Fluorescent probe of flower-like cyanine and its synthesis method and application

Abstract

The application discloses a kind of flower-like cyanin fluorescent probe and its synthesis method and application, belong to organic small molecule fluorescent probe field;The flower-like cyanin fluorescent probe molecular formula is C 22 H 17 ClN2O4, marked as N-3-I;The application provides a kind of novel flower-like cyanin fluorescent probe synthesis method with simple operation steps and fast process, the flower-like cyanin fluorescent probe synthesized can be reacted with sulfite under very mild conditions, and strong sensing process of response rapid, high sensitivity occurs;When there is sulfite in test sample, the color of fluorescent probe solution is significantly changed from original dark pink to colorless, can be visually realized in real time " naked eye " monitoring of sulfite content in traditional Chinese medicinal materials from vision, and monitoring level can reach nanomolar level;The flower-like cyanin fluorescent probe N-3-I can be applied to visual detection of sulfite and related test paper development, and it has important practical significance for safety evaluation of traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, specifically relating to a cyanin-like fluorescent probe, its synthesis method, and its application. Background Technology

[0002] Sulfites are commonly used bleaching agents and preservatives in the food industry. As a food additive, they play the following main roles in food: (1) preventing enzymatic and non-enzymatic browning of food, making the food bright and white; (2) inhibiting the growth of microorganisms, thereby extending the shelf life of food; (3) inhibiting the activity of oxidases in food, preventing oxidases from causing damage to nutrients and discoloration; (4) combining with colored substances contained in food, and playing a bleaching role due to their reducing properties.

[0003] However, as research has deepened, the toxicity of sulfites has received increasing attention. Numerous studies have shown that abnormal levels of SO2 in food can harm human health. Excessive sulfites entering the human circulation can cause kidney dysfunction, cardiovascular disease, and respiratory diseases. The toxicity of sulfites is mainly manifested in the following aspects: (1) Sulfites can react with substances such as amino acids and proteins, and can also combine with various vitamins to break them down into other products, thereby seriously damaging the nutrients in food and reducing the nutritional value of food. (2) Long-term exposure to low concentrations of sulfites can lead to headaches, dizziness, and general weakness. In severe cases, it can cause symptoms such as chronic pharyngitis, bronchitis, and decreased sense of smell and taste. (3) If animals eat food containing sulfites for a long time, they will experience adverse symptoms such as nerve inflammation and bone marrow atrophy, which will hinder the growth of animals. (4) Sulfites can also cause cell mutations and induce the oxidation of unsaturated fatty acids.

[0004] Therefore, the content of sulfite additives in food must be strictly tested and controlled. For example, the Codex Alimentarius Commission (CAC) stipulates that the sulfite content in dried fruits, vegetables, and wine should be below 100 mg / kg, 200 mg / kg, and 250 mg / kg, respectively. The U.S. Food and Drug Administration (FDA) stipulates that HSO3 in food and beverages... The content of sulfites should be less than 10 ppm (125 μmol / L), while the "Hygienic Standard for the Use of Food Additives" (GB2760-2015) clearly stipulates the allowable amount of sulfites in various foods. That is, the amount of sulfites used in food shall not exceed 30 mg / kg, and the Pharmacopoeia of the People's Republic of China (PRC) also stipulates that the sulfite content in Chinese medicinal materials shall not exceed 400 mg / kg.

[0005] Given the certain side effects of sulfites, there is an urgent need to develop a rapid and quantitative detection method for sulfites. Sulfites are oxyacid salts, and the anion in their structure is the sulfite ion (SO32-). 2- The detection of sulfite is actually the detection of sulfite ions (SO32-). 2- Traditional methods for detecting sulfites include ion chromatography, electrochemical methods, and capillary electrophoresis. However, these methods require complex pretreatment steps, high-end equipment, complex experimental operations, and long analysis times, limiting their application in food testing as they cannot meet the need for real-time monitoring of the target analyte. Photochemical sensors, on the other hand, have rapidly developed and attracted widespread attention in recent years due to their advantages such as low cost, simple method, rapid response, and large information capacity. Their main principle is to utilize the changes in the optical properties (absorption or emission) of the sensor unit as the output signal to achieve the detection of the analyte.

