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Reversible Fluorescent Probe for Selective Detection and Cell Imaging of Oxidative Stress Indicator Bisulfite Yajiao Zhang,†,‡,⊥ Lingmei Guan,∥,⊥ Huan Yu,†,‡ Yehan Yan,†,‡ Libo Du,∥ Yang Liu,∥ Mingtai Sun,*,†,‡ Dejian Huang,§ and Suhua Wang*,†,‡ †

Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China Department of Materials Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China § Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore ∥ State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡

S Supporting Information *

ABSTRACT: In this paper, we report a benzothiazolefunctionalized cyanine fluorescence probe and demonstrate that it is selectively reactive to bisulfite, an intermediate indicator for oxidative stress. The selective reaction can be monitored by distinct ratiometric fluorescence variation favorable for cell imaging and visualization. The original probe can be regenerated in high yield through the elimination of bisulfite from the product by peroxides such as hydrogen peroxide, accompanied by fluorescence turning on at 590 nm, showing a potential application for the detection of peroxides. We successfully applied this probe for fluorescence imaging of bisulfite in cancer cells (MCF-7) treated with bisulfite and hydrogen peroxide as well as a selective detection limit of 0.34 μM bisulfite in aqueous solution.

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been reported for the measurement of anions, biological molecules, and metal cations in physiological process,18 quite few fluorescent probes with reversible redox properties for sulfur dioxide and peroxides have been reported. A fluorescent sensor has been reported to monitor the reversible redox cycles mediated by reductants [NaHSO3, tris(2-carboxyethyl)phosphine (TCEP), or NaBH4] and oxygen or hydrogen peroxide by restoring fluorescence.19 So it is of importance to develop a reversible fluorescent probe for selectively monitoring the intracellular SO2/ROS redox cycle and visual detection of environmental sulfur dioxide. In general, the selective detection of bisulfite could be achieved through the specific nucleophilic addition to the aldehyde group20−25 or unsaturated carbon−carbon double bond.26−34 Recently, Liu et al. reported a sensitive fluorescent probe for circularly monitoring sulfites via a tandem reaction; the probe is based on coumarin dye conjugated with a selected ethyl cyanoacetate moiety and has been successfully applied to monitor the variation of sulfites in the living cells.35

ecently, it has been shown that bisulfite and sulfur dioxide can be endogenously generated through the oxidation of intracellular hydrogen sulfide or sulfur-containing amino acids by reactive oxygen species (ROS), which could result in oxidative stress and aged-related diseases.1−4 In addition, bisulfite can also be converted from gaseous sulfur dioxide (SO2), the main atmospheric pollutant that can endanger human health, cause acid rain, and damage aquatic and terrestrial ecosystems. It can be easily inhaled and turns into sulfite and bisulfite,5,6 which are widely used as food additives.7−12 Excessive intake of sulfur dioxide causes severe adverse effects and acute symptoms, such as flushing, hypotension, diarrhea, urticaria, and abdominal pain.13,14 Therefore, it is of great significance to develop a selective and sensitive fluorescent probe for the detection of sulfur dioxide and its derivative bisulfite in the point view of environmental protection and biological technology. The widely used methods for bisulfite detection include phosphorimetry, fluoremetry, electrochemistry, spectrophotometry, etc. Among these methods, fluoremetry is more attractive for both environmental and bioimaging applications because of its advantages of easy visualization, excellent sensitivity and selectivity, and cell imaging with spatial resolution.5,15−17 Although an increasing number of fluorescent probes have © 2016 American Chemical Society

Received: January 7, 2016 Accepted: March 31, 2016 Published: March 31, 2016 4426

