Accepted Manuscript

A distinctive near-infrared fluorescence turn-on probe for rapid, sensitive and

chromogenic detection of sulfite in food

Chong Duan, Jun-Feng Zhang, Yubo Hu, Lintao Zeng, Dongdong Su, Guang-Ming
Bao

PII: S0143-7208(18)31961-2
DOI: https://doi.org/10.1016/j.dyepig.2018.10.057
Reference: DYPI 7123

To appear in: Dyes and Pigments

Received Date: 4 September 2018

Revised Date: 9 October 2018
Accepted Date: 25 October 2018

Please cite this article as: Duan C, Zhang J-F, Hu Y, Zeng L, Su D, Bao G-M, A distinctive near-infrared
fluorescence turn-on probe for rapid, sensitive and chromogenic detection of sulfite in food, Dyes and
Pigments (2018), doi: https://doi.org/10.1016/j.dyepig.2018.10.057.

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 ACCEPTED MANUSCRIPT

1 A Distinctive Near-Infrared Fluorescence Turn-on Probe for Rapid, Sensitive

2 and Chromogenic Detection of Sulfite in Food

3 Chong Duan,a,b,# Jun-Feng Zhang,c,# Yubo Hu,c Lintao Zeng,*,a Dongdong Su,*,a
4 Guang-Ming Bao*,b

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a
5 School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin

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6 300384, PR China. E-mail: zlt1981@126.com (L. Zeng), chmsudd@foxmail.com (D. Su).
b
7 Institute of Veterinary Drug/Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi

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8 Agricultural University, Nanchang 330045, PR China. E-mail: bycb2005@gmail.com (G.-M. Bao)
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9 College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500,

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10 PR China.
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11 # These authors contributed equally to this work.
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12 Abstract

13 Sodium sulfite (Na2SO3) is widely used as a food additive, but the excessive residue of SO32- in

14 food can cause irritative effects and damages to the human body. In this study, we reported a novel

15 near-infrared (NIR) fluorescent probe (DCMQ) for visual detection of SO32- based on a

chromogenic reaction. The probe can quantitatively determine SO32- with high specificity and

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17 sensitivity, fast response (< 50 s) as well as a low detection limit (31.6 nM). A 1,4-Michael

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18 addition reaction was proposed for the sensing mechanism of this probe, which was confirmed by
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19 H NMR and HR-MS spectra. The probe has been successfully utilized to determine SO32- in food

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20 with good recovery, remarkable chromogenic effect and NIR fluorescence turn-on response.

21 Furthermore, the probe has been prepared as a reagent kit for instant on-site visual detection of

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22 SO32- in food. Therefore, this probe has great potential application for the detection of SO32- in
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23 food.

24 Keywords: fluorescent probe; chromogenic probe; near-infrared; colorimetric; sulfite;

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25 naked-eye detection
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26 INTRODUCTION

27 Sodium sulfite (Na2SO3) is a strong reducing agent, and has been widely used as a food additive to

28 prevent food from oxidation and keep bright food color [1,2]. Besides, a certain amount of SO32-

29 can block the normal physiological oxidation process of microorganisms and inhibit the

30 proliferation of microorganisms [3,4]. Therefore, Na2SO3 is often used as bleaching agent and

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31 preservative in the processing of agricultural products. Nevertheless, the excessive residue of

32 SO32- is easily hydrolyzed to corrosive sulfurous acid on the wet mucosa, which usually causes

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33 irritation and damage to the bronchus and lungs, resulting in respiratory tract inflammation [5,6].

34 To guarantee consumers' health, it is highly in demand to strictly control the residual SO32- in

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35 agricultural products.

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36 A number of methods have been developed for the detection of sulfite, including flow injection
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37 [7], electrochemical detection [8,9], ion chromatography [10,11] and others [12,13]. However,

38 these methods either depend on expensive instruments or require complicated pre-treatment

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39 processes. Therefore, these detection methods are not suitable for the rapid detection of

40 agricultural products in the wholesale market at this stage. Recently, small molecular fluorescent
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41 probes have attracted much attention due to their fascinating features such as simple, high
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42 sensitivity, and anti-interference ability through fluorescence signal response [14,15]. Several

43 fluorescent probes have been developed for the detection of harmful small molecules of food
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44 additives, such as benzoyl peroxide [16,17], formaldehyde [18], sodium sulfite[19,20] and sodium

