainly attributed to its two reductive derivatives: sulfite (SO32-) and bisulfite (HSO3-) (3 : 1 M/M, in neutral fluid).10 Therefore, it is meaningful and valuable to develop efficient methods for monitoring bisulfite/ROS redox cycle processes with different fluorescence signals, which is vital for biological research as well as clinical diagnoses. Fluorescent probes for real-time sensing and imaging are indispensable tools in life science and materials science, 11 providing an excellent detection technique that can identify appropriate signals immediately and provide useful information in a short time. To date, lots of fluorescent probes for SO32-/HSO3- detection have been reported and the reaction mechanisms can be sorted into two types, one is the nucleophilic addition to aldehydes/ketones, 12-17 which has a disadvantage as it may suffer from the interference of thiols, 18, 19 and the other is the nucleophilic addition to double bonds, especially the unsaturated compound addition reaction,20-21 which can avoid the thiols interference. Along with the rapid development of fluorescent probes for SO32-/HSO3-, considerable advances have been made in recent years. However, to the best of our knowledge, only a few fluorescent probes could realize reversible sensing for sulfur dioxide and peroxides.22

As a consequence, an ideal fluorescent probe for reversible monitoring the intracellular bisulfite/ROS is badly needed. It is worth pointing out that none ratiometric fluorescent probes for imaging the bisulfite /ROS redox cycle in living body has been reported. To address the above issues, a novel fluorescent probe was synthesized based on the naphthopyran and benzothiazolium cation as described in the Scheme 1. Cyanine dyes usually possess large Stokes shifts and excellent water solubility. And the hemicyanine fluorophore was reported to be able to capture nucleophilic agents via 1,2- or 1,4-addition.23,24 Therefore, we speculate that the potential reaction sites may respond to SO32-/HSO3- with ratiometric manner, and the original probe could be regenerated with reversible fluorescent responses towards H2O2. Scheme 1. Diagram of probe 1 for bisulphate/H2O2 detection.

EXPERIMENTAL SECTION Materials and Chemicals. All chemicals were purchased from commercial suppliers and used without further purification. All solvents were purified prior to use. Distilled water was used after passing through a water ultrapurification system. TLC analysis was performed using precoated silica plates. Hitachi U-3900 UV-vis spectropho-

