A Fluorescent Probe for Ratiometric Imaging of SO2 Derivatives in

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A Fluorescent Probe for Ratiometric Imaging of SO2 Derivatives in Mitochondria of Living Cells Haidong Li, Qichao Yao, Jiangli Fan, Chong Hu, Feng Xu, Jianjun Du, Jingyun Wang, and Xiaojun Peng Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b04530 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016

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Industrial & Engineering Chemistry Research

A Fluorescent Probe for Ratiometric Imaging of SO2 Derivatives in Mitochondria of Living Cells Haidong Lia, Qichao Yaoa, Jiangli Fana, Chong Hua, Feng Xua, Jianjun Dua, Jingyun Wangb, Xiaojun Peng*a a

State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China.

b

School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China.

ABSTRACT: When SO2 is inhaled through the respiratory tract into the body, it can be easily hydrated to sulfite (SO32-) and bisulfate (HSO3-) ions. They are widely used as essential preservatives for foods, beverages, and pharmaceutical products. Exposure to high doses of bisulfate induces a large number of respiratory diseases relevant to lung cancer, cardiovascular diseases, and many neurological disorders, as they involve in various physiology and pathological processes in mitochondria cell apparatus. In this work, a new fluorescent probe CYSO2 for bisulfate/sulfite based on hemicyanine dye is reported, which can used in solution detection with 56-fold fluorescence ratio (F467 nm/F580 nm) enhancement, fast response (completed within 90 sec) and excellent sensitivity (DL 2.67 nM). The effect have been exhibited in real sugar samples tests. CY-SO2 also displayed fluorescent imaging of breast cancer cells (MCF-7)

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successfully, demonstrating that the probe is a novel mitochondria-targeted ratiometric fluorescent probe to image cellular SO2 derivatives.

KEYWORDS: Fluorescent probe; Hemicyanine dye; Ratiometric fluorescence imaging; Fast response; SO2 derivatives; Mitochondria-targeted

1．．INTRODUCTION

When SO2 is inhaled through the respiratory tract into the body, it can be easily hydrated to sulfite (SO32-) and bisulfate (HSO3-) ions (3:1 M/M, in neutral liquids) based on pH dependent equilibrium.1,

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In daily life, SO2 derivatives have been widely used as enzyme inhibitor,

antimicrobial agent, pharmaceutical products and essential preservatives for foods, beverages. 3, 4 So, they are extensively used in a wide range of industries.5

However, numerous

epidemiological studies have showed that exposure to high doses of bisulfate not only induce a large number of respiratory disease6 but is also relevant to lung cancer, cardiovascular diseases, and many neurological disorders, such as strokes, migraine headaches and brain cancer.7-10 Generally, the SO2 derivetives can also be produced from L-cysteine in reactions catalyzed by aspartate aminotransferase-2 (AAT-2),11 which is constitutively expressed in cytosol and mitochondria of cell,12 and has been recognized to play a crucial role in various physiology and pathological processes.7, 13 Although many previous research work was about the physiological functions of sulfur dioxide, accurate molecular mechanisms of action remain elusive. Therefore, to fully understand SO2 derivatives biology, it is highly needed to develop efficient methods for monitoring cellular SO32-/ HSO3- concentration, which is vital for biological research as well as clinical diagnoses.


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To date, there are several methods had been developed to detect SO32-/ HSO3- in vitro or vivo, such as spectrophotometry,14-16 capillary electrophoresis,17 chromatography,18 and fluorescent probes.19-23 Compared with other techniques, fluorescence detection using small molecular probes, has regarded as a promising method in solution and living cells due to its high spatial and temporal resolution, sensitivity, selectivity, simple operation, and especially nondestructive characteristics.24-29 In recent years, some novel fluorescent sensors or probes have been developed for SO2 derivatives in vitro or vivo. For instance, in vitro, Chang30 constructed a sensor based on levulinate of resorufin, Fu synthesized a fluorescent chemodosimeter to based on complexation with amines,31 Guo reported a coumarin-hemicyanine dye based on a novel addition-rearrangement cascade reaction,21 and Duan developed a ratiometric fluorescence probe.32 Very recently, Chang and Yuan reported the first mitochondria-targetable ratiometric fluorescent probe for SO2 derivatives in living cells.33 In deed, it is of great worth to monitor celluar SO2 derivatives in mitochondria, where are the main source of intracellular endogenous SO2 generated by AAT-2,34-37 especially in ratiometric fluorescent way where probes could eliminate the interferences such as probe concentration, micro-environment conditions, and instrument efficiency to achieve quantitative detection in vitro and vivo via two different emission wavelengths.38-43 Up to now, to the best of our knowledge, there are few mitochondriatargeted ratiometric fluorescent probes for SO2 derivatives in vivo, 33, 44 and most of the reported sensors have much of improving needs for practical applications in response time and detection limit. It inspires us to develop a novel ratiometric fluorescent probe with high sensitivity, selectivity, rapid response and mitochondria-targeted. Herein we reported a novle fluorescent probe, CY-SO2 (Scheme 1), based on an 1,4-addition reaction of SO32-/ HSO3- in polymethine chain of hemicyanine dyes which has good solubility in