45 nitrite [21]. Most of these probe showed hypochromic effect and emission in the visible region,
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46 which suffered from the interference from background. A fluorescent probe with significant

47 hyperchromic effect and near-infrared fluorescence turn-on response towards analytes in food can
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48 minimize the interference from background, such as flavonoids with yellowish green fluorescence

49 and vitamin B2 with green fluorescence, and provides a visual manner to detect analyte with

50 naked eyes. This naked-eye detection fluorescent probe has several advantages including no need

51 for any auxiliary equipment, high resolution, simple operation, low cost, as well as real-time

52 visual detection [22,23]. Therefore, it is very attractive to design a high selective fluorescent probe

53 for SO32- with significant chromogenic reaction and NIR fluorescence enhancement.

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54 Herein, we reported a novel NIR fluorescence probe DCMQ for SO32- based on 1, 4-Michael

55 addition reaction. The probe displayed observable chromogenic reaction from yellow to purple

56 and NIR fluorescence turn-on response towards SO32- when quinolinium was interrupted by SO32-

57 via 1, 4-Michael addition reaction. Moreover, the novel probe showed fast response towards SO32-

58 (less than 50 s) with high sensitivity and selectivity, fast response and low detection limit.

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59 Importantly, the probe DCMQ was successfully used as a reagent kit to visually determine SO32-

60 in food samples under mild condition. Therefore, the unique probe can satisfy the need for

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61 visualizing SO32- in colorimetric and NIR fluorescence turn-on modes.

62

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63 EXPERIMENTAL SECTION
64 Materials and Chemicals

65
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Unless otherwise stated, all reagents were purchased from commercial suppliers
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66 (Aladdin-Reagent, Sigma-Aldrich, and TCI). All chemicals and solvents were of analytical grade

67 and used without further purification. The 1H NMR and 13

C NMR spectra were recorded on a
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68 Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the internal standard. The chemical

69 shift was recorded in ppm and the following abbreviations were used to explain the multiplicities:
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70 s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets. Mass

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71 spectra were measured on a HP-1100 LC-MS spectrometer. UV-vis spectra were recorded on

72 Hitachi UV-3310 spectrometer. Fluorescence spectra were recorded on a Hitachi FL-4500

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73 fluorometer. Confocal microscopy fluorescence images were acquired on a Nikon A1 confocal

74 laser-scanning microscope with a 100 objective lens. The pH values were determined by a
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75 PHS-3C pH meter, which was purchased from the Shanghai Yoke Instrument Co., Ltd.
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77 Synthesis of Compound 1
78 DCM was synthesized from commercially available starting materials according to literatures

79 [24-26]. 1H NMR (400 MHz, CDCl3) δ 8.96 (d, J = 7.6 Hz, 1H), 7.76–7.73 (m, 1H), 7.50–7.46 (m,

80 2H), 6.75 (s, 1H), 2.47 (s, 3H). DCM (134 mg, 0.64 mmol) and 3-quinolinaldehyde (101 mg, 0.64

81 mmol) were dissolved in 30 mL of ethanol, and then piperidine (5.8 µL, 0.064 mmol) was added

82 to the solution. The mixture was refluxed for 3 h. After the solution was cooled down to the room

83 temperature, some solid product was precipitated and collected. After washing with diethyl ether,
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84 the residue was then purified by column chromatography (dichloromethane/ethanol, 100:1, v/v) on

85 silica gel to give compound 1 as a yellow powder (168 mg, yield: 75.2%). 1H NMR (400 MHz,

86 CDCl3) δ 9.18 (s, 1H), 8.97 (dd, J =6.4, 1.2 Hz, 1H), 8.38 (s, 1H), 8.18 (d, J = 6.8 Hz, 1H), 7.94 (d,

87 J = 8.0 Hz, 1H), 7.84–7.79 (m, 3H), 7.67–7.62 (m, 2H), 7.54–7.50 (m, 1H), 7.11 (d, J = 12.8 Hz,
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88 1H), 7.00 (s, 1H). C NMR (125 MHz, DMSO-d6) δ 158.03, 153.43, 152.58, 150.18, 148.31,

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89 136.07, 135.82, 135.71, 131.16, 129.35, 129.16, 128.74, 127.95, 126.77, 125.25, 122.09, 119.57,

