Rapidly Responsive and Highly Selective Fluorescent Probe for Bisulfite Detection in Food

Article abstract of DOI:10.1021/acs.jafc.7b00353

The new fluorescent probe 4-hydroxy-3-((2E,4E)-5-phenylpenta-2,4-dienoyl)-2H-chromen-2-one (probe 1) was designed and synthesized for selective detection of sulfite. The fluorescence intensity of the probe was decreased only in the presence of HSO₃^- all other anions assessed resulted in an increased fluorescence response. Hence, probe 1 acts as a highly selective sensor for HSO₃^-. This sulfite sensitivity can also be readily monitored visually, as once treated with sulfite the solution shows a marked color change from yellow to colorless. Moreover, probe 1 can be conveniently used as a signal tool to determine the HSO₃^- levels in various sugar samples.

Full text of DOI:10.1021/acs.jafc.7b00353

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Article
Rapidly responsive and highly selective
fluorescent probe for bisulfite detection in food
jialin Wang, yanfeng Hao, hao Wang, shaoxiang yang, Hong-Yu Tian, Baoguo Sun, and yongguo Liu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00353 • Publication Date (Web): 16 Mar 2017
Downloaded from http://pubs.acs.org on March 17, 2017
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Rapidly responsive and highly selective fluorescent probe for bisulfite
detection in food
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Jialin Wang, Yanfeng Hao, Hao Wang, Shaoxiang Yang*, Hongyu Tian, Baoguo
Sun, and Yongguo Liu
Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Key
laboratory of Flavour Chemistry, Beijing Technology and Business University, No.11
Fucheng Road, Haidian District, Beijing 100048, P.R. China
* To whom correspondence should be addressed. Telephone: +86ꢀ10ꢀ68985382. Fax:
86ꢀ10ꢀ68985382. Eꢀmail: yangshaoxiang@th.btbu.edu.cn
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ABSTRACT: The new fluorescent probe 4ꢀhydroxyꢀ3ꢀ((2E, 4E) ꢀ5ꢀphenylpentaꢀ2,
4ꢀdienoyl)ꢀ2Hꢀchromenꢀ2ꢀone (probe 1) was designed and synthesized for selective
detection of sulfite. The fluorescence intensity of the probe was only decreased in the
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presence of HSO3 , all other anions assessed resulted in an increased fluorescence
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response. Hence, probe 1 acts as a highly selective sensor for HSO3 . This sulfite
sensitivity can also be readily monitored visually, as once treated with sulfite the
solution shows a marked color change from yellow to colorless. Moreover, probe 1
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can be conveniently used as signal tool to determine the HSO3 levels in various sugar
samples.
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KEYWORDS: fluorescent probe; bisulfite; Michaelꢀtype addition
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INTRODUCTION
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Bisulfite (HSO3 ) is widely used as an antioxidant, antimicrobial agent and an
enzyme inhibitor for foods, pharmaceutical products and beverages.1, 2 For example,
the addition of bisulfite to beer and wine has been a worldwide practice for centuries.3
However, it has been shown that bisulfite may be toxic in high doses, alongside
potentially triggering asthma attacks and allergic reactions.4 Given the reported
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harmful effects on people’s health, the threshold levels of HSO3 in food and
medicine are rigorously controlled. In particular, the United States Food and Drug
Administration require labeling of products that contain more than 10 µg/mL of
sulfite.5 Furthermore, in China, the total concentration of sulfur (calculated by SO2
content) in white granulated sugar is strictly regulated to be <0.1g/kg and <0.25g/kg
in red wine.6 For this reason, the development of rapid and convenient methods for
selective detection of bisulfite in food and drink products is important. To date,
titration,7 electrophoresis,8 capillary electrophoresis, spectrophotometry9 and
electrochemical detection10,11 methods are utilized to determine bisulfite content in
products such as, starch, fruit, sugar, beverages, wines and beers. However, most of
these methods require the use of multiple reagents, miscellaneous sample
pretreatments and are very time consuming. To this end, recently, fluorescent probes
have been suggested as an effective alternative tool for sensing and monitoring
bisulfite content; in particular, as such probes offer high sensitivity, facile use, are
inexpensive and have rapid analysis conditions.