[0006] Photochemical sensors can be categorized into fluorescent chemical sensors and colorimetric chemical sensors based on their signal detection methods. Fluorescent chemical sensors (fluorescent probes) primarily rely on fluorescence signals for detection, typically involving enhancement, quenching, or changes in emission wavelength. Colorimetric chemical sensors, on the other hand, utilize changes in hue, with chromogenic sensors, in particular, allowing for detection through visual observation, making them convenient for practical applications. Currently, single fluorescent or colorimetric chemical sensors are increasingly widely used in the detection of biomolecules due to their high selectivity, high sensitivity, and fast response time.

[0007] In 2022, GONG W, ZHANG C, ZHANG X, et al. published "Mitochondria-targetable colorimetric and far-red fluorescent sensor for rapid detection of SO2 derivatives in food samples and living cells" in *Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy*. The probe AQCB, based on a rhodamine analog backbone containing the 3-quinoline formaldehyde moiety, combined with the 1-methylquinoline moiety, produced a fluorescence-quenching receptor-π-receptor structure, thus synthesizing a novel HSO3. - Colorimetric far-infrared fluorescent probe. Quinoline, as a cationic surfactant, can improve membrane permeability and biocompatibility. It increases the intermolecular reaction rate and shortens the reaction time. When HSO3 It underwent a 1,4-nucleophilic addition reaction with the quinoline moiety to form an electron-rich enamine moiety, resulting in a donor-π-donor structure and producing strong purple fluorescence. AQCB exhibits good water solubility and reacts with HSO3 in PBS. It exhibits rapid response (<15 s), high sensitivity (LOD=49 nM), and selectivity. This probe possesses favorable cell membrane properties and can be successfully applied to HSO3 in live cell mitochondria. Imaging. It can not only quantitatively detect HSO3 in food samples. Furthermore, it can directly detect HSO3 in mitochondria. .

[0008]

[0009] In 2023, LI Y, WANG Y, LEI X, et al. reported in *Food Chemistry* on "Development of a responsive probe for colorimetric and fluorescent detection of bisulfite infod and animal serum samples in 100% aqueous solution." The probe, NPO, exhibited good water solubility, rapid reaction, and a detection limit as low as 0.25 μM, accompanied by a change in solution color from blue to yellow. The test strip experiment successfully detected HSO3. It enables real-time visual detection of SO2 gas. Furthermore, it has been successfully used for the detection of SO2 in the environment, as well as HSO3 in real food samples (sugar, liquor, dried edible fungi, etc.) and animal serum samples. The detection.

[0010] In 2021, ZHANG C, HAN L, LIU Q, et al. introduced "A colorimetric and far-redfluorescent probe for rapid detection of bisulfite / sulfite in full water-soluble based on biquinolinium and its applications" in Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. The probe DQ detects HSO3 through a 1,4 nucleophilic addition reaction. The detection exhibits high selectivity, with colors ranging from colorless to purplish-red in pure aqueous solutions, and significant far-red fluorescence enhancement. The probe DQ can be used to detect HSO3 on real samples such as paper strips, rice noodles, and sugar. HSO3 in live cell images .

[0011]

[0012] While the fluorescent probes disclosed in the aforementioned prior art possess advantages such as high sensitivity, good selectivity, and rapid detection, their applications are broad, encompassing the entire food, biological, and environmental safety fields. However, they fail to demonstrate excellent specificity and particularity when detecting sulfites in traditional Chinese medicine (TCM) materials. Furthermore, with the improvement of living standards in my country, the food safety of TCM materials has increasingly attracted significant attention. Therefore, there is an urgent need to research a fluorescent probe that is simple to operate, provides real-time and rapid detection, and is specifically designed for the detection of sulfites in TCM materials, providing strong technical support for self-monitoring by TCM material manufacturers and market supervision. Summary of the Invention

[0013] To address the problems existing in the prior art, the present invention aims to provide a cyanine-like fluorescent probe, its synthesis method, and its application. The cyanine-like fluorescent probe N-3-I can be applied to the visual detection of sulfites and the development of related test strips, and has important practical significance for the safety assessment of traditional Chinese medicine materials.

