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A New Red-emitting Fluorescence Probe for Rapid and Effective Visualisation of Bisulfite in Food Samples and Live Animals Fang Zhou, Yasmina Sultanbawa, Huan Feng, Yong-Lei Wang, Qing-Tao Meng, Yue Wang, Zhi-Qiang Zhang, and Run Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07110 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Journal of Agricultural and Food Chemistry

A New Red-emitting Fluorescence Probe for Rapid and Effective Visualization of Bisulfite in Food Samples and Live Animals Fang Zhou,† Yasmina Sultanbawa,‡ Huan Feng,† Yong-Lei Wang,# Qingtao Meng,*† Yue Wang,† Zhiqiang Zhang,† and Run Zhang*†, § †

School of Chemical Engineering, University of Science and Technology Liaoning, Anshan,

Liaoning, 114051, P. R. China ‡

Queensland Alliance for Agricultural and Food Innovation (QAAFI), The University of

Queensland, Australia #

Department of Materials and Environmental Chemistry, Arrhenius Laboratory, Stockholm

University, SE-106 91, Sweden §

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland,

Brisbane, 4072, Australia

ACS Paragon Plus Environment

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ABSTRACT

Development of new method for rapid and effective detection of bisulfite (HSO3–) in food samples and imaging of HSO3– intake in animals is of significant importance due to key roles of HSO3– in food quality assurance and community health. In this work, a new responsive fluorescence probe, EQC is reported for quantitative detection of HSO3– in food samples and visualization of HSO3– intake in animals. Upon addition of HSO3–, UV-vis absorption and red emission of EQC were significantly decreased within 120 seconds. The changes of absorption and emission spectra of EQC were rationalised by theoretical computation. The proposed reaction mechanism of EQC with HSO3– was confirmed by high-resolution mass spectrometry (HRMS) and spectroscopic titration measurements. EQC has the advantages of high sensitivity, selectivity (a detection limit of 18.1 nM), and fast response towards HSO3–, which enable rapid and effective HSO3– detection in buffer solution. The practical applications of EQC were demonstrated by the detection of HSO3– in food samples and the imaging of HSO3– intake in live animals.

KEYWORDS: fluorescence probe; bisulfite; visualization; food samples; live animals


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INTRODUCTION Bisulfite (HSO3–) has been widely used in textile and food industries. In food industry, this anion serves as an antimicrobial agent, additive in beverages, and an antioxidant for various foods. HSO3– is generally recognised as safe (GRAS) by the Food and Drug Administration (FDA).1 In animal bodies, HSO3– at low concentration (less than 450 μM) has been reported as a new messenger in the cardiovascular system for vasodilating, anti-hypertensive and anti-atherogenic effects.2,3,4 However, extensive intake of HSO3– could lead to harmful effects in cells and tissues, causing asthmatic attacks and allergic reactions in some individuals.5-7 Daily intake of HSO3– has been controlled and regulated in many countries and organizations.8,9 Lower 0.7 mg kg−1 of body weight is suggested jointly by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO),10,11 and daily intake of 10 ppm (125 µM) of HSO3– in food and beverages is allowed by the FDA.12,13 Strict management of HSO3– intake amount is necessary, but remains challenging due to the lack of quick and effective methods for quantitative detection of HSO3– in food samples and the intake amount in live bodies. To date, a variety of methods had been developed to detect of HSO3– including electrochemistry,14,15 chromatography,16 chemiluminescence,17,18 fluorescent and flow injection analysis.19-21 Of these methods, fluorescence analysis using responsive fluorescent probe has been recognised as one of the most promising approach for simple, rapid, sensitive and selective detection of target molecules and ions.22-29 For HSO3– detection in food samples and live organisms, a series of fluorescence probes have been developed by using various sensing reaction mechanisms, such as nucleophilic reaction with aldehyde,30-34 selective deprotection of levulinate,35-38 Michael-type additions39-42 and coordinative interactions.43,44 However, there are some unresolved issues for the responsive probes for HSO3– detection, such as short-wavelength


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excitation and emission, unsatisfactory detection limits, long response times (30 min to 10 h) and poor selectivity towards biothiols.45 These problems make it difficult to use these probes for the detection of HSO3– in practical food and biological samples.

