Fluorescent probe based on isosteviol as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to an isosteviol-based fluorescent probe as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving isosteviol and diethylene glycol bis (p-toluenesulfonate) into a solvent, and under the action of potassium carbonate, enabling carboxyl in the isosteviol to perform nucleophilic substitution on sulfonate groups in the diethylene glycol bis (p-toluenesulfonate) to obtain an intermediate; and dissolving the intermediate and hydroxyquinoline in a solvent, and replacing a sulfonate group in the intermediate with hydroxyl in hydroxyquinoline under an alkaline condition to obtain the fluorescent probe. The fluorescent probe can selectively recognize maleic acid, and the preparation process is simple and effective.

Description

Technical Field

[0001] The invention belongs to the technical field of maleic acid detection, and particularly relates to a fluorescent probe based on isosteviol and a preparation method and application thereof. Background Art

[0002] Fumaric acid is a commonly used food additive, and its derivatives can be used to treat multiple sclerosis, rheumatism, psoriasis, etc. Maleic acid is an inhibitor of the activity of sucrose cytosine hydrogenase and Krebs cycle glutathione coenzyme, and is often used in food and beverages as a new acidifier. However, maleic acid is more toxic than fumaric acid, and excessive accumulation of maleic acid in the human body may cause Fanconi syndrome, neurological diseases, cardiovascular diseases, and cancer. Since maleic acid can improve the glossiness of flour products such as starch and increase viscosity and elasticity, it is used by merchants to add maleic acid starch ester to edible starch. However, due to technical limitations, there are a lot of residues of maleic acid as a raw material, which makes edible starch a huge food safety hazard.

[0003] At present, the main methods for detecting maleic acid include high performance liquid chromatography, gas chromatography-mass spectrometry, ion mobility spectrometry, etc. These methods rely on expensive instruments and equipment, are costly, and have complex operation processes, making them difficult to be widely used. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a fluorescent probe based on isosteviol and a preparation method and application thereof.

[0005] The first object of the present invention is to provide a method for preparing a fluorescent probe based on isosteviol, comprising the following steps:

[0006] Step 1: dissolving isosteviol and diethylene glycol bis-p-toluenesulfonate in a solvent, and under the action of potassium carbonate, allowing the carboxyl group in isosteviol to nucleophilically replace the sulfonate group in diethylene glycol bis-p-toluenesulfonate to obtain an intermediate.

[0007] It should be noted that the natural sweetener steviol has many characteristics such as low price, easy availability, stability, etc., and often has multiple chiral sites; hydrolysis and rearrangement under acidic conditions produce isosteviol, which has multiple chiral centers and a special rigid skeleton and groove structure, making the isosteviol unit molecule an ideal skeleton for constructing molecular recognition fluorescent probes.

[0008] The invention uses steviol as a reaction raw material, dissolves steviol in a 10% sulfuric acid solution, performs a hydrolysis reaction at 70-80°C, performs suction filtration, and performs low-temperature recrystallization to form isosteviol. Then, isosteviol and diethylene glycol di-p-toluenesulfonate are dissolved in a solvent, and under the action of potassium carbonate, the oxygen atom of the carboxyl group in isosteviol acts as a nucleophilic reagent to attack the carbon atom of diethylene glycol di-p-toluenesulfonate; the carbon atom is connected to the sulfonate group to form a transition state; in the transition state, the sulfonate radical leaves as a good leaving group, undergoes a nucleophilic substitution reaction, generates a compound containing a tosyl group, and obtains a multifunctional ethoxyethyl intermediate. Finally, potassium carbonate and other solid residues are removed by filtration, and a crude product is obtained by concentrating the solution, and then purified by column chromatography to obtain a pure intermediate.

[0009] Preferably, the molar ratio of isosteviol to diethylene glycol di-p-toluenesulfonate is 3:4-5.

[0010] It should also be noted that potassium carbonate is used as a weak alkaline reagent to deprotonate the hydroxyl group of isosteviol to generate oxygen anions with strong nucleophilicity and improve its nucleophilicity. At the same time, potassium carbonate can promote the hydroxyl group of steviol to participate in the substitution reaction to generate an intermediate containing a tosyl group. The present invention uses a large amount of potassium carbonate and a non-polar solvent to promote the reaction equilibrium to move toward the direction of generating products. Preferably, the molar ratio of isosteviol to potassium carbonate is 3:4-5.