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In recent years, several fluorescent probes for HSO3 /SO3 have been developed
by a variety of approaches, such as, coordination to metal ions,12, 13 selective
deprotection of levulinate group,14ꢀ16 complexation with amines,17,18 selective reaction
with aldehyde19ꢀ21 and Michaelꢀtype Additions.22ꢀ31 Of these probe types, Michael
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Additionꢀtype probes are more effective as they produce both colorimetric and
fluorescent responses under mild conditions. To this end, we have developed a new
fluorescent probes based on Michael Addition reactions for the simple and effective
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detection of HSO3 . Here, we report the design, synthesis and sensing behaviors of the
novel fluorescent probe 4ꢀhydroxyꢀ3ꢀ((2E, 4E) ꢀ5ꢀphenylpentaꢀ2, 4ꢀ dienoyl)ꢀ 2H
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ꢀchromenꢀ2ꢀone (probe 1) which shows high selectivity and sensitivity for HSO3 . It
could be applied to detect bisulfite levels in real sugar samples with good recovery.
MATERIALS AND METHODS
Reagents and Instruments. The chemicals and reagents were purchased from
Beijing Huaxue Shiji Company (Beijing, P.R. China). The reagents were all
analytically pure. NMR spectra were obtained on a Bruker AV 300 MHz NMR (1H
13
NMR at 300 MHz, C NMR at 75 MHz) using TMS as internal standard. Chemical
shifts (δ) are given in ppm and coupling constants (J) in Hz. The high resolution mass
spectrum (HRMS) was performed at a Bruker Apex IV FTMS. UVꢀVis and
fluorescence spectra were recorded on a Shimadzu UVꢀ2600 spectrometer and a
Hitachi Fꢀ4600 fluorescence spectrometer with a temperature controller.
Preparation of Probe 1 (Scheme 1).
3ꢀAcetylꢀ4ꢀhydroxyxcoumarin
To a solution of 4ꢀhydroxycoumarin (8.10g, 0.05mol) in acetic acid (48 mL)
phosphorous oxychloride (18 mL) was added. The mixture was heated at reflux for
0.5 h. After cooling, the precipitate was collected and recrystallized from ethanol to
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give 3ꢀacetylꢀ4ꢀhydroxyxcoumarin (2, 8.87g, yield 87.0 %). H NMR (300 MHz,
DMSOꢀd6): δ 8.00 (dt, J = 7.8, 0.9, 1H), 7.79ꢀ7.85(m, 1H), 7.33ꢀ7.46(m, 2H), 2.66(s,
3H). 13C NMR (75 MHz, DMSOꢀd6): δ 205.99, 178.35, 154.53, 137.15, 125.58,
125.25, 117.30, 115.25, 101.91, 30.00.
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Probe 1. 4ꢀhydroxyꢀ3ꢀ((2E, 4E)ꢀ5ꢀphenylpentaꢀ2, 4ꢀdienoyl)ꢀ2Hꢀchromenꢀ2ꢀone
3ꢀacetylꢀ4ꢀhydroxyxcoumarin (2, 4.08 g, 0.02 mol) and cinnamaldehyde (2.90 g,
0.022 mol) were dissolved in chloroform (20 mL). A catalytic amount of piperidine
(one drop) was added and the reaction mixture was refluxed for 1.5 h. The chloroform
was distilled off and the residue was washed with methanol. The crude product was
recrystallized from 1, 4ꢀdioxane to give pure 4ꢀhydroxyꢀ3ꢀ((2E, 4E)ꢀ5ꢀphenylpentaꢀ2,
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4ꢀdienoyl)ꢀ2Hꢀchromenꢀ2ꢀone (probe 1, 3.94 g, yield 62.0 %). H NMR (300 MHz,
CDCl3): δ 19.02 (s, 1H), 8.10 (d, J = 0.75, 1H), 7.83ꢀ8.08(m, 2H), 7.66ꢀ7.71(m, 1H),
7.54 (d, J = 7.8, 2H), 7.21ꢀ7.43(m, 5H), 7.07ꢀ7.18(m, 2H). 13C NMR (75 MHz,
CDCl3): δ 191.93, 181.89, 160.35, 154.64, 147.94, 144.23, 136.04, 135.83, 129.87,
128.99, 127.75, 127.46, 125.91, 125.81, 124.41, 117.02, 116.50, 100.55. HRMS (ESI):
calcd. for [M]+ 318.0892, found 318.0895.
RESULTS AND DISCUSSION
Probe 1 was readily synthesized in a twoꢀstep reaction, whereby treatment of
4ꢀhydroxycoumarin with acetic acid provided the intermediate compound 2 which
was then reacted with cinnamaldehyde to give probe 1. This synthetic methodology
and purification by recrystallization were facile. The overall structure and purity of
probe 1 were confirmed by 1H NMR, 13C NMR and HRMS (Figs. S3ꢀS5).
