A fluorescent probe for detecting bisulfite ions and its preparation method and application

Abstract

The present invention discloses a fluorescent probe for detecting bisulfite ions, a preparation method thereof, and an application thereof, and relates to the field of analytical detection technology. The probe of the present invention utilizes levulinate as a recognition group, which can specifically undergo a nucleophilic addition reaction with sulfite ions, so that the probe can accurately and efficiently identify sulfite ions. The probe of the present invention is simple to synthesize, easy to prepare, and has high detection sensitivity. The probe of the present invention has low toxicity and good biocompatibility. The fluorescent probe of the present invention can achieve sensitive detection of sulfite ions and can be used for sulfite ion detection in food and the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a fluorescent probe for detecting bisulfite ions, and a preparation method and application thereof. Background Art

[0002] Sulfite is a major source of environmental pollution, and abnormal levels in the body are associated with a variety of diseases. Sulfite intake is strictly regulated. Therefore, there is an urgent need to develop rapid, accurate, sensitive, and non-invasive methods to monitor sulfite levels in physiological and pathological events, which is also of great significance for improving clinical diagnosis.

[0003] In this context, many analytical techniques have been developed over the past few decades for sulfite. Conventional methods for the detection of sulfite include electrochemical analysis, chromatography, flow injection analysis, chemiluminescence analysis, capillary electrophoresis and spectroscopy. These methods usually require complex procedures and expensive instrumentation, and cannot accurately assess the fluctuations of sulfite in biological systems. Among them, fluorescence and phosphorescence spectroscopy have attracted widespread attention in recent years due to their high sensitivity, high specificity, simplicity, fast response and high efficiency. Sulfite itself is non-fluorescent, and sulfite-responsive fluorescent probes are key tools for the detection of these species. Using responsive probes, sulfite probes cause changes in the emission signal of sulfite that can be recorded. Detection To date, hundreds of responsive probes, including molecular probes and nanoprobes, have been reported for sulfite, and most of these probes are used to analyze the levels of sulfite in biological systems.

[0004] In recent years, more and more high-efficiency and well-applicationed organic fluorescent materials have attracted widespread attention. However, with in-depth research, the characteristics of traditional organic fluorescent materials, such as poor biocompatibility and high cytotoxicity, have gradually attracted attention. Therefore, researchers have recently begun to focus on the design and synthesis of new environmentally friendly and low-toxic organic fluorescent materials. Isocarboxylic acid is an important terpene compound derived from turpentine. It has the characteristics of low cytotoxicity, high reactivity, good biocompatibility, excellent cell permeability and environmental protection. It is an ideal choice for the synthesis of new organic fluorescent materials. Professor Wang's research team's exploration of using isocarboxylic acid as a skeleton has attracted more and more attention. They found that isocarboxylic acid derivative probes can not only effectively make up for the defects of traditional organic small molecule probes (such as high toxicity, poor solubility, poor cell penetrability, etc.), but also have good cell penetrability and can be used for detection and imaging of living cells, thereby expanding the application potential of isocarboxylic acid in the biological field.

[0005] Another example is the isolongifolia alkanoyl-2-aminopyrimidine-based boron trifluoride fluorescent probe disclosed in the patent application with publication number CN109761917A, as well as its synthesis method and application. The probe can selectively react with boron trifluoride to produce obvious fluorescence changes under 365nm ultraviolet light irradiation. Another example is the isolongifolia alkanoyl-hexahydroquinazoline-2-amine Schiff base-based zinc ion fluorescent probe disclosed in the patent application with publication number CN106632084A. The prepared compound can only form a complex with zinc ions. The complex emits green fluorescence under 365nm ultraviolet light irradiation and can be used as a fluorescent probe for detecting zinc ion concentration.

[0006] However, there are no reports on the detection of sulfite ions. Summary of the Invention

[0007] In order to address the above shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a fluorescent probe that has an intramolecular charge transfer effect and uses levulinate as a recognition group to detect sulfite ions.