[0014] The technical solution of the present invention is as follows: One objective of this invention is to provide a cyanine-like fluorescent probe, characterized in that the cyanine-like fluorescent probe is labeled N-3-I, its solution is deep pink, and its molecular formula is C. 22 H 17 ClN₂O₄, chemical structural formula is shown in Figure I: (I).

[0015] The second objective of this invention is to provide a method for synthesizing a cyanin-like fluorescent probe, characterized by the following steps: (1) Cesium carbonate and 1,8-naphtholactam were dissolved in acetonitrile, and iodomethane was added dropwise. The mixture was stirred at room temperature for 3-5 hours under an argon atmosphere. The solvent was removed by rotary evaporation, and the resulting bright yellow powder was purified to obtain compound 1, whose structural formula is shown in II. (II); (2) Compound 1 obtained in step 1 was dissolved in anhydrous tetrahydrofuran at 0°C. Methyl magnesium chloride was added dropwise and stirred. Then, the mixture was refluxed in an oil bath at 50-70°C for 1-3 hours under an argon atmosphere. After the reaction was completed, the mixture was placed in an ice bath until the temperature dropped to 0°C. A small amount of water was added to quench the reaction. Then, perchloric acid was added to the reaction mixture and stirred thoroughly to obtain a yellow-green solution. The solution was then added to ice water, vacuum filtered, and dried to obtain a green powder, which is compound 2. Its structural formula is shown in III. (III); (3) Compound 2 and 5-indolecarboxaldehyde were mixed in a round-bottom flask, and then a mixture of ethanol and acetonitrile was added. The mixture was allowed to react overnight at room temperature in the dark. After the reaction was completed, the black precipitate was filtered, and the reaction mixture was purified by silica gel column chromatography to obtain the cyanin-like fluorescent probe N-3-I, the structural formula of which is shown in IV: (IV).

[0016] Further, step (1) involves the following reaction: .

[0017] Further, in step (1), the molar ratio of 1,8-naphtholactam, cesium carbonate, and iodomethane is 1:1.5:1.2. Furthermore, the separation and purification in step (1) is carried out by column chromatography, wherein the eluent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 23:1.

[0018] Furthermore, step (2) involves the following reaction: .

[0019] Further, in step (2), the molar ratio of compound 1 to methyl magnesium chloride is 1:4.5.

[0020] Furthermore, step (3) involves the following reaction: .

[0021] Furthermore, in step (3), the molar ratio of compound 2 to 5-indolecarboxaldehyde is 1:1.

[0022] Furthermore, in step (3), the volume ratio of ethanol to acetonitrile in the ethanol-acetonitrile mixed solution is 9:1.

[0023] Furthermore, the separation and purification in step (3) is carried out by column chromatography, wherein the eluent is a dichloromethane / methanol mixture with a volume ratio of 10:1.

[0024] The third objective of this invention is to provide an application of a cyanin-like fluorescent probe in the detection of sulfite content in traditional Chinese medicine materials.

[0025] Furthermore, when sulfites are present in the test sample of traditional Chinese medicine, the cyanin-like fluorescent probe can respond rapidly under very mild conditions, causing the solution to change significantly from its original deep pink color to colorless.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention designs a cyanine-like fluorescent probe as a novel sensor capable of rapid and sensitive detection of sulfite. The cyanine-like fluorescent probe exhibits good selectivity and specificity for sulfite, and can react with sulfite under very mild conditions. The sulfite / bisulfite ions selectively and rapidly react with the carbon-carbon double bond connecting the indole ring and benzoindole, disrupting the probe's large conjugated system and causing a blue shift in fluorescence. The probe can respond rapidly within 1 minute, with high sensitivity (detection limit up to 49 nM), good selectivity and specificity, and can effectively eliminate interference from other food additives in the sample. 2. Compared with existing fluorescent probe technologies, the synthesis method of the anthocyanin-like fluorescent probe N-3-I disclosed in this invention is simple, rapid, and easy to operate, and is environmentally friendly. When sulfite is present in the test sample, the color of the fluorescent probe solution changes significantly from its original deep pink to colorless, allowing for direct, visual, real-time "naked-eye" monitoring of sulfite content in traditional Chinese medicine materials, with a monitoring level reaching the nanomolar level. The anthocyanin-like fluorescent probe has no special limitations in use and can be used under normal room temperature and other mild conditions, demonstrating good practical applicability.

[0027] 3. The cyanin-like fluorescent probe N-3-I described in this invention can be applied to the safety assessment of sulfites in traditional Chinese medicine materials. This method uses a small amount of reagent, has a fast detection time, and high detection performance, thus it has broad application prospects in the development of visual detection and test strips for sulfites. Attached Figure Description

[0028] Figure 1 The 1H NMR spectrum of the cyanine-like fluorescent probe N-3-I synthesized in Example 1 of this invention; Figure 2 The UV spectral characteristics (A), fluorescence spectral characteristics (B), and linear relationship between fluorescence intensity (488 nm) and Angelica dahurica concentration (C) of the interaction between the cyanin-like fluorescent probe N-3-I and Angelica dahurica in Example 2 of this invention are shown in Figure 2. Figure 2 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 3The figures shown are: UV spectral characteristics (A), fluorescence spectral characteristics (B), and linear relationship between fluorescence intensity (488 nm) and licorice concentration (C) of the interaction between the cyanin-like fluorescent probe N-3-I and licorice in Example 2 of this invention. Figure 3 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 4 The figures shown are: UV spectral characteristics (A), fluorescence spectral characteristics (B), and linear relationship between fluorescence intensity (488 nm) and American ginseng concentration (C) of the interaction between the cyanin-like fluorescent probe N-3-I and American ginseng in Example 2 of this invention. Figure 4 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 5 The change factor of fluorescence intensity at 488 nm of the cyanine-like fluorescent probe N-3-I after reacting with Na2SO3 in different solvents during the performance test of this invention; Figure 6 The images show the UV and fluorescence spectra of the reaction between the cyanin-like fluorescent probe N-3-I and 10 equivalents of Na2SO3 in different solvents during the performance testing of this invention. Figure 6 A represents the ultraviolet spectrum. Figure 6 B represents the fluorescence spectrum (excitation light at 414 nm). Figure 6 C is a bar chart of fluorescence intensity at 488 nm for different solvents; Figure 7 The figures (A), (B), and (C) show the UV-Vis spectrum of the interaction between the cyanin-like fluorescent probe N-3-I and NaHSO3 in the performance test of this invention. Figure 7 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 8 The figures (A), (B), and (C) show the UV spectral characteristics of the interaction between the cyanin-like fluorescent probe N-3-I and Na2S2O4 in the performance test of this invention. Figure 8 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 9 The figures (A), (B), and (C) show the UV-Vis spectrum of the interaction between the cyanin-like fluorescent probe N-3-I and Na2S2O5 in the performance testing of this invention. Figure 9 The excitation light for the fluorescence spectrum in B is 414 nm. Figure 10This is a bar chart showing the fold change in fluorescence intensity at 488 nm after the cyanine-like fluorescent probe N-3-I reacted with different sulfur-containing compounds (100 μM) in the performance test of this invention. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken. Unless otherwise specified, the experimental methods in the following examples are conventional methods; The drying solvents used in the following examples are all of molecular sieve type 4A (sodium-A type molecular sieve) or molecular sieve type 3A (potassium-A type molecular sieve). 1 The H-s spectra were recorded on a JEOL ECZ600S (600 MHz) spectrometer using CDCl3 or CD3OD as solvents. Based on internal TMS (trimethylsilane) reference data, a front field chemical shift of one part per million was reported; The coupling constant (J) is expressed in Hertz (Hz), and the spin multiple states are expressed in s (singlet), d (doublet), t (triplet), and m (multiplex). Column chromatography was performed using thick-walled glass columns and silica gel (300-400 mesh); thin-layer chromatography (TLC) was performed using commercially available 0.25 mm silica gel plates under UV light. The ultraviolet absorption spectrum of the solution was obtained using a Shimadzu UV-1900 UV-Vis spectrophotometer. Fluorescence spectra were measured using a Spectrofluorometer FS5 fluorescence spectrometer. Mass spectra were recorded on a ThermoFisher high-performance liquid chromatography-mass spectrometry system.

[0031] Example 1 This embodiment provides a cyanin-like fluorescent probe, the synthesis method of which includes the following steps: S1. Cesium carbonate (0.48 g, 1.5 mmol) and 1,8-naphthyl lactam (0.17 g, 1 mmol) were dissolved in 8 mL of acetonitrile. Iodomethane (0.124 mL, 2 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h under an argon atmosphere. After removing the solvent and purifying, a bright yellow powder was obtained, which is compound 1. The eluent for chromatography was a mixture of petroleum ether and ethyl acetate with a volume ratio of 23:1. The specific reaction process is as follows: ; S2. Compound 1 (0.182 g, 1 mmol) obtained in step 1 was dissolved in anhydrous THF at 0°C. Methylmagnesium chloride (1.5 mL, 4.5 mmol) was added dropwise and stirred for 10 min. The mixture was refluxed in an oil bath at 60°C for 2 h under an argon atmosphere. After the reaction was completed, it was placed in an ice bath at 0°C. When the temperature dropped to 0°C, a small amount of water (0.5 mL) was added to quench the reaction. Then, perchloric acid (60%, 0.6 mL) was added to the reaction mixture and stirred thoroughly. The yellow-green solution was added to ice water (90 mL), vacuum filtered, and dried to obtain a green powder, which is compound 2. The specific reaction process is as follows: ; S3. Add mixed compound 2 (1.5 mmol, 0.27 g) and 5-indolecarboxaldehyde (1.5 mmol, 0.217 g) to a round-bottom flask, then add a mixed solution of ethanol and acetonitrile (volume ratio of ethanol to acetonitrile is 9:1). Incubate overnight at room temperature in the dark. After the reaction is complete, filter the black precipitate and purify the reaction mixture by silica gel column chromatography. The eluent for chromatography is a 10:1 volume ratio of dichloromethane / methanol mixed solution to obtain the cyanin-like fluorescent probe N-3-I.

[0032]

[0033] S4. Perform 1H NMR spectroscopy analysis on the cyanine-like fluorescent probe N-3-I, as shown in the attached spectrum. Figure 1 As shown.

[0034] This synthesis method can also be adjusted according to actual operating conditions, such as stirring S1 in an argon atmosphere for 3h or 5h, and refluxing S2 in an oil bath at 50℃ for 3h or in an oil bath at 70℃ for 1h.

[0035] Example 2 This embodiment utilizes the cyanin-like fluorescent probe from Example 1 to detect the sulfite content in actual samples of licorice root, angelica dahurica, and American ginseng, including the following steps: S1. Select the Chinese medicinal herbs Angelica dahurica, Glycyrrhiza uralensis and Panax quinquefolius as samples. Add water to the samples and place them at room temperature for 1 hour before taking the filtrate. S2. Under excitation light of 414 nm, the fluorescence spectra of different sample filtrates with different volumes (0-30 μL) added to the cyanin fluorescent probe N-3-I (10 μM) were measured, the maximum fluorescence intensity at the peak was recorded, and a linear relationship graph was plotted based on the obtained data.

[0036] As attached Figure 2 As shown in B-4B, the concentration of sulfite in the sample increases with the increase of sample volume, and the binding of the N-3-I probe with sulfite leads to an increase in the fluorescence value at 488 nm.

[0037] In summary, the binding of the N-3-I probe to sulfite can be used to determine whether the sulfite content in Chinese herbal medicine samples exceeds the standard. Therefore, the fluorescent probe N-3-I can be used to evaluate sulfite in Chinese herbal medicine samples.

[0038] Performance testing 1. Fluorescence spectroscopy test Fluorescence spectra of the cyanin-like fluorescent probe N-3-I described in Example 1 and 100 μM Na2SO3 were tested in solvents with different polarities and dielectric constants.