In order to obtain optimized fluorescent spectral response conditions for probe 1
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in the presence and absence of HSO3 , the response time and pH value were
investigated. The effect of pH was evaluated in the pH range from 2.0 from 12.0. As
shown in Figure 1, the free probe 1 and fluorescent spectral response of probe 1 to
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HSO3 were applicable over the entire measured range. A pH value of 7.4 showed
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good response to HSO3 and hence was selected for further experiments. As shown in
Figure 2a, the emission line of probe 1 is at 322 nm, which was related to the
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πꢀconjugation. The addition of HSO3 resulted in a redꢀshift in the absorption to 340
nm. The associated decrease in fluorescence intensity suggests that the πꢀconjugation
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network is interrupted by nucleophilic addition between probe 1 and HSO3 . The
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fluorescence response of probe 1 (10 ꢁM) in the absence and presence of HSO3 (10
ꢁM) at pH 7.4 is shown in Figure 2b. A timeꢀresponse study suggests that the reaction
is complete after about 26 min, under these particular experimental conditions (Figure
3a). We also found that the solution color changed from yellow to colorless when the
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amount of HSO3 reached 3 equiv. (Figure 3b). This confirms that the reaction
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between probe 1 and HSO3 is entirely completed by 26 min and it is suitable for the
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detection of HSO3 .
Under these optimized conditions, the fluorescence spectral response of probe 1 (10
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ꢁM) to various amounts of HSO3 were examined (Figure 4a). Probe 1 possesses an
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excellent linear function with HSO3 concentration in the range of 0−80 ꢁM, with a
correlation coefficient of R2=0.9947 and a detection limit for HSO3 of 1×10ꢀ8 M
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(Figure 4b). These figures are comparable to those of other reported fluorescent
probes for bisulfite, however, the advantage here is that probe 1 is very easy to
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synthesis (Table S2).
In addition, the solution color change from yellow to
colorless, which suggests that the πꢀconjugation is interrupted, highlights the easy by
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which HSO3 can be detected with the naked eye by using probe 1.
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The specific nature of probe 1 for HSO3 was evaluated in PBS (pH 7.4, 10 mM) as
shown in Figure 5a. A broad range of anionic analytes were assessed, including, F− ,
Cl− , Br−, I−, SCN−, NO2 , NO3 , HCO3 , CO3 , SO4 and HSO3 (10 equiv,
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2−
2−
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respectively). From these, only the addition of HSO3 resulted in a decrease in
fluorescence intensity
(Figure
5b),
all
other
anions
resulted
in
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an increased fluorescence signal indicating that probe 1 is highly selective for HSO3 .
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Figure S9 shows the spectral response of probe 1 toward HSO3 in the presence of
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other competing anions. The fluorescent intensity of probe 1 + chloride + HSO3 and
probe 1 + Br− + HSO3 increases by about 10% compared to probe 1 + HSO3 . The
other competing anions resulted in a change in the fluorescence intensity by less than
10%. The reason for these changes is perhaps probe 1 has greater chemical reactivity
in the Michael Additionꢀtype reaction than for other reactions. The coexistence of
other competing anions did not interfere with the detection. These competition
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experiments clearly revealed that probe 1 could be used to detect HSO3 under
complex conditions.
The obvious shifts in both absorption and emission spectra can be ascribed to the
Michael Addition of sulfite toward the C=C bond of the α, βꢀunsaturated ketone,
which shortens the conjugation structure of the probe (Scheme 2).31 To confirm this
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proposed mechanism, the reaction product probe 1ꢀSO3H was verified by H NMR
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(Figure S6) and HRMS (Figure S7). After HSO3 was added into the solution of probe
1, two peaks at 2.8 and 2.4 ppm were assigned to the methylene group. The addition
of sulfite to C=C results in the formation of a chiral C center and therefore two
protons at this carbon become inequivalent. The peak at 3.4 ppm was assigned to the
ꢀCHꢀS group which is the characteristic 1H NMR spectral shift of a carbonꢀsulfur
bond. The formation of probe 1−SO3H was further confirmed via mass spectrometry,
where the peak at m/z 399.05454 (calcd. 399.054397) corresponds to [MꢀSO3]−.
We know that bisulfite is used widely as a preservative and antioxidant in food
products, but that excess bisulfites may have negative health implications ꢀ therefore,
it is important to detect bisulfite in food products. In order to assess the performance
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of probe 1 in this respect, we used probe 1 to detect HSO3 in real food samples.