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tometer was employed to measure UV-vis spectra. Hitachi F−7000 fluorescence spectrophotometer was employed to measure fluorescence spectra. Shanhai Huamei Experiment Instrument Plants, China provided a PO-120 quartz cuvette (10 mm). 1H NMR and 13C NMR experiments were performed with a BRUKER AVANCE III HD 600 MHz and 151 MHz NMR spectrometer, respectively (Bruker, Billerica, MA). Coupling constants (J values) are reported in hertz. ESI determinations were carried out on AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA). The cell imaging experiments were measured by an Olympus FV1200 confocal laser scanning microscope. The zebrafish imaging experiments were measured by an Olympus FV1200 confocal laser scanning microscope. Preparation of Solutions of Probe 1 and Analytes. Stock solution of probe 1 (2 mM) was prepared in DMSO. Stock solutions (2 mM) of Cys, Hcy, GSH, H2S, CN−, CO32−, SO42−,F−, Cl−, Br−, I−, HPO42−, H2PO4−, NO3−, SCN−, HCO3−, S2O32−, N3−, AcO−, SO32− and HSO3−, were prepared by direct dissolution of proper amounts of sodium salts in deionized water. All chemicals used were of analytical grade. General fluorescence spectra measurements. All the detection experiments were measured in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25℃. The process was monitored by fluorescence spectrometer (λex= 400 nm, slit: 5 nm/10 nm). Confocal Fluorescence Imaging. The MCF-7 cells were grown in Dulbecco’s Modified Eagle’s medium, supplemented with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Cells were plated on 6 well plates and allowed to adhere for 24 h. Before the experiments, cells were washed with PBS 3 times. Firstly, we incubated probe 1 (10 µM) with MCF-7 cells for 30 min at 37℃ in PBS as a control experiment. Secondly, probe 1, which located MCF-7 cells were treated with bisulfite (50 µM) for another 30 min. Finally, all cells were washed with PBS for 3 times before imaging, and all images were carried out on a confocal microscope, separately. For bio-imaging in vivo, 5-day-old zebrafishes were prepared. Zebrafishes were fed with 10 µM of probe 1 in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 10-5% methylene blue; pH 7.5) at 28 °C for 20 min. Then incubated with Na2SO3 (50 µM) for 30 min, respectively, and finally incubated with H2O2 (50 µM) for 30 min. All the fishes were terminally anaesthetized using MS222, and images were carried out on a confocal microscope. Synthesis of 3H-benzo[f]chromene-2-carbaldehyde (Compound 2). 2-Hydroxy-1-naphthaldehyde (0.516 g, 3.0 mmol) and acrolein (0.5 mL, 7.5 mmol) were dissoloved in 1, 4-dioxane (60 mL) with K2CO3 (1.035g, 7.5 mmol). The mixture was heated at 105 °C for 72 h. After the reaction was completed, it was removed from the heating bath, poured into 150 mL of ice water, The resulting mixture was extracted three times with ether (40 ml). The combined organic layer was washed with saturated NaCl twice (40 ml), then dried over anhydrous Na2SO4, and concentrated under vacuum. Compound 2 was isolated using a silica gel chromatographic column eluted with EA/ PE (v/v, 1:5), resulting a light yellow solid (0.126 g, 20%). 1H NMR (600

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MHz, DMSO) δ 9.72 (s, 1H), 8.46 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 8.9 Hz, 1H), 5.06 (s, 2H). 13C NMR (151 MHz, DMSO) δ 190.9 (s), 155.4 (s), 138.3 (s), 134.5 (s), 130.8 (s), 129.9 (s), 129.4 (s), 129.2 (s), 128.5 (s), 125.1 (s), 122.1 (s), 117.9 (s), 114.3 (s), 63.0 (s). ESI–MS: m/z Calcd 211.0754, Found 211.0755 (Fig. S1). Synthesis of (E)-2-(2-(3H-benzo[f]chromen-2-yl)vinyl)-3methylbenzo[d]thiazol-3-ium iodide (Probe 1). Compound 2 (0.105 g, 0.5 mmol) and 2,3-dimethylbenzothiazolium iodid (0.204 g, 0.7 mmol) were dissolved in 10 mL EtOH with piperidine 30 µL. The reaction mixture was refluxed with stirring for 12 h and then cooled to r.t., the precipitate was filtered, washed with cold ethanol and dried in vacuum to afford the desired product as a dark brown solid (0.2 g, 84%). 1 H NMR (600 MHz, DMSO) δ 8.43 (d, J = 7.9 Hz, 1H), 8.32 (s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 15.8 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 15.8 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 5.38 (s, 2H), 4.31 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 170.77 (s), 153.89 (s), 144.86 (s), 141.56 (s), 132.80 (s), 132.10 (s), 129.46 (s), 128.80 (s), 128.51 (s), 128.32 (s), 127.70 (s), 127.43 (d, J = 13.5 Hz), 127.17 (s), 124.15 (s), 123.66 (s), 120.91 (s), 116.81 (s), 116.15 (s), 114.73 (s), 111.87 (s), 63.65 (s), 35.61 (s). ESI– MS: m/z Calcd. For: m/z Calcd 356.1104, Found 356.1116 (Fig. S2).