[0008] Another object of the present invention is to provide an application of the above fluorescent probe in the detection of sulfite ions.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A fluorescent probe for detecting bisulfite ions, having a structural formula as shown in Formula I:

[0011]

[0012] The present invention also provides a method for preparing the fluorescent probe, comprising the following steps:

[0013] (1) Vanillin undergoes a deprotonation reaction in the presence of a strong base, and then undergoes a substitution reaction with bromomethyl methyl ether to obtain the reactant raw material;

[0014] (2) reacting isolongiol with the reactant raw material obtained in step (1) under alkaline conditions to obtain product 1;

[0015] (3) reacting product 1 with 3-aminopyrazole under alkaline conditions to obtain product 2;

[0016] (4) reacting product 2 with hydrochloric acid to obtain product 3;

[0017] (5) The product 3 is reacted with levulinic acid to obtain the final product IFA-1, which is the isoflavone-based fluorescent probe.

[0018] In some embodiments of the present invention, in step (1), a strong base is added to vanillin under ice bath conditions to carry out a deprotonation reaction. After the reaction, bromomethyl methyl ether is added, the ice bath is removed, and the reaction is carried out at room temperature for 3 hours.

[0019] The strong base is sodium hydride,

[0020] The molar ratio of the vanillin, the strong base and the bromomethyl methyl ether is 1:1.2-3:1.2-3.

[0021] In the embodiment of the present invention, the molar ratio of vanillin, strong base and bromomethyl methyl ether is 1:1.2:1.2 as an example.

[0022] In some embodiments of the present invention, in step (2), the base used in the alkaline condition is potassium hydroxide.

[0023] The reaction conditions are: reflux reaction at 85-90°C for 4-8h to obtain product 1.

[0024] The molar ratio of the product 1, isolongifolia alkone and potassium tert-butoxide is 1:1.2-3:1.5-3.

[0025] In the embodiment of the present invention, the molar ratio of product 1, isolongiol and potassium tert-butoxide is 1:1.2:1.5 as an example.

[0026] In some embodiments of the present invention, in step (3), the base used in the alkaline condition is potassium tert-butoxide.

[0027] The reaction conditions are: reflux reaction at 85-90°C for 20-30h to obtain product 2.

[0028] The molar ratio of the product 2,3-aminopyrazole to potassium tert-butoxide is 1:1.5-3:1.5-3.

[0029] In the embodiment of the present invention, the molar ratio of the product 2, 3-aminopyrazole and potassium tert-butoxide is 1:1.5:1.5 as an example.

[0030] In some embodiments of the present invention, in step (4), the concentration of the hydrochloric acid is 1 mol / L.

[0031] The reaction conditions are: reflux reaction at 62-65°C for 2-6 hours to obtain product 3.

[0032] In some embodiments of the present invention, in step (5), in the presence of a coupling reagent and a catalyst, the product 3 is reacted with levulinic acid for 24 hours to obtain the final product IFA-1;

[0033] The coupling reagent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride,

[0034] The catalyst is p-dimethylaminopyridine,

[0035] The molar ratio of the product 3, coupling reagent, catalyst and levulinic acid is 1:1.2-3:0.5-3:1.5-3 to obtain the final product IFA-1.

[0036] In the embodiment of the present invention, the molar ratio of product 3, coupling reagent, catalyst and levulinic acid is selected as 1:1.2:0.5:1.5 as an example.

[0037] In the embodiment of the present invention, the molar ratio selected as an example is the optimal ratio. The same effect is achieved at other ratios, that is, the final compound (fluorescent probe) obtained can be used to specifically detect sulfite ions.

[0038] The specific steps are:

[0039] 1) Vanillin was dissolved in tetrahydrofuran, and 1.2 molar equivalents of sodium hydride were added in an ice bath. After reacting for several hours, 1.2 molar equivalents of bromomethyl methylimidazole were slowly added. The ice bath was removed, and the reaction was continued at room temperature for 3 hours to obtain the reactant raw material;

[0040] 2) The reactant of step (1) was dissolved in ethanol, 1.5 times the molar equivalent of potassium tert-butoxide and 1.2 times the molar equivalent of isolongifolia alkane were added, and the mixture was refluxed for 6 hours, and the product 1 was obtained by separation and purification;

[0041] 3) dissolving the product 1 of step (2) in ethanol, adding 1.5 times the molar equivalent of potassium tert-butoxide and 1.5 times the molar equivalent of 3-aminopyrazole, and refluxing for 30 hours, separating and purifying to obtain the product 2;

[0042] 4) dissolving the product 2 of step (3) in methanol, adding an appropriate amount of hydrochloric acid and refluxing for 3 hours, and then separating and purifying to obtain the product 3;

[0043] The product 3 of step (4) was dissolved in dichloromethane, and after adding 1.2 times the molar equivalent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 times the molar equivalent of p-dimethylaminopyridine, 1.5 times the molar equivalent of levulinic acid was added, and the reaction was carried out at room temperature for 18 hours to obtain a crude product. The separation and purification steps were as follows: after the reaction, extraction with water and dichloromethane was completed, the organic phase was taken, dried, and filtered; the organic solvent was removed by rotary evaporation, and the obtained solid was purified by silica gel chromatography to obtain the final product IFA-1.