Crystal sugar, granulated sugar and soft sugar, purchased from a local supermarket,
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were dissolved in deionized water. The sample solutions were added to the PBS (pH
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7.4, 10 mM) including probe 1. As shown in Table 1, probe 1 can determine HSO3
concentration in these sugar solutions with a good recovery, ranging from 90.4 % to
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109 %. The HSO3 levels in these samples were calculated to be 0.1, 1.5, and 7 mg/kg,
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respectively. To prove the accuracy of probe 1, the concentration of HSO3 in these
sugar samples was also analyzed by titration methods (Table S1),32 revealing good
agreement between the two methods. These results suggest overall that our proposed
methodology and the use of probe 1 is feasible and practical for the determination of
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HSO3 in food products.
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In summary, we have introduced a novel fluorescent probe for HSO3 . The function
of probe 1 relies on nucleophilic addition to break the πꢀconjugation, which we
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confirmed by H NMR and HRMS studies. Importantly, probe 1 exhibits high
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selectivity and sensitivity towards HSO3 . Once treated with sulfite, the solution color
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changed from yellow to colorless, indicating that HSO3 can be detected with the
naked eye in the presence of probe 1. Moreover, we have shown that probe 1 can be
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conveniently used as signal tool to determine the HSO3 levels in various sugar
samples.
ASSOCIATED CONTENT
Supporting Information
1H NMR and 13C NMR spectra of probe 1; 1H NMR, 13C NMR and HRMS spectra of
1
probe 1; H NMR and HRMS spectra of probe 1ꢀSO3H; the procedure of bisulfite
determination and sample analysis; plot of fluorescence intensity difference with
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HSO3 from 0 to 90 ꢁM; spectral response of probe 1 toward bisulfite in the presence
of other competing anions; the detection of sugar samples by titration method; and
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comparison of fluorescent probes for HSO3 .
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AUTHOR INFORMATION
Corresponding Author
*(S.X.Y.) Fax: (+86)10ꢀ68985382. Eꢀmail:yangshaoxiang@th.btbu.edu.cn
Funding
This work was supported by the national key research and development program
(2016YFD0400801), General funding project of Beijing Education Commission
Research Project (SQKM201610011003), the National Natural Science Foundation of
China (31571886).
Notes
The authors declare no competing financial interest.
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SO2 in white granulate sugar and crystal sugar. Food Sci. Technol. 2013, 38, 312−314
(in Chinese).
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324 FIGURE CAPTIONS
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Scheme 1. Synthesis of probe 1.
−
Scheme 2. The mechanism for probe 1 with HSO3 .
Figure 1. Fluorescent intensity of probe 1 (10 ꢁM) in the absence and presence of
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HSO3 (10 ꢁM) in different pH buffer solution, λex=377 nm, slites：5 nm/5 nm.
Figure 2. Absorption spectra (a) and fluorescence spectra (λex/λem=377/387 nm) (b) of
probe 1 (10 ꢁM) in the absence and presence of NaHSO（30 ꢁM）in the PBS (10 mM,
3
pH 7.4). “PBS” is phosphate buffer solution.
Figure 3. (a) The response of probe 1 (10 ꢁM) to NaHSO3 (3 equiv). (b) The color
changed of probe 1 (10 ꢁM) in the absence and presence of NaHSO3 (3 equiv).
Figure 4. (a) Fluorescence spectra (λex/λem=377/387 nm) of probe 1 (10 ꢁM) with
−
−
HSO3 . (b) The plot of fluorescence intensity difference with HSO3 from 0 to 90 ꢁM
in PBS (10 mM, pH 7.4). The test was repeated 3 times (Figure S8).
Figure 5. (a) Fluorescence spectra (λex/λem=377/387 nm) of probe 1 (10 ꢁM) in PBS
(pH 7.4, 10 mM) in the presence of various species. Slits: 5 nm/5 nm. (b) The
fluorescence intensity of various species. (0) probe 1, (1) F−, (2) Cl−, (3) Br−, (4) I−, (5)
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2−
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SCN−, (6) NO2 , (7) NO3 , (8) HCO3 , (9) CO32−, (10) SO4 , (11) HSO3 .
The test was repeated 3 times.
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Table 1. Results for the determination of HSO3 in various sugar samples.
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Scheme 1.
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Scheme 2.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Table 1.
ꢀ
Sample
HSO3 level Added
Found
Recovery
(µmol/L)
0.08
(µmol/L) (µmol/L) (%)
Crystal sugar
Granulated sugar
Soft sugar
0.03
0.05
0.03
0.05
0.03
0.05
0.120
0.127
1.110
1.067
0.520
0.517
109.1
97.7
95.7
90.4
94.5
90.7
1.13
0.52
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TOC Graphic
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