Scheme 1. Schematic illustration of the HSO3– by EQC fluorescence probe. The proposed sensing reaction mechanism of EQC towards HSO3– (A). The application of EQC for visualization of HSO3– in food samples by test strips (B) and imaging of HSO3– intake in live zebrafish and mouse (C). In this study, a red-emitting fluorescence probe, EQC was developed for HSO3– detection. EQC has a α,β-unsaturated ethylene unit that can selectively react with HSO3– (Scheme 1A). With this unique reaction, the donor--acceptor (D-π-A) based intramolecular charge transfer (ICT) system of EQC is corrupted. As a result, significant changes of absorption and emission spectra of EQC were obtained for HSO3– detection (Scheme 1B). The sensing mechanism of EQC towards HSO3– was confirmed by HRMS titration and Job’s plot measurements. Using EQC as the probe, quantitative and “naked-eye” detection of HSO3– in wine and sugar samples were demonstrated, suggesting potential application of EQC for HSO3– analysis in real food samples. EQC features fast response to HSO3–, high sensitivity and specificity and appropriate excitation/emission


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wavelength, which enable fluorescence imaging of HSO3– intake in live adult zebrafish and nude mouse (Scheme 1C). EXPERIMENTAL SECTION Reagents and Instruments 3-Formyl-N-ethyl-carbazole, quinaldine and iodoethane were purchased from Aladdin reagent Co. (Shanghai, China). Metal ions (nitrate salts), anions (sodium salts) and biothiols were purchased from Alfa Aesar. Wine and sugar samples were purchased from local food market of Anshan, China. Node mice (6-8 weeks) and adult zebrafish were obtained from the Experimental Animal Center of the Dalian Medical University, China. All the experiments of live nude mice and zebrafish were performed in compliance with the relevant local laws and institute guidelines, and also the institution internal ethics committee of the Dalian Medical University has approved the experiments. Unless otherwise stated, solvents and reagents were of analytical grade from commercial suppliers and were used without further purification. Deionized water was used throughout. 1H-NMR

and

13C-NMR

spectra were recorded with an AVANCE600MHZ spectrometer

(BRUKER) with chemical shifts reported as ppm (in DMSO, TMS as internal standard). Highresolution mass spectra (HRMS) were recorded on an Agilent 6530 QTOF spectrometer. Fluorescence spectra were measured with Perkin Elmer LS55 luminescence spectrometer (USA). Absorption spectra were measured with a Perkin Elmer Lambda 900 UV/VIS/NIR spectrophotometer (USA). Quartz cuvettes with a 1 cm path length and 3 mL volume were used in fluorescence and UV-vis spectrum measurements. The pH was recorded by OHAUS ST3100 digital pH-meter. Imaging of HSO3– in adult zebrafish and mice were performed using a