[0044] The fluorescent probe compound obtained by the present invention has a molecular formula of C 31 H39 N₃O₄ has a relative molecular mass of 515.27. It is a white solid powder that is insoluble in water but soluble in organic solvents such as dichloromethane, tetrahydrofuran, and dimethyl sulfoxide. The compound has excellent light and chemical stability and is non-toxic.

[0045] Under neutral conditions, sulfite ions react with the carbonyl group of the levulinate in the fluorescent probe to undergo a nucleophilic addition reaction. Further, the generated hydroxyl group undergoes an intramolecular cyclization reaction and is finally removed to form the fluorescent compound IFA-OH. The fluorescent compound IFA-OH after the response increases the electron energy on the fluorescent group due to intramolecular charge transfer. Under 304nm excitation light irradiation, the fluorescence intensity at 485nm increases with the concentration of sulfite. Therefore, the fluorescent probe of the present invention can be used to specifically detect sulfite ions.

[0046] The present invention also provides application of the fluorescent probe in detecting sulfite ions.

[0047] The present invention also provides application of the fluorescent probe in preparing a kit for detecting sulfite ions.

[0048] Compared with the prior art, the outstanding advantages of the probe provided by the present invention include:

[0049] (1) The probe of the present invention utilizes levulinate as a recognition group, which can specifically undergo a nucleophilic addition reaction with sulfite ions. Therefore, the probe can accurately and efficiently recognize sulfite ions.

[0050] (2) The probe of the present invention is simple to synthesize, easy to prepare, and has high detection sensitivity.

[0051] (3) The probe of the present invention has low toxicity and good biocompatibility.

[0052] (4) The fluorescent probe of the present invention can achieve sensitive detection of sulfite ions and can be used for sulfite ion detection in food and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The synthetic route of the fluorescent probe of the present invention is as follows;

[0054] Figure 2Figure 2 is a fluorescence spectrum of the fluorescent compound IFA-1 in different concentrations of sulfite (DMSO:H2O=1:9); wherein, a is the fluorescence spectrum of the fluorescent probe IFA-1 (10 μM) in response to different concentrations of sodium sulfite (0-600 μM), and b is the fluorescence relationship diagram of the fluorescent probe IFA-1 (10 μM) in response to different concentrations of sodium sulfite (0-600 μM). The solvent is PBS buffer (10% DMSO), and the excitation wavelength is 304 nm.

[0055] Figure 3 The graph shows the relationship between the ratio of the fluorescence intensity of the fluorescent probe of the fluorescent compound IFA-1 (10 μM) at 485 nm and different concentrations (0-7 μM) of sulfite ions, the solvent is PBS buffer (10% DMSO), and the excitation wavelength is 304 nm;

[0056] Figure 4 Figure 2 is a graph showing the fluorescence intensity changes of the fluorescent compound IFA-1 at different times after the addition of excess sulfite (DMSO:H2O=1:9); wherein, a is a fluorescence spectrum of the fluorescent probe IFA-1 (10 μM) and sodium sulfite (0-600 μM) after different response times (0-60 min), and b is a fluorescence relationship graph of the fluorescent probe IFA-1 (10 μM) and sodium sulfite (0-600 μM) after different response times (0-60 min). The solvent was PBS buffer (10% DMSO), and the excitation wavelength was 304 nm.

[0057] Figure 5 This is a bar graph of the selectivity test of the fluorescent probe IFA-1 (10 μM), i.e., the ratio of the fluorescence intensity at 485 nm between the probe response and various potential interferents. The yellow bar represents no sodium sulfite added, and the green bar represents the addition of sodium sulfite (600 μM). The solvent is PBS buffer (containing 10% DMSO), and the excitation light wavelength is 307 nm. DETAILED DESCRIPTION

[0058] The specific implementation of the present invention is further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0059] Example 1

[0060] The synthetic route of the fluorescent probe of the present invention, i.e. the fluorescent compound, is as follows Figure 1 shown.

