Preparation method and application of double positive charge fluorescent probe based on quinoline-hemicyanine structure

Abstract

The application discloses a preparation method and application of a double-positive-charge fluorescent probe based on a quinoline-hemicyanine structure, a sensing mechanism based on an ICT effect, a Michael acceptor type fluorophore (hemicyanine derivative) as a light-emitting group, preparation of a small-molecule fluorescent probe (probe 1) capable of detecting sulfur dioxide derivatives, modification of the structure of the probe 1, regulation of a site at which the probe and the sulfur dioxide derivatives undergo Michael addition reaction, realization of conversion of the quenching type fluorescent probe into the ratio type fluorescent probe (probe 2), improvement of the detection performance of the probe, and realization of fluorescent-colorimetric dual-mode detection of the sulfur dioxide derivatives. Due to addition of bisulfite to hydrogen at the para position of quinoline N, the conjugated system of the probe is increased, the ICT effect is also amplified, the probe is changed in different emission wavelengths, and the naked eye color also has a significant change, thereby providing a design idea for development of the fluorescent probe for specifically detecting the sulfur dioxide derivatives.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for preparing and applying a dual positively charged fluorescent probe based on a quinoline-hemicyanine structure. Background Technology

[0002] With the acceleration of human industrialization, sulfur dioxide has become a significant environmental pollutant. Sulfur dioxide derivatives are commonly used as preservatives and antioxidants in food, beverages, and pharmaceuticals. In the environment, sulfur dioxide forms a series of derivatives in water bodies, entering the biosphere cycle and severely impacting aquatic life and even human health. Epidemiological studies have confirmed that excessive intake of sulfur dioxide or its derivatives can cause a range of respiratory diseases, such as asthma and chronic bronchitis, and is even associated with certain neurological disorders, such as Alzheimer's disease and central nervous system tumors. Therefore, the detection of sulfur dioxide derivative content in food is crucial.

[0003] Currently, many countries have set maximum usage and maximum residue limits for sulfur dioxide derivatives (calculated as sulfur dioxide) in food and have developed various methods for sulfur dioxide detection. Liquid chromatography-tandem mass spectrometry or acid-base titration are commonly used to determine sulfur dioxide in food. These methods offer advantages such as accurate quantification, few interfering factors, and high stability. However, in actual food testing, rapid and convenient operating methods are often required for on-site testing, batch analysis of samples, or non-destructive testing. Therefore, the advantages of fluorescent probe methods, such as high sensitivity, short response time, in-situ detection, convenient operation, and visualization, meet the requirements for such practical sample testing. Developing suitable fluorescent probes to achieve these advantages is of great significance. In recent years, although some fluorescent probes for detecting sulfur dioxide derivatives have been reported, most fluorescent probes still have some drawbacks, such as complex synthesis routes, high cost, significant susceptibility to temperature and pH, and low stability. Therefore, developing a low-cost, environmentally resistant, and highly stable fluorescent probe for the detection of sulfur dioxide derivatives is of significant research importance.

[0004] Most previously reported fluorescent probes for detecting sulfur dioxide derivatives are single-channel probes with only one emission peak. While single-channel probes can ideally be used for quantitative detection, they face several challenges in real-world testing environments. For example, background interference from complex environments, instrument errors, and variations in probe concentration can all affect the accuracy of quantitative results. Therefore, ratiometric fluorescent probes are considered a superior solution in modern research. By introducing a fluorescence signal at an additional wavelength, ratiometric probes can eliminate the influence of probe concentration and instrument errors on the test results, thus improving quantitative accuracy in practical applications.

[0005] Hemicyanine dyes possess unique photophysical properties, including good water solubility, long absorption and emission wavelengths, large Stokes shifts, and high fluorescence quantum yields, making them frequently used by researchers to design fluorescent probes. However, hemicyanine dyes also have drawbacks such as short emission wavelengths and low selectivity during detection. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a quinoline-hemicyanine bipolar fluorescent probe, its preparation method, and its applications. In this invention, quinoline and hemicyanine are linked together via carbon-carbon double bonds to form a long-wavelength emitting fluorescent probe. Since methylation of quinoline leads to a redistribution of the electron cloud density throughout the molecule, and transforms quinoline from an electron acceptor to an electron donor, it facilitates the reaction of the probe with the nucleophilic sulfur dioxide derivative. Furthermore, methylation of quinoline also helps to generate molecules with stronger ICT effects after the probe reacts with sulfur dioxide derivatives, thereby achieving the purpose of ratiometric detection of sulfur dioxide derivatives. Because the quinoline-hemicyanine bipolar fluorescent probe possesses two molecules of positive charge after methylation, the binding of the quinoline-hemicyanine bipolar fluorescent probe with negative charge after hydrolysis is more convenient, which also facilitates the reaction of the quinoline-hemicyanine bipolar fluorescent probe with sulfur dioxide derivatives.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A method for preparing a dual positively charged fluorescent probe based on a quinoline-hemicyanine structure includes the following steps:

[0009] (1) Take 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole in a round-bottom flask, add anhydrous ethanol and iodomethane, heat to react, filter, wash and dry to obtain a pale yellow solid M1, which is 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole-3-iodide.