DOI: 10.1021/acs.analchem.6b00061 Anal. Chem. 2016, 88, 4426−4431

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Analytical Chemistry Different to coumarin, cyanine-based dyes not only possess CC double bonds specifically reactive to bisulfite but also exhibit low-energy fluorescence in the near-infrared region,36 which is favorable for cell imaging because of the low biological background fluorescence. Here, we synthesized a novel fluorescent probe, 2-(2′-hydroxyphenyl) benzothiazole cyanine (HBT-Cy), which shows dual emission bands at 450 and 590 nm upon a single excitation at 390 nm, derived from the benzothiazole and cyanine moieties, respectively. As shown in Scheme 1, one of the unsaturated CC double bonds is

ppm) was prepared from the reaction of HCOOH with concentrated H2SO4. CO2 gas (106 ppm) was obtained by the reaction of NaHCO3 with 1 M H2SO4. NO2 gas (106 ppm) was got by diluting the pure NO2 gas to the target concentration. H2S (106 ppm) gas was prepared from the reaction of Na2S with 6 M H2SO4. NH3 gas (105 ppm) was prepared from the reaction of NH4Cl with 1 M NaOH. Appropriate amounts of these gas samples (104 ppm) were bubbled into the probe solutions in PBS 7.0, and the fluorescence spectra were recorded. Standard SO2 gas was purchased from Nanjing Special Gas Factory Co. Ltd. In order to calibrate the concentration of SO2 accurately, we dissolved SO2 gas in ethanol and determined the concentration by measuring the UV absorbance at 277 nm (ε = 365 M−1 cm−1) and applying the Beer−Lambert law. The SO2 solution (103 ppm) was used immediately after determining its concentration. Cell Cytotoxicity Evaluated by MTT Assay. MCF-7 cells were prepared for cell viability studies in 96-well plates (1 × 104 cells per well). After 24 h of cell attachment, the substrate was replaced with Dulbecco’s modified Eagle’s (DMEM) basic medium, and then different concentrations of HBT-Cy (0−100 μM) were added into the 96-well plate. After 24 h of incubation at 37 °C in a humidity incubator containing 5% CO2, the supernatant was removed and 100 μL of 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/ mL) phosphate buffer solution was added into each well. After 4 h of incubation at 37 °C, excess MTT was carefully removed and then 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystals. The plate was shaken for 10 min, and the optical density at 490 nm was taken by a microplate reader. Each of the experiments was performed three times. Confocal Fluorescence Imaging. MCF-7 cells were prepared in confocal plates (1 × 105 cells per well) and treated with 5% CO2 at 37 °C in a humidity incubator for 24 h. For the control experiment, MCF-7 cells were incubated with 20 μM of probe HBT-Cy at 37 °C for 4 h in DMEM medium with 10% fetal bovine serum (FBS), then washed with 1 mL of PBS (pH 7.4) twice. For the detection of bisulfite, MCF-7 cells were incubated with 20 μM of probe HBT-Cy at 37 °C for 4 h in DMEM medium with 10% FBS, then treated with 50 μM of bisulfite for another 0.5 h. For the reversibility experiment, upon the above treatment bisulfite was further incubated with 50 μM of H2O2 for another 1 h. Cell imaging was performed after the cells were washed twice with 1 mL of PBS buffer solution. The cells were excited at 405 nm for the blue channel (425−510 nm) and at 520 nm for the red channel (560−650 nm) using laser confocal microscopy (OBSERVER Z1, ZEISS) and a 60× oil-immersion objective lens. Synthesis of 3-Benzothiazol-2-yl-4-hydroxybenzaldehyde (HBT-CHO). In a round-bottomed flask (25 mL) equipped with a magnetic stirrer, a solution of 2-aminothiophenol (0.628 g, 5 mmol) and SD-2 (4-hydroxybenzene1,3-dicarbaldehyde) (0.751 g, 5 mmol) in ethanol (10 mL) was prepared. The mixture was stirred at room temperature for 12 h. The solvent was removed under vacuum, and the crude product was purified by silica gel chromatography (DCM/PE = 2:1, v/v) to give the pure product. ESI-MS m/z calcd for C14H9NO2S [M − H]−, 254.03; found 254.01. Synthesis of (E)-3-(2-(3-(Benzothiazol-2-yl)-4-hydroxystyryl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1sulfonate (HBT-Cy). A mixture of HBT-CHO (0.382 g, 1.5 mmol), piperidine (0.33 mL, 1.5 mmol), and compound 1

Scheme 1. Structure of HBT-Cy and the Proposed Mechanism of HBT-Cy for the HSO3−/H2O2 (TBHP)Induced Redox Cyclea

a Inset image: left, visible-light and fluorescence image of 40 μM probe; right, visible-light and fluorescence image of 40 μM probe and 200 μM HSO3− in 50 mM pH 7.0 PBS.

