Water-soluble aie compounds, methods of making and using the same

Abstract

The present application relates to the technical field of fluorescent chemical sensor, and especially relates to a water-soluble AIE compound, a preparation method and application thereof.The water-soluble AIE compound has the general structure as shown in the following formula: wherein R is at least one of and.The compound has obvious AIE characteristics, and can be used for detection of butyrylcholinesterase; and the compound obtained by compounding the butyrylcholinesterase can also be used for detection of organic phosphorus compounds.

Description

Technical Field

[0001] This invention relates to the field of fluorescent chemical sensor technology, and in particular to a water-soluble AIE compound, its preparation method, and its application. Background Technology

[0002] Organic light-emitting materials (OLEDs) are widely used in fields such as light-emitting diodes (LEDs), organic light-emitting semiconductors (OLEDs), fluorescent probes, and anti-counterfeiting due to their ease of synthesis and modification. The fluorescence quenching phenomenon in traditional fluorescent materials during aggregation has limited their applications. The discovery of aggregation-induced emission (AIE) has solved the problem of aggregation-induced quenching (ACQ) in traditional fluorescent materials, thus expanding the application range of OLEDs.

[0003] Alternating current emission (AIE) materials, with their excellent biocompatibility, superior optical properties, and ease of synthesis, have gradually become a research focus in the fields of biodetection and imaging. Among them, hydrophilic AIE probes exhibit lower cytotoxicity. In aqueous solution, hydrophilic AIE molecules consume a large amount of excited-state energy through intramolecular motion, resulting in almost no luminescence. However, when in contact with biological systems, whether the single-molecule motion of AIE molecules is restricted in specific biological environments or they aggregate within the biological system, their fluorescence is significantly enhanced.

[0004] Butyrylcholinesterase (BC2) is a non-specific esterase synthesized in the liver. Changes in BC2 levels and activity are associated with the development of various diseases (such as malignant tumors and Alzheimer's disease). Therefore, accurate detection of BC2 levels and activity is crucial for the early diagnosis, disease progression monitoring, and treatment of these diseases. Furthermore, as a cholinesterase, BC2 can specifically bind to organophosphate compounds to form phosphorylated cholinesterase, thus inhibiting BC2 activity. Based on this characteristic, the detection system for BC2 can be further expanded to include the detection of organophosphate compounds.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide water-soluble AIE compounds, their preparation methods, and applications. The compounds of this invention have obvious AIE characteristics and can be used for the detection of butyrylcholinesterase. The complex obtained by combining butyrylcholinesterase can also be used for the detection of organophosphorus compounds.

[0007] To achieve the above-mentioned objectives of the present invention, a first aspect of the present invention provides a water-soluble AIE compound having the following general structural formula: ; Where R is , and At least one of them.

[0008] In a specific embodiment of the present invention, the water-soluble AIE compound comprises at least one of the following structures: (I) (II) (III).

[0009] The second aspect of this invention provides a method for preparing the water-soluble AIE compound provided in the first aspect of this invention, comprising the following steps: (a) Methyl 4-aminobenzoate or 4-aminobenzoic acid reacts with aldehydes in a first solvent; then 2,3-butanedione, an organic acid and a catalyst are added to carry out a second reaction to obtain an intermediate product; (b) The intermediate product is refluxed in a second solvent containing sodium hydroxide to obtain the water-soluble AIE compound; The aldehyde compounds include , and At least one of them.

[0010] In a specific embodiment of the present invention, the first solvent includes at least one of benzene, toluene, and xylene.

[0011] In a specific embodiment of the present invention, the organic acid includes at least one of acetic acid, oxalic acid and propionic acid.

[0012] In a specific embodiment of the present invention, the catalyst includes at least one of p-toluenesulfonic acid and ferric perchlorate.

[0013] In a specific embodiment of the present invention, the molar ratio of methyl 4-aminobenzoate or 4-aminobenzoic acid to the aldehyde compound is (0.8~1.2):1.

[0014] In a specific embodiment of the present invention, the molar ratio of the aldehyde compound to the 2,3-butanedione is 1:(0.4~0.6).

[0015] In a specific embodiment of the present invention, the molar ratio of the aldehyde compound to the catalyst is 1:(0.05~0.15).

[0016] In a specific embodiment of the present invention, in step (a), the temperature of the first reaction is 85~95°C and the time of the first reaction is 2~4 h.

[0017] In a specific embodiment of the present invention, in step (a), the temperature of the second reaction is 55~65°C, and the time of the second reaction is 10~14 h.

[0018] In a specific embodiment of the present invention, the second solvent includes water and tetrahydrofuran.

[0019] In a specific embodiment of the present invention, the molar ratio of sodium hydroxide to the intermediate product is not less than 5:1, such as (10~100):1.

[0020] In a specific embodiment of the present invention, in step (b), the reaction time is 10-14 h.

[0021] The third aspect of the present invention provides the use of the water-soluble AIE compound provided in the first aspect of the present invention in the preparation of fluorescent probes for the detection of butyrylcholinesterase or for the detection of organophosphorus compounds.

[0022] A fourth aspect of the present invention provides a fluorescent probe for detecting butyrylcholinesterase, comprising the water-soluble AIE compound and a solvent provided in the first aspect of the present invention; said solvent comprises either water or PBS buffer.

[0023] In a specific embodiment of the present invention, the concentration of the water-soluble AIE compound in the fluorescent probe is 10. -5 ~10 -7 mol / L.

[0024] In a specific embodiment of the present invention, the fluorescent probe further includes a carrier protein; the carrier protein includes acetylcholinesterase.

[0025] In a specific embodiment of the present invention, the molar ratio of the water-soluble AIE compound to the carrier protein in the fluorescent probe is 1:1.

[0026] The fifth aspect of the present invention provides a fluorescent probe for detecting organophosphorus compounds, comprising a water-soluble AIE compound, butyrylcholinesterase, and a solvent provided in the first aspect of the present invention; said solvent comprising either water or PBS buffer.

[0027] In a specific embodiment of the present invention, the concentration of the water-soluble AIE compound in the fluorescent probe is 10. -5 ~10 -7 mol / L.

[0028] In a specific embodiment of the present invention, the molar ratio of the water-soluble AIE compound to the butyrylcholinesterase is 1:1.