[0011] Preferably, the reaction temperature for preparing the intermediate is 60° C. to 70° C., and the reaction time is 12 h to 18 h.

[0012] Preferably, the solvent for preparing the intermediate is dichloromethane or acetonitrile.

[0013] Preferably, the diethylene glycol bis-p-toluenesulfonate is prepared by the following steps: dissolving diethylene glycol and p-toluenesulfonyl chloride in dichloromethane, adding a base in an ice-water bath, and stirring the reaction for 2h to 4h to form diethylene glycol bis-p-toluenesulfonate.

[0014] Preferably, the isosteviol is prepared by the following steps: using stevioside as a raw material, dissolving stevioside in a sulfuric acid solution, and performing a hydrolysis reaction at 70° C. to 80° C. to form isosteviol.

[0015] Step 2: dissolving the intermediate and hydroxyquinoline in a solvent, and under alkaline conditions, replacing the sulfonate group in the intermediate with the hydroxyl group in the hydroxyquinoline to obtain a fluorescent probe.

[0016] It should be noted that the present invention dissolves the intermediate and hydroxyquinoline in a solvent. Under alkaline conditions, the hydroxyl group in hydroxyquinoline is deprotonated to form a strongly nucleophilic alcohol anion; the alcohol anion attacks the positive center of the carbon atom in the intermediate, undergoes a nucleophilic substitution reaction, and removes the sulfonate group to form an ether bond. After the reaction is completed, potassium hydroxide is removed by filtration, the solution is concentrated to obtain a crude product, and then purified by column chromatography to obtain a pure final product. Preferably, the molar ratio of the intermediate to hydroxyquinoline is 2 to 3:3.

[0017] Preferably, the hydroxyquinoline is 8-hydroxyquinoline or 5-hydroxyquinoline.

[0018] The present invention adds an alkaline agent, firstly, to deprotonate the hydroxyl group of hydroxyquinoline to generate a strongly nucleophilic alcohol anion, and secondly, to promote the alcohol anion to participate in the substitution reaction to generate the target product. Preferably, the alkaline agent is potassium hydroxide, and the molar ratio of the intermediate to potassium hydroxide is 2 to 3:5.

[0019] Preferably, the reaction temperature for preparing the fluorescent probe is 70° C. to 80° C., and the reaction time is 24 h to 36 h.

[0020] Preferably, the solvent for preparing the fluorescent probe is dichloromethane or acetonitrile.

[0021] The third object of the present invention is to provide the use of the above fluorescent probe in detecting maleic acid.

[0022] Preferably, the method for detecting maleic acid is:

[0023] Dissolving the fluorescent probe in a solvent to obtain a fluorescent probe solution;

[0024] Adding the fluorescent probe solution to the solution to be tested, mixing evenly, to obtain a test solution;

[0025] The excitation wavelength was 325 nm and the fluorescence intensity change of the test solution at 350-450 nm was measured; the volume ratio of the fluorescent probe solution to the test solution was 1:1; the concentration of the fluorescent probe solution was 1×10 -5 mol / L, the concentration of the test solution is 5×10 -4 mol / L.

[0026] Alternatively, under an ultraviolet lamp, observe the fluorescence change of the test solution. When the test solution emits fluorescence, it indicates that the test solution contains maleic acid.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses the carboxyl group in isosteviol as a nucleophilic reagent, and reacts with the sulfonate group in diethylene glycol di-p-toluenesulfonate under the catalysis of potassium carbonate to undergo a nucleophilic substitution reaction; thereby, the natural properties of isosteviol are introduced into the fluorescent probe, and the biocompatibility and sensitivity of the fluorescent probe are enhanced. Hydroxyquinoline is used as a fluorescent group, and the hydroxyl group in hydroxyquinoline nucleophilically attacks the sulfonate group in the intermediate under alkaline conditions to generate a fluorescent probe. The hydroxyl group of hydroxyquinoline in the fluorescent probe can effectively absorb excitation light of a specific wavelength to generate fluorescence emission. In the presence of maleic acid, C=O and quinoline-N in the fluorescent probe form a stronger hydrogen bond with the carboxyl group of maleic acid, and the carboxyl group of maleic acid is closer to the quinoline group. The electron-withdrawing effect of the carboxyl group causes the electron cloud density of the proton of the quinoline group to decrease, thereby causing its chemical shift to shift to the low field. The C=O of the fluorescent probe forms a hydrogen bond with the carboxyl group of maleic acid, which corresponds to the fluorescence quenching caused by the photoelectron transfer process. As the concentration of maleic acid increases, the π-π accumulation between the fluorophore quinoline is enhanced, resulting in an exciton effect, thereby causing the appearance and continuous enhancement of the emission peak associated with the exciton, corresponding to the appearance and enhancement of the new fluorescence emission peak; thus, it becomes a fluorescent indicator signal for detecting maleic acid. The fluorescent probe prepared by the present invention has a fluorescent effect and a strong selective recognition effect on maleic acid.