selectively reactive with bisulfite through nucleophilic addition to produce the reduction product (HBT-CyO), accompanied by a distinct ratiometric fluorescence change. Upon treatment with peroxides like hydrogen peroxide and tert-butyl hydroperoxide (TBHP), the original probe HBT-Cy could be restored, leading to a fluorescence turn-on at 590 nm. The reversible reduction−oxidation cycle could be repeated and still remained the reactivity and spectral properties. Attractively, we also applied the probe HBT-Cy to detect gaseous SO2 with excellent selectivity, monitor HSO3− in a real sample water, and image the reversible redox cycle in living cells.

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EXPERIMENTAL SECTION Procedures for Spectra Measurement. The stock solution of HBT-Cy (10 mM) was prepared in ethanol. Interfering species [HSO3−, F−, Cl−, Br−, I−, CO32−, AcO−, NO2−, S2O82−, S2O32−, SCN−, HPO42−, HS−, CN−, vitamin C (Vc), cysteine (Cys), glutathione (GSH), bovine serum albumin (BSA)] and reactive oxygen species (NaClO, KO2, H2O2, TBHP, ·OtBu, 1O2, and HO·) were freshly prepared with a stock solution of 10.0 mM. Concentration of NaClO was determined by measuring its UV absorption immediately before use. tert-Butoxy radical (·OtBu) was generated by Fenton reaction between FeCl2 and TBHP.37 Singlet oxygen (1O2) was generated by the reaction of H2O2 and NaOCl.38 Hydroxyl radical (HO·) was obtained via the Fenton reaction of FeSO4· 7H2O and H2O2.39 Test solutions were prepared by placing 4 μL of the probe stock solution and appropriate aliquot of each substance stock solution into a test tube and diluting the solution to 2 mL with PBS 7.0. The fluorescence intensity changes upon addition of HSO3− after 15 min were detected by the fluorescence spectra in the range from 410 to 750 nm using a 390 nm excitation wavelength. Procedures for Gas Measurement. The procedures for all the gases were referred to the literatures.6,36 CO gas (106 4427


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Analytical Chemistry

nucleophilic addition by bisulfite is more favorable under neutral and acidic conditions. Therefore, it is appropriate to perform the detection and analysis in a PBS buffer solution at pH 7.0. Under this condition, the nucleophilic addition reaches equilibrium at 15 min, as evidenced by the reaction kinetic curves (Figure S11). It can be seen that, upon addition of bisulfite, the fluorescence intensity at 590 nm gradually decreases and reaches stability after 15 min. As shown in Figure 1a, the free HBT-Cy shows a main absorption band at 520 nm and a shoulder at 390 nm,

(2,3,3-trimethyl-1-(3-sulfonatepropyl)-3H-indolium) (0.442 g, 1.57 mmol) was dissolved in 10 mL of ethanol. The reaction mixture was stirred at 80 °C under a N2 atmosphere. An hour later, the solvent was removed under vacuum and the crude product was purified by silica gel chromatography (DCM/ MeOH = 20:1, v/v) to give the pure product. ESI-MS m/z calcd for C28H26N2O4S2 [M + H]+, 519.13; found 519.18. 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.37 (d, J = 13.6 Hz, 1H), 8.31 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.95 (s, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 7.4 Hz, 1H), 7.52−7.39 (m, 2H), 7.39−7.25 (m, 2H), 7.03 (d, J = 14.2 Hz, 1H), 6.54 (d, J = 9.0 Hz, 1H), 4.47 (s, 2H), 2.66 (s, 2H), 2.08 (s, 2H), 1.76 (s, 6H). Synthesis of (E)-3-(2-(4-Hydroxystyryl)-3,3-dimethyl3H-indol-1-ium-1-yl)propane-1-sulfonate (Cy-OH). 4-Hydroxy benzaldehyde (0.183 g, 1.50 mmol) was dissolved in ethanol (12 mL), the solution was stirred at N2 atmosphere for 15 min to remove the oxygen, then compound 1 (0.422 g, 1.5 mmol) and piperidine (0.33 mL, 1.5 mmol) were added into the solution. The reaction mixture was stirred at 80 °C under a N2 atmosphere. An hour later, the solvent was removed under vacuum and the crude product was purified by silica gel chromatography (DCM/EtOH = 10:1, v/v) to give the pure product. ESI-MS m/z calcd for C21H22NO4S [M + H]+, 386.13; found 386.14.