[0061] Dissolve vanillin (456 mg) in tetrahydrofuran, add sodium hydride (90 mg) under ice-water bath, then add bromomethyl methyl ether (412 mg), stir at room temperature for 3 h, and extract with ethyl acetate and water (3:1) three times. Combine the organic phases and spin dry to obtain the raw material without purification.

[0062] The raw material (332 mg) was dissolved in anhydrous ethanol (10 mL). Potassium hydroxide (130 mg) and isolongifolia alkalone (440 mg) were added to the mixture. The mixture was stirred at 85°C under reflux for 6 h. After vacuum distillation, the organic phases were extracted three times with ethyl acetate and water (3:1). The combined organic phases were then vacuum distilled to obtain a crude product. The crude product was further purified by column chromatography (silica gel, EA:PE = 1:10) to obtain compound 1 (955.8 mg). The product was a yellow oil with a yield of 90%.

[0063] The product was characterized by hydrogen nuclear magnetic resonance spectroscopy, and the results were: 1 H NMR (400MHz, Chloroform-d) δ7.47 (d, J=3.1Hz, 1H), 7.18 (d, J=8.4Hz, 1H), 7.10 (d, J=8.5, Hz, 1H ), 7.03 (d, J = 2.0Hz, 1H), 5.27 (s, 2H), 3.91 (s, 3H), 3.52 (s, 3H), 2.84 (d, J = 16.9Hz, 1H), 2.63 (d, J =16.9Hz, 1H), 1.98(d, J=2.1Hz, 1H), 1.91-1.73(m, 3H), 1.66(d, J=12.0Hz, 1H), 1.52(d, J=12.5Hz, 1H), 1.31(d, J=9.8Hz, 1H), 1.24(s, 3H), 1.15-1.08(m, 1H), 1.07(s, 3H), 0.87(d, J=6.1Hz, 6H). The synthesized product can be determined to be the final product by nuclear magnetic analysis.

[0064] Example 2

[0065] Compound 1 (354 mg) and 3-aminopyrazole (83 mg) were dissolved in ethanol (10 mL), and potassium tert-butoxide (188 mg) was added, and the mixture was stirred at reflux at 88°C for 28 h. After rotary evaporation, the organic phases were combined and extracted three times with ethyl acetate and water (3:1). The organic phase was dried and the crude product was further purified by column chromatography (silica gel, EA:PE=1:8) to obtain compound 2 (86.2 mg), a yellow oil, with a yield of 60%.

[0066] The product was characterized by hydrogen nuclear magnetic resonance spectroscopy, and the results were: 1H NMR (400MHz, Chloroform-d) δ7.98 (d, J=2.3Hz, 1H), 7.34 (d, J=8.4Hz, 1H), 7.02 (d, J=7.1Hz, 2H) , 6.57 (d, J = 2.3Hz, 1H), 5.38-5.28 (m, 2H), 3.91 (s, 3H), 3.57 (s, 3H), 2.72 (d, J = 16.1Hz, 1H), 2.66 -2.61 (m, 1H), 2.22 (d, J = 16.1Hz, 1H), 2.04-1.91 (m, 1H), 1.79 (d, J = 2.3Hz, 2H), 1.71 (d, J = 3.9Hz, 1H), 1.58 (d, J=12.5Hz, 1H), 1.47 (s, 3H), 1.31-1.24 (m, 2H), 1.00 (s, 3H), 0.75 (d, J=11.2Hz, 6H).

[0067] Example 3

[0068] Compound 2 (86.2 mg) was dissolved in methanol, and 3 drops of dilute hydrochloric acid (3%) were added to bring the concentration to 1 mol / L. The mixture was then refluxed and stirred at 65°C for 3 h. After vacuum distillation, the mixture was extracted three times with ethyl acetate and water (3:1). The combined organic phases were vacuum distilled to obtain a crude product, which was further purified by recrystallization to obtain compound 3 (72 mg). A white powder was obtained in a 90% yield.