[0029] In a specific embodiment of the present invention, the organophosphorus compound includes at least one of dichlorophen and methyl parathion.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention synthesizes a novel class of water-soluble AIE compounds with distinct AIE properties, suitable for the detection of butyrylcholinesterase. Furthermore, when these AIE compounds are combined with acetylcholinesterase, they enable highly specific and extremely sensitive detection of butyrylcholinesterase. The AIE compounds of this invention, when combined with butyrylcholinesterase, can also be used to detect organophosphate compounds. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 The fluorescence spectra (A) of Compound I prepared in Example 1 of this invention in THF / H2O mixed solvents with different THF contents, and the trend graph (B) showing the change of the emission wavelength to fluorescence intensity ratio (I / I0) with the THF content in the mixed solvent; the excitation wavelength was 390 nm, and the concentration of Compound I was 10. -5 mol / L; Figure 2 The fluorescence spectrum of compound I prepared in Example 1 of this invention and the fluorescence spectrum after adding butyrylcholinesterase thereto; excitation wavelength: 390 nm, concentration of compound I: 10 -6 The concentration of butyrylcholinesterase was 0.5 mg / mL. Figure 3 The relationship between the fluorescence intensity enhancement ratio ((I-I0) / I0) of compound I prepared in Example 1 of this invention in response to butyrylcholinesterase and the concentration of butyrylcholinesterase; excitation wavelength: 390 nm, concentration of compound I: 10 -6 mol / L; Figure 4 The fluorescence spectra (A) of compound II prepared in Example 2 of this invention in THF / H2O mixed solvents with different THF contents, and the trend graph (B) showing the change of the emission wavelength to fluorescence intensity ratio (I / I0) with the THF content in the mixed solvent; the excitation wavelength was 408 nm, and the concentration of compound II was 10. -5 mol / L; Figure 5 The fluorescence spectrum of compound II prepared in Example 2 of this invention and the fluorescence spectrum after adding butyrylcholinesterase thereto; excitation wavelength: 408 nm, concentration of compound II: 10-6 The concentration of butyrylcholinesterase was 0.5 mg / mL. Figure 6 The relationship between the fluorescence intensity enhancement ratio ((I-I0) / I0) of compound II prepared in Example 2 of this invention in response to butyrylcholinesterase and the concentration of butyrylcholinesterase; excitation wavelength: 408 nm, concentration of compound II: 10 - 6 mol / L; Figure 7 The fluorescence spectra (A) of compound III prepared in Example 3 of this invention in THF / H2O mixed solvents with different THF contents, and the trend graph (B) showing the change of the emission wavelength to fluorescence intensity ratio (I / I0) with the THF content in the mixed solvent; the excitation wavelength was 400 nm, and the concentration of compound III was 10. -5 mol / L; Figure 8 The fluorescence spectrum of compound III prepared in Example 3 of this invention and the fluorescence spectrum after adding butyrylcholinesterase thereto; excitation wavelength: 400 nm, concentration of compound III: 10 -6 The concentration of butyrylcholinesterase was 0.5 mg / mL. Figure 9 The relationship between the fluorescence intensity enhancement ratio ((I-I0) / I0) of compound III prepared in Example 3 of this invention in response to butyrylcholinesterase and the concentration of butyrylcholinesterase; excitation wavelength: 400 nm, concentration of compound III: 10 - 6 mol / L; Figure 10 This describes the relationship between the fluorescence intensity of the complex of compound III and butyrylcholinesterase prepared in Example 4 of this invention in response to diclofenac and the concentration of diclofenac; excitation wavelength: 400 nm, concentration of compound III: 10. -6 The concentration of butyrylcholinesterase was 10 mol / L. -6 mol / L; Figure 11 This describes the relationship between the fluorescence intensity of the complex of compound III and butyrylcholinesterase prepared in Example 4 of this invention in response to methyl parathion and the concentration of methyl parathion; excitation wavelength: 400 nm, concentration of compound III: 10. - 6 The concentration of butyrylcholinesterase was 10 mol / L. -6 mol / L; Figure 12 The binding force between compound III prepared in Example 3 and butyrylcholinesterase was calculated using molecular docking technology; Figure 13The fluorescence intensity enhancement ratio of compound III prepared in Example 3 of this invention to different proteins; excitation wavelength: 400 nm, concentration of compound III: 10 -5 mol / L, the protein concentration is 0.5 mg / mL; Figure 14 The fluorescence intensity of the complex of compound III and acetylcholinesterase prepared in Example 6 of this invention was enhanced in response to different proteins; excitation wavelength: 400 nm, concentration of compound III: 10 -5 The concentration of acetylcholinesterase is 10 mol / L. -5 mol / L, the protein concentration is 0.5 mg / mL; Figure 15 This describes the relationship between the fluorescence intensity enhancement ratio ((I-I0) / I0) of the complex of compound III and acetylcholinesterase prepared in Example 7 of this invention and the concentration of butyrylcholinesterase; excitation wavelength: 400 nm, concentration of compound III: 10 -6 The concentration of acetylcholinesterase is 10 mol / L. -6 mol / L; Figure 16 This invention demonstrates the anti-interference performance of the compound III and acetylcholinesterase complex prepared in Example 8 of this invention in detecting butyrylcholinesterase in different biological components; excitation wavelength: 400 nm; concentration of compound III in the sample to be tested: 10. -6 The concentration of acetylcholinesterase is 10 mol / L. -6 mol / L. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0034] In the description of this invention, it should be noted that the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0035] The first aspect of this invention provides a water-soluble AIE compound having the following general structural formula: ; Where R is , and At least one of them.

[0036] In a specific embodiment of the present invention, the water-soluble AIE compound comprises at least one of the following structures: (I) (II) (III).

[0037] The second aspect of this invention provides a method for preparing the water-soluble AIE compound provided in the first aspect of this invention, comprising the following steps: (a) Methyl 4-aminobenzoate or 4-aminobenzoic acid reacts with aldehydes in a first solvent; then 2,3-butanedione, an organic acid and a catalyst are added to carry out a second reaction to obtain an intermediate product; (b) The intermediate product is refluxed in a second solvent containing sodium hydroxide to obtain the water-soluble AIE compound; The aldehyde compounds include , and At least one of them.

[0038] In a specific embodiment of the present invention, the synthetic route of water-soluble AIE compounds is illustrated using methyl 4-aminobenzoate as the starting material. The synthetic route and conditions for synthesizing water-soluble AIE compounds using 4-aminobenzoic acid as the starting material are consistent.

[0039] In a specific embodiment of the present invention, the molar ratio of methyl 4-aminobenzoate or 4-aminobenzoic acid to the aldehyde compound is (0.8~1.2):1, specifically it can be a range of 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1 or any two of them, preferably 1:1.

[0040] In specific embodiments of the present invention, the first solvent includes at least one of benzene, toluene, and xylene, preferably toluene. In some embodiments, the mass of the first solvent can be 3 to 10 times the mass of the reactants, but is not limited thereto; the amount of the first solvent can be conventionally adjusted to ensure that the reactants react uniformly and stably in the reaction system.

[0041] In a specific embodiment of the present invention, in step (a), the temperature of the first reaction is 85~95°C, specifically 85°C, 88°C, 90°C, 92°C, 95°C or any combination thereof; the time of the first reaction is 2~4h, specifically 2h, 2.5h, 3h, 3.5h, 4h or any combination thereof.

[0042] In a specific embodiment of the present invention, the molar ratio of the aldehyde compound to the 2,3-butanedione is 1:(0.4~0.6), specifically it can be a range of 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6 or any two of these, preferably 1:0.5.

[0043] In a specific embodiment of the present invention, the organic acid includes at least one selected from acetic acid, oxalic acid, and propionic acid, preferably acetic acid. The organic acid provides an acidic environment for the reaction system to achieve ring closure, nucleus formation, and pyrrole formation.

[0044] In a specific embodiment of the present invention, the mass of the organic acid is 20 to 50 times the mass of the 2,3-butanedione, specifically it can be 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, or any combination thereof.

[0045] In a specific embodiment of the present invention, the catalyst includes at least one of p-toluenesulfonic acid and ferric perchlorate, preferably ferric perchlorate.

[0046] In a specific embodiment of the present invention, the molar ratio of the aldehyde compound to the catalyst is 1:(0.05~0.15), specifically it can be a range of 1:0.05, 1:0.08, 1:0.1, 1:0.12, 1:0.15 or any two of them, preferably 1:0.1.

[0047] In a specific embodiment of the present invention, in step (a), the temperature of the second reaction is 55~65°C, specifically 55°C, 58°C, 60°C, 62°C, 65°C or any combination thereof; the time of the second reaction is 10~14 h, specifically 10 h, 11 h, 12 h, 13 h, 14 h or any combination thereof.

[0048] In a specific embodiment of the present invention, step (a) may include: adding methyl 4-aminobenzoate or 4-aminobenzoic acid, aldehyde compounds, and a first solvent to a reaction vessel, heating to 85-95°C and stirring for 2-4 h; then lowering the temperature of the reaction system to 55-65°C, adding 2,3-butanedione, organic acid, and catalyst, and then stirring for 10-14 h at 55-65°C; and performing post-processing after the reaction to obtain an intermediate product.

[0049] In a specific embodiment of the present invention, the post-processing of step (a) includes: removing the solvent in the reaction system by means of rotary evaporation or the like to obtain a solid substance; adding acetonitrile at 40-50°C to dissolve the solid substance, allowing it to cool naturally to room temperature, and filtering to collect the precipitated solid substance. The steps of adding acetonitrile, cooling, and filtering can be repeated 1-3 times.

[0050] In a specific embodiment of the present invention, the second solvent comprises water and tetrahydrofuran. Further, the volume ratio of the water to the tetrahydrofuran is 1:(0.5~2), specifically it can be a range of 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2 or any two of these, preferably 1:1.

[0051] In a specific embodiment of the present invention, the mass of the second solvent can be 10 to 50 times the mass of the reactants, but is not limited thereto; the amount of the second solvent can be conventionally adjusted to ensure that the reactants react uniformly and stably in the reaction system.