90 117.64, 117.41, 116.11, 115.45, 107.84. HRMS (ESI): calcd for C23H14N3O+, 348.1059; found,

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91 348.1146.
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93

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Synthesis of DCMQ
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95 Compound 1 (150 mg, 0.43 mmol) and methyl trifluoromethanesulfonate (196 µL, 1.72 mmol)

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96 were dissolved in dry dichloromethane (10 mL). The reaction solution was stirred overnight at
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97 room temperature. After the completion of the reaction, some solid products were precipitated and

98 collected. The crude product was purified by column chromatography (dichloromethane/methanol,

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99 20:1, v/v) on silica gel to give DCMQ as a red brown powder (120 mg, yield: 54.3%). 1H NMR

100 (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.54 (s, 1H), 8.78 (d, J = 6.4 Hz, 1H), 8.55 (d, J = 7.2 Hz,
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101 1H), 8.44 (d, J = 6.4 Hz, 1H), 8.32 (t, J = 6.2 Hz, 1H), 8.11 (t, J = 6.2 Hz, 1H), 8.02–7.96 (m, 2H),
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102 7.88–7.85 (m, 1H), 7.80 (d, J = 6.4 Hz, 1H), 7.7 (t, J = 7.8 Hz, 1H), 7.07 (s, 1H), 4.67 (s, 3H). 13C

103 NMR (100 MHz, DMSO-d6) δ 156.66, 153.14, 152.38, 150.36, 144.44, 138.17, 136.44, 131.80,
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104 131.22, 131.12, 129.58, 129.37, 127.05, 125.26, 125.17, 122.43, 119.87, 119.82, 119.49, 117.53,

105 117.07, 115.92, 108.95, 63.03, 46.27. HRMS (ESI): calcd for C24H16N3O+, 362.1288; found,
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106 362.1291.

107 Food Sample Treatment and Measurement

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108 Food samples (granulated sugar, vermicelli) were purchased from supermarkets and used as real

109 food samples for analysis. Vermicelli (2.5 g) was placed in a round bottom flask, then 40 mL

110 deionized water was added and stirred for 1 h. The mixture solution was centrifuged at 8000

111 rpm/min for 30 min, and supernatant was collected for analysis. Granulated sugar (2.5 g) was

112 dissolved in 40 mL deionized water for analysis. Appropriate amount of HEPES and NaOH were

113 added to keep the solution of pH 7.4. The levels of SO32- in these food samples were measured as

114 the following procedure: aliquots of food samples solution were incubated with DCMQ solution
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115 (10 µM, pH 7.4, HEPES solution with 60% DMSO) at 37 °C for 10 min, and the fluorescence

116 spectrum was recorded. Then, aliquots of food samples were spiked with different concentrations

117 of SO32- (1.0 µM, 2.0 µM, 3.0 µM and 4.0 µM), and the fluorescence spectra were recorded. The

118 results shown in Tables 1 and Table 2 were reported as the average values from three experiments.

119 All experiments meet the relevant laws and institutional guidelines. No hazard or risk was

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120 associated with this work.

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121 RESULTS AND DISCUSSION

122 Design and Synthesis of Probe DCMQ

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123 Dicyanomethylene-benzopyran (DCM) is known as a photostable and easy-to-prepare precursor

124 of fluorophore [27,28], and its derivatives often exhibit emission in the NIR region [29,30]. These

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125 attractive features motivated us to construct NIR fluorescent probes for the detection of SO32-
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126 based on DCM and quinolinium. Since the strong electron-withdrawing quinolinium can quench

127 the fluorescence of DCMQ and increase the water solubility, hence it was chosen as a building
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128 block to prepare DCMQ (Scheme 1) and its structural analogue 3 (Scheme S1). It was envisioned

129 that DCMQ and 3 would be attacked by the nucleophilic SO32-, which would destroy the
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130 quinolinium skeleton and form a typical intermolecular charge transfer (ICT) system. Thus, the
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131 two compounds would display a significant fluorescence enhancement and a visible chromogenic

132 reaction. The probe DCMQ and compound 3 were was prepared through conventional reactions,
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133 and the synthesis scheme was illustrated in Scheme 1 and S1 respectively. The structures of

134 DCMQ and compound 3 were fully characterized by 1H NMR, 13C NMR and HR-MS. Detailed
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135 synthesis procedures and structure characterizations were described in the Experimental Section
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136 and the Supporting Information (Fig. S5-S14).

