A NIR Fluorescent Probe Benzopyrylium Perchlorate-based for Visual Sensing and Imaging of SO2 Derivatives in Living Cells

Endogenous sulfur dioxide (SO2), as a gas signal molecule, has a certain physiological functions. Understanding the role of endogenous SO2 in human physiology and pathology is of great significance to the biological characteristics of SO2,which bring challenges to develop fluorescent probes with excellent performance. Herein, we rationally designed and constructed a novel near-infrared bioprobe benzaldehyde-benzopyrylium (BBp) by employing the nucleophilic addition benzopyrylium perchlorate fluorophore and benzaldehyde moiety by means of C = C/C = O group that serves as both fluorescence reporting unit. Probe BBp exhibit excellent sensing performance with fluorescent “On − Off”rapid response (100 s) and long-wavelength emission (670 nm). With the treatment of HSO3−, the color of BBp solution obviously varies from purple to colorless, and the fluorescent color varies from red to colorless. By the fluorescence and colorimetric changes, probe BBp was capable of sensitive determination HSO3− with low limits of detection (LOD) of 0.43 μM, realizing visual quantitative monitoring SO2 derivative levels. Due to the low phototoxicity and good biocompatibility, it was successfully applied to monitor SO2 derivatives and fluorescence imaging in HepG2 and HeLa living cells. Hopefully, this work supplies a new strategy for designing NIR fluorescent probes for quantitative determination SO2 derivatives in biological samples.


Introduction
Endogenous sulfur dioxide (SO 2 ) is discovered as an important endogenous gasotransmitter after hydrogen sulfide (H 2 S), carbon monoxide (CO), and nitric oxide (NO) [1,2]. Endogenous SO 2 can be produced endogenous reactivating species (RSS) by the metabolism of sulfur, a class of sulfur-containing compounds that play a crucial role in redox homeostasis,cellular signaling, and metabolic regulation [3][4][5][6]. Under physiological conditions, SO 2 is readily hydrated and transformed to its derivatives sulfite (SO 3 2− ) and bisulfite (HSO 3 − ) [7][8][9]. SO 2 in the body can be inhaled not only through the respiratory tract, but also through diet and medication. Increasing evidences demonstrated that endogenous SO 2 and its derivatives (HSO 3 − / SO 3 2− ) take part in some physiological processes such as vasorelaxant, antioxidative effects, and regulating cardiovascular structure and function [10][11][12][13]. In a manner of speaking, the SO 2 biological function can be replaced to HSO 3 − / SO 3 2− . It is noteworthy that abnormal changes in endogenous SO 2 levels are associated with a series of physiological diseases, such as lung injury, cancer, respiratory, abnormal glucose metabolism, and other diseases [14][15][16][17][18]. Considering the contradictory nature of endogenous SO 2 , we deem that it is necessary to deeply explore the intracellular role of SO 2 derivatives, to provide valuable information for pathological mechanism of cell cellular regulatory, which prompted us to focus on developing analytical tools for effectively monitor SO 2 derivatives in living cells.
Compared with traditional analytic technique, fluorescence assay is one of attractive bioanalytical tool. Fluorescent probe technology has been proved more practical due to its many outstanding properties such as non-invasive, high temporal resolution, high sensitivity as well as real-time detectability, which are widely used in biochemical analysis, environmental analysis, biological imaging, medical diagnosis and other fields [19][20][21][22][23]. Recently, with regard to SO 2 1 3 derivatives determination and recognition, a variety of functional and practical fluorescent probes with highly selective and sensitive have been built and these probes have achieved satisfactory results in sensing and fluorescence imaging in living cells and in vivo, whose different recognition mechanism mainly including intramolecular charge transfer (ICT) effect, Michael addition, resonance energy transfer (FRET), and nucleophilic addition with aldehyde groups [24][25][26][27][28]. Among these probes, their excitation and emission wavelengths are mainly take advantage of the UV-Visible region (Table S1), only a few probes displayed large stokes shifts and rapid responses [29][30][31], which are essential for sensitive detection SO 2 derivatives in living cells. Near-infrared (NIR) fluorescent probes can effectively avoid the interference of spontaneous fluorescence, self-absorption as well as low phototoxicity in biological systems, which is beneficial to improve the biological imaging efficiency [32][33][34][35][36]. Needless to say, there is still ample sufficient space to exploit a novel near-infrared fluorescent probe for sensing SO 2 derivative with tailored property.
Benzopyrylium salt is an ideal fluorescent skeleton with large shift and long wavelength emission [37,38]. Based on this, we selected benzopyrylium perchlorate as fluorophore, benzaldehyde unit as SO 2 derivative recognition unit, and reasonably designed and fabricated a new near infrared biological probe benzaldehyde-benzopyrylium (BBp), to achieve sensitive and rapid detection of SO 2 derivative levels. The sensing mechanism as presented in Scheme 1, based on irreversible nucleophilic addition of HSO 3 − toward -C = O and -C = C-, with HSO 3 − treatment, it reacts with the probe BBp of the aldehyde group and the furan ring double bond, the emission intensity at 670 nm was gradually decreased and the response time reach the maximum within 100 s, building a near-infrared fluorescence "On-Off" sensitive and quick response SO 2 derivative sensing platform. More importantly, the NIR probe BBp presented low optical toxicity and good cell permeability, the probe BBp had been successfully applied for sensing of SO 2 derivatives and fluorescent imaging in HepG2 and HeLa living cells.