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SPECTRAL Ami Imaging Systems (Spectral Instruments Imaging, LLC, Tucson, AZ) with an excitation filter 465 nm and an emission filter 610 nm. Synthesis and characterization of EQC 3-Formyl-N-ethyl-carbazole (223.1 mg, 1 mmol), 1-ethyl-2-methylquinolinium iodide (0.172 g, 1 mmol),46,47 and catalytic amount of piperidine (two drops) were added into 20 mL ethanol. The reaction mixture was heated to 80 oC for 8 hours under argon atmosphere to afford a red precipitate. The precipitate was filtrated, washed with cold ethanol and dried under vacuum to form EQC in 81% yield. 1H NMR (DMSO-d6, 600 MHz), δ (ppm) 8.99 (d, J = 10.68 Hz, 1H), 8.86 (s, 1H), 8.66 (d, J = 10.80 Hz, 1H), 8.55 (t, J = 9.30 Hz, 2H), 8.33 (t, J = 9.48 Hz, 1H), 8.26 (t, J = 9.30 Hz, 1H), 8.17 (m, 2H), 7.92 (t, J = 8.91 Hz, 1H), 7.85 (d, J = 18.6 Hz, 1H), 7.80 (d, J = 10.32 Hz, 1H), 7.71 (d, J = 9.78 Hz, 1H), 7.55 (d, J = 9.06 Hz, 1H), 7.33 (t, J = 8.91 Hz, 1H), 5.20 (q, J = 8.46 Hz, 2H), 4.53 (q, J = 8.40 Hz, 2H), 1.62 (t, J = 8.31 Hz, 3H), 1.37 (t, J = 8.34 Hz, 3H). 13C NMR (DMSO-d6, 150Hz), δ (ppm) 156.1, 150.4, 143.9, 142.3, 140.8, 138.6, 135.3, 130.8, 129.1, 128.3, 128.2, 127.2, 126.5, 123.4, 123.2, 122.7, 121.3, 121.2, 121.4, 119.3, 114.9, 110.4, 40.7, 37.9, 14.6, 14.3. HRMS-API (positive mode, m/z) for [EQC]+: Calcd. 377.2012, found, 377.20198. Mp: 256.9 oC-258.2 oC. Visualization of HSO3– in food samples White wine and sugar samples were purchased and prepared to demonstrate the feasibility of EQC for the detection of HSO3- in food samples. The white wine sample was 10-fold diluted with deionized water, and sugar sample solution was prepared by dissolving 1.0 g of sugar in deionized water and diluting to 10 mL. After mixing the EQC (10 µM) and food samples spiked with HSO3(0, 10, 20 and 30 µM, respectively) for 2 min, the emission intensity at 598 nm of each sample was measured. For visualization of HSO3– in food samples, test paper were prepared by soaking


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Whatman filter paper with a acetonitrile solution of EQC (20 µM) for 2 minutes and then the EQC stained filter paper were dried in air for further application. The prepared test paper were simply immersed in the aqueous solution of food samples for 5 seconds, followed by recording the color and fluorescence changes after 2 min incubation at room temperature. Visualization of HSO3– in live adult zebrafish Adult zebrafish was incubated with 40 µM EQC for 30 min. After washing three times with PBS, the zebrafish was further incubated with fresh culture medium containing HSO3– (1 mM) for another 10 min. The zebrafish was rinsed with PBS three times and then was subjected to imaging with an excitation filter 465 nm and an emission filter 610 nm. Zebrafish incubated with 40 µM EQC for 3 h was applied as the control group. Imaging data were acquired and processed using Amiview Living Image 2.0 software (PerkinElmer, USA). Visualization of HSO3– in live nude mice The nude mice (6-8 week old mouse) were anesthetized by isoflurane in a flow of oxygen during all of the experiments. For visualization of HSO3– in live mice, EQC (40 M, 125 L) was subcutaneously injected into mice, followed by the injection of 8.3 mM HSO3– at the same area. Imaging for the injection area were recorded every 3 min within 15 min with an excitation filter 465 nm and an emission filter 610 nm. Imaging data were acquired and processed using Amiview Living Image 2.0 software (PerkinElmer, USA). RESULTS AND DISCUSSION The fluorescence probe for HSO3– was designed using a nucleophilic addition reaction between α,β-unsaturated ethylene of EQC and HSO3–.48 Specifically, EQC has distinct red emission signal due to an intramolecular charge transfer (ICT) process.49 The donor-π-


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acceptor (D-π-A) structure of EQC is corrupted after a nucleophilic addition reaction with HSO3– (Scheme 1A).50 As the result, UV-vis absorption and emission spectra are changed for HSO3– detection.51 The “D-π-A” structure of EQC and the corruption of ICT process were then rationalised by theoretical computation.52,53 The molecular geometries of EQC and EQC-SO3 were first optimized (Figures S1, S2, Tables S1, S2), followed by analysis of frontier molecular orbital distributions. As shown in Figure 1, optimized molecular geometry of EQC clearly showed π-conjugation between carbazole and quinolinium moieties through a C=C double bond (α,β-unsaturated ethylene). This π-conjugation structure was corrupted in EQC-SO3, nucleophilic addition reaction product of C=C double bond of EQC with HSO3–. Based on the optimized ground-state geometries, frontier molecular orbitals of HOMO and LUMO distributions and corresponding electric energies of EQC and EQC-SO3 were calculated. In EQC, HOMO is mainly localized on the carbazole moiety with small distribution on quinolinium, while LUMO is mainly localized on quinolinium with small distribution on carbazole. ICT from carbazole to quinolinium is clearly observed in this “D-π-A” structure. In ECQ-SO3, HOMO is totally dominated by carbazole moiety and LUMO is dominated by quinolinium moiety, respectively. Therefore, UV-vis absorption and fluorescence of ECQ-SO3 are mainly attributed to carbazole and quinolinium moieties. As the result, decreases in UV-vis absorption at visible range and red-emitting fluorescence of EQC are expected after its reaction with HSO3– in aqueous solution.


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Figure 1. Theoretical computation of EQC and EQC-SO3. Optimized molecular geometries of EQC and EQC-SO3, their HOMO and LUMO orbital distributions, and the corresponding electric energies. As shown in Scheme S1, EQC was readily synthesized by a condensation reaction between 3-formyl-9-ethyl carbazole and 1-ethyl-2-methylquinolinium iodide in the presence of catalytic amount of piperidine. The chemical structure of EQC was confirmed by 1HNMR, 13CNMR

and high resolution mass spectrometry (HRMS) (Figures S3-S5).

The nucleophilic addition reaction mechanism of EQC towards HSO3– was then investigated by Job’s plot analysis and HRMS titration analysis. As shown in Figure S6, the changes of fluorescence intensities at 598 nm against the mole fraction of HSO3– clearly showed a maximum value at around 0.5, indicating the 1:1 stoichiometry nucleophilic reaction between EQC and HSO3–.54,55 The products of the reaction between EQC and


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HSO3– were further confirmed by HRMS analysis. EQC exhibited a molecular ion peak at m/z = 377.2020 (Figure S5), which can be assigned to the peak of [EQC]+ (Calcd. m/z = 377.2018). Upon addition of HSO3– in PBS buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4), the peak of [EQC]+ was absent and a new peak at m/z = 481.1553 was observed (Figure S7). This new peak is assigned to the molecular ion peak of the product, EQC-SO3 (Calcd. m/z = 481.1556).

Figure 2. (A) UV-vis absorption spectra of EQC (10 μM) in the presence of different concentrations of HSO3– (0-2.0 mM) in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4) (inset: absorbance of EQC at 453 nm as a function of HSO3– concentration). (B) Absorption spectra and (C) color changes of the EQC (10 μM) in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4) in the presence of various competitive species (1.5 mM): a) Blank, b) HSO3–, c) Br–, d) Cl–, e) F–, f) HSO4–, g) S2–, h) NO2–, i) NO3–, j) 1O2, k) OH–, l) ONOO–, m) P2O74–, n) PO43–, o) SO32–, p) SO42–, q) HCO3–, r) Pi, s) PPi, t) H2O2, u) Cys, v) Hcy, w) GSH, x) HOCl, y) AcO–. In the presence of HSO3–, changes of UV-vis spectra were first measured in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4). As shown in Figure 2A, EQC exhibited an


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absorption band centred at 453 nm, which is assigned to the typical ICT-based absorption process of EQC.56-60 Upon addition of HSO3–, π-conjugation of EQC was corrupted after nucleophilic addition reaction between C=C bond and HSO3–. As a result, the absorption peak at 453 nm was gradually decreased with the increase of HSO3– concentration from 0 to 2 mM (Figure 2A).61,62 UV-vis response selectivity of EQC towards HSO3– was then evaluated. Significant decrease of UV-vis absorption band at 453 nm was noticed in the presence of HSO3–. In sharp contrast, no obvious changes of absorption spectra were noticed upon addition of other analytes, including Br–, Cl–, F–, HSO4–, S2–, NO2–, NO3–, 1O2, OH–, ONOO–, P2O74–, PO43–, SO32–, SO42–, HCO3–, Pi, PPi, H2O2, Cys, Hcy, GSH, HOCl and AcO– (Figure 2B). The specific UV-vis response of EQC towards HSO3– was further confirmed by colorimetric assay,63 where color change was only observed upon the addition of HSO3– (Figure 2C). The results of UV-vis and colorimetric analysis indicate that EQC is highly selective toward HSO3– over other anions. The fluorescence responses of EQC towards HSO3– was then evaluated in PBS buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4). As anticipated, EQC exhibited red fluorescence (Φ = 0.192) with the emission peak centred at 598 nm (λex = 460 nm) (Figure 3A). The fluorescence emission was found to be stable over 30 h incubation in PBS buffer solution (Figure S8). After reaction with HSO3–, EQC in PBS buffer solution showed a significant decrease in emission (Figure 3A). Maximum decrease of the emission intensity at 598 nm was obtained upon addition of 1.5 mM HSO3– (Figure 3B). The fluorescence quantum yield of EQC-SO3 was determined to be 0.011. Accordingly, the red fluorescence color of the EQC solution faded in the presence of HSO3– (Figure S9). The changes of fluorescence


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intensity of EQC exhibited good linearity towards the concentration of HSO3– from 0 to 100 µM (Figure S10). According to the concentration corresponding to three standard deviations of the background signal (LOD = 3σ/k),64-66 the detection limit was then calculated to be 18.1 nM. Specific fluorescence response of EQC towards HSO3– was then evaluated. As shown in Figure 3C, significant decrease in fluorescence intensity of EQC at 598 nm was observed in the presence of HSO3–, while negligible decrease of fluorescence intensity was obtained upon addition of other competitive species, including Br–, Cl–, F–, HSO4–, S2–, NO2–, NO3–, 1O

2,

OH–, ONOO–, P2O74–, PO43–, SO32–, SO42–, HCO3–, Pi, PPi, H2O2, HOCl, AcO–, Hcy,

Cys and GSH. To further evaluate the selectivity of EQC for the detection of HSO3–, the fluorescence response of EQC towards HSO3– has been studied in the presence of different competitive species. As shown in Figure 3C, very limited effects of these species on the fluorescence detection of HSO3– was observed, indicating high specificity of EQC towards HSO3– detection. The specific fluorescence response of EQC to HSO3– (100 equiv. of EQC) was then verified by fluorescence color changes, where red fluorescence emission was decreased exclusively in the presence of HSO3– (Figure S9). Time-dependent fluorescence response of EQC was then investigated in PBS buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4) in the absence and presence of different amounts of HSO3–. As shown in Figure 3D, free EQC exhibited a strong and stable emission centered at 598 nm. Upon addition of HSO3–, significant decrease of fluorescence intensity was noticed within 100 s, suggesting that the nucleophilic addition reaction between EQC and HSO3– could be completed within 2 min.


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Figure 3. (A) Fluorescence spectra of EQC (10 μM) in the presence of different concentrations of HSO3– (0-2.0 mM) in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4). (B) Fluorescence intensities of EQC at 598 nm as a function of HSO3– concentration. (C) Fluorescence responses of EQC (10 μM) to various analytes (1.5 mM) in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4). 1. Blank, 2. Br–, 3. AcO–, 4. Cl–, 5. F–, 6. HSO4–, 7. S2–, 8. NO2–, 9. NO3–, 10. 1O2, 11. OH–, 12. ONOO–, 13. P2O74–, 14. PO43–, 15. SO32–, 6. SO42–, 17. HCO3–, 18. Pi, 19. PPi, 20. H2O2, 21. Cys, 22. Hcy, 23. GSH, 24. HOCl, 25. HSO3–. (D) Time-profile fluorescence quenching at 598 nm of EQC in the presence of (■) 0 mM, (●) 0.5 mM, (▲) 0.9 mM, and (▼) 1.5 mM HSO3– in PBS aqueous buffer (CH3CN: PBS = 1:9, 20 mM, pH = 7.4). Excitation was performed at 460 nm. The effect of pH on the fluorescence response of EQC towards HSO3– was then examined. As shown in Figure S11, EQC showed stable fluorescence emission at the pH range from


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4.0 to 11.5. In the presence of HSO3–, remarkable decrease of fluorescence intensity at 598 nm of EQC was noticed in pH 7.0-11.5, suggesting the feasibility of HSO3– detection at neutral and weakly basic solution. HSO3– is an important food addictive that has been widely used in food industry, but excess addition of HSO3– to foods may cause several health issues.67,68 Employing EQC as the fluorescence probe, the levels of HSO3– in food samples, including sugar and white wine were measured. EQC was added into the food samples and then the emission intensity at 598 nm was tested. As shown in Table 1, the HSO3– concentration was determined to be 26.12 ± 1.03 and 17.01 ± 0.41 μM in 10 times diluted white wine and prepared sugar samples,69 respectively. To confirm the accuracy of EQC for the detection of HSO3– in food samples, the HSO3–concentration in white wine sample was then determined using a standard method (GB/T 5009.34-2016).69 As shown in Table S3, the concentrations of HSO3– in white wine were determined to be 91.43, 62.32, and 24.18 μM after 3, 4, and 10-times dilution, respectively. The HSO3- concentration determined by BG/T 5009.34-2016 is very close the one measured using EQC probe, suggesting high accuracy of EQC for the detection of HSO3–. For the HSO3– spiked food samples, recoveries for the HSO3– detection were found to be in the range from 96.68% to 102.28%. The good recovery of the presented method indicates high precision and accuracy in HSO3– detection in food samples using EQC as a fluorescence probe.

The practical application of EQC for on-site “naked-eye” detection of HSO3− levels was then demonstrated. A test paper was prepared by soaking Whatman filter paper into an acetonitrile solution of EQC (20 µM) for 2 minutes, followed by drying of the filter paper in the air. For visual detection of HSO3− in aqueous solution, the test papers were immersed into pure water solution


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containing different concentrations of HSO3− for 5 seconds. As shown in Figure 4, the EQCinfiltrated test paper presented immediate and clear color changes from orange to paler shades of orange. Under 365 nm UV light, obvious fluorescence color changes of test paper were observed. The performance of the EQC-based test paper for the “naked-eye” visual detection of HSO3− has been further demonstrated in practical food samples. Exposure the test paper to white wine and sugar samples with different concentrations of HSO3−, clearly color and fluorescence changes were observed under daylight and 365 nm UV light, respectively (Figure S12,13). The results indicated that the EQC-based test paper has the potential to be used in the “naked-eye” detection of HSO3− in practical food samples. Table 1. Results for the determination of HSO3− in food samples Food samples

White wine

Sugar

Bisulfite level (μM)

26.12 ± 0.64

17.01 ± 0.41

Bisulfite added (μM)

Bisulfite found (μM)

Recovery (%)

10

36.03 ± 0.37

99.76

20

44.59 ± 0.43

96.68

30

55.72 ± 0.28

99.29

10

26.64 ± 0.21

98.63

20

37.85 ± 0.25

102.28

30

46.89 ± 0.62

99.74

We then investigated capability of EQC for the detection of HSO3− intake in animals through oral feeding (zebrafish) and local injection (nude mouse). Live zebrafish were stained with EQC (40 μM) for 30 min, followed by feeding with HSO3– (1 mM) for another 10 min. As shown in Figure 5, zebrafish incubated with EQC exhibited strong fluorescence signal. This fluorescence signal was significantly quenched after further feeding the zebrafish with HSO3– for 10 min. Decrease of


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red fluorescence signal in zebrafish could be ascribed to the formation of EQC-SO3 after a nucleophilic addition reaction between EQC and HSO3–. The images suggest that EQC can be employed as the probe for fluorescence visualization of HSO3– intake through oral feeding of zebrafish.

Figure 4. Photographs of color (A) and fluorescence (B) responses of the EQC-based test papers towards different HSO3– concentrations in aqueous media: (1) 0 mM, (2) 0.10 mM, (3) 0.5 mM and (4) 1.0 mM.

Figure 5. Fluorescence imaging of HSO3– intake in live zebrafish. (a) Free zebrafish; (b) zebrafish was incubated with EQC (40 μM) for 30 min; (c) the zebrafish (b) was then fed with HSO3– (1 mM) for another 10 min. Imaged with an excitation filter 465 nm and an emission filter 610 nm. The capability of EQC for visualization of HSO3– intake in mouse was then investigated. EQC (40 μM) was subcutaneously injected into 6-8 week old nude mouse, followed by the injection of 1 mM HSO3– at the same region. Fluorescence images were recorded at different time courses after injection. As shown in Figure 6, intense fluorescence signal was observed after EQC injection.


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Further injection of HSO3–, the fluorescence signal gradually decreased and reached to minimal intensity after 15 min. The fluorescence imaging of HSO3– in live mouse demonstrates potential applications of EQC as a probe for visualization of HSO3– intake in vivo.

Figure 6. Fluorescence visualization of HSO3– intake in mice. (a) Control group; (b) 40 μM EQC was subcutaneously injected into the left hind limbs of the mouse; then the HSO3– (1 mM) was injected into the same areas of interest: (c) 5 min; (d) 10 min and (e) 15 min post of injection. (f) The mean fluorescence intensity of interested area at different time showing in (a-e). The mice were imaged with an excitation filter 465 nm and an emission filter 610 nm. In conclusion, a new red-emitting fluorescence probe, EQC, has been developed for HSO3– detection. EQC can sensitively detect HSO3– over other anions and small molecules through a unique nucleophilic addition. The reaction mechanism between EQC and HSO3– was confirmed by HRMS analysis, spectroscopic titration studies, and theoretical


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computations. EQC shows a high sensitivity and specificity, reliability at physiological pH, fast-response and red-emitting, which enabled the detection of HSO3– in food samples and HSO3– intake in live animals. The test paper was successfully prepared for “naked-eye” visualization of HSO3– in white wine and sugar. It is expected that the EQC can be widely used as a quality control tool for HSO3– detection in agriculture and food industries. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis and characterization of EQC, spectrometric and colorimetric analysis of HSO3–, and analysis of HSO3– in food samples. AUTHOR INFORMATION Corresponding Author *Q. Meng, Tel.: +86-412-5929627. E-mail: [email protected] *R. Zhang, Tel.: + 61 7 3346 3806, Fax: + 61 7 3346 3978. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. Funding This work was financially supported by the Natural Science Foundation of Liaoning Province (No. 201602400), National Natural Science Foundation of China (No. 21601076), Australian Research


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Council (DE170100092), and The University of Queensland Early Career Researcher grant (UQECR1833837). The authors also acknowledged the facilities and the assistance of Queensland Node of the Australian National Fabrication Facility (ANFF-Q), the University of Queensland. Notes The authors declare no competing financial interest. REFERENCES (1) Isaac, A.; Livingstone, C.; Wain, A. J.; Compton, R. G.; Davis, J. Electroanalytical methods for the determination of sulfite in food and beverages Research article. Trends Anal. Chem. 2006, 25, 589–598. (2) Finkel, T.; Holbrook, N. J. Oxidative stress and the biology of ageing. Nature. 2000, 408 239−247. (3) Liu, X.; Yang, Q.; Chen, W.; Mo, L.; Chen, S.; Kang, J.; Song, X. A ratiometric fluorescent probe for rapid, sensitive and selective detection of sulfur dioxide with large Stokes shifts by single wavelength excitation. Org. Biomol. Chem. 2015, 13, 8663–8668. (4) Luo, J.; Song, G.; Xing, X.; Shen, S.; Ge, Y.; Cao, X. A simple but effective fluorescent probe for the detection of bisulfate, New J. Chem. 2017, 41, 3986–3990. (5) Chao, J.; Li, Z.; Zhang, Y.; Huo, F.; Yin, C.; Liu, Y.; Li, Y.; Wang, J. Single fluorescent probe for multiple analyte sensing: efficient and selective detection of CN–, HSO3– and extremely alkaline pH. J. Mater. Chem. B 2016, 4, 3703–3712. (6) Sang, N.; Yun, Y.; Yao, G.; Li, H.; Guo, L.; Li, G. SO2-Induced Neurotoxicity Is Mediated by Cyclooxygenases-2-Derived Prostaglandin E2 and its Downstream Signaling Pathway in Rat Hippocampal Neurons. Toxicol. Sci. 2011, 124, 400–413. (7) Zhang, W.; Liu, T.; Huo, F.; Ning, P.; Meng, X.; Yin, C. Reversible Ratiometric Fluorescent Probe for Sensing Bisulfate/H2O2 and Its Application in Zebrafish. Reversible Ratiometric


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