[0029] The fluorescent probe prepared by the present invention can detect maleic acid by fluorescence changes under ultraviolet light. When the fluorescent probe is added to the solution to be tested, when the test solution emits fluorescence, it indicates that the test solution contains maleic acid. At the same time, the fluorescent probe prepared by the present invention has two emission wavelengths. After adding maleic acid, the fluorescence in the wavelength range of 350nm to 400nm decreases, and the fluorescence intensity in the wavelength range of 400nm to 450nm increases. The present invention realizes a more sensitive detection of maleic acid concentration through the dual signals of the change in fluorescence intensity and the change in the fluorescence color of the solution; and the fluorescent probe of the present invention can detect maleic acid in starch-rich foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The fluorescence spectrum changes of the fluorescent probe 1 prepared in Example 1 of the present invention under different concentrations of maleic acid.

[0031] Figure 2 The fluorescence spectrum changes of the fluorescent probe 1 prepared in Example 1 of the present invention under different concentrations of fumaric acid.

[0032] Figure 3 The fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 of the present invention under different concentrations of maleic acid.

[0033] Figure 4 The fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 of the present invention under different concentrations of fumaric acid.

[0034] Figure 5 The fluorescent probe 1 prepared in Example 1 of the present invention and the photo of the fluorescence color change under ultraviolet light after maleic acid and fumaric acid were added to the fluorescent probe 1; wherein a is the fluorescent probe 1, b is the addition of maleic acid, and c is the addition of fumaric acid.

[0035] Figure 6 The fluorescent probe 2 prepared in Example 2 of the present invention and the photo of the fluorescence color change under ultraviolet light after maleic acid and fumaric acid were added to the fluorescent probe 2; wherein a is the fluorescent probe 2, b is the addition of maleic acid, and c is the addition of fumaric acid.

[0036] Figure 7 The nuclear magnetic hydrogen spectra of the fluorescent probe 1, the fluorescent probe 1-maleic acid complex and the fluorescent probe 1-fumaric acid complex prepared in Example 1 of the present invention; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex.

[0037] Figure 8 The nuclear magnetic hydrogen spectra of the fluorescent probe 2, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex prepared in Example 2 of the present invention; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex.

[0038] Fig. 9 The three-dimensional structures of the fluorescent probe 1, the fluorescent probe 1-maleic acid complex and the fluorescent probe 1-fumaric acid complex prepared in Example 1 obtained by B3LYP / 6-31G (d, p) calculation of the present invention; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex.

[0039] Fig.10 The three-dimensional structure diagram of the fluorescent probe 2, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex prepared in Example 2 obtained by B3LYP / 6-31G (d, p) calculation of the present invention; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex.

[0040] Fig.11 The fluorescent probe 1 prepared in Example 1 of the present invention is used to determine maleic acid in starchy food; wherein a is potato, b is sweet potato, and c is corn.

[0041] Fig.12 The fluorescent probe 2 prepared in Example 2 of the present invention is used to measure maleic acid in starchy food; wherein a is potato, b is sweet potato, and c is corn. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0043] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0044] Example 1

[0045] This embodiment provides a method for preparing a fluorescent probe based on isosteviol.

[0046] Step 1: Preparation of intermediates:

[0047] 1.1) Dissolve 5 g of stevioside in 250 mL of 10% sulfuric acid solution and react at 75°C for 7 h to generate an off-white solid. After filtration, recrystallize in low-temperature ethanol to obtain light yellow isosteviol crystals with a yield of 67%. The reaction route is shown in Reaction Formula 1:

[0048]

[0049] The H-NMR spectrum data of isosteviol are as follows: 1 HNMR (500MHz, Chloroform-d) δ2.64 (dd, J=18.6, 3.8Hz, 1H), 2.17 (dtd, J=13.5, 3.4, 1.5Hz, 1H), 1.93-1.33 (m, 13H), 1.25 (s, 3H, 1.23-1.13 (m, 3H), 1.03 (td, J=13.6, 4.2Hz, 1H), 0.98 (s, 3H), 0.92 (td, J=13.2, 4.4Hz, 1H), 0.79 (s, 3H), indicating that the present invention successfully synthesized isosteviol.