Synthesis of Probe BBp
Benzopyrylium perchlorate was fabricated according to the method of reference [37]. The mixture of benzopyrylium perchlorate (356 mg, 1 mmol) and p-benzaldehyde (134 mg, 1.2 mmol) were added into a 50 mL two-bottle, and dissolved in 10 mL acetic acid. The mixture was stirred and reflow for 12 h, and the reaction process was monitored by TLC. After the reaction, the black solid compound as probe BBp (0.216 g, yield: 44%) was obtained by filtration and purification of dichloromethane/ethanol (V/V, 100/1) by chromatography column. 1

Sample Preparation Process
The analysis of solution preparation process: Na 2 HPO 4 , NaH 2 PO 4 , KCl and NaCl were used as raw materials, and phosphate buffer solution (PBS, 0.01 M, pH 7.4) was prepared according to the standard method. The analytical solution of probe BBp was prepared in DMF solution (100.0 μM).The test solution 0.01 M of NaHSO 3 , amino acids, anionic and cationic salts was prepared with ultrapure water. All the experimental water is ultra-pure water. Scheme 1 Sensing mechanism of NIR probe BBp to HSO 3 − Feasibility experiment analysis process: take 10 μL probe BBp analysis solution and different amounts of test solution NaHSO 3 (0 − 100 μM) into a 1.5 mL centrifuge tube, then dilute the solution to 1000 μL with PBS buffer solution (0.01 M, pH 7.4), and incubate for 3 min. Finally, the absorption spectrum data were collected. Under the same experimental conditions, the selectivity experiment is carried out: 10 μL probe BBp solution was mixed with 50 μM amino acids, biological ion and other substances, and diluted to 1000 μL with PBS buffer solution (0.01 M, pH 7.4). After incubation for 3 min, fluorescence spectra were determined under excitation wavelength of 530 nm. All analytical experiments were carried out at room temperature.