137

138 Insert Scheme 1

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140 Spectral Response of DCMQ towards SO32-

141 Firstly, we measured the UV-vis absorption spectra and fluorescence spectra of DCMQ in

142 DMSO-HEPES buffer solution (6/4, v/v, pH = 7.4). As shown in Fig. 1a, DCMQ exhibited a weak

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143 absorption band centred at 421 nm. Upon the addition of increasing amount of SO32-, a new

144 absorption band appeared at 582 nm and regularly increased, while the absorption band at 421 nm

145 gradually decreased. When the concentration of SO32- reached 16.0 µM, DCMQ exhibited a

146 remarkable red shift (~ 161 nm) in the UV-vis absorption spectra together with a noticeable color

147 change from yellow to purple (Fig. 1a). As expected, DCMQ also exhibited a dramatic

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148 fluorescence response towards SO32- (Fig. 1b). Upon the addition of SO32-, a fluorescence band at

149 675 nm emerged and gradually increased. Compared to non-fluorescent DCMQ, a great

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150 fluorescence enhancement at 675 nm was observed in the presence of SO32- (Φ= 0.38 in DMSO

151 with relative to Cy 5.5) [31], indicating that DCMQ could act as a NIR fluorescence turn-on probe

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152 for SO32- with high contrast ratio. In addition, a large Stokes shift (~ 93 nm) was also conducive to

153 reduce measurement errors caused by excitation and scattered light, thereby significantly

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154 improving the signal fidelity in real sample detection. In contrast, compound 3 showed almost no
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155 spectral change when treated with SO32- (Fig. S1).

156
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157 Insert Fig. 1

158
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159 By plotting the absorbance of DCMQ at 582 nm versus the concentration of SO32-, a good
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160 linear relationship (R2 = 0.9984) was obtained with the concentration ranging from 0 to 12.0 µM.

161 Similarly, the fluorescence intensity of DCMQ at 675 nm is also linearly related to the
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162 concentration of SO32- (Fig. 1d). According to the linear calibration curve (Fig. 1d), the detection

163 limit of DCMQ for SO32- is calculated to be 31.6 nM (approximately 0.002mg/L expressed as SO2)
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164 based on 3σ/k, which is much lower than the threshold level of SO32- in foods (< 10 mg/L) [32].
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165 Therefore, we have established a NIR fluorescent probe for quantitative determination of SO32-

166 with high sensitivity and observable chromogenic reaction.

167 The time-coursed fluorescence responses of DCMQ towards SO32- were also examined, as

168 shown in Fig. 2. The fluorescence intensity of DCMQ (10 µM) at 675 nm promptly increased and

169 reached a plateau within 50 second after the addition of 5 equiv of SO32-, suggesting that DCMQ

170 could serve as a “fast response” fluorescent probe for instant on-site detection of SO32- in food. In

171 addition, the pH stability of DCMQ and its applicable pH range were investigated. The results

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172 showed that probe DCMQ and its product were stable at pH 5.5 to 8.5, indicating its broad pH

173 range for the detection of SO32- in food assay.

174

175 Insert Fig. 2

176

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177 We next investigated the selectivity of probe DCMQ for SO32- and various relevant analytes

178 such as F−, Cl−, NO2−, NO3−, AcO−, HCO3−, HPO4−, SO42−, S2O72−, CN−, H2O2, ClO−, HS−,

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179 N2H4•H2O, NH3•H2O, HOC2H4NH2, C6H5NH2, n-C3H7NH2 and biothiols (Cys, Hcy, GSH) (100.0

180 µM). As shown in Fig. 3 and Fig. S2, the addition of SO32- to DCMQ led to a significant colour

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181 and fluorescence changes, which could be observed by the naked eyes. By contrast, other small

182 molecular species except HSO3- caused negligible changes in the fluorescence intensity and no

183

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obvious color change was observed. Thus, the probe DCMQ can simultaneously determine SO32-
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184 and HSO3-. In addition, the detection of SO32- by probe DCMQ in the presence of various analytes