[0010] (2) A mixture of 3-quinoline formaldehyde and 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole-3-iodide was refluxed in the presence of piperidine and anhydrous ethanol. After the reaction was completed, the reaction mixture was cooled to room temperature, rotary evaporated, filtered, washed, and dried to obtain pure benzo[e]indole salt, i.e., fluorescent probe 1 based on the quinoline-hemicyanine structure; the structural formula of probe 1 is shown below:

[0011] The structural formula of probe 1 is shown below:

[0012] ;

[0013] (3) The benzo[e]indole salt obtained in step (2) was dissolved in ultra-dry dichloromethane, and methyl trifluoromethanesulfonate (TfOMe) was added and mixed under nitrogen atmosphere protection. The mixture was stirred at room temperature. After the reaction was completed, the reaction system was cooled to room temperature, and the reaction mixture was poured into a vacuum filter funnel for filtration. The filter cake was washed with diethyl ether and dried to obtain a double positively charged fluorescent probe based on the quinoline-hemicyanine structure, namely probe 2. The structural formula of probe 2 is shown below:

[0014] .

[0015] Furthermore, in step (1), the molar ratio of 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole to iodomethane is 1:2.362.

[0016] Furthermore, the reaction temperature in step (1) is 60 °C and the reaction time is 2 h.

[0017] Furthermore, in step (2), the molar ratio of 3-quinoline carboxaldehyde and 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole-3-iodide is 1:1, and the reflux reaction time is 2h.

[0018] Furthermore, in step (3), the molar ratio of benzo[e]indole salt to methyl trifluoromethanesulfonate is 1:4.44, and the reaction time at room temperature is 3 hours.

[0019] The optimal excitation wavelength of the dual positively charged fluorescent probe based on the quinoline-hemicyanine structure prepared by the method of the present invention is 370 nm, the optimal emission wavelength is 615 nm, and the dual positively charged fluorescent probe based on the quinoline-hemicyanine structure has a Stokes shift of 245 nm.

[0020] This invention also provides the application of a dual positively charged fluorescent probe based on a quinoline-hemicyanine structure in the detection of trace sulfur dioxide derivatives. Specifically, probe 2 is dissolved in 4 mL of chromatographically pure DMSO solution to make a stock solution with a concentration of 2 mM. The probe 2 stock solution is added to 2 mL of PBS solution with pH=7.4 to make a final concentration of 10 μM. The solution is continuously irradiated under a laser at 370 nm for 3 min. Then, the sulfur dioxide derivative solution is rapidly added. The fluorescence intensity of the system is recorded every 2 s. Within 10 s after the addition of the sulfur dioxide derivative, the fluorescence intensity of probe 2 at 615 nm rapidly decreased by 6.8 times, and the fluorescence intensity at 575 nm rapidly increased by 32 times, and remained stable for the next 10 minutes.

[0021] Furthermore, probe 2 was dissolved in 4 mL of chromatographically pure DMSO solution to prepare a 2 mM stock solution. This stock solution was then added to 2 mL of PBS solution at pH 5-8 to achieve a final concentration of 10 μM. Simultaneously, SDS was added to bring the final concentration to 50 μM. Using 370 nm as the excitation wavelength and 615 nm as the emission wavelength, the fluorescence intensity of probe 2 significantly increased within 20 s and remained stable thereafter. After the addition of SDS, the fluorescence intensity of probe 2 at 615 nm increased by 1.7 times, while the fluorescence intensity at 575 nm remained unchanged. Subsequently, the addition of a sulfur dioxide derivative resulted in a 91-fold change in the ratio of fluorescence intensity at 575 nm to that at 615 nm. The addition of SDS facilitated an increase in the fold change in the fluorescence intensity ratio of probe 2, thus improving its sensitivity.

[0022] The detection limit of probe 2 of the present invention for sulfur dioxide derivatives is 0.80 μM.

[0023] The beneficial effects of this invention are as follows: Based on the sensing mechanism of the ICT effect, this invention uses a Michael acceptor fluorophore (hemicyanine derivative) as the luminescent group to prepare a small molecule fluorescent probe (probe 1) capable of detecting sulfur dioxide derivatives, and has been successfully applied to the detection of sulfur dioxide in actual red wine. By modifying the structure of probe 1 and controlling the site of the Michael addition reaction between the probe and the sulfur dioxide derivative, a quenching-type fluorescent probe was transformed into a ratiometric fluorescent probe (probe 2), improving the detection performance of the probe and achieving dual-mode fluorescence-colorimetric detection of sulfur dioxide derivatives. The fluorescence of the probe can quantitatively detect sulfur dioxide derivatives, while the visual color change can qualitatively determine the presence of sulfur dioxide derivatives in the solution. Due to the addition of bisulfite to the N-hydrogen of quinoline, the conjugated system of the probe increases, and the ICT effect is amplified, achieving the switching of different emission wavelengths of the probe, and a significant change in visual color is also observed. Meanwhile, the probe has a Stokes shift of 245 nm, effectively avoiding the influence of the probe's excitation wavelength on the fluorescence emission peak. Even with a 50-fold increase in the concentration of interfering substances, the addition of a sulfur dioxide derivative still resulted in a 91-fold increase in fluorescence intensity. Figure 10 As shown, this meets the detection requirements of the probe in actual samples. Most importantly, the fluorescence intensity of this probe increased by 1.7 times after the addition of SDS, as... Figure 14 As shown, this enhances the sensitivity of the probe in detecting sulfur dioxide derivatives. After rationally designing the structure of probe 2, the detection limit for sulfur dioxide derivatives also decreased from 3.51 μM to 0.80 μM. This lays a solid foundation for the future development of portable devices for detecting sulfur dioxide derivatives and provides design ideas for developing fluorescent probes that specifically detect sulfur dioxide derivatives. The design strategy of this invention can provide a reference for the modification and detection of other similar molecules. Attached Figure Description

[0024] Figure 1 It is probe 1. 1 H NMR (600 MHz, DMSO-d6).

[0025] Figure 2 It is probe 1. 13 C NMR (151 MHz, DMSO-d6).

[0026] Figure 3 It is probe 2. 1 H NMR (600 MHz, DMSO-d6).

[0027] Figure 4 It is probe 2. 13 C NMR (151 MHz, DMSO-d6).

[0028] Figure 5 The images show the absorption spectra (A) of probe 1 molecules at different concentrations in PBS buffer (10 mM, pH=7.4) and the fluorescence emission spectra (B) of probe 1 (10 μM) in PBS buffer. Excitation wavelength: 380 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0029] Figure 6 The fluorescence spectra (A) of probe 1 (10 μM) gradually added to sodium bisulfite and (B) of probe 1 (10 μM) mixed with SDS (100 μM) and then with NaHSO3 (0-100 μM) are shown. Excitation wavelength: 380 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0030] Figure 7 The images show the absorption spectra (A) of probe 2 molecules at different concentrations in PBS buffer (10 mM, pH=7.4) and the UV absorption and fluorescence emission spectra (B) of probe 2 (10 μM) in PBS buffer. Excitation wavelength: 370 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0031] Figure 8 Figure 1 shows the UV-Vis absorption spectrum (A) and fluorescence spectrum (B) of probe 2 (10 μM) with the gradual addition of sodium bisulfite, and the linear relationship between the concentration of sodium bisulfite (0-20 μM) and the fluorescence intensity of probe 2 (10 μM) at 575 nm and 615 nm (D). Figure 2 (B) shows the excitation wavelength: 510 nm, slit width: 10 / 10 nm, voltage: 650 V. Figure 3 (C) shows the excitation wavelength: 370 nm, slit width: 10 / 10 nm, voltage: 650 V. Inset: Photographs of 10 μM probe 2 before and after the reaction with 100 μM sodium bisulfite under sunlight (A) and a 365 nm (B) UV lamp.

[0032] Figure 9 This is the fluorescence spectrum of probe 2 (10 μM) against a related interfering substance (200 μM) in a PBS system, where 1: blank; 2: F - , 3:Cl - , 4: Br - , 5:I - , 6: CO3 2- ,7:AcO - ,8:SO4 2- ,9:S2O3 2- ,10:NO2 - ,11: NO3 - ,12:GSH,13:Cys,14:HPO42- ,15:ClO - ,16:H2O2,17:HSO3 2- (20 μM), 18: K + , 19: Ca 2+ , 20:Mg 2+ , 21: Fe 3+ , 22: Al 3+ , 23: Cu 2+ , 24: Fe 2+ Excitation wavelength: 370 nm, excitation wavelength: 510 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0033] Figure 10 The time-scan test was performed before and after the addition of NaHSO3 to probe 2 (10 μM) in PBS buffer. (A) Excitation wavelength: 380 nm, emission wavelength: 590 nm, slit width: 10 / 10 nm, voltage: 650 V. (B) Excitation wavelength: 510 nm, emission wavelength: 575 nm, slit width: 10 / 10 nm, voltage: 650 V.