[0052] In a specific embodiment of the present invention, the molar ratio of sodium hydroxide to the intermediate product is not less than 5:1, such as (10~100):1, specifically 10:1, 20:1, 30:1, 50:1, 80:1, 100:1, etc.

[0053] In a specific embodiment of the present invention, in step (b), the reaction time is 10 to 14 hours, specifically 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or any combination thereof.

[0054] In a specific embodiment of the present invention, step (b) may include: adding the intermediate product and tetrahydrofuran in the second solvent to the reaction vessel, heating and refluxing to dissolve the intermediate product; then dissolving sodium hydroxide in water in the second solvent to obtain an aqueous sodium hydroxide solution, adding the aqueous sodium hydroxide solution dropwise to the aforementioned reaction system, heating and refluxing for 10-14 h; and performing post-treatment after the reaction to obtain the water-soluble AIE compound.

[0055] In a specific embodiment of the present invention, the post-processing of step (b) includes: removing tetrahydrofuran and part of the water in the reaction system by vacuum concentration such as rotary evaporation to obtain a concentrated solution; adding the concentrated solution dropwise to a saturated sodium chloride aqueous solution to precipitate the precipitate, filtering and washing the filter cake with a saturated sodium chloride aqueous solution and tetrahydrofuran, collecting the filter cake and drying it.

[0056] The third aspect of the present invention provides the use of the water-soluble AIE compound provided in the first aspect of the present invention in the preparation of a fluorescent probe for detecting butyrylcholinesterase.

[0057] The water-soluble AIE compound of this invention is soluble in water or PBS buffer, resulting in a solution with low fluorescence intensity. Upon addition of butyrylcholinesterase to this solution, the fluorescence intensity of the system significantly increases compared to the initial value, thereby enabling the detection of butyrylcholinesterase. Furthermore, the water-soluble AIE compound provides rapid detection of butyrylcholinesterase with a low detection limit, a response time of less than 3 seconds, and a detection limit as low as 0.00068 U / mL.

[0058] A fourth aspect of the present invention provides a fluorescent probe for detecting butyrylcholinesterase, comprising the water-soluble AIE compound and a solvent provided in the first aspect of the present invention; said solvent comprises either water or PBS buffer.

[0059] In a specific embodiment of the present invention, the concentration of the water-soluble AIE compound in the fluorescent probe is 10. -5 ~10 -7 mol / L, specifically 10 -5 mol / L, 10 -6 mol / L, 2×10 -6 mol / L, 5×10 -6 mol / L, 8×10 -6 mol / L, 10 -7 The range of mol / L or any combination thereof, preferably 10 mol / L. -6 mol / L.

[0060] In a specific embodiment of the present invention, the method for detecting butyrylcholinesterase using a fluorescent probe includes: adding a solution of butyrylcholinesterase to be tested to the fluorescent probe, and detecting butyrylcholinesterase based on the fluorescence change of the fluorescent probe before and after adding the solution of butyrylcholinesterase to be tested.

[0061] In a specific embodiment of the present invention, the concentration of butyrylcholinesterase in the test butyrylcholinesterase solution is 0.1~1 mg / mL.

[0062] In a specific embodiment of the present invention, the fluorescent probe further includes a carrier protein; the carrier protein includes acetylcholinesterase.

[0063] Fluorescent probes containing carrier proteins enable the specific detection of butyrylcholinesterase. Specifically, the water-soluble AIE compound of this invention exhibits the lowest binding energy to butyrylcholinesterase, a slightly higher binding energy to acetylcholinesterase, and a higher binding energy to other common proteins found in organisms than to these two proteins (butyrylcholinesterase and acetylcholinesterase). Furthermore, the fluorescence enhancement factor when the water-soluble AIE compound binds to butyrylcholinesterase is higher than that when the water-soluble AIE compound binds to acetylcholinesterase. Therefore, this invention pre-assembles a water-soluble AIE compound with acetylcholinesterase to form a complex. When the detection system contains butyrylcholinesterase, due to the lower binding energy between the water-soluble AIE compound and butyrylcholinesterase, the water-soluble AIE compound dissociates from the acetylcholinesterase complex and forms a lower-energy complex with butyrylcholinesterase, thus achieving the detection of butyrylcholinesterase. When the detection system contains other proteins, due to the difference in binding energy, the water-soluble AIE compound cannot dissociate from the acetylcholinesterase complex and bind to other proteins, thereby avoiding interference from other proteins in the detection. Furthermore, because the water-soluble AIE compound has extremely high detection sensitivity for butyrylcholinesterase, even after preparing the complex with acetylcholinesterase, it still maintains extremely high detection sensitivity for butyrylcholinesterase, with a detection limit as low as 0.00096 U / mL.

[0064] In a specific embodiment of the present invention, the molar ratio of the water-soluble AIE compound to the carrier protein in the fluorescent probe is 1:1. Further, the concentration of both the water-soluble AIE compound and the carrier protein in the fluorescent probe is 10. -4 ~10 -6 mol / L.

[0065] In a specific embodiment of the present invention, the method for preparing a fluorescent probe containing a carrier protein includes: dissolving a water-soluble AIE compound in a solvent to obtain a first solution, dissolving the carrier protein in a solvent to obtain a second solution; adding the first solution to the second solution, stirring at room temperature for 1-3 hours, to obtain a fluorescent probe containing the carrier protein. The concentration of the water-soluble AIE compound in the first solution is 10%. -2 ~10 -5 mol / L; in the second solution, the concentration of the carrier protein is 10 mol / L. -2 ~10 -5 mol / L.

[0066] In a specific embodiment of the present invention, a fluorescent probe containing a carrier protein can be used to specifically detect butyrylcholinesterase in simulated serum, and can be used to assess the butyrylcholinesterase content in serum to reflect the health status of an organism. The simulated serum includes butyrylcholinesterase, serum albumin, γ-globulin, fibrinogen, glucose, cholesterol, and urea. The present invention is used to verify that the simulated serum used has the following mass ratio: albumin:γ-globulin:fibrinogen:glucose:urea:cholesterol = 400:300:19:6.7:1:4, the albumin concentration is 20 mg / mL, and the butyrylcholinesterase concentration is 2.5 U / mL.

[0067] In a specific embodiment of the present invention, a fluorescent probe containing a carrier protein is used in a method for detecting samples containing butyrylcholinesterase, comprising: ① Obtain a standard curve of fluorescence enhancement intensity ratio and butyrylcholinesterase concentration for the fluorescent probe containing carrier protein in response to butyrylcholinesterase using conventional methods, such as Y=133.82X+21.46, where X is the concentration of butyrylcholinesterase, ranging from 0 to 0.4 U / mL, and Y is the fluorescence enhancement intensity ratio (I-I0) / I0, ranging from 20 to 80; ② Test the initial fluorescence intensity I0 of the fluorescent probe containing the carrier protein; dilute the sample to be tested by N times to obtain a diluted sample; take a certain volume of the diluted sample and add it to the fluorescent probe containing the carrier protein, then test the fluorescence intensity I of the system and calculate (I-I0) / I0; If (I-I0) / I0 is within the range of the above standard curve, the concentration of butyrylcholinesterase in the diluted sample can be calculated according to the standard curve, and then the concentration of butyrylcholinesterase in the test sample can be calculated by combining the dilution factor N. If (I-I0) / I0 is below or above the range of the standard curve, then decrease or increase the dilution factor N until the (I-I0) / I0 of the corresponding test is within the range of the standard curve, and then calculate according to the above method.

[0068] The present invention also provides the application of the water-soluble AIE compound provided in the first aspect of the present invention in the preparation of fluorescent probes for the detection of organophosphorus compounds.

[0069] Organophosphorus compounds can bind to cholinesterase to form phosphorylated cholinesterase, thereby inhibiting cholinesterase activity. Based on this principle, the AIE compound of this invention, which can detect butyrylcholinesterase, is mixed with butyrylcholinesterase to prepare a complex. When the analyte contains an organophosphorus compound, the organophosphorus compound will destroy butyrylcholinesterase, thereby causing the luminescence of the complex to quench, thus achieving the detection of organophosphorus compounds.