185 (including reactive species) is still effective (Fig. S3), which indicates that probe DCMQ has high
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186 selectivity for SO32- without any interference. Thus, DCMQ has great potential in the detection of

187 SO32- in real samples as reagent kit.

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188
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189 Insert Fig. 3

190
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191 The Sensing Mechanism of DCMQ to SO32-

To investigate the sensing mechanism of DCMQ for SO32-, 1H NMR and HR-MS analysis were
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192

193 performed. The partial 1H NMR spectra of DCMQ before and after treatment with SO32- were
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194 shown in Fig. 4. After treatment with 10 equiv. of SO32-, the chemical shifts of Ha and Hb at 9.97

195 ppm and 4.67 ppm were shifted to 4.97 ppm and 3.36 ppm, respectively, which suggested that the

196 quinolinium skeleton was broken by nucleophilic SO32- according to 1,4-Michael addition reaction.

197 HR-MS spectra (Fig. S10) also confirmed the formation of DCMQ-SO3 adduct (calcd for

198 DCMQ-SO3−: 442.0867, found 442.0881, Fig. S10). The sensing mechanism of DCMQ for SO32-

199 was depicted in Scheme 1. The SO32− attacked the quinolinium skeleton to form DCMQ-SO3

200 adduct based on 1,4-Michael addition reaction. Consequently, the electron-withdrawing

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201 quinolinium group of DCMQ was transformed into a strong electron-donating group. Therefore,

202 DCMQ exhibited a significant color change from yellow to purple along with remarkable NIR

203 fluorescence enhancement.

204

205 Insert Fig. 4

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206

207 Detection of SO32- in Real Food by DCMQ

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208 Considering the favorable sensing capability of the probe DCMQ for SO32-, we next examined

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209 the practicability of probe DCMQ for determination of SO32- in real food samples. Herein,

210 commercial available granulated sugar and vermicelli were dissolved in 10 mM HEPES solution

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211 (pH = 7.4) and filtered for analysis. As shown in Fig. 5 and Fig. 6, DCMQ exhibited noticeable

212 changes in both UV-vis absorption spectra and fluorescence spectra after addition of food samples.
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213 The concentration of SO32- in filtrate was directly analyzed by probe DCMQ according to

214 standard calibration curves (Fig. 1b and Fig. 1d). As shown in Table 1, the level of SO32- in
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215 granulated sugar and vermicelli were determined to be 21.27 mg/kg and 8.72 mg/kg, respectively,
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216 which were consistent with the results (21.02 mg/kg and 9.12 mg/kg) obtained from the

217 fluorescence calibration curve. Moreover, we measured the concentrations of SO32- in food
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218 samples by a standard method [33] (Iodine for sulfite) and compared the results with those

219 determined by probe DCMQ. As shown in Table 1, the results determined by two assays were in
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220 good agreement with each other, which validated the accuracy of this fluorescent probe. Beside,

221 we used internal standard method to determine SO32- in these food samples. These food samples
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222 were separately spiked with different concentrations of SO32- (0 µM, 1.0 µM, 2.0 µM, 3.0 µM , 4.0
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223 µM, 5.0 µM and 6.0 µM), and then we examined the UV-vis absorption spectra and fluorescence

224 spectra responses of DCMQ towards these samples (shown in Fig. 5 and Fig. 6). As shown in

225 Table 2, good recoveries (91% ~ 109%) were achieved for these SO32- spiked samples, which

226 indicated that the concentration of SO32- in real food samples could be accurately determined by

227 fluorescent probe DCMQ.

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229 Insert Fig. 5

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230

231 Insert Fig. 6

232

233 Insert Table 1

234

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235 Insert Table 2

236

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237 As mentioned above, DCMQ displayed a noticeable colour change from yellow to purple and a

238 significant fluorescence enhancement in the presence of SO32-, thus we tentatively prepare a kit for

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239 real-time and visual detection of SO32-. DCMQ (10 µM) was loaded into a 96-well plate, and then

240 different concentrations of SO32- (0 µM, 2.0 µM, 4.0 µM, 6.0 µM, 8.0 µM, 10.0 µM and 12.0 µM)

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241 were added and incubated with DCMQ (10 µM) for 1 min. As shown in Fig. 7a, the color of
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242 DCMQ solution in 96-well plate turned into purple in the presence of SO32-, which became much