[0034] Figure 11 The images show the UV-Vis absorption spectrum (A) and fluorescence spectrum (B) of probe 2 (10 μM) gradually added to SDS. Insets: photographs of 10 μM probe 2 reacted with 10–100 μM SDS under sunlight (A) and a 365 nm UV lamp (B). Excitation wavelength: 370 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0035] Figure 12 The fluorescence emission spectrum is obtained by mixing probe 2 (10 μM) with SDS (50 μM) in PBS buffer and then adding NaHSO3 (0-20 μM). Excitation wavelength: 370 nm, slit size: 10 / 10 nm, voltage: 650 V.

[0036] Figure 13 This is a graph showing the change in fluorescence intensity of probe 2 (10 μM) over time before and after adding 50 μM SDS to a PBS (pH=7.4) system. Excitation wavelength: 370 nm, emission wavelength: 615 nm.

[0037] Figure 14 The fluorescence emission spectra of the reaction between probe 2 (10 μM) and sulfur dioxide derivatives of different concentrations (0-75 μM) are shown in Figure (A) and Figure (F) of the reaction between probe 2 and sulfur dioxide derivatives. 575 / F 615 The relationship between the value and the concentration of sulfur dioxide derivatives (0-20 μM) (B) (F)575 The fluorescence emission intensity at 575 nm after adding a sulfur dioxide derivative to the probe and exciting it at 510 nm was: F 615 (The fluorescence emission intensity at 615 nm after the probe is excited at 370 nm with the sulfur dioxide derivative) and the F emission intensity of the reaction between probe 2 and the sulfur dioxide derivative. 575 / F 615 Linear relationship between the value and the concentration of sulfur dioxide derivatives (20-38 μM) (C), with a slit width of 10 nm. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above-described invention.

[0039] Example 1: Synthesis of Probe 1

[0040] (1) 1,1,2,3-Tetramethyl-1-hydro-benzo[e]indole (10 g, 47.8 mmol) was placed in a 100 mL round-bottom flask, and 15 mL of anhydrous ethanol and iodomethane (7 mL, 112.9 mmol) were added. The mixture was heated to 60 °C and reacted for 2 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was filtered through a vacuum funnel and washed three times with ice-cold ethanol to obtain a pale yellow filter cake. The filter cake was dried in a vacuum drying oven for 12 hours to obtain 9.3 g of pale yellow solid M1. The product did not require further purification and was stored directly in a brown glass bottle for later use.

[0041] (2) A mixture of 3-quinoline carbaldehyde (2 g, 12.5 mmol) and 1,1,2,3-tetramethyl-1-hydro-benzo[e]indole-3-iodide (4.39 g, 12.5 mmol) was refluxed for 2 hours in the presence of piperidine (120 μL, 1.2 mmol) and 25 mL of anhydrous ethanol. The reaction progress was monitored by thin-layer chromatography. The reaction mixture was then cooled to room temperature. Excess solvent was removed by rotary evaporation. The precipitate was filtered, washed three times with cold ethanol and diethyl ether, and dried in a vacuum oven for 12 hours to give pure benzo[e]indole salt as a red-orange powder (3.2 g). The yield was 52.2%. The synthetic route is shown below:

[0042]

[0043] 1 H NMR (600 MHz, DMSO- d 6) δ 9.70 (d, J= 2.2 Hz, 1H), 9.29 – 9.24 (m,1H), 8.71 (d, J = 16.6 Hz, 1H), 8.49 (d, J = 8.5 Hz, 1H), 8.34 (d, J = 8.9 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.17 (d, J = 8.9 Hz, 1H), 8.14 (dd, J = 8.2, 3.6 Hz, 2H), 8.05 (d, J = 16.6 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.77(dt, J = 10.0, 4.9 Hz, 2H), 4.39 (s, 3H), 2.09 (s, 6H). 13 C NMR (151 MHz, DMSO d 6) δ 182.33, 150.58, 148.65, 148.43, 139.40, 138.52, 138.26, 133.33, 132.05, 130.93, 130.00, 129.26, 129.01, 128.46, 127.86, 127.77, 127.37, 127.10, 126.56, 123.32, 114.31, 113.44, 54.00, 40.06, 35.47, 24.84. (Characteristics results are attached.) Figures 1-2 ).

[0044] Example 2: Synthesis of Probe 2

[0045] The benzo[e]indole salt (probe1) (0.2 g, 0.41 mmol) prepared in Example 1 was dissolved in ultra-dry dichloromethane (10 mL), and methyl trifluoromethanesulfonate (TfOMe) (206 μL, 1.82 mmol) was added under nitrogen atmosphere and mixed. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction system cooled to room temperature, the reaction mixture was poured into a vacuum filter funnel and filtered. The filter cake was washed with diethyl ether and then dried in a vacuum drying oven to obtain a bright orange solid (0.17 g, yield 63.69%). The synthetic route is shown below:

[0046]

[0047] 1 H NMR (600 MHz, DMSO- d 6) δ 10.21 (s, 1H), 9.96 (s, 1H), 8.66 (d, J = 12Hz, 2H), 8.53 (t, J = 6 Hz, 2H), 8.41 (t, J = 6 Hz, 1H), 8.37 (d, J = 12 Hz, 1H), 8.27 (d, J = 12 Hz, 1H), 8.19 (dd, J = 18, 6 Hz, 2H), 8.10 (d, J = 18 Hz, 1H), 7.86(t, J = 6 Hz, 1H), 7.79 (t, J = 12 Hz, 1H), 4.73 (s, 3H), 4.40 (s, 3H), 2.07 (s, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 182.28, 151.39, 146.70, 146.66, 144.23, 140.00, 139.59, 139.07, 138.88, 137.55, 134.06, 133.86, 131.81, 131.63, 131.45, 130.55, 129.19, 129.09, 128.86, 128.23, 127.05, 124.35, 124.06, 122.22, 120.12, 120.08, 117.94, 117.44, 114.12, 54.79, 54.53, 46.49, 36.33, 36.30, 25.33, 24.95. (Characteristics results are attached.) Figures 3-4 ).

[0048] Implementation Results Example

[0049] Preparation of probe 1 stock solution: Accurately weigh 3.9 mg of probe 1 (M = 490.0906 g / mol) solid, dissolve it in 4 mL of chromatographically pure DMSO to prepare a 2 mM stock solution.

[0050] Preparation of probe 2 stock solution: Weigh 5.2 mg of probe 2 (M=654.0661 g / mol) solid and dissolve it in 4 mL of chromatographic grade DMSO solution to make a stock solution with a concentration of 2 mM.

[0051] Spectroscopic performance testing of probe 1

[0052] The prepared stock solution was diluted in 2 mL of PBS buffer to a final concentration successively from 2 μM to 10 μM for UV-Vis absorption and fluorescence emission spectroscopy determination. UV-Vis absorption spectroscopy test conditions: absorption wavelength range: 250 nm to 800 nm; scan step size: 2 nm. Fluorescence spectroscopy test conditions: excitation wavelength: 380 nm; emission wavelength scan range: 400 nm to 740 nm; scan rate: 2400 nm / min; slit width: 10 nm / 10 nm; photomultiplier tube negative voltage (PMT Voltage): 650 V.

[0053] Depend on Figure 5 As shown in A, with the increase of probe concentration, probe 1 exhibits two absorption peaks at 380 nm and 430 nm. Figure 5 As shown in B, the optimal excitation wavelength for probe 2 is 380 nm, and the optimal emission wavelength is 590 nm. This results in a Stokes shift of 210 nm for the probe, effectively avoiding interference from the excitation wavelength on the emission peak. According to... Figure 5 A indicates that a final concentration of 10 μM for probe 1 is suitable. Subsequent experiments all used a 10 μM probe 2. (e.g.) Figure 5 B).

[0054] Feasibility of probe 1 for the detection of sulfur dioxide derivatives

[0055] The feasibility of this study was investigated by analyzing the changes in the fluorescence emission spectra of probe 1 before and after the addition of SDS, followed by the addition of sodium bisulfite. The fluorescence emission... Figure 6 As shown in A, before the addition of SDS to probe 1, the emission peak at 590 nm gradually quenched with the addition of sodium bisulfite, and the fluorescence intensity changed by a factor of 9. Figure 6As shown in Figure B, after the addition of SDS, the fluorescence intensity of probe 1 at 590 nm increased to approximately 2900. With the addition of sodium bisulfite, the emission peak at 590 nm gradually quenched, and the fluorescence intensity changed by a factor of 13. This indicates that probe 1 reacted with sodium bisulfite to produce a new fluorescent substance, and the addition of SDS increased the fluorescence fold in the detection of sulfur dioxide derivatives. These experiments preliminarily verify that probe 1 may be able to achieve fluorescence detection of sulfur dioxide derivatives and that the addition of SDS may enable probe 1 to achieve more sensitive detection of sulfur dioxide derivatives.

[0056] Probe 2 spectral performance test

[0057] The prepared stock solution was diluted in 2 mL of PBS buffer to a final concentration of 2 μM to 10 μM for UV-Vis absorption and fluorescence emission spectroscopy determination. UV-Vis absorption spectroscopy conditions: Absorption wavelength range: 250 nm to 800 nm; Scan step size: 2 nm. Fluorescence spectroscopy conditions: Excitation wavelength: 370 nm; Emission wavelength scan range: 390 nm to 720 nm; Excitation wavelength: 510 nm; Emission wavelength scan range: 530 nm to 900 nm; Scan rate: 2400 nm / min; Slit width: 10 nm / 10 nm; Photomultiplier tube negative voltage (PMT Voltage): 650 V.

[0058] Depend on Figure 7 As shown in A, with the increase of probe concentration, the two absorption peaks of probe 2 at 300 nm and 370 nm also gradually increase, and a strong absorption is observed at 370 nm at a longer wavelength. It is speculated that these two absorption peaks correspond to the quinoline derivative structure and the probe structure, respectively. Figure 7 As shown in B, the optimal excitation wavelength for probe 2 is 370 nm, and the optimal emission wavelength is 615 nm. This results in a Stokes shift of 245 nm for the probe, effectively avoiding interference from the excitation wavelength on the emission peak. According to... Figure 7 A indicates that a final concentration of 10 μM for probe 2 is suitable. All subsequent experiments used a 10 μM probe 2 concentration. (e.g.) Figure 7 A).

[0059] Feasibility of probe 2 for the detection of sulfur dioxide derivatives

[0060] The feasibility of the experiment was explored by analyzing the changes in the UV-Vis absorption and fluorescence emission spectra of probe 2 before and after the addition of the sulfur dioxide derivative. Figure 8As shown in Figure A, with the addition of sodium bisulfite, the absorption peaks of probe 2 at 300 nm and 370 nm gradually disappeared, and a new strong absorption peak gradually appeared at 510 nm. The isoabsorption point was located at 480 nm, indicating that probe 2 reacted chemically with the sulfur dioxide derivative to form a new substance. As can be seen from the inset, the solution of probe 2 was yellow under sunlight, turning red after the addition of the sulfur dioxide derivative; the experimental phenomenon is consistent with the changes in the UV-Vis absorption spectrum. These experiments suggest that probe 2 and sodium bisulfite may have undergone a Michael addition reaction, resulting in a change in the conjugated structure of probe 2.

[0061] fluorescence emission Figure 8 B and Figure 8 As shown in C, with the addition of sodium bisulfite, the emission peak at 615 nm gradually quenched, and the fluorescence intensity changed by an 8-fold. The emission peak at 575 nm rapidly increased, and the fluorescence intensity changed by a 74-fold. Figure 8 As shown in Figure D, the emission peaks of probe 2 at 575 nm and 615 nm are linearly related to the sodium bisulfite concentration. This preliminarily confirms that probe 2 has a ratiometric response to sodium bisulfite. As can be seen from the inset, the probe fluorescence color changes from orange-yellow (left) to pink (right). This indicates that probe 2 produces a new fluorescent substance after reacting with sodium bisulfite. These experiments preliminarily verify that probe 2 may be able to achieve fluorescence detection of sulfur dioxide derivatives.

[0062] 5. Selectivity of probe 2 for sulfur dioxide derivatives

[0063] The actual food environment is complex. Considering that food often contains various anions and cations, the anti-interference ability of probe 2 was investigated, focusing on common anions and cations and reactive sulfides. 50 equivalents of interfering ion solution and 20 μM sodium bisulfite solution were added to a series of cuvettes, and the fluorescence intensity ratio at 575 nm and 615 nm was compared with that of the blank solution. Figure 9 As shown, the fluorescence intensity of the probe increased 9.7 times after the addition of GSH. The concentration of GSH was 1 mM, which is 50 times the concentration of sodium bisulfite. Therefore, GSH has minimal interference with the detection of sulfur dioxide derivatives by probe 2. In the presence of other relevant interfering substances, the fluorescence intensity did not change significantly, indicating that the probe is not easily affected by potential substances and is stable. The fluorescence intensity increased 91 times after the addition of sodium bisulfite to the probe 2 system, demonstrating that probe 2, which relies on fluorescence intensity ratios for detection, has high anti-interference capability.

[0064] The above tests on interfering substances by the probe demonstrate that probe 2 has good anti-interference performance, laying the foundation for the application of the probe in actual food testing.

[0065] 6. Reaction time of probe 2 to sulfur dioxide derivatives

[0066] Food safety regulations often require quick results; therefore, the reaction time between probe 2 and the sulfur dioxide derivative is a crucial influencing factor. To investigate the reaction time of probe 2, the probe solution was added to 2 mL of PBS solution to a final concentration of 10 μM, and irradiated under a 370 nm laser for 3 min. Then, the sulfur dioxide derivative solution was rapidly added. The fluorescence intensity of the system was recorded every 20 s. Figure 10 As shown, within 10 seconds of adding the sulfur dioxide derivative, the fluorescence intensity of probe 2 decreased rapidly by 6.8 times at 615 nm and increased rapidly by 32 times at 575 nm, remaining stable for the next 10 minutes. This allows for real-time and rapid detection of sulfur dioxide derivatives, with significant changes in fluorescence intensity, demonstrating its potential as a ratiometric fluorescent probe.

[0067] 7. Investigation of the spectral properties of probe 2 and SDS

[0068] This invention introduces a common anionic surfactant, sodium dodecyl sulfate (SDS), to accelerate the Michael addition reaction between probe 2 and sulfur dioxide derivatives. The interaction between SDS and probe 2 was studied using spectroscopic methods such as UV-Vis absorption spectroscopy and fluorescence spectroscopy, as well as electron microscopy methods such as SEM (scanning electron microscopy) and TEM (transmission electron microscopy), and the changes in the reaction between probe 2 and sulfur dioxide derivatives after the addition of SDS were investigated.

[0069] like Figure 11 As shown in Figure A, with the addition of SDS, the absorbance of probe 2 gradually decreased at all wavelengths, indicating that probe 2 and SDS gradually dispersed in the solution. Figure 11 As can be seen from B, when the excitation wavelength remains at 370 nm, the fluorescence emission intensity of probe 2 gradually increases with the addition of SDS, but the emission wavelength remains unchanged at 590 nm. This indicates that the addition of SDS does not affect the emission wavelength of probe 2. Figure 11 As shown in the illustration, the addition of SDS did not change the visual color of probe 2, but it did change its fluorescence color. This is likely because the presence of SDS weakens the polarity of the solution microenvironment, thereby enhancing the ICT (intramolecular charge transfer) effect of probe 2, leading to enhanced fluorescence. Based on the experimental results, 10 μM probe 2 and 50 μM SDS were used as the basic conditions for subsequent experiments.

[0070] 8. Investigation of the spectroscopic properties of probe 2 and sulfur dioxide derivatives after the addition of SDS

[0071] The spectroscopic properties of probe 2 and sulfur dioxide derivatives in the presence of SDS were investigated, such as... Figure 12 As shown, after adding 20 μM sodium bisulfite solution to 10 μM probe 2 and 50 μM SDS, the fluorescence emission intensity of probe 2 at 590 nm decreased by 9.5 times, while the fluorescence emission intensity at 575 nm increased by 6 times, proving that SDS promoted the reaction of probe 2 with sulfur dioxide derivatives.

[0072] 9. Response time of probe 2 after adding SDS

[0073] The interaction time between probe 2 and SDS is also one of the factors affecting probe detection performance. Figure 13 It can be seen that using 370 nm as the excitation wavelength and 615 nm as the emission wavelength, the fluorescence intensity of probe 2 significantly increased within 20 s and remained stable thereafter. The addition of SDS increased the fluorescence intensity of probe 2 at 615 nm by 1.7 times, which is beneficial for increasing the fold change in the fluorescence intensity ratio of probe 2 and improving its sensitivity.

[0074] 10. Working curve of probe 2 for sulfur dioxide derivatives

[0075] Based on the above experimental data, the working curves of the system's reaction with different concentrations of sulfur dioxide derivatives were further investigated. Since probe 2 is a ratiometric fluorescent probe, the ratio of fluorescence intensity at emission wavelengths corresponding to different excitation wavelengths before and after the addition of the sulfur dioxide derivative was used for further investigation. Figure 14 As shown in Figure A, with the gradual increase of the concentration of the sulfur dioxide derivative, the fluorescence emission intensity ratio of the probe 2 system gradually increases. There is a sudden jump in the fluorescence emission intensity ratio when the concentration of the sulfur dioxide derivative is 20-40 μM. Subsequently, the change in fluorescence intensity ratio decreases with further addition of the sulfur dioxide derivative. When the concentration of the sulfur dioxide derivative reaches 60 μM, the difference in fluorescence emission intensity reaches equilibrium and no longer increases. This curve is similar to the "S"-shaped curve in a monoprotic acid-base titration experiment, indicating that the reaction between the probe system and the sulfur dioxide derivative also has a sudden jump stage and a stoichiometric point. Simultaneously, the F... 575 / F 615 (F) 575 The fluorescence emission intensity at 575 nm after adding a sulfur dioxide derivative to the probe and exciting it at 510 nm was: F 615 The fluorescence emission intensity at 615 nm after the probe is excited at 370 nm with the sulfur dioxide derivative was linearly fitted to the concentration of the sulfur dioxide derivative. Figure 14 B and Figure 14As shown in Figure C, the sulfur dioxide derivative concentrations exhibit a good linear relationship between 0-20 μM and 20-38 μM. The working curve for 0-20 μM is y = 0.191x + 0.172, with R0... 2 =0.9535. Using the limit of detection formula: LOD = 3σ / k (where σ is the standard deviation of ten blank solutions and k is the slope of the linear equation), the lowest limit of detection for the fluorescence spectrometry method for detecting sulfur dioxide derivatives is calculated to be 1.55 μM. The working curve corresponding to 20-38 μM is y = 0.369x - 4.896, R0 2 =0.9197. Using the formula for calculating the limit of detection (LOD): LOD = 3σ / k (where σ is the standard deviation of ten blank solutions and k is the slope of the fitted linear equation), the lowest detection limit for the fluorescence spectrometry method for detecting sulfur dioxide derivatives is calculated to be 0.80 μM. Experimental results indicate that probe 2 can be used for the detection of trace amounts of sulfur dioxide derivatives.