RESULTS AND DISCUSSION Design of probe 1 and the proposed detection mechanism. Probe 1 was synthesized by two steps with high yield (Scheme 2). In our research, an ideal fluorescent probe should satisfy the following properties: high selectivity; rapidly response; ratiometric detection; good permeability and biocompatibility. Bearing these facts in mind, herein, a novel naphthapyrones chromophore was efficiently synthesized. With the aldehyde-functionalized chromophore in hand, a ratiometric fluorescent probe for SO32-/HSO3- was developed based on the fundamental notion of the nucleophilic addition reaction. Benefited from the strong electron withdrawing moiety of benzothiazole moiety, probe 1 featured ultrafast responses towards SO32-/HSO3- while maintained high sensitivity and selectivity properties. The sensing mechanism were characterized by 1H NMR and electrospray ionization mass spectrometry (ESI-MS), which was consistent with the change appeared in the absorption and fluorescence spectrum. Scheme 2. Synthesis Route for Probe 1

Sensing Properties of Probe 1 towards SO32-/HSO3-. Probe 1 was steady in a broad pH range (2.0-9.0). However, upon the addition of SO32-/HSO3-, probe 1 displayed the best response in the pH range of 7.0-9.0 (Figure. S3). Thus, to study the spectroscopic response of probe 1 towards SO32/HSO3-, pH 7.4 was selected for the following experiments. Fluorescence titration experiments were carried out firstly. As shown in Figure 1A, the free probe displayed a near-infrared


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fluorescence emission at 630 nm. However, the addition of an increasing amount of SO32-/HSO3- (0-30 µM) elicited a gradual decrease of the fluorescence intensity at 630 nm, accompanied with a distinct increase at 520 nm. These results indicated that the π-conjugation of probe 1 was interrupted. Then, the corresponding UV-vis spectral changes were also studied (Figure 1B). Probe 1 (50 µM) shows a strong absorption maximum at 470 nm and a shoulder at 350 nm. Upon addition of SO32/HSO3- (0-150 µM) to the buffered solution of probe 1, the two absorption bands gradually decreased, with an obvious color change from violet to colorless, suggesting that probe 1 can be used to detect SO32-/HSO3- with the “naked-eye”.

Figure 1. (A) Fluorescent spectral changes of probe 1 (10 µM) upon addition of SO32-/HSO3- (0 - 30 µM) in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. (B) UV-vis absorption spectra of Probe 1 (50.0 µM) in the presence of different amounts of SO32-/HSO3- (0 - 150 µM) in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. λex = 400 nm , slit: 5 nm/10 nm. Sensing Properties of the addition towards H2O2. After the demonstration of probe 1 as a suitable chemosensor for the detection of SO2 derivatives, we further investigated some possible applications such as the reversibility of the nucleophilic addition product induced by SO32-/HSO3-. In the course of our research, it was found that hydrogen peroxide could effectively restore the fluorescence intensity. As show in Figure 2A, an addition of 50 µM H2O2 to the solution could induce big fluorescence variation. A dramatic fluorescence enhancement was observed at 630 nm and the emission peak at 520 nm decreased simultaneously. The relative fluorescent intensity (I520/I630) was calculated as 0.65, indicating that the SO32-/HSO3- added system of probe 1 effectively restored by the addition of H2O2. Fig. 2B shows the change of the UVVisible spectrum upon addition of H2O2. The absorption band at 470 nm gradually increased, which accompanied by a clear visual change in the color of the experimental solution from colorless to violet. In other words, when oxidized by H2O2, the color of the addition product can be changed almost the same as the original probe. Moreover, the probe 1 with HSO3- addition product showed a negligible response to some reactive oxygen species (HClO, OH., TBHP, KO2) and some metal ions (Cu2+, Zn2+, Na+, K+, Mg2+, Ca2+, Hg2+, Ba2+, Mn2+, Al3+) even at the concentrations up to 1 mM. By contrast, noticeable fluorescence ratio change was obtained in the presence of H2O2 (Fig. S4). Meanwhile, it was also found that, after treatment with 50 µM of H2O2, the fluorescence ratio of I630/I520 increased steadily and nearly reached a plateau within 60 min, which was resulted from the red fluorescence enhancement and the green fluorescence decline. (Fig. S5).