[0039] The cyanin-like probe N-3-I described in Example 1 was dissolved in dimethyl sulfoxide and placed in various solvents including petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, acetonitrile, ethyl acetate, ethanol, and methanol. The fluorescence spectrum after the reaction with 100 μM Na2SO3 was measured, and a bar graph based on the highest fluorescence value at 488 nm was plotted. The fluorescence spectrometer conditions were: λex = 414 nm / λem = 488 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 1, Bdw = 4.

[0040] As attached Figure 5 As shown, the cyanin-like fluorescent probe N-3-I exhibits the largest emission peak at 488 nm in the spectrum. Among them, the fluorescence emission peak of probe N-3-I at 488 nm is the highest in PBS solution. At the same time, the degree of change of probe N-3-I in PBS solution is obvious after the addition of 100 μM Na2SO3. Therefore, based on the results of fluorescence spectroscopy, the detection effect of probe N-3-I on Na2SO3 in PBS solution is better than that in other solvents.

[0041] 2. Ultraviolet absorption spectroscopy test Example 1: Ultraviolet absorption spectra of the cyanin-like fluorescent probe N-3-I under different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5.

[0042] S1. Dissolve the cyanin-like fluorescent probe N-3-I described in Example 1 in dimethyl sulfoxide, and dilute it with PBS buffer solution to prepare a 10 μM probe buffer solution; S2. Take several portions of the above probe buffer solution and add Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 solutions respectively to make their concentrations 0-4 μM, 0-7 μM, 0-4 μM, and 0-6 μM. Then, measure the ultraviolet absorption spectra of the cyanine fluorescent probe N-3-I after reacting with amines of different concentrations.

[0043] As attached Figure 6 A - Figure 9 As shown in Figure A, in the ultraviolet spectrum, the cyanin-like fluorescent probe N-3-I exhibits the largest absorption value at 520 nm. After reacting with Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 respectively, the absorption peak of the probe at 520 nm gradually decreases with the increase of the concentration of sulfite substances. At the same time, the color of the solution changes from the original dark pink to colorless, indicating that probe N-3-I can achieve "naked-eye" detection of sulfite in a relatively intuitive way.

[0044] 3. Fluorescence spectroscopy test Fluorescence spectra of the cyanin-like fluorescent probe N-3-I described in Example 1 under different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5: S1. Dissolve the cyanin-like fluorescent probe N-3-I described in Example 1 in dimethyl sulfoxide, and dilute it with PBS buffer solution to prepare a 10 μM probe buffer solution; S2. Take several portions of the above probe buffer solution and add Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 solutions respectively to make their concentrations 0-4 μM, 0-8 μM, 0-3 μM, and 0-4 μM. Under the condition of excitation light of 414 nm, measure the fluorescence spectra of the cyanin-like fluorescent probe N-3-I after reacting with different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5. The fluorescence spectrometer conditions are: λex = 414 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 1, Bdw = 4.

[0045] like Figure 6 B- Figure 9 As shown in Figure B, the behavior of the cyanin-like fluorescent probe N-3-I in the fluorescence spectrum is consistent with that in the ultraviolet spectrum, exhibiting similar sensitivity and selectivity. When the excitation light is 414 nm, the fluorescence of probe N-3-I at 488 nm gradually increases with the addition of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5. from Figure 6 C- Figure 9As can be seen from C, the fluorescence intensity of the cyanin-like fluorescent probe N-3-I has a good linear relationship with the concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 (the linear correlation coefficients are R1, R2, and R3, respectively). 2 =0.9931, R 2 =0.9997, R 2 =0.9964, R 2 =0.9952), the surface probe N-3-I can perform quantitative detection of sulfites. Using the formula, the detection limits for Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 can be calculated to be 1.182 nM, 1.046 nM μM, 0.422 nM, and 0.495 nM, respectively.

[0046] from Figure 10 It can be seen that the cyanin-like fluorescent probe N-3-I exhibits good detection performance for these four sulfites. Specifically, probe N-3-I shows a stronger and more significant fluorescence change for tetravalent sulfur sulfites, with an approximately 100-fold increase in fluorescence intensity for Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5. However, the increase in fluorescence intensity for other valence sulfides is only slight. This indicates that the selectivity of probe N-3-I for sulfites is related to the valence of sulfur in the sulfite. 4. Spiked recovery test Using the cyanine fluorescent probe N-3-I described in Example 1, the actual sample was spiked with sulfur dioxide for recovery testing.

[0047] S1. Cut the samples of licorice, angelica and American ginseng into small pieces and soak them at room temperature to obtain the filtrate. Under excitation light of 414 nm, add different volumes (0-30 μL) of the supernatant of different Chinese herbal medicine samples to the N-3-I buffer solution (10 μM) of the cyanin fluorescent probe and measure the changes before and after reacting at room temperature for 2-3 min. S2. Obtain the fluorescence value from the fluorescence spectrum in S1, substitute it into different standard curves to calculate the SO2 content, and then calculate the spiked recovery rate according to the spiked recovery formula as follows: P (recovery rate) = (spiked sample amount - measured sample amount) / spiked amount; The calculation results are shown in Table 1: Table 1. Spiked recovery results of probe N-3-I with different samples

[0048] As shown in Table 1, the average recovery rate of the cyanobacterial fluorescent probe N-3-I for spiked Chinese medicinal materials was 89%-103%, all within the good range. The relative standard deviation (RSD) was relatively low, and the repeatability was good, indicating that the cyanobacterial fluorescent probe N-3-I has a high-quality detection capability in actual sample detection.

[0049] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A cyanin-like fluorescent probe, characterized in that, The cyanin-like fluorescent probe is labeled N-3-I, and its solution is dark pink. Its molecular formula is C1. 22 H 17 ClN₂O₄, chemical structural formula is shown in Figure I: （Ⅰ）。 2. A method for synthesizing the cyanin-like fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Cesium carbonate and 1,8-naphtholactam were dissolved in acetonitrile, and iodomethane was added dropwise. The mixture was stirred at room temperature for 3-5 hours under an argon atmosphere. The solvent was removed by rotary evaporation, and the resulting bright yellow powder was purified to obtain compound 1, whose structural formula is shown in II. （Ⅱ）； (2) The compound 1 obtained in step 1 was dissolved in anhydrous tetrahydrofuran at 0°C. Methyl magnesium chloride was added dropwise while stirring. Then, the mixture was refluxed in an oil bath at 50-70°C for 1-3 hours under an argon atmosphere. After the reaction was completed, the mixture was placed in an ice bath until the temperature dropped to 0°C. A small amount of water was added to quench the reaction. Then, perchloric acid was added to the mixture to ensure complete reaction. A yellow-green solution was obtained and added to ice water. The solution was then vacuum filtered and dried to obtain a green powder, which is compound 2. Its structural formula is shown in III. （Ⅲ）； (3) Compounds 2 and 5-indolecarboxaldehyde were mixed in a round-bottom flask, and a mixed solution of ethanol and acetonitrile was added. The mixture was allowed to react overnight at room temperature in the dark. After the reaction was completed, the black precipitate was filtered out, and the reaction mixture was purified by silica gel column chromatography to obtain the cyanin-like fluorescent probe N-3-I, the structural formula of which is shown in IV: (IV); The molar ratio of 1,8-naphtholactam, cesium carbonate, and iodomethane in step (1) is 1:1.5:1.2; the separation and purification in step (1) is carried out by column chromatography, wherein the eluent is a mixed solution of petroleum ether / ethyl acetate with a volume ratio of 23:

1. In step (2), the molar ratio of compound 1 to methyl magnesium chloride is 1:4.5; In step (3), the molar ratio of compound 2 to 5-indolecarboxaldehyde is 1:1; In step (3), the volume ratio of ethanol to acetonitrile in the ethanol-acetonitrile mixed solution is 9:

1. The separation and purification in step (3) is carried out by column chromatography, wherein the eluent is a dichloromethane / methanol mixture with a volume ratio of 10:

1.

3. The application of the cyanin-like fluorescent probe according to claim 1 in the detection of sulfite content in traditional Chinese medicine materials.

4. The application of the cyanin-like fluorescent probe according to claim 3, characterized in that, When sulfites are present in the test samples of traditional Chinese medicine materials, the cyanin-like fluorescent probe can respond rapidly under very mild conditions, causing the solution to change significantly from its original deep pink color to colorless.

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