[0050] 1.2) 2.653 g of diethylene glycol and 9.533 g of p-toluenesulfonyl chloride were dissolved in 25 mL of dichloromethane, and 11.22 g of potassium hydroxide was added in portions under ice-water bath conditions, and then magnetic stirring was performed for 3 h. Insoluble matter was removed by suction filtration, and the resulting solution was concentrated and recrystallized with methanol to obtain colorless diethylene glycol bis-p-toluenesulfonate crystals with a yield of 73%. The reaction route is shown in Reaction Formula 2:

[0051]

[0052] The H NMR spectrum data of diethylene glycol bis(p-toluenesulfonate) are as follows: 1HNMR (400MHz, chloroform-d) δ7.79 (d, J=8.4Hz, 4H), 7.37 (d, J=8.1Hz, 4H), 4.23-4.02 (m, 4H), 3.73-3.58 (m, 4H), 2.46 (s, 6H). This indicates that the present invention successfully synthesized diethylene glycol bis-p-toluenesulfonate.

[0053] 1.3) 0.955 g of isosteviol and 1.865 g of diethylene glycol bis-p-toluenesulfonate were dissolved in 50 mL of acetonitrile; 0.622 g of potassium carbonate was added and reacted at 70° C. for 12 h; the mixture was filtered and the resulting solution was concentrated to obtain a crude product, which was purified by column chromatography with a volume ratio of petroleum ether to ethyl acetate of 3:1 to obtain 2-(2-(benzenesulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecane-6a,9-methylcyclo[a]naphthalene-4-carboxylate, recorded as an intermediate, with a yield of 89%. The reaction route is shown in Reaction Formula 3:

[0054]

[0055] The H NMR data of 2-(2-(benzenesulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecanoyl-6a,9-methylcyclo[a]naphthalene-4-carboxylate are as follows: 1H NMR (400 MHZ, chloroform-d) δ7.91-7.72 (m, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.28-4.04 (m, 4H), 3.81-3.55 (m, 4H), 2.60 (dd, J = 18.6, 3.7 Hz, 1H), 2.46 (s, 3H), 2.30-2.08 (m, 1H), 1.94-1.84 (m, 1H), 1.82 (s, 1H), 1.81-1.72 (m, 2H), 1.69 (h, J = 4.0, 3.2 Hz, 3H), 1.65 (t, J = 3.4 Hz, 1H), 1.62-1.45 (m, 3H ),1.45-1.32(m,3H),1.32-1.22(m,2H),1.20(s,3H),1.14(dd,J=12.1,2.3z,1H),1.04(dd,J=13.5,4.2Hz,1H),0.99(s,2H),0.91(td,J=13.2,4.2Hz,1H),0.69(s,3H), indicating that the present invention successfully synthesized 2-(2-(benzenesulfonyloxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecane-6a,9-methylcyclo[a]naphthalene-4-carboxylate.

[0056] Step 2: Preparation of fluorescent probes:

[0057] 1.4 g of the intermediate and 0.435 g of 8-hydroxyquinoline were dissolved in 30 mL of acetonitrile, and then 0.28 g of potassium hydroxide was added to obtain a mixture; the mixture was then refluxed and heated at 80° C. for 24 h. After the reaction was completed, the precipitate was filtered out to obtain a brown-black oily liquid, which was then concentrated and purified by column chromatography. The solvent system was a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate was 2:1 to obtain 2-(2-(quinoline-8-oxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecane-6a,9-methylcyclo[a]naphthalene-4-carboxylate with a yield of 55%, i.e., fluorescent probe 1. The reaction route is shown in Reaction Formula 4:

[0058]

[0059] The H NMR data of 2-(2-(quinolin-8-oxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecanoyl-6a,9-methylcyclo[a]naphthalene-4-carboxylate are as follows: 1H NMR (400MHZ, chloroform-d) δ8.93 (dd, J=4.2, 1.8Hz, 1H), 8.12 (dd, J=8.3, 1.8Hz, 1H), 7.51-7.37 (m, 3H ),7.13(dd,J=7.6,1.3z,1H),4.42(t,J=5.3Hz,2H),4.30-4.14(m,2H),4.05(dd,J=5.8,4.7Hz, 2H),3.83(ddd,J=5.7,4.0,1.7Hz,2H),2.58(dd,J=18.6,3.7Hz,1H),2.23-2.10(m,1H),1.92- 1.30(m,13H),1.26-1.05(m,6H),1.04-0.92(m,4H),0.85(td,J=13.3,4.4Hz,1H),0.67(s,3H).