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water, long emission wavelength.45-48 As an azide group introduced in the dye, the strongly electron-withdrawing merit lets the addition fast (completed within 90 sec) and sensitive to SO32/ HSO3- (detection limmit 2.67 nM). On the other hand, the bulkiness of SO32-/ HSO3- was prevented it to attack toward C-2 atom of fluorescent probe CY-SO2 because of steric effect. Upon the addition of HSO3- to a solution (10 mM, pH 7.4) of CY-SO2, in turn, HSO3- will interrupt the π-conjugation of probe CY-SO2, which resulted in the blue shift of emission wavelength with a large shift (~113 nm), providing excellent ratiometric merit. Due to rational molecule design, CY-SO2 permeates into live cells and localized in mitochondria, as the demonstration of fluorescent imaging in breast cancer cells (MCF-7). 2．．RESULTS AND DISCUSSION

2.1 Design Strategy. The probe CY-SO2 could be easily synthesized via three steps, which was shown in the Supporting Information section. Proposed sensing mechanism of CY-SO2 was displayed in Scheme 1. In order to verify the correctness of CY-SO2 and CY-HSO3 (Scheme 1) were characterized by nuclear magnetic resonance (1H,

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C NMR) and high resolution mass

spectrum (HRMS) (see Supporting Information). 2.2 Spectropic Responses. With CY-SO2 probe in hand, firstly, we tested the spectroscopic properties of CY-SO2 (10 µM) in stimulated physiological media (10 mM PBS buffer, pH 7.4, containing 25 µM CTAB). Free CY-SO2 probe has one prominent absorption at 443 nm (ε: 1.75× 104 M-1 cm-1) and a corresponding emission maximum at 580 nm with moderate fluorescence intensity as shown in Figure 1. Upon the addition of NaHSO3, the absorption intensity at 443 nm was gradually decreased accompanied by a new absorption peak appeared at 325 nm with a clear isobestic point at 348 nm, which the color of the solution changed from


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yellow to colorless, allowing colorimetric detection of SO32-/ HSO3- by naked eyes (Figure 1 and Figure S1). In the corresponding fluorescence spectrum, free CY-SO2 probe displayed relatively moderate fluorescence intensity owing to the electron-withdrawing effects of both sides. In contrast, when NaHSO3 was added to the solution of CY-SO2 (10 µM), a remarkable fluorescence signal gradually enhanced at 467 nm (ΦF = 0.024) and decreased at 580 nm (ΦF = 0.004) upon excitation at 400 nm (Figure 1b). Such a large shift in emission (~113 nm) indicates probe CYSO2 is promising dual emission ratiometric detection for SO32-/ HSO3- with a 33-fold fluorescence intensity (467 nm) and 56-fold ratio (F467 nm/F580 nm) enhancement. Furthermore, we also plotted the function of the intensity ratio (F467

nm/F580 nm)

and different NaHSO3

concentrations (Figure S3), which implied the great fluorescent signal changes of probe CY-SO2 (Figure S2), could be caused by low analyte concentration and further smoothly increased until a maximum reach a plateau with 10 µM NaHSO3. Moreover, in order to research the sensitivity of probe CY-SO2 to NaHSO3, titration of NaHSO3 at low concentration were carried out. Intriguingly, the fluorescence titration curve showed that the fluorescence ratios (F467 nm/F580 nm) of probe CY-SO2 (10 µM) increased excellent linearly (R2=0.9933) with the concentration of NaHSO3 ranging from 1 to 5 µM (Figure 2). Importantly, the detection limit (3σ/k) toward HSO3was calculated to be 2.67 nM, to the best of our knowledge, which is the most sensitive fluorescence probe for HSO3- in PBS buffer (0.01 M, pH 7.4, containing 25 µM CTAB), by far. 2.3 Recognition Mechanism. We envisioned a 1,4-addition reaction occurred between CYSO2 and HSO3- rather than a 1,2-addition reaction, which was verified by the 1H NMR analysis with different amounts of NaHSO3 in DMSO-d6 solution (Figure S8). In the presence of NaHSO3, all of the 1H NMR signals shifted upfield due to the nucleophilic attack of HSO3-