Cells Viability Determination and Fluorescence Imaging
The cells viability was determined by CCK8 method. Firstly, 8000 of HeLa cells and HepG2 cells were cultured in 96-well plates at 5% CO 2 and 37 ℃ for 24 h. Different amounts of probe BBp (0 -30 μM) were added and incubated for 24 h. Whereafter, adding 10 μL CCK8 dealt with each well and continued incubation for 2 h, and then the cells were washed with PBS solution several times. Finally, absorbance was collected by ThermoFisher, Japan. Its relative cell viability (%) was calculated by the following formula: Among them, OD treated and OD control represent the optical density values measured in the presence and absence of probe BBp.
The HeLa cells and HepG2 cells were cultured in glass plates for 24 h before imaging experiment. The two kinds of cells were first handled with probe BBp (10 μM) for further incubation for 10 min, and then the cells were washed with PBS solution for several times, and then with different concentrations of HSO 3 − (10 and 50 μM) were treated for 10 min. Finally, fluorescence images were collected by fluorescence confocal image. The emission blue channel wavelength range of the collected image data is λ em = 566 − 697 nm (λ ex = 561 nm).

Design and Characterization of Fluorescent Probe BBp
The benzaldehyde-benzopyrylium (BBp) fluorescence probe was synthesized by employing the main p-phthalaldehyde and benzopyrylium perchlorate by means of the nucleophilic addition reaction as depicted in Fig. S1. Benzopyrylium perchlorate is an ideal long-wavelength emission skeleton and benzaldehyde unit as SO 2 derivative recognition unit, which ensures that the probe conjugate system exhibits near-infrared emission (670 nm). Meanwhile, aldehyde group and the furan ring double bond are an excellent recognizing sites for detection SO 2 derivatives (HSO 3 − /SO 3 2− ) because of its high reaction performance, which could meet the requirements for the sensitive determination under the visible light excitation (530 nm). The fluorescent probe BBp was characterized by 1 H NMR, 13

Optical Properties of HSO 3 − Sensing by Probe BBp
The sensing ability of probe BBp to HSO 3 − was investigated by fluorescence and colorimetric performance under optimum conditions. Firstly, the UV-Vis spectra of the probe BBp (10 μM) was estimated in PBS buffer solution (0.01 M, pH 7.4, containing 1% DMF) at room temperature. As shown in Fig. 1A, the original probe BBp solution exhibited a maximal absorption band centered at 560 nm and has another a weak absorption peak at 300 nm. In contrast, Upon treatment with HSO 3 − (50 μM), the main absorption peak at 560 nm was decreased significantly, while a new generated absorption band at 340 nm. It was observed that the color of the corresponding solution remarkable changed from purple to colorless under visible light (illustrated in Fig. 1A), indicating that the probe BBp could achieve the visual detection of HSO 3 − levels by colorimetric method. This results speculated that HSO 3 − could destroy the conjugate system of the probe, causing a steady decrease in the long absorption wavelength.
Furthermore, under excitation at 530 nm, the initial probe BBp showed a strong emission peak at 670 nm, and its near-infrared fluorescence emission intensity gradually weakened with the increase of HSO 3 − concentration (0 -100 μM) and the color of fluorescence changes from red to colorless green as seen on the inset (Fig. 1B). Meanwhile, the results of spectral properties suggest that this phenomenon may be attributed to the nucleophilic addition reaction between the aldehyde group and double bonds of furan ring in the probe BBp and HSO 3 − . The fluorescence intensity ratio (F / F 0 ) of probe BBp exhibited an excellent linear relationship between the HSO 3 − concentration (5 -60 μM) with a relatively high coefficient of determination (R 2 = 0.991) (F represents the fluorescence intensity of probe BBp changing with different concentrations of HSO 3 − , F 0 represents the fluorescence intensity of initial probe BBp) (Fig. 1C). The LOD is calculated according to the equation LOD = 3σ/ k, where σ is the standard deviation of blank sample N = 10, k is the slope of the linear regression equation), the calculated detection of limit was 0.43 μM. The results confirmed that probe BBp enables a sensitive and quantitative determination of HSO 3 − by means of NIR fluorescence "On-Off". The fluorescence probe BBp that we construct has a good linear range and a relatively low LOD compared with previously reported fluorescent probes for detection HSO 3 − levels (Table S1). This long emission probe BBp is comparable and performs better than other methods. It is worth mentioning that the fairly low LOD are much lower than the level of tissues and organs in living organisms generation endogenous SO 2 levels [39,40], illustrating that the probe BBp is feasible for the actual determination of SO 2 derivatives in vivo.
In addition, time-dependent is an important parameter in analysis and detection. The response time fluorescence intensity of the reaction between probe BBp and HSO 3 − with different concentrations (0 -50 μM) was recorded. It can be seen from Fig. 1D that the reaction probe BBp with HSO 3 − proceeded resulting in the fluorescence intensity gradually decrease and remain unchanged after 100 s at room temperature. The probe BBp has a fast response to HSO 3 − in the buffer solution. The long-wavelength probe BBp could meet well the requirement for real-time monitoring of HSO 3 − levels in living cells. It follows that probe BBp has the advantages of low detection limit, high sensitivity and fast response, affirming that probe BBp has excellent sensing performance for detecting SO 2 derivatives.