243 stronger with the concentration of SO32- increased. Accordingly, the fluorescence of DCMQ
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244 solution (10 µM) changed from non-fluorescence to strong red fluorescence under the irradiation

245 of a 365 nm UV lamp (Fig. 7b). Then, we employed DCMQ to determine SO32- in real granulated
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246 sugar and vermicelli. As shown in Fig. 7c, the probe displayed observable purple even after
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247 incubation with low dose (62.5 mg/mL) of granulated sugar. Thus, we can utilize the color of

248 DCMQ solution (10 µM) to rapidly and conveniently determine the concentration of SO32- in real
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249 granulated sugar and vermicelli. Moreover, the fluorescence image in Fig. 7d provided another

250 manner to visually determine the concentration of SO32- in real food. Therefore, we have prepared

a convenient reagent kit for real time and visual detection of SO32- in food.
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251

252
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253 Insert Fig. 7

254 CONCLUSION

255 In summary, we have developed a NIR fluorescent probe for the detection of SO32- in food. The

256 probe displayed a remarkable colorimetric and NIR fluorescence turn-on response towards

257 SO32-with high contrast ratio based on 1,4-nucleophilic addition reaction. Beside, some good
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258 merits of the probe were achieved including fast response (in less than 50 s), chromogenic reaction,

259 high selectivity and sensitivity, as well as low limit of detection (31.6 nM). These features

260 combined with its high contrast ratio in fluorescence and absorbance, enable the probe to serve as

261 a reagent kit for real time visual detection of SO32- in food samples. This visual assay has the

262 advantages of convenient use, instant on-site rapid detection, thus has great potential application

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263 for instant on-site detection of SO32- in food. This work not only presents a distinctive fluorescent

264 probe for SO32- with fast response and a chromogenic reaction, but also provides a new perspective

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265 for the development of robust chromogenic probes for exciting applications in food analysis,

266 medical diagnosis and environmental monitoring.

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267

268

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269 AUTHOR INFORMATION
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270 Corresponding Author

271 *E-mail: zlt1981@126.com (L. Zeng), chmsudd@foxmail.com (D. Su). Phone: +86-22-6021-4259.
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272 Fax: 86-22-6021-4252. bycb2005@gmail.com (G.-M. Bao). Fax: +86-791-83828018.

273
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274 Notes
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275 The authors declare no competing financial interest.

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276 ACKNOWLEDGEMENT

277 We gratefully appreciate the financial support from the National Key Research and Development
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278 Program of China (No.2017YFD0501406), the National Natural Science Foundation of China
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279 (Nos. 21203138, 31560712, 21708029, 21461011), and the Natural Science Foundation of Tianjin

280 (No. 17JCYBJC19600).

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352 two-photon fluorescent probe with near-infrared emission for hydrogen sulfide imaging in
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353 biosystems. Chem Commun 2013; 49: 3890-2

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354 [28] Li M, Wu XM, Wang Y, Li YS, Zhu WH, James TD. A near-infrared colorimetric

355 fluorescent chemodosimeter for the detection of glutathione in living cells. Chem Commun
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365 [32] EU Directive 95/2. European Parliament and Council Directive No 95/2/EC on Food

366 Additives Other than Colours and Sweeteners, European parliament and council of EU,

367 Brussels 1995.

368 [33] Sun Y, Zhao D, Fan SW, Duan L, Li RF. Ratiometric fluorescent probe for rapid detection of

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370 3405-3409.

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Scheme and Figure Captions
Scheme 1. Design of the near-infrared fluorescence turn-on probe DCMQ for SO32-

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Fig. 1. (a) UV-vis absorption spectra changes of DCMQ (10 µM) in DMSO/HEPES solution (v/v

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= 6/4, pH 7.4, 10 mM) upon addition of increasing amount of SO32- (0-16.0 µM). (b) Linear
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relationship between absorbance of DCMQ (10 µM) versus concentrations of SO32-. (c) The

fluorescence spectra changes of DCMQ in DMSO/HEPES solution (v/v = 6/4, pH 7.4, 10 mM)
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upon addition of increasing amount of SO32- (0-16.0 µM). (d) Linear relationship between

fluorescence intensity of DCMQ (10 µM) versus concentrations of SO32-. Each spectrum was
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recorded after incubation with SO32- for 1 min. The excitation wavelength was 576 nm. Slits: 5/5
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nm.

Fig. 2. Time-dependent fluorescence response (F675) of DCMQ (10 µM) towards SO32- (50.0 µM)
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in DMSO/HEPES solution (v/v = 6/4, pH 7.4, 10 mM). The excitation wavelength was 576 nm.

Slits: 5/5 nm.

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Fig. 3. (a) UV-vis absorption spectra and (b) fluorescence spectra responses of DCMQ (10 µM)

toward various analytes (100.0 µM) in DMSO/HEPES solution (v/v = 6/4, pH 7.4, 10 mM) at

25 °C. Each spectrum was recorded after 5 min. The excitation wavelength was 576 nm. Slits: 5/5

nm.

Fig. 4. 1H NMR spectra of DCMQ treated with different concentrations of SO32- (a) 0.2 eq. (b) 0.5

eq. (c) 1.0 eq. (d) 2.0 eq.

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Fig. 5. (a) The UV-vis absorption spectra of DCMQ (10 µM, pH 7.4) in the presence of different

concentrations of SO32- in granulated sugar. (b) Linear relationship between absorbance of DCMQ

(10 µM) versus concentrations of SO32-. (c) The fluorescence spectra of DCMQ (10 µM, pH 7.4)

in the presence of different concentrations of SO32- in granulated sugar. (d) Linear relationship

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between fluorescence intensity of DCMQ (10 µM) versus concentrations of SO32-. The spectrum

of DCMQ (10 µM, pH 7.4) was recorded after incubation with the sample for 1 min. The

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excitation wavelength was 576 nm. Slits: 5/5 nm. Error bars are ± SD, n= 3.

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Fig. 6. (a) The UV-vis absorption spectra of DCMQ (10 µM, pH 7.4) in the presence of different

concentrations of SO32- in vermicelli. (b) Linear relationship between absorbance of DCMQ (10

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µM) versus concentrations of SO32-. (c) The fluorescence spectra of DCMQ (10 µM, pH 7.4) in
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the presence of different concentrations of SO32- in vermicelli. (d) Linear relationship between

fluorescence intensity of DCMQ (10 µM) versus concentrations of SO32-. The spectrum of
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DCMQ (10 µM, pH 7.4) was recorded after incubation with the sample for 1 min. The excitation

wavelength was 576 nm. Slits: 5/5 nm. Error bars are ± SD, n= 3.
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Fig. 7. DCMQ (10 µM) as a reagent kit for determination of SO32-. (a) The color and (b)

fluorescence images of DCMQ reagent (10 µM) in the presence of different concentrations of

SO32-. (c) The color and (d) fluorescence images of DCMQ reagent (10 µM) in the presence of
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different concentrations of food samples.

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Tables
Table 1 Results for Na2SO3 in granulated sugar and vermicelli determined by the probe DCMQ

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and titration method.
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Probe DCMQ Probe DCMQ Titration method Titration method
Sample b
(µΜ) (mg/kg) (µΜ) (mg/kg)
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4.22 ± 0.11 (A582) 21.27 ± 0.55

Granulated sugar 4.40 ± 0.32 22.18 ± 1.61
4.17 ± 0.09 (F675) 21.02 ± 0.45
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1.73 ± 0.06 (A582) 8.72 ± 0.30

Vermicelli 2.04 ± 0.30 10.28 ± 1.51
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1.81 ± 0.10 (F675) 9.12 ± 0.50

a
371 Based on I2 oxidation (Na2SO3 + I2 + H2O = Na2SO4 + 2HI), starch as indicator.
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b
372 The concentration of food samples in water was 62.5 mg/mL.

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374 Table 2 Determination of SO32- in real food samples by DCMQ.