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toward C-4 interrupting π-π conjugation and weakening its electro-withdrawing characteristic. When N+ was converted to N in the indolenium, the signal at 1.53 ppm (Ha) and 4.86 ppm (Hb) of the ethyl group were significantly shifted upfield to 1.50 ppm and 3.64 ppm, respectively. The signal at 7.58 ppm (Hd) gradually decreased and finally disappeared, while new signal at 5.02 ppm appeared upon the addition of NaHSO3. Furthermore, the signal at 1.98 ppm (Hc) of the two methyl group were also shifted upfield and split into 1.79 ppm (Hc1) and 1.60 ppm (Hc2), which became nonequivalent hydrogen after the formation of CY-HSO3. What's more, for a further confirmation, the mass spectrometry analysis of the mixuture of CY-SO2 and NaHSO3 was carried out, where one apparent peak at m/z 447.1509 (calcd. 447.1496 for C24H23N4O3S-) corresponding to [M + SO3]- was shown in the high resolving mass spectrum data (Figure S9). Thus, these results were in agreement with proposed sensing mechanism of CY-SO2 depicted in Scheme 1. 2.4 Response Speed and Selectivity. The time-dependent (0-500 s) fluorescence intensity changes (467 nm) of CY-SO2 (10 µM) in the presence of NaHSO3 (10 µM) displayed the probe could promptly response to NaHSO3 within 90 s (Figure 3) with a pseudo-first-order rate constant k 2.14×103 M-1 S-1 (Firgure S6b). In the selectivity test, the probe was treated with a variety of ions, ROS (reactive oxygen species), reducing substance and biological mercaptan (1 mM unless otherwise stated). For the representative ions including F-, Cl-, Br-, I-, NO2-, NO3-, SCN-, HPO42-, CH3COO-, CO32-, HCO3-; ROS including NaClO, H2O2; reducing substance including Ascorbic Acid (AA); biological mercaptan containing Glutathione (GSH), Cysteine (Cys) commonly found in biological systems, only NaHSO3 could lead a remarkable fluorescence emission ratios (F467

nm/F580 nm)

variation of CY-SO2 (10 µM) along with clear

change (Figure S2), while implied that interferents did not trigger minor change in fluorescence


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behavior (Figure 4). However, considering sulfate radical (S2-) may be a potential interfering species, the fluorescence responses of CY-SO2 (10 µM) to differing amounts of Na2S were tested in PBS buffer (10 mM, pH 7.4, containing 25 µM CTAB). The results indicated that even high Na2S concentrations (6 equivalent) did not induce an obvious fluorescence change in the test system (Figure S11). In addition, the fluorescence ratios intensity of CY-SO2 (10 µM) was largely unaffected by other coexistent anions, reactive species, ROS or biological mercaptan (Figure S10). The above results showed that the probe CY-SO2 has a highly selective fluorescent probe for SO32-/ HSO3- in PBS buffer (10 mM, pH 7.4, containing 25 µM CTAB), implying the possibility of quantitative detection without troublesome sample pretreatment. 2.5 Real Sample Testing. To further evaluate the performance of CY-SO2 for the actual sample testing, we investigated the applicability of this probe for SO2 derivatives in solution, subsequently. As we all know, the residue of bisulfate is left in the sugar industry, which is of great necessary to detect HSO3- content in real samples. Then, the proposed method was used to detect HSO3- content in soft sugar and granulated sugar were purchased from a supermarket. As shown is in Table S1, the probe CY-SO2 could able to detect the low HSO3- concentration in sugar solution. Moreover, to verify the accuracy of this method, malachite green’s method49 was adopted as comparative approach (Figure S12, S13, and S14). All measured values were listed in Table S1. The HSO3- levels in these samples were 9.40 mg/kg and 9.34 mg/kg, corresponding to 10.53 mg/kg and 9.58 mg/kg via malachite green's method. Satisfactorily, comparison results indicated CY-SO2 could be qualified to measure HSO3- content in real samples. However, malachite green’s method cannot be applied to the recognition of HSO3- in living cells due to its strong carcinogenic, teratogenic and mutagenic side effects.