[0076] Detection of sulfur dioxide derivatives in actual samples using probe 2

[0077] To evaluate the practicality of the constructed system, sulfur dioxide derivatives in red wine were determined according to the following method: (20 μL of red wine was taken and diluted to a total volume of 4 mL with PBS buffer. This solution was used as the actual sample solution. 10 μL was added to the reaction system of probe 2 (10 μM) each time, followed by the addition of a standard solution of sulfur dioxide derivatives, and the total volume was diluted to 2 mL with PBS buffer. The fluorescence emission intensity of each sample at 575 nm and 615 nm was measured. The ratio of the fluorescence intensity at the two locations was substituted into the previously fitted linear relationship equation between the fluorescence intensity difference and the concentration of sulfur dioxide derivatives to calculate the content of sulfur dioxide derivatives.) A certain amount of standard solution was added to each system for experimental verification. The results are shown in Table 1. Sulfur dioxide derivatives were not detected in red wine. The recoveries obtained by spiked recovery ranged from 97.4% to 122.9%, with relative standard deviations ranging from 0.37% to 1.40%. The experimental results show that the method has high accuracy and can be used for the detection of sulfur dioxide derivatives in red wine.

[0078] Table 1. Detection of sulfur dioxide derivatives in red wine (n=5)

[0079]

[0080] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Use of the bispositively charged fluorescent probe 2 based on the quinoline-hemicyanine structure in the detection of sulfur dioxide derivatives for non-diagnostic and therapeutic purposes, characterized in that: The sulfur dioxide derivative is sodium bisulfite; the structure of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure is as follows: 。 2. Use according to claim 1, characterized in that: The double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure is dissolved into 4 mL of chromatographically pure DMSO solution to become a stock solution with a concentration of 2 mM; the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure stock solution is added into 2 mL of PBS solution with pH=7.4 to have a final concentration of 10 μM, and irradiated with laser at 370 nm for 3 min, and then the sulfur dioxide derivative solution is rapidly added, and the fluorescence intensity of the system is recorded every 2 s, within 10 s after the addition of the sulfur dioxide derivative, the fluorescence intensity of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure at 615 nm is rapidly reduced by 6.8 times, and the fluorescence intensity at 575 nm is rapidly increased by 32 times, and both remain stable within the next 10 min.

3. Use according to claim 1, characterized in that: The double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure is dissolved into 4 mL of chromatographically pure DMSO solution to become a stock solution with a concentration of 2 mM; the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure stock solution is added into 2 mL of PBS solution with pH=7.4 to have a final concentration of 10 μM, and SDS is added to have a final concentration of 50 μM, and the fluorescence intensity of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure is significantly enhanced within 20 s with 370 nm as the excitation wavelength and 615 nm as the emission wavelength, and remains stable thereafter, and the fluorescence intensity of the probe 2 at 615 nm is increased by 1.7 times after the addition of SDS, and the fluorescence intensity at 575 nm remains unchanged, and then after the addition of the sulfur dioxide derivative, the ratio of the fluorescence intensity at 575 nm to that at 615 nm changes by 91 times, and the addition of SDS is conducive to increasing the multiple of the fluorescence intensity ratio change of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure, and improves the sensitivity of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure.

4. Use according to any one of claims 1 to 3, characterized in that: The detection limit of the double positive charge fluorescent probe 2 based on quinoline-hemicyanine structure for the sulfur dioxide derivative is 0.80 μM.

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