Figure. 2 (A) Fluorescent spectral changes upon addition of H2O2 (50 µM) in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C, λex = 400 nm , slit: 5 nm/10 nm. (B) UV-vis absorption spectra of probe-HSO3- in the presence H2O2 (200µM) in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. Fluorescence Reversibility and LOD of Probe 1. In a further experiment, we carried out the reversibility study of probe 1 towards SO32-/HSO3- upon addition of H2O2. As shown in Fig. 3A, three times supplement of SO32-/HSO3- and H2O2 caused negligible intensity attenuation, which provided a unique approach for monitoring SO32-/HSO3- and H2O2 in vitro and in vivo. Next, we performed fluorescence titration experi2ment of probe 1 for SO3 /HSO3 . Probe 1 (10 µM) was treated with various concentrations of SO32-/HSO3- (0-30 µM), and the fluorescence ratio of I520/I630 presented an excellent linear relationship (R2 = 0.9934) towards increased SO32-/HSO3concentration (Fig. 3B). Moreover, the detection limit for SO32-/HSO3- was calculated to be 95 nM based on IUPAC (CDL=3 Sb/m) 25-27.

Figure. 3 (A) Reversibility study of probe 1 (10 µM) towards SO32-/HSO3- (30 µM) upon addition of H2O2 (50 µM). (B) Relative emission intensity at I520/I630 of probe 1 (10 µM) with SO32-/HSO3- (6-24 µM) in DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. λex = 400 nm , slit: 5 nm/10 nm. Kinetic Study and Selectivity of Probe 1. The kinetic analysis of probe 1 towards 10 equivs of SO32-/HSO3- displayed that the reaction was completed within 40 s (Fig. S6). In the selectivity test, the probe was treated with various biological relevant anions and small molecules (10 equals). Upon excitation at 400 nm, Only SO32-/HSO3- leading to a big relative fluorescent intensity change at 520 nm and 630 nm (I520/I630≈37), while other selected anions showed negligible change in the emission spectra of probe 1 (Fig. S7).

Proposed Mechanism. The sensing mechanism of probe 1 for SO32-/HSO3- was carefully examined by the NMR titration experiment. Clearly, the proton signal at δ 4.32 (a1) and δ 8.33 (d1) shifted forward (a2, d2, at δ 4.05 and δ 7.35 ppm) after reaction with SO32-/HSO3-, and the characteristic proton signal of the double bond in probe 1 (b1, c1, at δ 7.40, 7.38, 7.26, 7.24 ppm) shifted to (b2, c2 at δ 5.00, 4.98, 4.87, 4.84 ppm), respectively (Fig. S8). Furthermore, the mixture of SO32-/HSO3- and probe 1 was characterized by mass spectrom-



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etry. As shown in Fig. S9, two dominant peaks at m/z 356.11 and 438.08 were attributed to [probe 1]+ and [probe 1- HSO3-]+, respectively. Thus, the sensing mechanism of probe 1 towards SO32-/HSO3- was based on the nucleophilic addition, as shown in Scheme 1. Cellular Imaging. Inspired by the excellent properties of probe 1 for reversible sensing SO32-/HSO3- and H2O2 in vitro, the ability of probe 1 for cell imaging were evaluated with MCF-7 cells. Firstly, cytotoxicity of probe 1 was conducted by MTT assay (Fig. S10), and the result showed that probe 1 was suitable for living cells below 10.0 µM. Hence, the following experiment was conducted with probe 1 at the concentration of 10 µM. In Fig. 4, MCF-7 cells were incubated with probe 1 (10 µM) for 30 min at 37℃ in PBS buffer. On the excitation of 488 nm, probe 1 exhibited red fluorescence in the red channel, but negligible fluorescence in green channel. These data was consistent with fluorescence spectra results of probe 1. In a control experiment, upon incubation SO32-/HSO3- to probe 1loaded MCF-7 cells 0.5 h, the cells displayed a stronger green fluorescence enhancement and a weaker red fluorescence. These results demonstrated that the probe was capable of detecting exogenous SO32-/HSO3- in living cells. Besides, when H2O2 was supplemented with the above stained MCF-7 cells for another 30 min, the initial red fluorescence enhanced and the green fluorescence faded, indicating that probe 1 underwent a redox activity in living cells, and it can be regenerated by hydrogen peroxide indeed.

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exogenous SO32-/HSO3-, accompanied with decreased red fluorescence in the red channel. What is more, when the above-mentioned zebrafish pre-treated with H2O2, green fluorescence changes disappeared and the red fluorescence restored. These imaging findings demonstrate that probe 1 can effectively enable redox cycle process in living body.

Figure 5. Confocal images of Probe 1 response to exogenous SO32-/HSO3- in zebrafish. (a1-d1): Zebrafish was incubated with Probe 1 (10 µM) for 30 min; (a2-d2): Zebrafish was incubated with Probe 1 (10 µM) for 30 min, then incubated with SO32-/HSO3- (50 µM) for 30 min. Probe 1 loaded Zebrafish incubated with 50 µM of NaHSO3 for 30 min, and then incubated with 50 µM of H2O2 for another 30 min. From left to right: Green channel: λem = 520±20 nm (λex = 405 nm), Red channel: λem = 650±20 nm (λex = 488 nm), Bright field, Merge. Scale bar: 400 µm.

CONCLUSIONS In summary, a reversible and ratiometric fluorescent probe was explored to sense bisulfite and hydrogen peroxide in vitro and in vivo. The ratiometric fluorescence response was based on the π-conjugation interruption along with the cancelation of an ICT process. In the reversibility process of the SO32-/HSO3--probe 1 system induced by H2O2 in vitro, we successfully demonstrated the reversible redox properties of probe 1 for SO32-/HSO3- and H2O2. Besides, further cells and zebrafish experiments clearly revealed that probe 1 could be used for monitoring redox process in biological system. Moreover, this will prompt us to explore the reversible process of other bioactive molecules in the next step.

ASSOCIATED CONTENT Supporting Information Figure 4. Confocal images of probe 1 responds to exogenous SO32-/HSO3- and H2O2 in MCF-7 cells. (a1-d1): MCF-7 cells were incubated with probe 1 (10 µM) for 30 min; (a2-d2): MCF-7 cells were incubated with probe 1 (10 µM) for 30 min, then incubated with NaHSO3 (50 µM) for 30 min; (a3d3): Probe 1 loaded MCF-7 cells incubated with 50 µM of HSO3- for 30 min, and then incubated with 50 µM of H2O2 for another 30 min. From left to right: Green channel: λem = 520±20 nm (λex = 405 nm), Red channel: λem = 650±20 nm (λex = 488 nm), Bright field, Merge. Scale bar: 20 µm. Zebrafish Imaging. To further display the reversibility advantage of probe 1-SO32-/HSO3--H2O2 system, zebrafish imaging experiment was performed to examine the redox cycle processes in vivo. As is shown in Fig. 5, when zebrafish was incubated with the probe 1 for 30 min, an obviously red fluorescence signals can be achieved while none fluorescence signals could obtained in the green channel. By contrast, the green fluorescence increased obviously with the treatment of

Structure characterizations of probe 1, pH dependent spectra, kinetic analysis of probe 1, and data for investigation of the sensing mechanism. The material is available and free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

* E-mail: [email protected]. ORCID

Caixia Yin: 0000-0001-5548-6333 Author Contributions This manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 21472118), talents Support Program of Shanxi Province (No. 2014401) and Scientific Instrument Center of Shanxi University (No. 2015).

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