[0070] The fifth aspect of the present invention provides a fluorescent probe for detecting organophosphorus compounds, comprising a water-soluble AIE compound, butyrylcholinesterase, and a solvent provided in the first aspect of the present invention; said solvent comprising either water or PBS buffer.

[0071] In a specific embodiment of the present invention, the molar ratio of the water-soluble AIE compound to the butyrylcholinesterase is 1:1. Further, in the fluorescent probe, the concentration of both the water-soluble AIE compound and the butyrylcholinesterase is 10. -5 ~10 -7 mol / L, preferably 10 -6 mol / L.

[0072] In a specific embodiment of the present invention, the preparation method of the fluorescent probe for detecting organophosphorus compounds includes: dissolving a water-soluble AIE compound in a solvent to obtain a third solution; adding the third solution to butyrylcholinesterase, stirring at room temperature for 3-5 min, and obtaining the fluorescent probe. The concentration of the water-soluble AIE compound in the third solution is (0.1-10) × 10⁻⁶. -6 mol / L.

[0073] In a specific embodiment of the present invention, the organophosphorus compound includes at least one of dichlorophen and methyl parathion.

[0074] In a specific embodiment of the present invention, the method for detecting organophosphorus compounds using a fluorescent probe includes: adding a solution of the organophosphorus compound to be tested to the fluorescent probe, and detecting the organophosphorus compound based on the fluorescence change of the fluorescent probe before and after adding the solution of the organophosphorus compound to be tested.

[0075] In a specific embodiment of the present invention, the concentration of the organophosphorus compound in the test solution is 10. -5 ~10 -7 mol / L. Further, the solvent in the test organophosphorus compound solution includes at least one of acetone, acetonitrile, and dimethyl sulfoxide.

[0076] The following is some product information used in the specific embodiments of the present invention: Butyrylcholinesterase (BuChE): Shanghai Aladdin Biochemical Technology Co., Ltd.; Acetylcholinesterase (AChE): Shanghai Aladdin Biochemical Technology Co., Ltd.; Serum albumin: Sigma-Aldrich; Trypsin: Shanghai Aladdin Biochemical Technology Co., Ltd.; Pepsin: Shanghai Aladdin Biochemical Technology Co., Ltd.; Lipase: Shanghai Aladdin Biochemical Technology Co., Ltd.; Lysozyme: Shanghai Aladdin Biochemical Technology Co., Ltd.; γ-globulin: Shanghai Aladdin Biochemical Technology Co., Ltd.; Fibrinogen: Shanghai Aladdin Biochemical Technology Co., Ltd.; PBS buffer: Cell culture grade PBS (1X), manufactured by Coring Biotechnology, pH 7.2~7.4.

[0077] Example 1 This embodiment provides a water-soluble AIE compound I, with the following structural formula: (I).

[0078] A method for preparing water-soluble AIE compound I includes the following steps: 4-Aminobenzoate (0.01 mol), 4-pyridinecarboxaldehyde (0.01 mol), and toluene (15 mL) were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 3 h. The temperature of the reaction system was then lowered to 60 °C, and 2,3-butanedione (0.005 mol), ferric perchlorate (0.001 mol), and glacial acetic acid (15 mL) were slowly added to the reaction system using a syringe. After the addition was complete, stirring was continued for 12 h. After the reaction was completed, the solvent was evaporated to obtain a solid. The solid was dissolved in acetonitrile at 40–50 °C (e.g., 45 °C), allowed to cool naturally to room temperature, and the precipitated solid was collected by filtration. The steps of adding acetonitrile, cooling, and filtration were repeated three times to obtain the intermediate product.

[0079] An intermediate product (0.001 mol) and 25 mL of tetrahydrofuran were added to a 100 mL round-bottom flask equipped with a magnetic stirrer, and the mixture was heated under reflux to dissolve the intermediate product. 2.00 g of NaOH was weighed and dissolved in 25 mL of water to obtain an aqueous NaOH solution. This NaOH solution was gradually added dropwise to the aforementioned round-bottom flask, and the mixture was heated under reflux for 12 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran and a small amount of water. The concentrated reaction solution was then added dropwise to a saturated sodium chloride solution, and the precipitated product was collected by filtration. The filter cake was washed with saturated sodium chloride solution and tetrahydrofuran, and then dried to obtain the compound shown in Formula I.

[0080] The compound of formula I prepared was characterized, and the data are as follows: 1H NMR (400 MHz, D2O, ppm): δ 8.04 – 7.98 (m, 4H), 7.67 (d, J = 8.2Hz, 4H), 6.62 (d, J = 6.5 Hz, 8H), 5.86 – 5.72 (m, 2H). 13 C NMR (101 MHz, 90% D2O + 10% THF-d8, ppm): δ 173.14, 163.75,148.52, 140.78, 140.68, 135.12, 133.77, 133.51, 130.62, 124.13, 121.75,97.59. ESI-MS (negative ion mode, m / z) calcd. for C 30 H 18 N4O4 2- [M] 2- : 249.07, found: 249.21. The characterization results above indicate that the synthesized compound has the structure shown in Formula I.

[0081] The AIE properties of compound I were verified as follows: Weigh 5.44 mg of compound I, dissolve it in an appropriate amount of water, transfer it to a 10 mL volumetric flask, and dilute to 10 mL with water to obtain a concentration of 1×10⁻⁶. -3 An aqueous solution of compound I at mol / L. Take 1 mL of 1×10⁻⁶ mol / L. -3 An aqueous solution of compound I at a concentration of mol / L was added to a 10 mL volumetric flask and diluted to 10 mL with water to obtain a solution with a concentration of 1×10⁻⁶. -4 An aqueous solution of compound I at a concentration of mol / L was prepared. Eleven sample vials were prepared and labeled 1-11. 300 μL of a 1×10⁻⁶ mol / L solution was added to each of the 1-10 sample vials. - 4 A 1×10⁻⁶ mol / L aqueous solution of compound I was added to sample vial #11. -3An aqueous solution of compound I was prepared at a concentration of 1 mol / L. Then, water was added sequentially to sample vials 1-11 at concentrations of 2700 μL, 2400 μL, 2100 μL, 1800 μL, 1500 μL, 1200 μL, 900 μL, 600 μL, 300 μL, 0 μL, and 0 μL, respectively. Finally, tetrahydrofuran (THF) was added sequentially to sample vials 1-11 at concentrations of 0 μL, 300 μL, 600 μL, 900 μL, 1200 μL, 1500 μL, 1800 μL, 2100 μL, 2400 μL, 2700 μL, and 2970 μL, respectively. This resulted in a concentration of compound I of 1 × 10⁻⁶ mol / L in each of the sample vials. -5 A mixed solution of compound I in THF / H2O with THF volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% respectively.

[0082] Fluorescence emission spectroscopy was performed on the THF / H2O mixed solutions of compound I in sample vials 1-11, with an excitation wavelength of 390 nm, and the results were as follows. Figure 1 The fluorescence spectra of compound I in THF / H2O mixed solvents with different THF contents are shown, along with a trend graph of the emission wavelength to fluorescence intensity ratio (I / I0, where the fluorescence intensity of sample vial 1 is I0 and the fluorescence intensity of other sample vials is I) as a function of THF content. Figure 1 It can be seen that fluorescence begins to enhance when the volume fraction of THF exceeds 10%; when the volume fraction of THF reaches 50%, the fluorescence intensity reaches its maximum value, which is 90 times stronger than the fluorescence intensity I0 of sample vial 1. This fluorescence enhancement is due to aggregation, that is, compound I has obvious AIE properties.

[0083] The response of compound I to butyrylcholinesterase was verified as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 An aqueous solution of compound I at a concentration of mol / L was prepared. 10 mg of butyrylcholinesterase was weighed, dissolved in water, transferred to a 1 mL volumetric flask, and diluted to 1 mL with water to obtain an aqueous solution of butyrylcholinesterase with a concentration of 10 mg / mL.

[0084] Take two sample vials, label them 1 and 2 respectively, and add 1 mL of a 1×10⁻⁶ solution to each vial. -6 A mol / L aqueous solution of compound I was prepared by adding 50 µL of water to sample vial 1 and 50 µL of a 10 mg / mL aqueous solution of butyrylcholinesterase to sample vial 2. Fluorescence emission spectroscopy was then performed on the solutions in sample vials 1 and 2 at an excitation wavelength of 390 nm to obtain the following results: Figure 2 The fluorescence response spectrum of compound I to butyrylcholinesterase is shown in the figure. As can be seen from the figure, the fluorescence intensity of the solution after the addition of butyrylcholinesterase was significantly enhanced compared with the initial value, with a fluorescence enhancement factor of 8 times.

[0085] The detection limit of compound I for butyrylcholinesterase was determined as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 An aqueous solution of compound I at a concentration of mol / L, and an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL. The activity of the butyrylcholinesterase used was 7.5 U / mg.

[0086] Take 1 mL of a concentration of 1×10 -6 Fluorescence spectroscopy was performed on an aqueous solution of compound I at a concentration of mol / L. Subsequently, an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL was added dropwise using a pipette, and fluorescence spectroscopy was performed again. The volume added each time was fixed at 0.3 µL. This process was repeated three times. The initial fluorescence intensity was recorded as I0, and other fluorescence intensities were recorded as I. A graph was then plotted with the fluorescence intensity enhancement ratio ((I-I0) / I0) on the ordinate and the concentration of butyrylcholinesterase in the solution on the abscissa to determine the linear relationship between the butyrylcholinesterase concentration and the fluorescence intensity enhancement ratio ((I-I0) / I0). Figure 3 As shown. Then, the detection limit was calculated using the formula Detection Limit = 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit of compound I for butyrylcholinesterase was 0.015 U / mL.

[0087] Example 2 This embodiment provides a water-soluble AIE compound II, with the following structural formula: (II).

[0088] The preparation method of water-soluble AIE compound II includes the following steps: 4-Aminobenzoate (0.01 mol), 3-quinoline carbaldehyde (0.01 mol), and toluene (15 mL) were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 3 h. The temperature of the reaction system was then lowered to 60 °C, and 2,3-butanedione (0.005 mol), ferric perchlorate (0.001 mol), and glacial acetic acid (15 mL) were slowly added to the reaction system using a syringe. After the addition was complete, stirring was continued for 12 h. After the reaction was completed, the solvent was evaporated to obtain a solid. The solid was dissolved in acetonitrile at 40–50 °C (e.g., 45 °C), allowed to cool naturally to room temperature, and the precipitated solid was collected by filtration. The steps of adding acetonitrile, cooling, and filtration were repeated three times to obtain the intermediate product.

[0089] An intermediate product (0.001 mol) and 25 mL of tetrahydrofuran were added to a 100 mL round-bottom flask equipped with a magnetic stirrer, and the mixture was heated under reflux to dissolve the intermediate product. 2.00 g of NaOH was weighed and dissolved in 25 mL of water to obtain an aqueous NaOH solution. This NaOH solution was gradually added dropwise to the aforementioned round-bottom flask, and the mixture was heated under reflux for 12 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran and a small amount of water. The concentrated reaction solution was then added dropwise to a saturated sodium chloride solution, and the precipitated product was collected by filtration. The filter cake was washed with saturated sodium chloride solution and tetrahydrofuran, and then dried to obtain the compound shown in Formula II.

[0090] The compound represented by Formula II was characterized, and the data are as follows: 1 H NMR (400 MHz, D2O, ppm): δ 8.35 (s, 2H), 7.87 (d, J = 8.0 Hz, 4H), 7.75 (d, J = 8.4 Hz, 2H), 7.60 (t, J = 7.6 Hz, 2H), 7.50 – 7.39 (m, 6H), 7.01 (d, J = 8.1 Hz, 4H), 6.16 (s, 2H). 13 C NMR (101 MHz, 90% D2O + 10% THF-d8, ppm): δ 172.68, 149.19,145.24, 140.91, 135.55, 133.43, 132.98, 132.74, 130.81, 130.02, 127.77,127.58, 126.32, 124.17, 96.52. ESI-MS (negative ion mode, m / z) calcd. for C 38 H 22 N4O4 2- [M] 2- 299.08, found: 299.53. The characterization results above indicate that the synthesized compound has the structure shown in Formula II.

[0091] The AIE properties of compound II were verified as follows: Weigh 6.44 mg of compound II, dissolve it in an appropriate amount of water, transfer it to a 10 mL volumetric flask, and dilute to 10 mL with water to obtain a concentration of 1×10⁻⁶. -3 An aqueous solution of compound II at mol / L. Take 1 mL of 1×10 mol / L solution. -3 An aqueous solution of compound II at a concentration of mol / L was added to a 10 mL volumetric flask and diluted to 10 mL with water to obtain a solution with a concentration of 1×10⁻⁶. -4 An aqueous solution of compound II at a concentration of mol / L was prepared. Eleven sample vials were prepared and labeled 1-11. 300 μL of a 1×10⁻⁶ mol / L solution was added to each of the 1-10 sample vials. -4 Add 30 μL of a 1×10⁻⁶ mol / L aqueous solution of compound II to sample vial #11. -3 An aqueous solution of compound II was prepared at a concentration of 1 mol / L. Then, water was added sequentially to sample vials 1-11 at concentrations of 2700 μL, 2400 μL, 2100 μL, 1800 μL, 1500 μL, 1200 μL, 900 μL, 600 μL, 300 μL, 0 μL, and 0 μL, respectively. Finally, tetrahydrofuran (THF) was added sequentially to sample vials 1-11 at concentrations of 0 μL, 300 μL, 600 μL, 900 μL, 1200 μL, 1500 μL, 1800 μL, 2100 μL, 2400 μL, 2700 μL, and 2970 μL, respectively. This resulted in a concentration of compound II of 1 × 10⁻⁶ mol / L in each of the sample vials. -5 A mixed solution of compound II in THF / H2O with THF volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% respectively.

[0092] Fluorescence emission spectroscopy was performed on the THF / H2O mixed solutions of compound II in sample vials 1-11, with an excitation wavelength of 408 nm, and the results were as follows. Figure 4The fluorescence spectra of compound II in THF / H2O mixed solvents with different THF contents are shown, along with a trend graph of the emission wavelength to fluorescence intensity ratio (I / I0, fluorescence intensity of sample vial 1 is I0, fluorescence intensity of other sample vials is I) as a function of THF content. Figure 4 It can be seen that fluorescence begins to enhance when the volume fraction of THF exceeds 20%; when the volume fraction of THF reaches 90%, the fluorescence intensity reaches its maximum value, which is 70 times stronger than the fluorescence intensity I0 of sample vial #1. This fluorescence enhancement is due to aggregation, meaning that compound II exhibits significant AIE properties.

[0093] The response of compound II to butyrylcholinesterase was verified as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 An aqueous solution of compound II at a concentration of mol / L was prepared. 10 mg of butyrylcholinesterase was weighed, dissolved in water, transferred to a 1 mL volumetric flask, and diluted to 1 mL with water to obtain an aqueous solution of butyrylcholinesterase with a concentration of 10 mg / mL.

[0094] Take two sample vials, label them 1 and 2 respectively, and add 1 mL of a 1×10⁻⁶ solution to each vial. -6 A mol / L aqueous solution of compound II was prepared. 50 µL of water was added to sample vial #1, and 50 µL of a 10 mg / mL aqueous solution of butyrylcholinesterase was added to sample vial #2. Fluorescence emission spectroscopy was performed on the solutions in sample vials #1 and #2 at an excitation wavelength of 408 nm to obtain the following results: Figure 5 The fluorescence response spectrum of compound II to butyrylcholinesterase is shown in the figure. It can be seen from the figure that the fluorescence intensity of the solution after the addition of butyrylcholinesterase was significantly enhanced compared with the initial value, with a fluorescence enhancement factor of 32 times.

[0095] The detection limit of compound II for butyrylcholinesterase was determined as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 An aqueous solution of compound II at a concentration of mol / L, and an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL. The activity of the butyrylcholinesterase used was 7.5 U / mg.

[0096] Take 1 mL of a concentration of 1×10 -6Fluorescence spectroscopy was performed on an aqueous solution of compound II at a concentration of mol / L. Subsequently, an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL was added dropwise using a pipette, and fluorescence spectroscopy was performed again. The volume added each time was fixed at 0.3 µL; this process was repeated three times. The initial fluorescence intensity was recorded as I0, and other fluorescence intensities were recorded as I. A graph was then plotted with the fluorescence intensity enhancement ratio ((I-I0) / I0) as the ordinate and the concentration of butyrylcholinesterase in the solution as the abscissa to determine the linear relationship between the butyrylcholinesterase concentration and the fluorescence intensity enhancement ratio ((I-I0) / I0). Figure 6 As shown. Then, the detection limit was calculated using the formula Detection Limit = 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit of compound II for butyrylcholinesterase was 0.0070 U / mL.

[0097] Example 3 This embodiment provides a water-soluble AIE compound III, with the following structural formula: (III).

[0098] The preparation method of water-soluble AIE compound III includes the following steps: 4-Aminobenzoate (0.01 mol), 2-naphthaldehyde (0.01 mol), and toluene (15 mL) were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 3 h. The temperature of the reaction system was then lowered to 60 °C, and 2,3-butanedione (0.005 mol), ferric perchlorate (0.001 mol), and glacial acetic acid (15 mL) were slowly added to the reaction system using a syringe. After the addition was complete, stirring was continued for 12 h. After the reaction was completed, the solvent was evaporated to obtain a solid. The solid was dissolved in acetonitrile at 40–50 °C (e.g., 45 °C), allowed to cool naturally to room temperature, and the precipitated solid was collected by filtration. The steps of adding acetonitrile, cooling, and filtration were repeated three times to obtain the intermediate product.

[0099] Add 0.001 mol of the intermediate product and 25 mL of tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer, and heat under reflux to dissolve the intermediate product. Weigh 2.00 g of NaOH and dissolve it in 25 mL of water to obtain an aqueous NaOH solution. Gradually add the NaOH solution dropwise to the aforementioned round-bottom flask, and heat under reflux for 12 h. After the reaction is complete, concentrate the reaction solution under reduced pressure to remove tetrahydrofuran and a small amount of water. Add the concentrated reaction solution dropwise to a saturated sodium chloride solution, precipitate the product, filter and collect the filter cake. Wash the filter cake with saturated sodium chloride solution and tetrahydrofuran, and dry to obtain the compound shown in Formula III.

[0100] The compound represented by Formula III was characterized, and the data are as follows: 1 H NMR (400 MHz, Methanol-D4, ppm): δ 8.05 – 7.90 (m, 4H), 7.82 –7.63 (m, 8H), 7.47 – 7.36 (m, 6H), 7.36 – 7.28 (m, 4H), 6.62 (s, 2H). 13 C NMR (101 MHz, 90% D2O + 10% THF-d8, ppm): δ 172.87, 137.64,135.97, 135.48, 135.10, 133.75, 132.65, 131.57, 129.57, 129.16, 127.86,127.36, 127.20, 126.75, 126.35, 124.06, 121.94, 95.40. ESI-MS (negative ion mode, m / z) calcd. for C 40 H 24 N2O4 2- [M] 2- : 298.09, found: 298.39. The characterization results above indicate that the synthesized compound has the structure shown in Formula III.

[0101] The AIE properties of compound III were verified as follows: Weigh 6.42 mg of compound III, dissolve it in an appropriate amount of water, transfer it to a 10 mL volumetric flask, and dilute to 10 mL with water to obtain a concentration of 1×10⁻⁶. -3 An aqueous solution of compound III at mol / L. Take 1 mL of 1×10⁻⁶ mol / L. -3 An aqueous solution of compound III at a concentration of mol / L was added to a 10 mL volumetric flask and diluted to 10 mL with water to obtain a solution with a concentration of 1×10⁻⁶. -4 An aqueous solution of compound III at a concentration of mol / L was prepared. Eleven sample vials were prepared and labeled 1-11. 300 μL of a 1×10⁻⁶ mol / L solution was added to each of the 1-10 sample vials. -4 Add 30 μL of a 1×10⁻⁶ mol / L aqueous solution of compound III to sample vial #11. -3An aqueous solution of compound III was prepared at a concentration of 1 mol / L. Then, water was added sequentially to sample vials 1-11 at concentrations of 2700 μL, 2400 μL, 2100 μL, 1800 μL, 1500 μL, 1200 μL, 900 μL, 600 μL, 300 μL, 0 μL, and 0 μL, respectively. Finally, tetrahydrofuran (THF) was added sequentially to sample vials 1-11 at concentrations of 0 μL, 300 μL, 600 μL, 900 μL, 1200 μL, 1500 μL, 1800 μL, 2100 μL, 2400 μL, 2700 μL, and 2970 μL, respectively. This resulted in a concentration of compound III of 1 × 10⁻⁶ mol / L in each of the sample vials. -5 A mixed solution of compound II in THF / H2O with THF volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% respectively.

[0102] Fluorescence emission spectroscopy was performed on the THF / H2O mixed solutions of compound III in sample vials 1-11, with an excitation wavelength of 400 nm, and the results were as follows. Figure 7 The fluorescence spectra of compound III in THF / H2O mixed solvents with different THF contents are shown, along with a trend graph of the emission wavelength to fluorescence intensity ratio (I / I0, fluorescence intensity of sample vial 1 is I0, fluorescence intensity of other sample vials is I) as a function of THF content. Figure 7 It can be seen that fluorescence begins to enhance when the volume fraction of THF exceeds 20%; when the volume fraction of THF reaches 80%, the fluorescence intensity reaches its maximum value, which is 700 times stronger than the fluorescence intensity I0 of sample vial #1. This fluorescence enhancement is due to aggregation, meaning that compound III exhibits significant AIE properties.

[0103] The response of compound III to butyrylcholinesterase was verified as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 A mol / L aqueous solution of compound III. Weigh 10 mg of butyrylcholinesterase, dissolve it in water, transfer it to a 1 mL volumetric flask, and dilute to 1 mL to obtain an aqueous solution of butyrylcholinesterase with a concentration of 10 mg / mL.

[0104] Take two sample vials, label them 1 and 2 respectively, and add 1 mL of a 1×10⁻⁶ solution to each vial. -6 A mol / L aqueous solution of compound III was prepared by adding 50 µL of water to sample vial #1 and 50 µL of a 10 mg / mL aqueous solution of butyrylcholinesterase to sample vial #2. Fluorescence emission spectroscopy was then performed on the solutions in sample vials #1 and #2 at an excitation wavelength of 400 nm to obtain the following results: Figure 8 The fluorescence response spectrum of compound III to butyrylcholinesterase is shown in the figure. It can be seen from the figure that the fluorescence intensity of the solution after the addition of butyrylcholinesterase was significantly enhanced compared with the initial value, with a fluorescence enhancement factor of 110 times.

[0105] The detection limit of compound III for butyrylcholinesterase was determined as follows: The concentration obtained by preparing the solution as described above is 1×10⁻⁶. -6 An aqueous solution of compound III at a concentration of mol / L, and an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL. The activity of the butyrylcholinesterase used was 7.5 U / mg.

[0106] Take 1 mL of a concentration of 1×10 -6 Fluorescence spectroscopy was performed on an aqueous solution of compound III at a concentration of mol / L. Subsequently, an aqueous solution of butyrylcholinesterase at a concentration of 10 mg / mL was added dropwise using a pipette, and fluorescence spectroscopy was performed again. The volume added each time was fixed at 0.4 µL; this process was repeated three times. The initial fluorescence intensity was recorded as I0, and other fluorescence intensities were recorded as I. A graph was then plotted with the fluorescence intensity enhancement ratio ((I-I0) / I0) as the ordinate and the concentration of butyrylcholinesterase in the solution as the abscissa to determine the linear relationship between the butyrylcholinesterase concentration and the fluorescence intensity enhancement ratio ((I-I0) / I0). Figure 9 As shown in the figure. Then, the detection limit was calculated using the formula 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit of compound III for butyrylcholinesterase was 0.00068 U / mL.

[0107] Example 4 This embodiment provides a fluorescent probe for detecting the organophosphorus pesticides dichlorvos and methyl parathion, the preparation method of which includes: The concentration of 1×10 was prepared according to the method in Example 3. -6 An aqueous solution of compound III at a concentration of mol / L. Weigh 4.40 mg of butyrylcholinesterase and add 10 mL of a 1×10⁻⁶ mol / L solution. -6 A mol / L aqueous solution of compound III was stirred at room temperature for 3-5 min to obtain a complex of compound III and butyrylcholinesterase, which is a fluorescent probe for detecting organophosphorus pesticides dichlorvos and methyl parathion.

[0108] The detection limit of the complex of compound III and butyrylcholinesterase for the organophosphorus pesticide dichlorvos is determined as follows: Weigh 2.20 mg of diclofenac, dissolve it in dimethyl sulfoxide (DMSO), transfer it to a 10 mL volumetric flask, and dilute to 10 mL with DMSO to obtain a concentration of 1×10⁻⁶. -3A DMSO solution of dichlorvos at a concentration of mol / L. A 1 mL sample of the complex of compound III and butyrylcholinesterase was analyzed for fluorescence spectroscopy. Subsequently, 1 × 10⁻⁶ mol / L dichlorvos was added dropwise to the solution using a pipette. -3 A DMSO solution of dichlorvos (mol / L) was prepared and its fluorescence spectrum was measured. The volume added each time was fixed at 10 µL; this process was repeated three times. The initial fluorescence intensity was recorded as I0, and all other fluorescence intensities were recorded as I. The average fluorescence intensity was plotted on the ordinate, and the concentration of dichlorvos in the solution was plotted on the abscissa to determine the linear relationship between dichlorvos concentration and fluorescence intensity. Figure 10 As shown. Then, the detection limit was calculated using the formula Detection Limit = 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit of the compound III complex with butyrylcholinesterase for dichlorvos was 0.528 µg / mL.

[0109] The detection limit of the complex of compound III and butyrylcholinesterase for the organophosphorus pesticide dichlorvos is determined as follows: Weigh 2.63 mg of methyl parathion, dissolve it in DMSO, transfer it to a 10 mL volumetric flask, and dilute to 10 mL with DMSO to obtain a concentration of 1×10⁻⁶. -3 A DMSO solution of methyl parathion (mol / L). Take 1 mL of the complex of compound III and butyrylcholinesterase for fluorescence spectroscopy, then add 1×10⁻⁶ mol / L solution dropwise using a pipette. -3 A DMSO solution of methyl parathion (mol / L) was prepared and its fluorescence spectrum was measured. The volume added each time was fixed at 10 µL; this process was repeated three times. The initial fluorescence intensity was recorded as I0, and all other fluorescence intensities were recorded as I. The average fluorescence intensity was plotted on the ordinate, and the concentration of methyl parathion in the solution was plotted on the abscissa to determine the linear relationship between the concentration of methyl parathion and the fluorescence intensity. Figure 11 As shown. Then, the detection limit was calculated using the formula Detection Limit = 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit for the complex of compound III and butyrylcholinesterase with respect to methyl parathion was 1.76 µg / mL.

[0110] Example 5 The binding energies of compound III prepared in Example 3 and different proteins were calculated using molecular docking technology, specifically including the following steps: The molecular structure of compound III was drawn using ChemDraw software, and then the lowest energy configuration was calculated using Chem3D and saved as a PDB file. AutoDock Tools software was used to perform hydrogenation on the compound III molecule and to detect torsion centers and torsion bonds, and then the result was exported as a ligand file in PDBQT format.

[0111] Download PDB files for human serum albumin (HSA), lipase, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), lysozyme, trypsin, pepsin, and γ-globulin from the PDB database. Use Pymol or Discovery Studio software to remove redundant ligands from the protein PDB files, retaining only the protein structure. Use AutoDock Tools software to remove water molecules, add hydrogen, and balance the charge of the protein. Then, output the receptor file in PDBQT format.

[0112] Open the ligand and receptor files in AutoDock Tools software, set the docking box size to include the entire receptor region, ensuring that all possible binding regions of the ligand are covered; set the docking parameters, generating 20 configurations each time, and export the parameter file; use Autodock Vina to sequentially dock compound III with the protein to obtain the binding energy between compound III and the protein. The binding energies of compound III with different proteins are shown in Table 1 below. Figure 12 The binding force between compound III prepared in Example 3 and butyrylcholinesterase was calculated using molecular docking technology.

[0113] Table 1. Binding energies of Compound III to different proteins

[0114] The fluorescence response of compound III to different proteins was tested, as follows: Weigh 6.42 mg of compound III and dissolve it in 10 mL of PBS buffer to prepare a solution with a concentration of 1×10⁻⁶. -3 Prepare PBS solutions of compound III at mol / L. Weigh 0.5 mg of each protein (human serum albumin, lipase, acetylcholinesterase, butyrylcholinesterase, lysozyme, trypsin, pepsin, γ-globulin) and dissolve them in 0.99 mL of PBS buffer to prepare the corresponding protein PBS solutions.

[0115] Take 9 sample vials, numbered 1 to 9. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of the PBS solution for each of the 8 proteins to sample vials 2 through 9 respectively. Add 10 µL of 1×10⁻⁶ PBS solution to each of the 9 sample vials sequentially. - 3 A PBS solution of compound III at a concentration of mol / L was used to ensure that the concentration of compound III in each sample vial was 1 × 10⁻⁶. -5The protein concentration was 0.5 mg / mL in all sample vials except for sample vial #1, which contained no protein. Fluorescence emission spectroscopy was performed on the solutions in sample vials #1 through #9 at an excitation wavelength of 400 nm, yielding the following results: Figure 13 The fluorescence response spectra of compound III to different proteins are shown, where the fluorescence intensity of sample vial 1 is represented as I0, and the fluorescence intensity of the other sample vials is represented as I. As can be seen from the figure, the fluorescence intensity of the mixed solution of compound III and butyrylcholinesterase is significantly enhanced compared to sample vial 1. Furthermore, compound III also exhibits a significant fluorescence-enhancing response to albumin, acetylcholinesterase, and γ-globulin, making specific detection of butyrylcholinesterase impossible.

[0116] Based on the above results, the binding energy ranking of compound III with the aforementioned proteins follows this order: compound III has the lowest binding energy with butyrylcholinesterase, followed by acetylcholinesterase, and then other proteins, i.e., butyrylcholinesterase < acetylcholinesterase < other proteins. This result indicates that when butyrylcholinesterase is present in the solution system, the probe preferentially binds to butyrylcholinesterase, followed by acetylcholinesterase, and finally to other proteins. Furthermore, Figure 13 The fluorescence enhancement of compound III for acetylcholinesterase was lower than that for butyrylcholinesterase. Therefore, to ensure the probe's specific recognition of butyrylcholinesterase, acetylcholinesterase was chosen as the carrier protein, and compound III was assembled with the carrier protein acetylcholinesterase into a complex to serve as the probe. When other proteins such as albumin and γ-globulin are present in the detection system, the binding energy between compound III and acetylcholinesterase is lower than that between compound III and other proteins; therefore, compound III will not dissociate from the cavity of acetylcholinesterase to bind to other proteins. However, when butyrylcholinesterase is present in the detection system, the binding energy between compound III and butyrylcholinesterase is even lower, causing compound III to dissociate from the cavity of acetylcholinesterase and form a lower-energy complex with butyrylcholinesterase. Furthermore, since the fluorescence enhancement factor when compound III binds to butyrylcholinesterase is higher than that when compound III binds to acetylcholinesterase, when butyrylcholinesterase is present in the detection system, the fluorescence intensity of the solution will be significantly enhanced after adding the complex of compound III and acetylcholinesterase to the solution. This can avoid interference from other proteins and achieve a specific response to butyrylcholinesterase.