Figure 1. (a) UV−vis spectra of HBT-Cy (50 μM) upon addition of HSO3− (0−100 μM) in PBS 7.0 solution. (b) Ratiometric fluorescence spectra of HBT-Cy (20 μM, λex = 390 nm) in the presence of different concentrations of HSO3− (0−50 μM) in PBS 7.0 solution. The inset photos show the visible-light and fluorescence color changes of HBTCy (40 μM) in the different concentrations of HSO3− (0, 10, 30, 60, 100, 200 μM, respectively).

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RESULTS AND DISCUSSION Design and Synthesis of Fluorescence Probe HBT-Cy. The probe was synthesized from salicylaldehyde, paraformaldehyde, and 2-aminothiophenol, as shown in Scheme S1 (see the Supporting Information). It can be seen that the probe consists of a cyanine dye fluorophore moiety and a benzothiazole fluorophore moiety. The two functional moieties have specific binding site for bisulfite40−42 and various biological activities.43−46 Probe HBT-Cy has two well-resolved emission wavelengths in the visible region, which can cancel various negative effects often observed in single-wavelength probes.47−49 The fluorescence quantum yield at 590 nm was measured to be 11.1% using rhodamine B in ethanol solution (ΦF = 0.71) as a reference (Figure S1). The molecular structure of probe HBT-Cy and the synthetic intermediates were characterized by 1H NMR and electrospray ionization mass spectrometry (ESI-MS) (Figures S2−S4). For comparison, a similar probe (Cy-OH) without the benzothiazole functional group was also synthesized, as shown in Scheme S2. The structure of the probe was characterized by spectra and ESI-MS (Figures S5−S8). The similar probe has a single emission band at 565 nm with a quantum yield of 0.34% (Figure S9), which is much shorter and lower than those of the benzothiazole-functionalized probe, respectively. The comparison indicates that the functionalization with benzothiazole greatly improves the analytical performance by enhancing the quantum yield, red-shifting the emission band, and introducing the second emission band. Spectral Properties of HBT-Cy. The influence of pH on the properties of HBT-Cy was first examined in the absence and presence of HSO3− because nucleophilic addition was sensitive to pH.50 The results show that pH does have a vital effect on the probe (Figure S10). It can be seen that the fluorescence intensity of HBT-Cy at 590 nm in the absence of bisulfite gradually increases in the pH region from 5.0 to 7.0. However, the fluorescence intensity keeps at a low value in the presence of bisulfite below pH 7.0. This result suggests that the

respectively. Upon addition of bisulfite, both the two absorption bands decrease, showing an obvious color change from red to colorless (Figure 1a, inset) and the potential for colorimetric determination of bisulfite. More interestingly, the probe shows a distinct ratiometric fluorescent response toward HSO3− with two emission bands at 450 and 590 nm upon single excitation at 390 nm. As shown in Figure 1b, the emission band at 590 nm decreases gradually accompanied by a slight increase at the 450 nm band after addition of HSO3−. Moreover, a significant fluorescence color change from rose-red to blue could be observed under a 365 nm UV lamp (Figure 1b, inset). This could be attributed to the 1,4-addition reaction at the unsaturated bond of cyanine moiety by HSO3−, which breaks the conjugated structure of the cyanine dye. The fluorescence intensity of HBT-Cy decreases in proportion to the concentration of HSO3− (Figure S12). The fluorescence ratio (F450/F590) is also linearly proportional to the amounts of HSO3− (Figure S13) with a detection limit (S/N = 3) of 0.34 μM. The result indicates that probe HBT-Cy is potentially useful for the quantitative determination of HSO3−. The nucleophilic addition product by bisulfite was confirmed by ESI-MS and 1H NMR spectra (Figures S14 and S15). Selectivity and Interference. The selectivity of the probe toward HSO3− over other species (including F−, Cl−, Br−, I−, CO32−, AcO−, NO2−, S2O82−, S2O32−, SCN−, HPO42−, Vc, Cys, GSH, BSA, HS−, and CN−) was examined by monitoring the fluorescence intensity in PBS 7.0 media (Figure S16). It can be seen that bisulfite shows the most efficient fluorescence quenching effect at 590 nm. Hydrosulfide also shows a slight quenching effect, which could be caused by the 1,2-addition reaction at the C-2 atom.51 Other biologically relevant molecules do not lead to obvious fluorescence changes even at higher concentrations up to 500 μM. In addition, the fluorescence response of HBT-Cy to HSO3− in the presence of other interfering species is nearly identical to that in the 4428