[0060] The NMR carbon spectrum data are as follows: 13 C NMR (101MHz, CDCl3) δ222.20,177.20,154.67,149.29,140.42,135.87,129.55,12 6.68,121.57,120.01,109.36,77.34,77.03,76.71,69.40,69.29,68.38,63.14,57 .17,54.73,54.31,48.65,48.40,43.83,41.49,39.79,39.42,38.00,37.91,37.32,28.89,21.61,20.29,19.85,18.91,13.25, indicating that the present invention successfully synthesized the 2-(2-(quinoline-8-oxy)ethoxy)ethyl intermediate.

[0061] Example 2

[0062] This embodiment provides a method for preparing a fluorescent probe based on isosteviol.

[0063] 1.4 g of the intermediate and 0.435 g of 5-hydroxyquinoline were dissolved in 30 mL of acetonitrile, and then 0.691 g of potassium carbonate was added to obtain a mixture; the mixture was then refluxed at 80° C. for 24 h. After the reaction was completed, the precipitate was filtered out to obtain a brown-black oily liquid, which was then concentrated and purified by column chromatography. The solvent system was a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate was 2:1 to obtain 2-(2-(isoquinoline-5-oxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecane-6a,9-methylcyclo[a]naphthalene-4-carboxylate with a yield of 47%, i.e., fluorescent probe 2.

[0064] The H NMR spectrum data of 2-(2-(isoquinolin-5-oxy)ethoxy)ethyl (4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecyl-6a,9-methylcyclo[a]naphthalene-4-carboxylate are as follows: 1 H NMR (400 MHz, chloroform-d) δ 9.21 (d, J = 1.0 Hz, 1H), 8.53 (d, J = 5.8 Hz, 1H), 8.15-7.95 (m, 1H), 7.64-7.40 (m, 2H), 7.03 (dd, J = 7.4, 1.2 Hz, 1H), 4.39-4.16 (m, 4H), 4.05-3.95 (m, 2H), 3.84 (ddd, J = 5. .5,4.0,1.3Hz,2H),2.59(dd,J=18.5,3.7Hz,1H),2.24-2.14(m,1H),1.90-1.30(m,13H),1. 19(s,3H),1.17-1.06(m,3H),1.06-0.94(m,4H),0.86(td,J=13.3,4.4Hz,1H),0.70(s,3H).

[0065] The NMR carbon spectrum data are as follows: 13C NMR (101MHz, CDCl3) δ222.22,178.02,153.60,151.81,144.25,130.29,128.53,127.38,118.73,114. 22,108.73,77.34,77.02,76.70,69.47,69.41,68.12,63.08,57.14,54.67,54.27,48.66,48.40,43.8 6, 41.47, 39.75, 39.41, 38.01, 37.89, 37.30, 28.90, 21.61, 20.28, 19.83, 18.91, 13.27. This indicates that the present invention successfully synthesized 2-(2-(isoquinoline-5-oxy)ethoxy)ethyl (4R, 4aS, 6aR, 9S, 11aR, 11bS)-4,9,11b-trimethyl-8-oxotetrahydrotetradecane-6a,9-methylcyclo[a]naphthalene-4-carboxylate.

[0066] Experimental testing

[0067] 1. Fluorescence test

[0068] The fluorescent probe 1 prepared in Example 1 and the fluorescent probe 2 prepared in Example 2 were dissolved in ethanol to a concentration of 1×10 -5 mol / L fluorescent probe 1 solution and fluorescent probe 2 solution; and measure two 1.5mL portions of fluorescent probe 1 solution and two 1.5mL portions of fluorescent probe 2 solution; then add gradient concentrations of fumaric acid and maleic acid solutions to the fluorescent probe 1 solution and the fluorescent probe 2 solution for titration; the gradient concentrations are 0.01mM, 0.05mM, 0.1mM, 0.2mM, 0.5Mm, 0.8mM, 1mM, 2mM, 3mM, 4mM and 5mM, and the corresponding fluorescence intensity is tested each time. The fluorescence during titration is measured in a standard rectangular quartz cuvette using a Horiba Fluoro Max-4 fluorescence spectrophotometer, and the corresponding emission values ​​during the entire titration process are recorded.