SO32- level found (µΜ)

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Sample Added (µΜ) Found (µΜ) Recovery (%)

3.0 2.85 95
4.22 (A582)
6.0 5.96 99
Granulated sugar
3.0 3.49 97
4.17 (F675)
6.0 5.49 91
3.0 3.22 107
1.73 (A582)
6.0 6.56 109
Vermicelli
3.0 2.85 95
1.81 (F675)
6.0 6.04 100

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Scheme and Figure Graphics
Scheme 1

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Fig. 1
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0.20
(a) (b)
0.16
0.16

Absorbance
16 µM
0.12
0.12

A582
[SO32-]
0.08
0.08 0
0.04
0.04 y = 0.0136x + 0.0033
0.00 R2 = 0.9984
0.00
400 450 500 550 600 650 700 0 2 4 6 8 10 12 14 16
Wavelength (nm) [SO32-] (µM)
Fluorescence Intensity

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800
(c) (d)
600
600 16 µM

[SO32-] 400

F675

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400
0
200 200
y = 52.3539x + 10.2421
R2 = 0.9953

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0 0
630 660 690 720 750 0 2 4 6 8 10 12 14 16
Wavelength (nm) [SO32-] (µM)

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Fig. 2 AN
800

600
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F675

400

200
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0
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0 20 40 60 80 100
Time (s)
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Fig. 3
Fluorescence Intensity

0.20
(a) 800 (b)
SO32- SO32-
0.16
Absorbance

600
0.12
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400
0.08

GSH 200 GSH

0.04 Others Others

0.00 0
400 450 500 550 600 650 700 630 660 690 720 750
Wavelength (nm) Wavelength (nm)

Fig. 4

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Fig. 5
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0.16
0.16 (a) (b)
6.0 µM
Absorbance

0.12 [SO32-] 0.12

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A582

0
0.08
0.08
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0.04
y = 0.0138x + 0.0582
0.00 R2 = 0.9998
0.04
400 450 500 550 600 650 700 0.0 2.0 4.0 6.0
Wavelength (nm) [SO32-]
Fluorescence Intensity

600
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(c) (d)
600
6.0 µM 500

400 [SO32-]
F675

400
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0
200 300
y = 51.3871x + 214.5071
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200
0 R2 = 0.9993
630 660 690 720 750 0.0 2.0 4.0 6.0
Wavelength (nm) [SO32-]

Fig. 6

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0.16
(a) (b)
6.0 µM 0.12

Absorbance
0.12
[SO32-]

A582
0.08 0 0.08

0.04
0.04 y = 0.0143x + 0.0247
R2 = 0.9994
0.00
400 450 500 550 600 650 700 0.0 2.0 4.0 6.0
Wavelength (nm) [SO32-]
Fluorescence Intensity

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500 500
(c) (d)
400 400
6.0 µM
300
[SO32-]

F675
300

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200 0
200
100
y = 52.8612x + 95.6987
100

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0 R2 = 0.9987
640 680 720 760 0.0 2.0 4.0 6.0
Wavelength (nm) [SO32-]

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Fig. 7
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Graphic for table of contents

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Highlights
• A novel NIR fluorescence probe DCMQ for SO32- is developed.

• The probe showed fast response toward SO32- (˂ 50 s) with high selectivity and low

detection limit.

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The probe displayed observable chromogenic reaction from yellow to purple and NIR

fluorescence turn-on response toward SO32-.

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• The probe DCMQ was successfully used as a reagent kit to visually determine SO32-

in food samples.

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Statement on the manuscript’s significance

The significance of this manuscript was listed below:

(1) Sulfite (SO32−) is widely used as bleaching agent and food preservative.

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However, SO32- excess can irritate and damage human body. Therefore, the threshold

levels of SO32− in food and drugs are strictly controlled. In this study, we reported a

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fluorescent probe for visual detection of SO32- based on specific chemical reaction,

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which displayed a colorimetric and ratiometric fluorescence response towards SO32-

with high sensitivity, fast response (< 50 s) as well as a low detection limit (31.6 nM).

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The probe has been successfully utilized to determine SO32- in food with good
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recoveries, remarkable chromogenic effect and NIR fluorescence turn-on response.
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Furthermore, the probe has been prepared as a reagent kit for instant on-site visual
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detection of SO32- in food.

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(3) A 1,4-Michael addition reaction was proposed for the sensing mechanism of

this probe, which was confirmed by 1H NMR and HR-MS spectra. The probe
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exhibited a significant NIR fluorescence turn-on response along with remarkable

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chromogenic effect. This work created a new method to visually determine SO32- in
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food based on specific chemical-reaction.

(3) This work not only developed a simple, rapid and sensitive fluorescent probe

for visual detection of SO32- in food, but also provide a new perspective to devise

novel fluorescent probe for detection of some species in food. Therefore, this work

represents an important progress and will advance research to food chemistry.