Article pubs.acs.org/JAFC
Ratiometric Fluorescent Probe for Rapid Detection of Bisulfite through 1,4-Addition Reaction in Aqueous Solution Yue Sun, Dong Zhao, Shanwei Fan, Lian Duan,* and Ruifeng Li School of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China S Supporting Information *
ABSTRACT: A ratiometric fluorescent probe based on a positively charged benzo[e]indolium moiety for bisulfite is reported. The bisulfite underwent a 1,4-addition reaction with the C-4 atom in the ethylene group. This reaction resulted in a large emission wavelength shift (Δλ = 106 nm) and an observable fluorescent color change from orange to cyan. The reaction could be completed in 90 s in a PBS buffer solution and displayed high selectivity and sensitivity for bisulfite. A simple paper test strip system was developed to detect bisulfite rapidly. Probe 1 was used to detect bisulfite in real samples with good recovery. KEYWORDS: benzo[e]indolium, bisulfite sensing, chemosensor, ratiometric fluorescence
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HSO3− in 100% aqueous media. Recently, Zhang reported a ratiometric fluorescent probe for HSO3− via the Michael addition reaction;18 however, 1 mM CTAB was added as cosolvent, and the detection was really time-consuming (1 h). Yu reported a water-soluble near-infrared probe for HSO3− ratiometric sensing with a satisfactory detection limit of 0.25 nM and a short response time (90 s),13 but two excitation waves were needed to achieve the ratiometric fluorescent change. As such, the design of more efficient ratiometric fluorescent probes that can perform in 100% aqueous media remains a great challenge. Cyanine dyes are widely used in fluorescent labeling of biomolecules, photodynamic therapy, optical data storage, and molecular recognition.23 A series of anion probes containing cyanine dye has been reported to detect CN−,23−27 HS−,28 HSO3−,16,20 and HSO4−29 because of their long emission wavelengths, potential ratiometric fluorescence, and specific binding site, as well as satisfactory water solubility. The nucleophilic attack caused by CN−, HS−, and HSO3− toward these probes through 1,2-addition reactions or an addition−rearrangement reaction interrupts the π-conjugation and blocks the intramolecular charge transfer process, which result in ratiometric fluorescent changes. In this work, a ratiometric fluorescent probe 1 for HSO3− detection in PBS buffer was introduced (Scheme 1). A positively charged benzo[e]indolium moiety in probe 1 was used as the fluorophore, and an unsaturated CC double bond was incorporated as the binding site for HSO3−. In our previous study,26 probe 1 was designed to detect CN− in water by taking advantage of the 1,2-addition reaction (Scheme 1). However, the use of PBS buffer solution resulted in excellent selectivity and sensitivity to HSO3− as well as a short response time. Probe 1 displayed an emission maximum at 571 nm in PBS buffer (pH 7.4, 10 mM), and HSO3− was expected to be detectable via a nucleophilic attack toward the C-4 atom through a 1,4-
INTRODUCTION Bisulfite (HSO3−) has been widely used as an antimicrobial agent, an enzyme inhibitor, and an antioxidant for foods, beverages, and pharmaceutical products.1,2 However, extensive intake of HSO3− would induce harmful effects to tissue, cells, and biomacromolecules, causing asthmatic attacks and allergic reactions in some individuals.3 Furthermore, inhaled SO2 can be hydrated to produce sulfurous acid in the respiratory tract and subsequently forms its derivatives HSO3−,4,5 which may pose health risks to humans. Given the reported harmful effects on people’s health, the threshold levels of HSO3− in food and medicine have been rigorously controlled in many countries. For instance, the total concentration of sulfur (calculated by SO2) in white granulated sugar is strictly regulated as 120-fold (from 0.12 to 15.4). To our knowledge, the notable fluorescent spectral change caused by 0.1 equiv of HSO3− (1 μM) and obtaining the maximal spectral signal with 1 equiv of HSO3− (10 μM) were impressive and unprecedented compared with those of many reported HSO3− probes. In similar conditions, the ratio changes produced an excellent linear function with HSO3− concentration between 0 and 10 μM (Figure S6 in the Supporting Information), and the detection limit for HSO3− was 5.6 × 10−9 M (R2 = 0.9935). The absorption spectrum of probe 1 (20 μM) showed two strong bands at 310 and 360 nm (Figure 2B). The decrease in band intensity upon the addition of HSO3− also suggests that the π-conjugation was broken. This phenomenon leads to a solution color change from yellow to colorless. The noticeable color change indicates that HSO3− can be detected with the naked eye by using probe 1. The ratiometric sensing selectivity of probe 1 for HSO3− was further examined in PBS buffer (pH 7.4, 10 mM) as shown in Figure 3. Among the added representative anions, including HS−, CN−, F−, Cl−, Br−, I−, AcO−, ClO4−, NO3−, N3−, SO42−, HSO42−, SCN−, PO43−, H2PO4−, and HPO42− (100 equiv, respectively), only HS− caused an intensity change because of the 1,2-addition reaction with a fluorescent color change from orange to colorless. The other anions did not induce any significant fluorescence change, and the I465/I571 values varied in a limited range between 0.15 and 1.2. By contrast, treatment of 1 with HSO3− resulted in an obvious ratiometric fluorescent response. Probe 1 was originally synthesized to detect CN− in water, and the pH value of the solution changed from 7.2 to 9.4
Figure 3. Emission ratio I465/I571 of 1 (10 μM) in PBS buffer (pH 7.4, 10 mM) in the presence of various species. λex = 400 nm. Slits: 5 nm/5 nm. (Inset) Corresponding fluorescent color changes under a handle UV lamp (365 nm): 1, probe alone; 2, HSO3− (10 μM); 3, HS−; 4, CN−; 5, F−; 6, Cl−; 7, Br−; 8, I−; 9, AcO−; 10, ClO4−; 11, NO3−; 12, N3−; 13, SO42−; 14, HSO4−; 15, SCN−; 16, PO43−; 17, HPO43−; 18, H2PO43− (3−27, 1000 μM).
with the addition of CN−. However, the constant neutral pH condition suppressed the necleophilic addition of CN− to 1 as the protonation of CN− [pKa (HCN) = 9.2].30,31 Competition experiments of probe 1 were also ascertained by various species mentioned previously in the same environment. The signaling of 1 toward HSO3− (1 equiv) was not affected by the presence of 100 equiv of coexisting species (Figure S7 in the Supporting Information). The phenomenon suggests that probe 1 could be used as an efficient signaling probe for HSO3− in aqueous media. There was no spectral change when HSO3− was added to the solution of 1,2,3,3-tetramethylbenz[e]indolium iodide because of the steric effect. Hence, we proposed a 1,4-addition reaction occurred between 1 and HSO3− rather than a 1,2-addition reaction, and 1H NMR analysis was carried out to demonstrate the proposed addition mechanism (Figure 4). After HSO3− was
Figure 4. 1H NMR spectral change of 1 (3 × 10−3 M) in the absence (A) and presence of 1 equiv of NaHSO3 (B) in DMSO-d6/D2O = 9:1 (v/v).
added into the solution of 1, all of the 1H NMR signals shifted upfield because the nucleophilic attack of HSO3− toward C-4 disturbed the π-conjugation and weakened its electronwithdrawing characteristic. The proton signal (Ha, at δ 4.34) of the methyl group connected with N+ was significantly shifted upfield to δ 3.05. The proton signal (Hb, at δ 2.03) of the two methyl groups was also shifted upfield and divided into two 3407
dx.doi.org/10.1021/jf5004539 | J. Agric. Food Chem. 2014, 62, 3405−3409
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single signals (Hb′, at δ 1.87 and 1.48), which become nonequivalent after the formation of 1−SO3H. Moreover, the proton signal (Hc, at δ 7.7−8.5) appeared at δ 5.05 after the HSO3− addition. Furthermore, the formation of 1−SO3H was further confirmed via mass spectrometry analysis, where the peak at m/z 460.1193 (calcd 460.1200) corresponding to [M + SO3]− was clearly observed (Figure S8 in the Supporting Information). To demonstrate the practical application of probe 1 for the detection of HSO3−, a preliminary paper test strip system was developed, as shown in Figure 5. After the neutral filter papers
Table 2. Results of This Method Compared with a Titration Method for HSO3− from Sugar Samples sample
titration method (mg/kg)
this method (mg/kg)
granulated sugar soft sugar crystal sugar
17.5 10.1 14.3
17.21 10.70 14.58
In summary, a ratiometric fluorescent probe for HSO3− was introduced. The probe showed high selectivity and sensitivity to HSO3− in aqueous media with a short response time. The sensing mechanism was based on the 1,4-addition, which was confirmed via 1H NMR titration and HRMS studies. Moreover, a simple test paper prepared using probe 1 for the rapid monitoring of HSO3− was developed, which could be used as a convenient signaling tool for HSO3− determination in chemical analyses. The aqueous solution of probe 1 could also be used to determine HSO3− levels in sugar samples.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
NMR, MS, and other fluorescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 5. Photographs of the test paper exposure to various species under UV light (365 nm): 1, probe alone; 2, HSO3− (10 μM); 3, HS−; 4, CN−; 5, F−; 6, Cl−; 7, Br−; 8, I−; 9, AcO−; 10, ClO4−; 11, NO3−; 12, N3−; 13, SO42−; 14, HSO4−; 15, SCN−; 16, PO43−; 17, HPO43−; 18, H2PO43−.
Corresponding Author
*(L.D.) Fax: (+86)351-6018564. E-mail:
[email protected]. cn.
had been dipped into the solution of probe 1 (10 μM) and dried, yellow emission test papers were obtained under UV light (365 nm). When the various anion solutions (0.1 M) were dripped onto the test paper, only HSO3− induced a fluorescent color change from yellow to cyan. Although HS− and CN− also caused color changes from yellow to blue, they could be easily distinguished by the naked eye. Finally, probe 1 was used to detect HSO3− in real samples. Granulated sugar, soft sugar, and crystal sugar purchased from a supermarket were used in the sample analysis. Sample solution was prepared by dissolving 5.0 g of sugar in deionized water and diluting to 10 mL. Aliquots of the sugar solution were added directly to the PBS buffer (pH 7.4, 10 mM) containing 1 (10 μM), and the emission intensities at 465 and 571 nm were recorded. Table 1 shows that probe 1 was able to determine
Funding
This work was supported by the National Natural Science Foundation of China (21301126), Shanxi Province Science Foundation for Youths (2013021009-3), and Youth Foundation of Taiyuan University of Technology (2012L018 and 2012L062). Notes
The authors declare no competing financial interest.
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Table 1. Results for the Determination of HSO3− in Various Samples HSO3− level (μmol/L)
added (μmol/L)
found (μmol/L)
recovery (%)
granulated sugar
4.13
3 5
7.26 9.21
101.8 100.8
soft sugar
2.57
3 5
5.49 7.48
98.6 98.8
crystal sugar
3.50
3 5
6.32 8.24
97.2 97.0
sample
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