[0069] The product was characterized by hydrogen nuclear magnetic resonance spectroscopy, and the hydrogen nuclear magnetic resonance spectrum results were: 1 H NMR (400MHz, Chloroform-d) δ7.98 (d, J=2.3Hz, 1H), 7.06 (d, J=8.1Hz, 1H), 7.03-6.95 (m, 2H), 6 .57 (d, J=2.4Hz, 1H), 3.92 (s, 3H), 2.72 (d, J=16.1Hz, 1H), 2.62 (d, J=1.8Hz, 1H), 2.22 (d, J=16. 1Hz, 1H), 2.02-1.92 (m, 1H), 1.79 (d, J=2.0Hz, 2H), 1.70 (d, J=12.0Hz, 1H), 1.57 (d, J=12.6Hz, 1 H), 1.46 (s, 3H), 1.33-1.27 (m, 1H), 1.20 (d, J=10.7Hz, 1H), 1.00 (s, 3H), 0.74 (d, J=11.1Hz, 6H).

[0070] Example 4

[0071] Compound 3 (72 mg) and levulinic acid (29 mg) were dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (38 mg) and p-dimethylaminopyridine (DMAP) (20 mg) were added. The mixture was stirred at room temperature for 24 hours. After vacuum distillation, the mixture was extracted three times with ethyl acetate and water (3:1). The combined organic phases were then vacuum distilled to obtain a crude product. The crude product was further purified by column chromatography (silica gel, MeOH:DCM = 1:10) to obtain the final product, compound IFA-1 (70 mg). It was obtained as a white powder in an 80% yield.

[0072] The product was characterized by nuclear magnetic resonance hydrogen spectrum and high-performance mass spectrometry. The results of the nuclear magnetic resonance hydrogen spectrum were as follows: 1 H NMR (400MHz, Chloroform-d) δ7.23 (d, J=8.0Hz, 1H), 7.12-7.01 (m, 2H), 6.35 (s, 1H), 3.85 (s , 3H), 2.96-2.87 (m, 4H), 2.68 (d, J=16.1Hz, 1H), 2.59 (s, 1H), 2.42 (s, 3H), 2.25 (s, 3H), 2.14 (d, J=16.0Hz, 1H), 1.95 (d, J=10.4Hz, 1H), 1.80 (s, 1H), 1.76-1.64 (m, 2H), 1.56 (d, J=12.6H z, 1H), 1.44 (s, 3H), 1.27 (d, J = 9.8Hz, 1H), 1.19 (s, 1H), 0.98 (s, 3H), 0.72 (d, J = 12.6Hz, 6H). 13 C NMR (101MHz, Chloroform-d) δ206.22, 170.85, 161.35, 154.23, 151.26, 148.40, 142.62, 140.81, 129.11, 123.19, 122.46, 114.38, 114.02, 94.38 ,58.45,56.16,55.44,48.05,45.51,39.34,37.99,37.40,32.55,30.51 ,29.91,28.33,27.91,25.91,25.31,24.78,23.01,14.78.ESI-HRMS: m / z calcd.for[C 32 H 39 N3NaO4] + :552.2841, found552.2838([M+Na] + ).

[0073] Example 5

[0074] Spectral test of the fluorescent probe compound obtained in the present invention in detecting sulfite ions:

[0075] 5.2 mg of solid IFA-1 was dissolved in 10 mL of dimethyl sulfoxide to prepare a fluorescent probe stock solution with a concentration of 1 mM. Before testing, the fluorescent probe was diluted with distilled water to a concentration of 10 μM. The total test volume was 1 mL (containing 10% dimethyl sulfoxide).

[0076] 1. Fluorescence detection of sulfite ions at different concentrations by IFA-1 fluorescent probe

[0077] The prepared IFA-1 fluorescent probe dispersion was used to detect SO3 2- 5.2 mg of solid IFA-1 was dissolved in 10 mL of dimethyl sulfoxide to prepare a mother solution with a concentration of 1 mM. SO3 2- The solution was prepared by dissolving solid sodium sulfite in pure water to a 10 mM stock solution. During testing, the fluorescent probe was diluted with water to a final concentration of 10 μM, SO3 2- The solution was diluted to a final concentration of 0μM, 1μM, 3μM, 5μM, 7μM, 10μM, 15μM, 20μM, 30μM, 60μM, 100μM, 200μM, and 400μM. The total volume of the test system was 1mL (containing 10% dimethyl sulfoxide), the test temperature was 25°C, the excitation wavelength was 310nm, and the measured fluorescence spectrum was shown in the figure below. Figure 2 shown.

[0078] With SO3 2- As the concentration of α-aminobutyric acid increases, the fluorescence intensity at 482 nm gradually increases. Figure 2 The figure is a relationship diagram between the ratio of the fluorescence intensity of the fluorescent probe of Example IFA-1 at 482nm and different concentrations of sulfite ions. Figure 3 It can be seen that there is a good linear relationship between the fluorescence intensity ratio and the sulfite ion concentration.

[0079] 2. Fluorescence detection of IFA-1 fluorescent probe after adding excess sulfite ions for different time periods

[0080] The prepared IFA-1 fluorescent probe dispersion was used to detect SO3 2- 5.2 mg of solid IFA-1 was dissolved in 10 mL of dimethyl sulfoxide to prepare a mother solution with a concentration of 1 mM. The response time was 0-40 min, the total volume of the test system was 1 mL (containing 10% dimethyl sulfoxide), the test temperature was 25 ° C, the excitation wavelength was 310 nm, and the test results were as follows: Figure 4 It can be observed that as the response time increases, the fluorescence intensity of the emission peak at 482 nm increases.

[0081] 3. Selective testing of IFA-1 fluorescent probe for different potential interferents:

[0082] 5.04 mg of solid IFA-1 was dissolved in 10 mL of dimethyl sulfoxide to prepare a stock solution with a concentration of 1 mM. The test system included a fluorescent probe with a final concentration of 10 μM and potential interferents with different final concentrations: Cl - 、F - Br - NH 4+ 、NO 2- 、SO4 2- :3mM; Cys, GSH: 2mM; H2O2: 200μM; SO3 2- : 600μM. (Using excess sulfite) The total amount of the test system is 1mL, the temperature is 25℃, the excitation wavelength is 310nm, and the fluorescence ratio bar graph is as follows Figure 5 As shown by Figure 5 It can be seen that the fluorescent probe of the present invention has good selectivity.

[0083] The above examples are preferred embodiments of the present invention, but the embodiments of the invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting bisulfite ions, characterized in that: The structural formula is as shown in Formula Ⅰ: Formula I.

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Vanillin undergoes a deprotonation reaction in the presence of a strong base, and then undergoes a substitution reaction with bromomethyl methyl ether to obtain the reactant raw material; (2) reacting isolongifolia ketone with the reactant raw material obtained in step (1) under alkaline conditions to obtain product 1; (3) Product 1 is reacted with 3-aminopyrazole under alkaline conditions to obtain product 2; (4) reacting product 2 with hydrochloric acid to obtain product 3; (5) reacting the product 3 with levulinic acid to obtain the final product IFA-1, i.e., the fluorescent probe according to claim 1; The reaction route is as follows: 。 3. The preparation method according to claim 2, characterized in that In step (1), vanillin is deprotonated by adding a strong base under ice bath conditions. After the reaction, bromomethyl methyl ether is added, the ice bath is removed, and the reaction is carried out at room temperature for 3 hours. The strong base is sodium hydride, The molar ratio of the vanillin, the strong base and the bromomethyl methyl ether is 1:1.2-3:1.2-3.

4. The preparation method according to claim 2, characterized in that In step (2), the base used in the alkaline condition is potassium hydroxide, The reaction conditions are: reflux reaction at 85-90°C for 4-8h to obtain product 1. The molar ratio of the reactant raw material obtained in step (1), isolongifolia alkone and potassium hydroxide is 1:1.2-3:1.5-3.

5. The preparation method according to claim 2, characterized in that In step (3), the base used in the alkaline condition is potassium tert-butoxide, The reaction conditions are: reflux reaction at 85-90°C for 20-30h to obtain product 2. The molar ratio of the product 1,3-aminopyrazole and potassium tert-butoxide is 1:1.5-3:1.5-3.

6. The preparation method according to claim 2, characterized in that In step (4), the concentration of hydrochloric acid is 1 mol / L, The reaction conditions are: reflux reaction at 62-65°C for 2-6 hours to obtain product 3.

7. The preparation method according to claim 2, characterized in that In step (5), in the presence of a coupling reagent and a catalyst, the product 3 reacts with levulinic acid for 22-24 hours to obtain the final product IFA-1; The coupling reagent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, The catalyst is p-dimethylaminopyridine, The molar ratio of the product 3, coupling reagent, catalyst and levulinic acid is 1:1.2-3:0.5-3:1.5-3 to obtain the final product IFA-1.

8. Use of the fluorescent probe according to claim 1 in detecting sulfite ions, wherein the use is not for the purpose of disease diagnosis or treatment.

9. Use of the fluorescent probe according to claim 1 in preparing a kit for detecting sulfite ions.

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