[0069] Figure 1 The fluorescence spectrum changes of the fluorescent probe 1 prepared in Example 1 under different concentrations of maleic acid. Figure 1 As shown, when excited at 325 nm, the fluorescent probe 1 exhibits fluorescence emission at a wavelength of 400 nm. As the concentration of the maleic acid solution increases from 0.01 mM to 5 mM, the fluorescence intensity at 400 nm gradually decreases, and at the same time, a new emission peak appears at a wavelength of 488 nm, and its fluorescence intensity gradually increases, forming a typical ratio fluorescence signal.

[0070] Figure 2The fluorescence spectrum changes of the fluorescent probe 1 prepared in Example 1 under different concentrations of fumaric acid. Figure 2 As shown, when fumaric acid was added to the fluorescent probe 1 solution, no significant fluorescence change was observed with the increase in the concentration of the fumaric acid solution, and the Stokes shift of the fluorescent probe 1 was 88 nanometers.

[0071] Figure 3 The fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 under different concentrations of maleic acid. Figure 3 As shown, fluorescent probe 2 also exhibits a maleic acid response similar to fluorescent probe 1. When excited at 325nm, fluorescent probe 2 exhibits fluorescence emission at a wavelength of 364nm; as the concentration of maleic acid solution increases from 0.01mM to 5mM, the fluorescence intensity at 364nm gradually decreases, and a new emission peak appears at a wavelength of 442nm, and its fluorescence intensity gradually increases, forming a typical ratio fluorescence signal.

[0072] Figure 4 The fluorescence spectrum changes of the fluorescent probe 2 prepared in Example 2 under different concentrations of fumaric acid. Figure 4 As shown, there is no significant fluorescence change after adding fumaric acid to the solution of fluorescent probe 2. The Stokes shift of fluorescent probe 2 is 78 nm.

[0073] Figure 5 The fluorescent probe 1 prepared in Example 1 and the photos taken under ultraviolet light after maleic acid and fumaric acid were added to the fluorescent probe 1, respectively; wherein a is the fluorescent probe 1, b is the addition of maleic acid, and c is the addition of fumaric acid. Figure 5 It can be seen that both the fluorescent probe 1 and the fluorescent probe with fumaric acid added have no fluorescence changes, and only the fluorescent probe 1 with maleic acid added shows green fluorescence under ultraviolet light.

[0074] Figure 6 The following are photos of the fluorescent probe 2 prepared in Example 2 and the fluorescent probe 2 after maleic acid and fumaric acid were added to the fluorescent probe 2 under ultraviolet light; wherein a is the fluorescent probe 2, b is the addition of maleic acid, and c is the addition of fumaric acid. Figure 6 It can be seen that both the fluorescent probe 2 and the fluorescent probe with fumaric acid added have no fluorescence changes, and only the fluorescent probe 2 with maleic acid added shows light blue fluorescence under ultraviolet light.

[0075] This indicates that the fluorescent probe prepared by the present invention has a fluorescent effect and a selective recognition effect on maleic acid.

[0076] 2. Nuclear magnetic resonance hydrogen spectrum test

[0077] In order to further explore the exact mechanism of the fluorescence spectrum change of the fluorescent probe 1 prepared in Example 1 and the fluorescent probe 2 prepared in Example 2 in maleic acid solution, the present invention respectively dissolves Example 1 and Example 2 in deuterated DMSO as blank solutions; mixes them with maleic acid and fumaric acid in a molar ratio of 1:1 and then dissolves them in deuterated DMSO to obtain four test solutions, recorded as fluorescent probe 1-maleic acid complex, fluorescent probe 1-fumaric acid complex, fluorescent probe 2-maleic acid complex and fluorescent probe 2-fumaric acid complex, and performs nuclear magnetic resonance testing. The results are as follows Figure 7 and Figure 8 shown.

[0078] Figure 7 The H NMR spectra of the fluorescent probe 1, the fluorescent probe 1-maleic acid complex and the fluorescent probe 1-fumaric acid complex prepared in Example 1; wherein a is the fluorescent probe 1, b is the fluorescent probe 1-maleic acid complex, and c is the fluorescent probe 1-fumaric acid complex. Figure 7 It can be seen that compared with the H NMR spectrum of fluorescent probe 1, the protons of the quinoline group in fluorescent probe 1 after complexing with maleic acid show downfield shifts due to its deshielding effect, specifically ΔδH1=0.1ppm, ΔδH2=0.25ppm, ΔδH3=0.17ppm, ΔδH4=0.12ppm, ΔδH5=0.12ppm, ΔδH6=0.15ppm. In the H NMR spectrum of the fluorescent probe 1-fumaric acid complex, the chemical shift has almost no change.

[0079] Figure 8 The NMR hydrogen spectra of the fluorescent probe 2, the fluorescent probe 2-maleic acid complex and the fluorescent probe 2-fumaric acid complex prepared in Example 2; wherein a is the fluorescent probe 2, b is the fluorescent probe 2-maleic acid complex, and c is the fluorescent probe 2-fumaric acid complex. Figure 8 As shown, the isoquinoline protons of fluorescent probe 2 were downfield shifted after mixing with maleic acid, and the juice was (ΔδH1=0.21ppm, ΔδH2=0.06ppm, ΔδH3=0.19ppm, ΔδH4=0.14ppm, ΔδH5=0.13ppm, ΔδH6=0.15ppm; while the fluorescent probe 2-fumaric acid complex did not change significantly.

[0080] Therefore, the change in downfield shift may be due to the fact that fluorescent probe 1 and fluorescent probe 2 form a complex with maleic acid through intermolecular hydrogen bonds, which affects the chemical environment of the fluorophore protons and shifts their signals to the downfield. This indicates that the fluorescent probe prepared by the present invention can form a complex with maleic acid, thereby affecting the chemical environment of the fluorophore protons and having a fluorescent effect and selective recognition effect on maleic acid.

[0081] 3. Density functional theory research

[0082] The present invention uses Gaussian16 software, utilizes density functional theory method, uses IEFPCM solvent model and GD3BJ dispersion correction at B3LYP / 6-31G (d, p) level to calculate the three-dimensional structure diagram of fluorescent probe 1-maleic acid complex, fluorescent probe 1-fumaric acid complex, fluorescent probe 2-maleic acid complex and fluorescent probe 2-fumaric acid complex.

[0083] Fig. 9 The three-dimensional structures of fluorescent probe 1, fluorescent probe 1-maleic acid complex and fluorescent probe 1-fumaric acid complex calculated by B3LYP / 6-31G (d, p); wherein a is fluorescent probe 1, b is fluorescent probe 1-maleic acid complex, and c is fluorescent probe 1-fumaric acid complex. Fig. 9 As shown, fluorescent probe 1 forms intermolecular hydrogen bonds in maleic acid solution, as shown by the red arrows. Compared with the three-dimensional structure diagram of fluorescent probe 1 in fumaric acid solution, C=O and quinoline-N in fluorescent probe 1 form stronger hydrogen bonds with the carboxyl group of maleic acid, and the carboxyl group of maleic acid is closer to the quinoline group. The electron-withdrawing effect of the carboxyl group causes the electron cloud density of the proton of the quinoline group to decrease, thereby shifting its chemical shift to the low field. C=O of fluorescent probe 1 forms a hydrogen bond with the carboxyl group of maleic acid, which corresponds to the fluorescence quenching caused by the photoelectron transfer process. With the increase of maleic acid concentration, the π-π accumulation between the fluorophore quinoline is enhanced, resulting in an exciton effect, thereby appearing and continuously enhancing the emission peak associated with the exciton, corresponding to the appearance and enhancement of the new fluorescence emission peak. This shows that the fluorescent probe prepared by the present invention has a fluorescence effect and a strong selective recognition effect on maleic acid.

[0084] Fig.10 The three-dimensional structures of fluorescent probe 2, fluorescent probe 2-maleic acid complex and fluorescent probe 2-fumaric acid complex calculated by B3LYP / 6-31G (d, p); wherein a is fluorescent probe 2, b is fluorescent probe 2-maleic acid complex, and c is fluorescent probe 2-fumaric acid complex. Fig.10 As shown, the formation of intermolecular hydrogen bonds in the fluorescent probe 2-maleic acid complex is indicated by the red arrows. Similarly, the complexation of fluorescent probe 2 with maleic acid causes the protons of the isoquinoline to shift downfield, and the fluorescence intensity changes proportionally.

[0085] 4. Detection of maleic acid in starch samples

[0086] The present invention extends this detection system to the detection of maleic acid in starch samples. The present invention chops up 10g of starch-rich food, wherein the starch-rich food is potato, sweet potato or corn; and puts it in a 500mL beaker, and then adds 200mL of ethanol to obtain a mixture; stirs the mixture for 12h, and then filters the mixture and collects the filtrate. Measure 30mL of the filtrate and divide it into three parts, one as a blank control, and add 1.75mg of maleic acid and fumaric acid to the other two parts to obtain three test solutions; respectively dissolve the fluorescent probe 1 prepared in Example 1 and the fluorescent probe 2 prepared in Example 2 in ethanol to prepare a concentration of 1×10 -5 mol / L fluorescent probe 1 solution and fluorescent probe 2 solution; then 1.5mL of fluorescent probe 1 solution and fluorescent probe 2 solution were added to the three test solutions respectively to obtain test solutions, and the fluorescence spectra of these six test solutions were measured.

[0087] Fig.11 The fluorescent probe 1 prepared in Example 1 is used to determine maleic acid in starchy food; wherein a is potato, b is sweet potato, and c is corn. Fig.11 As shown, compared with the fluorescence spectrum of the fluorescent probe 1 mixed with starch food, no significant fluorescence change was observed after the fluorescent probe 1 was mixed with starch food added with fumaric acid; after the fluorescent probe 1 was mixed with starch food added with maleic acid, the fluorescence intensity in the wavelength range of 400nm to 420nm was weakened.

[0088] Fig.12 The fluorescent probe 2 prepared in Example 2 is used to determine maleic acid in starchy food; wherein a is potato, b is sweet potato, and c is corn. Fig.12 As shown, compared with the fluorescence spectrum of fluorescent probe 1 mixed with starch food, no significant fluorescence change was observed after fluorescent probe 2 was mixed with starch food added with fumaric acid; after fluorescent probe 2 was mixed with starch food added with maleic acid, the fluorescence intensity at 364nm weakened, and at the same time, a new emission peak appeared at a wavelength of 442nm, and its fluorescence intensity increased.

[0089] This indicates that the fluorescent probe of the present invention can perform qualitative detection of maleic acid in starchy foods such as potatoes, sweet potatoes and corn.

[0090] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic creative concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0091] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a fluorescent probe based on isosteviol, characterized in that: The following steps are involved: Dissolving isosteviol and diethylene glycol bis-p-toluenesulfonate in a solvent, and under the action of potassium carbonate, allowing the carboxyl group in isosteviol to nucleophilically replace the sulfonate group in the diethylene glycol bis-p-toluenesulfonate to obtain an intermediate; The intermediate and hydroxyquinoline are dissolved in a solvent, and under alkaline conditions, the hydroxyl group in the hydroxyquinoline replaces the sulfonate group in the intermediate to obtain a fluorescent probe.

2. The method for preparing a fluorescent probe according to claim 1, characterized in that: The molar ratio of the intermediate to hydroxyquinoline is 2-3:

3.

3. The method for preparing a fluorescent probe according to claim 1, characterized in that: The hydroxyquinoline is 8-hydroxyquinoline or 5-hydroxyquinoline.

4. The method for preparing a fluorescent probe according to claim 1, characterized in that: The molar ratio of isosteviol to diethylene glycol di-p-toluenesulfonate is 3:4-5; The molar ratio of isosteviol to potassium carbonate is 3:4-5.

5. The method for preparing a fluorescent probe according to claim 1, characterized in that: The reaction temperature for preparing the fluorescent probe is 70° C. to 80° C., and the reaction time is 24 h to 36 h.

6. The method for preparing a fluorescent probe according to claim 1, characterized in that: The reaction temperature for preparing the intermediate is 60° C. to 70° C., and the reaction time is 12 h to 18 h.

7. The method for preparing a fluorescent probe according to claim 1, characterized in that: The alkaline condition is achieved by adding an alkaline reagent, and the alkaline reagent is potassium hydroxide; The molar ratio of the intermediate to potassium hydroxide is 2-3:

5.

8. The method for preparing a fluorescent probe according to claim 1, characterized in that: The solvents for preparing the fluorescent probe and the intermediate are both dichloromethane or acetonitrile.

9. A fluorescent probe prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the fluorescent probe according to claim 9 in detecting maleic acid.

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