[0117] Example 6 This embodiment provides a method for preparing a complex of compound III and acetylcholinesterase, including: The concentration of 1×10 was prepared according to the method in Example 5. -3Prepare a PBS solution of compound III at a concentration of 1 mol / L. Weigh 2.8 mg of acetylcholinesterase and dissolve it in 0.99 mL of PBS buffer to prepare an acetylcholinesterase PBS solution. Add 10 µL of a 1×10⁻⁶ mol / L solution of compound III to the acetylcholinesterase PBS solution. -3 A PBS solution of compound III at a concentration of mol / L was used to ensure that the concentrations of both compound III and acetylcholinesterase in the system were 1 × 10⁻⁶. -5 The concentration was mol / L, and then stirred at room temperature for 2 h to obtain a complex of compound III and acetylcholinesterase.

[0118] The fluorescence response of the complex of compound III and acetylcholinesterase to different proteins in this embodiment was tested as follows: Weigh 1 mg of each protein (human serum albumin, lipase, acetylcholinesterase, butyrylcholinesterase, lysozyme, trypsin, pepsin, and γ-globulin) and dissolve them in 0.1 mL of PBS buffer to prepare the corresponding protein PBS solutions. Take nine sample vials, numbered 1-9. Add 1 mL of the compound III / acetylcholinesterase complex to each vial. Add 50 µL of PBS buffer to vial 1, and add 50 µL of protein PBS solution to vials 2-9 respectively. Except for vial 1, which contains no protein, the protein concentration in all other vials is 0.5 mg / mL. Perform fluorescence emission spectroscopy on the solutions in vials 1-9 at an excitation wavelength of 400 nm to obtain the results shown below. Figure 14 The fluorescence response spectra of the compound III complex with acetylcholinesterase to different proteins are shown below. The fluorescence intensity of sample vial 1 is represented as I0, and the fluorescence intensity of the other sample vials is represented as I. The figure shows that the fluorescence intensity of the sample vial containing butyrylcholinesterase is significantly enhanced compared to sample vial 1, while the fluorescence intensity of the other sample vials is not significantly enhanced compared to sample vial 1. This result indicates that the compound III complex with acetylcholinesterase can achieve a specific response to butyrylcholinesterase.

[0119] Example 7 This embodiment provides a method for preparing a complex of compound III and acetylcholinesterase, including: The concentration of 1×10 was prepared according to the method in Example 5. -3 A 1 mol / L solution of compound III in PBS was then diluted 10-fold with PBS buffer to obtain a concentration of 1 × 10⁻⁶. -4 Prepare a PBS solution of compound III at a concentration of mol / L. Weigh 2.8 mg of acetylcholinesterase and dissolve it in 9.9 mL of PBS buffer to prepare an acetylcholinesterase PBS solution.

[0120] Take 0.99 mL of acetylcholinesterase in PBS and add 10 µL of a 1×10⁻⁶ solution. -4 A PBS solution of compound III at a concentration of mol / L was used to ensure that the concentrations of both compound III and acetylcholinesterase in the system were 1 × 10⁻⁶. -6 The concentration was mol / L, and then stirred at room temperature for 2 h to obtain a complex of compound III and acetylcholinesterase.

[0121] The detection limit of butyrylcholinesterase by the complex of compound III and acetylcholinesterase in this embodiment is tested as follows: Weigh 10 mg of butyrylcholinesterase, dissolve it in water, transfer it to a 1 mL volumetric flask, and dilute to 1 mL to prepare an aqueous solution of butyrylcholinesterase with a concentration of 10 mg / mL. The activity of the butyrylcholinesterase used is 7.5 U / mg. Perform fluorescence spectroscopy on 1 mL of the complex of compound III and acetylcholinesterase. Then, use a pipette to add 10 mg / mL of the butyrylcholinesterase aqueous solution dropwise, and perform fluorescence spectroscopy again. The volume added each time is fixed at 0.67 µL; repeat the operation three times. Record the initial fluorescence intensity as I0, and other fluorescence intensities as I. Then, plot the fluorescence intensity enhancement ratio ((I-I0) / I0) on the ordinate and the concentration of butyrylcholinesterase in the solution on the abscissa to determine the linear relationship between the butyrylcholinesterase concentration and the fluorescence intensity enhancement ratio ((I-I0) / I0). Figure 15 As shown. Then, the detection limit was calculated using the formula Detection Limit = 3σ / k, where σ is the standard deviation of the lowest response value during testing and k is the slope of the linear relationship. The calculated detection limit of the complex of compound III and acetylcholinesterase for butyrylcholinesterase was 0.00096 U / mL.

[0122] Example 8 This embodiment provides a method for preparing a complex of compound III and acetylcholinesterase, including: Weigh 6.42 mg of compound III and dissolve it in 1 mL of PBS buffer to prepare a solution with a concentration of 1×10⁻⁶. -2 Prepare a PBS solution of compound III at a concentration of mol / L; dissolve 28 mg of acetylcholinesterase in 0.99 mL of PBS buffer to prepare an acetylcholinesterase PBS solution. Take 0.99 mL of the acetylcholinesterase PBS solution and add 10 µL of a 1×10⁻⁶ mol / L solution. -2 A PBS solution of compound III at a concentration of mol / L was used to ensure that the concentrations of both compound III and acetylcholinesterase in the system were 1 × 10⁻⁶. -4 The concentration was mol / L, and then stirred at room temperature for 2 h to obtain a complex of compound III and acetylcholinesterase.

[0123] The interference resistance of the compound III complex with acetylcholinesterase in this embodiment when detecting butyrylcholinesterase in different biological components is as follows: Take 7 sample vials and label them 1 through 7; Add 1 mL of PBS buffer to sample vial #1; Weigh out common small molecules found in biological tissues (adenosine-5'-monophosphate, acetylthiocholine chloride, disodium adenosine-5'-triphosphate, cholesterol, adenine sulfate, heparin sodium, L-arginine hydrochloride, L-aspartic acid, L-aspartic acid, L-cysteine, L-glutamic acid, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tyrosine, L-valine, N-acetylneuraminic acid), and prepare a mixed solution using PBS buffer as the solvent. The concentration of each small molecule is 0.5 mg / mL. Take 1 mL of this solution and add it to sample vial No. 2. Weigh out common proteins found in biological tissues (human serum albumin, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin) and prepare a mixed solution with a concentration of 0.5 mg / mL for each protein. Take 1 mL of this solution and add it to sample vial #3. Weigh common proteins and small molecules found in biological tissues (human serum albumin, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, γ-globulin, adenosine-5'-monophosphate, acetylthiocholine chloride, adenosine-5'-triphosphate disodium salt, cholesterol, adenine sulfate, heparin sodium, L-arginine hydrochloride, L-aspartic acid, L-aspartic acid, L-cysteine, L-glutamic acid, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tyrosine, L-valine, N-acetylneuraminic acid), and prepare a mixed solution using PBS buffer as the solvent. The concentration of each protein and small molecule is 0.5 mg / mL. Take 1 mL of this solution and add it to sample vial No. 4. Weigh albumin, γ-globulin, fibrinogen, glucose, and urea, and prepare a mixed solution using PBS buffer as a solvent. The mass ratio of albumin:γ-globulin:fibrinogen:glucose:urea:cholesterol is 400:300:19:6.7:1:4. The concentration of albumin is 20 mg / mL. Take 1 mL of this solution and add it to sample vial No. 5. Weigh butyrylcholinesterase, albumin, γ-globulin, fibrinogen, glucose, cholesterol, and urea. Prepare a mixed solution using PBS buffer as the solvent, wherein the mass ratio of albumin:γ-globulin:fibrinogen:glucose:urea:cholesterol is 400:300:19:6.7:1:4. The concentration of albumin is 20 mg / mL, and the concentration of butyrylcholinesterase is 0.5 mg / mL. Take 1 mL of this solution and add it to sample vial No. 6. Weigh 2.5 mg of butyrylcholinesterase and dissolve it in PBS buffer. Then, bring the volume up to 5 mL with PBS buffer to prepare a 0.5 mg / mL butyrylcholinesterase PBS solution. Add 1 mL of this solution to sample vial #7.

[0124] Add 10 µL of the compound III complex with acetylcholinesterase to each of the seven sample vials, and then perform fluorescence emission spectroscopy on the solutions in sample vials 1-7 to obtain the results shown below. