Colorimetric and Ratiometric Fluorescence Detection of HSO3− With a NIR Fluorescent Dye

Bisulfite (HSO3−) has been widely used in food and industry, which has brought convenience to human life, but also seriously endangered human health. In this work, the probe PBI was designed and synthesized to detect bisulfite (HSO3−) through nucleophilic addition reaction. The probe PBI showed a selective reaction to HSO3− and can quantitatively detect HSO3−. At the same time, the color of the probe PBI changed significantly, which provided a simple method for the naked eye to identify HSO3−. Finally, it was successfully applied to the fluorescence imaging of HSO3− in living cells.


Introduction
In food, HSO 3 − can be used as a bleaching agent, preservative, and antioxidant [1][2][3]. HSO 3 − is a bleaching agent to improve food color and antibacterial effects, and widely used in food processing [4]. Then it can be used as a preservative to inhibit microbial activity and prevent food spoilage [5]. In terms of antioxidants, HSO 3 − can prevent or delay food oxidation, improve food stability and extend storage life [6]. As a large amount of HSO 3 − may cause tissue damage in individuals, it is necessary to strictly control the amount of HSO 3 − in food [7][8][9]. HSO 3 − is a reducing agent that plays the key role in industries such as dyes, papermaking, leather making, and chemical synthesis [10][11][12][13]. HSO 3 − can bleach cotton fabrics and organic substrate [14], treat chromiumcontaining waste water [15], and be used as an electroplating additive [16]. In the physiological system, HSO 3 − is mainly produced by the oxidation of cysteine and glutathione, and this process is mediated through reactive oxygen species (ROS) [17,18]. Toxicological studies had shown that low concentrations (< 450 μM) of HSO 3 − have a significant promoting effect on the vasodilation of the cardiovascular system [19]. However, when the expression of HSO 3 − rises in the vivo, it can cause a series of diseases [20][21][22][23]. Therefore, it is of great significance to study new methods of HSO 3 − detection. Some conventional analysis techniques for the detection of bisulfite have been developed, such as spectrophotometry [24,25], chemiluminescence measurements [26,27], chromatography [28], electrochemistry [29,30], and phosphorimetry [31]. However, the detection process of these methods is more complicated, and some of them are not sensitive enough. So far, many fluorescent probes have been developed for the detection of HSO 3 − , because they have obvious advantages, including admirable sensitivity, in-situ detection ability and easy visualization with the naked eye [32][33][34]. These probes mainly react with HSO 3 − by using several kinds of reaction mechanisms. For example, the nucleophilic reaction with aldehydes [35], hydrogen bonding [36], and the nucleophilic addition reaction with the carbon-carbon double bond [37,38].
In this work, a new colorimetric and ratiometric fluorescent probe PBI for detecting HSO 3 − was designed based on the nucleophilic addition reaction (Scheme 1). Through experiments, the detection properties of the probe PBI and its application in biological fluorescence imaging have been studied.

The Parameters of the Solution in Spectral Tests
Configure the PBI with concentration of 1 × 10 −3 mol/L (dissolved in DMSO). Remove 30 μL of PBI solution and add it to 3000 μL solution containing 2400 μL PBS and 600 μL DMSO, the final concentration of PBI in the test system was 1 × 10 −5 mol/L.
The concentrations of anion ions, metal ions and small biomolecules used in the detection were 0.01 mol/L initially. The concentration of HSO 3 − was 0.05 mol/L.

Synthesis and Methods
The synthetic route was shown in Scheme 1. Compound 3 was prepared according to previously reported method [39].

Synthesis of Compound 2
Compound 1 (402.75 mg, 2.2 mmol) was added to 3-methyl-2-butanone (20 mL), then concentrated sulfuric acid (0.5 mL) was mixed to get a white turbid liquid. The mixture was heated up to 125 °C and reacted for 8 h. After the reaction, the mixture was cooled to room temperature, a solid precipitated out, and suction filtration to get the compound 2 (310 mg, 60.2%). The specific characterization of compound 2 ( 1 H NMR, 13

Detection Properties of Probe PBI
For better experimental results, we must first select the most suitable experimental system. The DMSO content was changed from 10%, 20%, to 60% in the test system, and HSO 3 − was added to test under the same conditions. From Fig. 1a, when the DMSO content increased from 10 to 60%, the I 397 /I 646 decreased. The I 397 /I 646 reached the maximum as the DMSO content was 10%, but in comparison, when the DMSO content was 20%, the ∆I 397 /I 646 was the largest before and after the reaction. So we chose the test system as V water :V DMSO = 8:2.  first period and have no influence on the overall reaction time of probe with HSO 3 − . The probe should have a wide range of pH for better detection of HSO 3 − . Test system separately was prepared with DMSO and deionized water of different pH, the pH ranges from 1 to 14, and fluorescence intensity of probe PBI was recorded in the absence and presence of HSO 3 − . As shown in Fig. 1d, in the pH range of 4 to 8, great difference of the I 397 /I 646 of probe PBI with and without HSO 3 − was obtained, so the optimal pH test range for the probe PBI to detect HSO 3 − was 4-8. Such result illustrated that probe PBI can detect HSO 3 − in a wide pH range and have potential applications in real sample detection.
The UV-vis spectral response of probe PBI to HSO 3 − was tested firstly. As shown in Fig. 2a, probe PBI showed the absorption maximum at 520 nm originally. Upon addition of HSO 3 − to the solution, the absorption peak at 520 nm gradually decreased, and the absorption peak progressively increased at 350 nm. When the HSO 3 − concentration in the test system was 0.07 mmol/L −0.22 mmol/L, the A 350 /A 520 showed a good linear relationship with the HSO 3 − concentrations (Fig. 2b), this means that in this interval, we can achieved quantitative detection of HSO 3 − . After adding HSO 3 − , the color of PBI solution changed from pink to colorless under daylight (Fig. 2c), the change was so obvious that it provides an easy way to detect HSO 3 − with the naked eye.
Fluorescence spectra of probe PBI over various concentrations of HSO 3 − were recorded. As for PBI, We used double excitation mode that was to choose 350 nm and 520 nm as the excitation wavelength. As shown in Fig. 3a, after the titration of HSO 3 − , the fluorescence intensity of PBI at 392 nm increased gradually, while fluorescence intensity of PBI progressively decreased at 646 nm. In order to more directly express the relationship between fluorescence intensity and concentration of HSO 3 − , the I 397 /I 646 was plotted as a function of the concentration of HSO 3 − (Fig. 3b). In the range of 0.04 mmol/L −0.19 mmol/L, the linear increase of I 397 / I 646 could be used in the quantitative detection of HSO 3 − . Upon addition of HSO 3 − , The color also changed significantly under portable UV lamps, gradually changing from pink to blue purple (Fig. 3c), which can be more convenient for HSO 3 − detection in practical applications.

Selectivity and Anti-interference Ability Studies of Probe PBI
The special selectivity of fluorescent probes for analytes was higher than other substances is an important feature of it. To understand the selectivity of probe PBI toward HSO 3 − , we conducted a series of controlled experiments with anions. As can be seen from Fig. 4, The A 350 /A 520 and I 397 /I 646 with only the above anions added had no big difference compared with that of probe PBI only except HSO 3 − , which showed all these anions could not respond to probe PBI. The above anions and HSO 3 − were added to probe PBI solution at the same time, The A 350 /A 520 and I 397 /I 646 were significantly increased compared with that of probe PBI only. Obviously, probe PBI responds to HSO 3 − only, and when other anions and HSO 3 − coexist, there was no interference on the detection of HSO 3 − with probe PBI. − with probe PBI was also explored. From the Figs. S10 and S11, it is clear that probe PBI can specifically select HSO 3 − in the presence of cations and amino acids, and thus probe PBI had good selectivity and strong anti-interference ability.

The Reaction Mechanism of Probe to Detect HSO 3 −
The reaction mixture of PBI and NaHSO 3 was analyzed by ESI-MS spectroscopy to explore the recognition mechanism of probe PBI to HSO 3 − . As shown in Fig. S12, m/z = 516.1957 (theoretical molecular weight = 516.1952) [M + HSO 3 + H] + was the peak after the carbon-carbon double bond nucleophilic addition reaction between probe PBI and HSO 3 − . So we proposed the detection mechanism of probe PBI for HSO 3 − : in the condition of HSO 3 − , six-membered ring of probe PBI was broken, and HSO 3 − was added to the position of the original carbon-carbon double bond (Scheme 1).

The Detection of HSO 3 − in Living Cells
In order to explore the potential application of probe PBI in the detection of HSO 3 − in living cells, fluorescence confocal imaging experiments were performed in HeLa cells with probe PBI, and the images were captured under a confocal fluorescence microscope. We studied the feasibility of the probe PBI to detect exogenous HSO 3 − in HeLa cells. The bright field pictures showed the position in the living cells (Fig. 5a, d). The sole probe PBI emitted clear red fluorescence (Fig. 5b), when the cells were treated with HSO 3 − , the red fluorescence disappeared (Fig. 5e), and they overlaid well (Fig. 5c, f). The results indicated that probe PBI can effectively detect exogenous HSO 3 − , which can be used to detect HSO 3 − in the living cells.

Comparison of Probe PBI with Some Reported Probes Toward HSO 3 −
As shown in Table 1, in terms of excitation wavelength/ emission wavelength, detection medium, response type, pH, detection limit and cell application, probe PBI had good analytical performance compared with other HSO 3 − fluorescent probes reported recently. 40−45 The result showed that probe PBI was a ratiometric fluorescent probe with a low detection limit (208 nmol/L). Moreover, biological experiments have demonstrated the application of probe PBI in monitoring intracellular HSO 3 − .

Conclusion
In summary, we have developed a new colorimetric and ratiometric fluorescence probe PBI to detect HSO 3 − . The reaction mechanism of probe PBI to detect HSO 3 − was attributed to the nucleophilic addition reaction. When the probe was added with HSO 3 − , the absorption and fluorescence emission changed significantly, and the color change was so obvious that it provided an easy way to detect HSO 3 − with the naked eye. In addition, probe PBI shows good selectivity and strong anti-interference ability for HSO 3 − . The probe PBI can in HeLa cells, which would be potential candidates to track HSO 3 − in live cells.