Sensing Mechanism Study
In order to validate the mechanism by which the probes BBp sensitively sense HSO 3 − levels, we collected the evidence through HRMS analysis. According to the characteristic peak of mass spectrum, the mass charge ratio (m/z) corresponding to the main characteristic peak at 372.1958 attributed to the compound mass peaks of probe BBp [M], while the calculated value was 372.1958 ( Fig. 2A). In addition, when a certain amount of HSO 3 − was added into the probe BBp, the characteristic peak emerged at m/z 534.1269 [M + H]-was clearly obtained (calca. = 534.1267), it was mainly attributed to the reaction product of recognition site 1 ( furan ring double bond) and recognition site 2 (aldehyde group) in probe BBp with HSO 3 − ( Fig. 2B). It can be seen that the theoretical value is consistent with the experimental value. Combined with the spectral results, we speculated that the irreversible nucleophilic addition reaction between the furan ring double bond and aldehyde group in the probe BBp and HSO 3 − was reasonable [41][42][43][44] (Fig. 2C). It was further confirmed that the destruction of conjugated system by irreversible nucleophilic addition reaction was the main reason that the fluorescence of probe BBp changed from red to colorless. Taken together, HRMS spectral analysis have well supported our speculative sensing mechanism.

Anti-interference Ability and pH Effect
Anti-interference capability is an important factor to evaluate the effectiveness of probes in analyzing biological samples. To this end, the chemoselectivity of probe BBp for detection HSO 3 − was studied. Several exogenous substances including amino acids (100 μM of Cys, Hcy and GSH), physiological anions and cations (50 μM of Na + , K + , Ca 2+ , Mg 2+ , Cu 2+ , Fe 3+ , Cl − , I − , F − , H 2 PO 4 − , CO 3 2− , HCO 3 − , and HS − ) were selected as bio-related substances and those ions that can coexist with HSO 3 − . As shown in Fig. 3A, the introduction of these potential interferes has no effect on the fluorescence intensity of probe BBp. When HSO 3 − / SO 3 2− were added, the fluorescence intensity of the probe BBp decreased significantly. Except for HS − and mercaptan molecules coexist with HSO 3 − at a higher concentration can cause mild fluorescence intensity change. This is mainly because HSO 3 − / SO 3 2− can undergo nucleophilic addition reaction with the double bond and aldehyde group in the furan ring [26,38,44], but its influence on the detection of HSO 3 − by probe BBp in biological system is negligible. The presence of these high concentration of exogenous interferences has little interference, making clear that the NIR probe BBp has high selectivity and good anti-interference ability to HSO 3 − . The anti-interference experiments suggested that the probe could be used for determination SO 2 derivatives in complex biological systems.
Subsequently, the influence of pH fluctuations on the detection performance of probe BBp was evaluated. We adjusted the PBS buffer of probe BBp to different changed in the range of pH 3.5 -10.5, revealing that probe BBp had excellent acid-alkali resistance (Fig. 3B). It shows that the probe realized the determination of SO 2 derivatives under physiological pH conditions. Whereafter, the photostability of probe BBp was assessed under UV light irradiation at 365 nm. The emission intensity of the synthesized probe BBp displays hardly fluctuation upon continuous irradiation for 60 min (Fig. S5), which confirmed that the synthesized probe BBp has better photostability. Based on the above research, the excellent performance of probe BBp has the ability to determine the content of SO 2 derivatives in real biological samples.