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Analytical Chemistry absence of these species, showing that the presence of other substances does not interfere the reaction between the probe molecules and bisulfite. The results illustrate that probe HBTCy does have specific response toward HSO3− over other common interfering species. Fluorescence Reversibility of the Probe Mediated by Peroxides. We extensively investigated the reversibility of the nucleophilic addition product (HBT-CyO) by ROS including KO2, H2O2, TBHP, NaClO, ·OtBu, 1O2, and HO· in PBS 7.0 solution at 37 °C. As can be seen in Figure 2a, hydrogen

Figure 3. 1H NMR spectra of (A) HBT-Cy in DMSO-d6 and (B) upon addition of 2 equiv of HSO3− in D2O and (C) subsequent addition of 2 equiv of H2O2 in D2O. a, b, and c denote the protons of the origin probe HBT-Cy; a′, b′, and c′ denote the protons of addition product HBT-CyO; a″, b″, and c″ denote the protons of the restored probe.

Figure 2. (a) Fluorescence recovery ratio of HBT-CyO with the addition of different ROS during 2 h at 37 °C. Bars represent fluorescence intensity at 0.5, 1.0, 1.5, and 2.0 h, respectively. Fluorescence recovery ratio = (F590/F450)/(F590/F450)0, where F590/ F450 is the fluorescence intensity ratio of HBT-CyO (20 μM HBT-Cy + 50 μM HSO3−) after addition of different species of ROS (50 μM) and (F590/F450)0 is the fluorescence intensity of HBT-Cy (20 μM). (b) Ratiometric fluorescence response of HBT-Cy (20 μM) to redox cycles of HSO3− and H2O2. HBT-Cy reacted with 50 μM of HSO3−. Fifteen minutes later, the solution was treated with 50 μM of H2O2 at 37 °C. After 60 min, the fluorescence returned to the starting levels and another 50 μM of HSO3− was added to the mixture. The redox cycle was repeated five times. All data were acquired in 50 mM PBS at pH 7.0 (λex = 390 nm). The inset photos show the corresponding fluorescence colors of HBT-Cy, HBT-CyO, and then subsequent addition of H2O2.

When the 1,4-addition product was subsequently treated with 1 equiv of H2O2 (Figure S17), both the proton signals assigned to HBT-Cy and HBT-CyO can be observed in the 1H NMR spectrum, indicating that a fraction of HBT-CyO has been restored to the initial probe. When the concentration of H2O2 was increased to 2 equiv, the corresponding 1H NMR spectrum obtained is nearly identical to that of the initial probe, suggesting the full recovery of the initial probe from addition product by H2O2 oxidation. This is further confirmed by the ESI-MS spectrum and 1H NMR (Figures S18 and S19). Therefore, the 1,4-addition reaction mechanism and the reversibility of the probe by alternate addition of HSO3− and H2O2 can be confirmed according to the above experimental results and analysis. Living Cell Imaging of Bisulfite. The ability of HBT-Cy for imaging reversible redox cycles of HSO3− and H2O2 in living cells was evaluated with MCF-7 cells. As shown in Figure 4a, the cells incubated with the probe HBT-Cy for 4 h at 37 °C present intense red intracellular fluorescence and inconspicuous blue fluorescence. Upon addition of HSO3− and culturing for 30 min, the red fluorescence decreased significantly and the blue fluorescence increased (Figure 4b). The results suggested that probe HBT-Cy is cell-permeable and capable of monitoring HSO3− in living cells. In addition, when H2O2 was added to the above stained cells for another 1 h, the red fluorescence was restored and the blue fluorescence became weak again (Figure 4c), indicating that probe HBT-Cy still possesses the redox activity in living cells. To demonstrate the detection of endogenous bisulfite in living cells, we synthesized a SO2 donor which can release SO2 by reacting with Cys (Figure S20). Its ESI-MS spectrum is shown in Figure S21. It can be seen that the cell fluorescence in the blue channel increased and the red channel decreased after being treated with SO2 donor (25 μM) and Cys (250 μM) (Figure 5), consistent with that of adding bisulfite. This result is indicative of the fact that HBT-Cy can be utilized to sense endogenous bisulfite in living cells. To further evaluate whether or not the probe HBT-Cy can detect the changes of bisulfite levels in living cells, we conducted the cell experiments incubated with HBT-Cy for 4 h, followed by incubation with different concentrations of

peroxide and tert-butyl hydroperoxide are the most effective oxidants to restore the fluorescence intensity by up to 92% of recovery ratio, whereas other oxidants including KO2, NaClO, · OtBu, HO·, and 1O2 only exhibit limited recovery rate. Further experiments show that the restored probe still possesses the same reactivity to HSO3− as the original one, as shown in Figure 2b; such a reversible redox cycle could be repeated four times without much degradation of the reactivity and fluorescence properties. The color of the addition product after oxidation by H2O2 is similar to that of the original probe HBT-Cy, which can further verify the reversible redox activity of the probe. Reaction Mechanism of the Probe with Bisulfite and Peroxides. The sensing mechanism of HBT-Cy for bisulfite was carefully examined by 1H NMR spectral changes of the probe after subsequent addition of HSO3− and H2O2, as shown in Figure 3. Clearly, the proton signal at δ 1.76 (Ha) of the two methyl groups CH3 shifts forward and splits into two signals (Ha′, at δ 1.22 and 1.57) after reaction with HSO3−. The proton signals (Hb, at δ 6.54, and Hc, at δ 7.03), which are assigned to the double bond of HBT-Cy, also shift to δ 5.05 (Hb′) and δ 4.86 (Hc′), respectively. Moreover, a new signal appeared at δ 9.95 (Hd′), which can be explained by the nucleophilic attack toward the C-4 atom by HSO3−. These results suggested that 1,4-addition product indeed generated after reaction with HSO3−. 4429


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exogenous and endogenous HSO3− as well as monitoring the redox cycle in the presence of H2O2 in living cells. Gaseous SO2 Detection and Real Sample Analysis. It is well-known that SO2 and HSO3− quickly reach equilibrium in aqueous solution, so this method could be used to detect gaseous SO2 in aqueous solution. For this measurement, we added different concentrations of SO2, which was dissolved in ethanol, to the HBT-Cy in PBS 7.0 solution. The emission intensity at 590 nm decreases and the emission at 450 nm increases with the addition of SO2 from 0 to 500 ppm (Figure S24). There was an excellent dose−response relationship between the fluorescence intensity ratio (F450/F590) and the SO2 concentration (Figure S25). Comparing with the effect of adding HSO3−, we can demonstrate that probe HBT-Cy could be used as a ratiometric fluorescent probe for the detection of gaseous SO2. We then evaluated the selectivity of HBT-Cy toward other common gases. Various gas species (CO, CO2, H2S, NO2, and NH3, 104 ppm) were syringed into the HBT-Cy solutions, and the fluorescence spectra were recorded after 15 min. As shown in Figure 6a, there is no obvious fluorescence

Figure 4. Confocal fluorescence imaging of (a) living MCF-7 cells incubated with probe HBT-Cy (20 μM), (b) HBT-Cy (20 μM) loaded MCF-7 cells further incubated with 50 μM of HSO3− for another 0.5 h, and (c) HBT-Cy loaded MCF-7 cells incubated with 50 μM of HSO3− for another 0.5 h, and then incubated with 50 μM of H2O2 for another 1 h in the blue channel, bright-field, red channel, and overlay images (left to right). Ex@405 nm for the blue channel (425−510 nm); Ex @520 nm for the red channel (560−650 nm).

Figure 6. (a) Ratiometric fluorescent effect of various gas species to probe HBT-Cy (20 μM). (b) Color responses of probe HBT-Cy to different gas species under daylight and a 365 nm UV lamp in PBS 7.0 solution. The final concentration of SO2 was 500 ppm; CO, CO2, H2S, NO2, and NH3 were 104 ppm, respectively. Figure 5. Confocal fluorescence imaging of (a) living MCF-7 cells incubated with probe HBT-Cy (20 μM), (b) HBT-Cy (20 μM) loaded MCF-7 cells further incubated with SO2 donor (25 μM) and Cys (250 μM) for 30 min in the blue channel, bright-field, red channel, and overlay field (left to right). Ex@405 nm for the blue channel (425− 510 nm); Ex @520 nm for the red channel (560−650 nm).

change that could be observed when compared with that of adding SO2 gas. Figure 6b shows that the solution color remains unchanged for other gas species, except the one with SO2 gas, which turns to colorless from red and its fluorescence color changes from rose-red to blue under a 365 nm UV lamp. These results clearly demonstrate that the probe HBT-Cy can be applied for the visual detection of SO2 gas on the basis of fluorometry and colorimetry. To validate the application of the probe HBT-Cy for detecting HSO3− in real samples, spike and recovery tests were carried out in matrixes such as tap water, lake water, and rainwater. All the experiments and measurements were repeated three times. The results are summarized in Table S1 and suggest that probe HBT-Cy has a potential for the detection of HSO3− in real water samples with satisfactory recovery.

NaHSO3 (0, 5, 10, 20, 50 μM) for another 30 min. The intracellular fluorescence imaging showed that the blue fluorescence increased and the red fluorescence gradually decreased as the amounts of NaHSO3 increased (Figure S22). The results suggested that HBT-Cy was able to detect the changes of bisulfite concentrations in cells. It was reported that the average physiological concentrations of sulfur dioxide (calculated by sulfite) in thoracic aortic tissues and plasma were 127.76 ± 31.34 and 16.77 ± 8.24 μM, respectively;52 therefore, we can ensure that HBT-Cy is capable of monitoring the fluctuation of endogenous bisulfite levels in living cells. The cytotoxicity of HBT-Cy to MCF-7 cells was determined by an MTT assay with the concentrations of probe HBT-Cy ranging from 0 to 100 μM. As shown in Figure S23, nearly 100% cell viability remained after incubation with 20 μM of HBT-Cy for 24 h, revealing that probe HBT-Cy is almost nontoxic for MCF-7 cells. Therefore, all results elucidated that HBT-Cy was cell membrane permeable, nontoxic, and capable of detecting

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CONCLUSIONS In summary, we have synthesized a reversible colorimetric and ratiometric probe HBT-Cy. The probe employs a cyanine dye moiety which is reactive to sulfur dioxide and its derivative bisulfite sensing. Probe HBT-Cy is easy to use and has high selectivity and ratiometric fluorescence response to SO2/ HSO3− by the mechanism of nucleophilic addition. The cell imaging experiments indicate that HBT-Cy has the potential to sense exogenous and endogenous bisulfite as well as possessing 4430


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the redox activity in biological system. We also successfully applied HBT-Cy for the visual detection of gaseous SO2 and the determination of HSO3− in real water samples. The results demonstrate the diverse applications of the probe and suggest its potential for further imaging of oxidative stress in living cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00061. Details about the experiments, synthesis (Schemes S1− S3), additional figures including fluorescence quantum yields, 1H NMR, ESI-MS, UV−vis, and fluorescence spectra, calibration curves, and confocal imaging (Figures S1−S25), and recovery results for HSO3− spiked in real water samples (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 86-551-65591812. Fax: 86551-65591156. *E-mail: [email protected]. Phone: 86-551-65591812. Fax: 86551-65591156. Author Contributions ⊥

Y.Z. and L.G. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (Grant Nos. 21302187, 21475134, 21507135, and 91439101). S.W. and D.H. thank the National Natural Science Foundation of China for Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund 21228702.

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