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2.6 Influence of pH and Stability. Figure S4 displayed that CY-SO2 exhibited minimal change in emission intensity (F467 nm and F580 nm) at pH 3.0-9.0. Then, the emission intensity (F467 nm)

and fluorescence intensity ratios (F467 nm/F580 nm) became apparent enhancement upon the

addition of NaHSO3 (Figure S5a and S5b), which indicated that this probe would not be disturbed by cellular pH micro-environment. Furthermore, the probe CY-SO2 could remain stable as long as 60 min in PBS buffer (10 mM, pH 7.4) (Figure 5), showing the possibility of this probe fluorescence imaging for a long time. 2.7 Intracellular Imaging. Encouraged by the above favorable properties of the probe CYSO2 for monitor SO2 derivatives, including excellent sensitivity, high selectivity and rapid response in the physiological pH range, we then evaluated the potential applications of CY-SO2 in live-cell imaging assays. As shown in Figure 6, after incubation of MCF-7 cells with CY-SO2 (5 µM) for 30 min at 37 oC, nearly no intracellular fluorescence signals in the green channel (450 nm - 510 nm) were collected by the confocal microscope FV1000 (Figure 6a); on the contrary, obvious fluorescence signals at the red channel of 540 nm - 600 nm were also gathered (Figure 6b), which indicated that the probe CY-SO2 had remarkable member permeability. The ratio of the two emissions generated from green channel to red channel was around 0.2. Furthermore, the MCF-7 cells incubated with CY-SO2 (5 µM) for 30 min at 37 oC, then treating the cells with 100 µM NaHSO3 at 37 oC for 15 min (Figure 6f-j). As expected, the green fluorescence channel gradually became brightened and the red fluorescence channel became darkened, respectively. Notably, the emission ratio was severely increased, resulting in the ratio value to be about 1.4. Then, obvious changes in ratiometric fluorescence responses generated from green channel and red channel in living cells were observed (Figure 6e and 6j). Exhilaratingly, the little changes in HSO3- levels were also clearly observed via the ratiometric fluorescence imaging, indicating that


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our designed probe CY-SO2 has fine resolution in bio-imaging. These results implied that CYSO2 is capable of monitoring HSO3- changes through the ratiometric fluorescence imaging in living cells. 2.8 Cytotoxicity. To estimate cytotoxicity of probe CY-SO2, we performed 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays in MCF-7 cells with 0, 1, 5 and 10 µM probe for 12 h, respectively. The experimental data were shown in Figure S15, which clearly demonstrated that our proposed probe was low toxicity to cultured cells at the concentration of 5 µM for 30 min under the experimental conditions. 2.9 Mitochondrial-localization. In order to research where the probe CY-SO2 located in living cells, we carried out the colocalization experiment via containing MCF-7 cells with Mito Tracker Green FM (a commercial green-fluorescent mitochondrial dye). After incubating with 5 µM CY-SO2 for 30 min, the MCF-7 cells were stained with 1 µM Mito Tracker Green FM for 10 min at 37 oC. As shown in Figure 7, the cells distribution of CY-SO2 and Mito Tracker Green FM demonstrated that probe CY-SO2 with bright red emission mainly localized in the mitochondria with a higher co-localization coefficient (Pearson’s correlation) of 0.98 and not in the nucleus or lysosomes (Figure S16 and S17). Hence, colocalization experiment clearly confirmed that the probe CY-SO2 could localize in the mitochondria, where is one of the main organelle for generation of the endogenous SO2 in vivo, implying the potential capacity of CYSO2 for monitoring endogenous SO2 in living cells. 3．．CONCLUSIONS

In summary, we have developed a novel water-soluble fluorescent probe CY-SO2 based on hemicyanine dye platform. This probe exhibited an excellent linear ratiometric fluorescence


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response, excellent sensitivity (2.67 nM), fast response (90 sec.), high selectivity for HSO3- over other biologically-relevant ions, reactive species, ROS and biological mercaptans in broad pH range. The real sample detecting HSO3- content in sugar exhibited the excellent monitor effects. The sensing mechanism of CY-SO2 was confirmed to be an 1,4-addtion reaction via 1H NMR titration and high resolving mass spectrum. In addition, CY-SO2 probe could be capable of monitoring HSO3- changes through the ratiometric fluorescence imaging with mitochondriatargeted in living cells. So CY-SO2 might be developped to a good SO2-derivative probe for chemical and biological applications. ASSOCIATED CONTENT Supporting Information Synthesis, additional spectroscopic, time courses, MS spectra, pH titration, MTT results, Confocal ratio image , 1H-NMR and 13C-NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *

Fax: +86 0411-84986306; Email address: [email protected].

Author Contributions All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was financially supported by NSF of China (21136002 and 21421005).


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Scheme 1 Structure of probe CY-SO2 and Proposed Sensing Mechanism for HSO3-


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Figure 1 The spectroscopic properties of CY-SO2 (10 µM) in PBS buffer (10 mM, pH 7.4， containing 25 µM CTAB). (a) UV-vis absorption responses of CY-SO2 (10 µM) in the presence of different concentrations of NaHSO3. (b) Fluorescence emission intensity changes of CY-SO2 (10 µM) towards different concentrations of NaHSO3 (titration concentration: 0-16 µM). Each measurement was performed after 3 min of mixing. λex = 400 nm, slit: 10/10 nm.


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Figure 2 Fluorescence ratio (F467 nm/F580 nm) enhancement of CY-SO2 (10 µM) as a function of NaHSO3 (1-5 µM) in PBS buffer (10 mM, pH 7.4, containing 25 µM CTAB). λex = 400 nm, slit: 10/10 nm.


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Figure 3 Time-dependent fluorescence intensity of CY-SO2 (10 µM) at 467 nm after adding 10 µM NaHSO3 in PBS buffer (10 mM, pH 7.4, containing 25 µM CTAB). Time range: 0-500 s, λex = 400 nm, slit: 10/10 nm.


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Figure 4 Fluorescence responses (F467 nm/F580 nm) of CY-SO2 (10 µM) toward various analytes (1 mM unless otherwise stated).1, blank; 2, F-; 3, Cl-; 4, Br-; 5, I-; 6, NO2-; 7, NO3-; 8, SCN-; 9, HPO42-; 10, CH3COO-; 11, CO32-; 12, HCO3-; 13, ClO-; 14, H2O2; 15, Glutathione (GSH); 16, Cysteine (Cys); 17, Ascorbic Acid (AA); 18, S2- (60 µM); 19, HSO3- (10 µM). Each measurement was performed after 3 min of mixing. λex = 400 nm, slit: 10/10 nm.


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Figure 5 The stability of time dependence of CY-SO2 (10 µM) with 1 equiv NaHSO3 (red line) or not (black line) in PBS buffer (10 mM, pH 7.4, containing 25 µM CTAB) were measured with a spectrophotometer every 6 min from 0 to 60 min. λex = 400, slit: 10/10 nm.


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Figure 6 Confocal fluorescence imaging of MCF-7 cells. (Top) the MCF-7 cells were incubated with CY-SO2 (5 µM) for 30 min at 37 oC. (a) Confocal image of CY-SO2 (5 µM) on green channel (450-510 nm). (b) Confocal image of CY-SO2 (5 µM) on red channel (540-600 nm). (c) Bright image. (d) Merged image of (a), (b) and (c). (e) Ratio image (green channel/ red channel). (Bottom) the MCF-7 cells were incubated with CY-SO2 (5 µM) for 30 min at 37 oC, and then treated with 100 µM NaHSO3 at 37 oC for 15min. (f) the emission was collected at 450-510 nm with green pseudocolor. (g) the emission was collected at 540-600 nm with red pseudocolor. (h) Bright image. (i) Merged image of (f), (g) and (h). (j) Ratio image (green channel/ red channel). Excitation wavelength at 405 nm, scale bar = 20 µm.


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a)

e)

b)

f)

c)

g)

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d)

h)

Figure 7 Colocalization fluorescence imaging of MCF-7 cells incubated with CY-SO2 (5 µM) for 30 min and Mito Tracker Green FM (1.0 µM) for 10 min at 37 oC. (a) Confocal image from Mito Tracker Green FM on green channel (λex = 488 nm). (b) Confocal image from CY-SO2 on red channel (λex = 405 nm). (c) Bright image. (d) Merged image of (a), (b) and (c). (e) Correlation plot of the intensities of CY-SO2 and Mito Tracker Green FM (Rr = 0.98). (f), (g) and (h) Normalized intensity profile of regions of interest (ROIs) across MCF-7 cells. Scale bar = 20 µm.


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A Fluorescent Probe for Ratiometric Imaging of SO2 Derivatives in Mitochondria of Living Cells Haidong Lia, Qichao Yaoa, Jiangli Fana, Chong Hua, Feng Xua, Jianjun Dua, Jingyun Wangb, Xiaojun Peng*a a

State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China.

b

School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China.


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A Fluorescent Probe for Ratiometric Imaging of SO2 Derivatives in

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