Cytotoxicity and Intracellular Fluorescence Imaging
Based on the analysis performance evaluation results of the probe, the designed probe BBp exhibited superior recognition capability, especially including good sensitivity, excellent photostability and low phototoxicity. Consequently, the fluorescence imaging capability of the probe BBp for sensing SO 2 derivatives in living cells was estimated. HeLa cells and HepG2 cells were selected as imaging model cells. Firstly, the cytotoxicity of different doses of probe BBp towaord HeLa cells and HepG2 cells were analyzed by the standard CKK8 assay. As presented in Fig. S6, the survival rate of HeLa cells and HepG2 cells reached 80% even if the high-concentration of probe BBp (30 μM) (the cell survival rate was calculated by Formula 1). The cytotoxicity experiments were confirmed that the probe BBp had relatively low toxicity and good biocompatibility for living cells. These results suggested that the probe BBp meets the requirements for the analysis and detection in biological systems.
Subsequently, the NIR probe BBp was used to monitor HSO 3 − levels and imaging in living cells. Firstly, the intake time-dependence imaging of living cells uptake probe BBp was performed. As described in Fig. 4, the probe BBp (10 μM) was incubated with HeLa cells and HepG2 cells at different periods of time. The intracellular red fluorescence obvious appeared in the red channel with increasing time, and the red fluorescence became significantly brighter to stable until 20 min after incubation (Fig. 4A-D), suggesting that the probe BBp displays an excellent cell uptake ability and intracellular stability. These results demonstrated that 10 min is suitable for imaging incubation experiments and probe BBp has pontential applications in real-time monitoring of SO 2 in living cells.
Inspired by the excellent imaging results, the effectiveness of the probe BBp for imaging the SO 2 derivatives levels in living was further carried out. As expected, when HepG2 cells and HeLa cells were incubated with 10 μM probe BBp only for 10 min, obvious red red fluorescence were observed from the cells in the red channel (Fig. 5A). However, when HeLa cells and HepG2 cells were pre-treated with different concentrations of exogenous HSO 3 − (10 μM and 50 μM), and significantly became blurred fluorescence imaging is observed (Fig. 5B, C), indicating that the intracellular probe BBp can be efficiently quenched by exogenous HSO 3 − . The change from red fluorescence to non-fluorescent signal observed in cells further explained that the probe BBp can be an ideal tool for the effectively monitoring SO 2 in living cells.

Conclusion
In summary, a near-infrared fluorescent "On-Off" probe BBp was rationally designed to achieve sensitive detection of HSO 3 − levels in living cells. Based on the nucleophilic addition reaction mechanism, the fluorescence intensity decreased gradually at 670 nm, and the color of the corresponding solution was observed to change from red to colourless under UV lamp (356 nm), realizing visual recognition of HSO 3 − . In vitro experiments, the probe BBp with rapid response, high selectivity, and sensitivity, and the detection of limit was as low as 0. 43 μM. The recognition mechanism was verified by HRMS. A fluorescence "On-Off" sensitive sensing SO 2 derivatives platform was successfully constructed. Due to the advantages of biocompatibility and good cell permeability, it was successfully used to monitor exogenous HSO 3 − and fluorescence imaging in HepG2 and HeLa cells. We believe that the rational design strategy using benzopyrylium perchlorate as a long-wave fluorophore can be exploited to construct long-wavelength fluorescent probes, which are ideal further applications for bioassay and imaging applications.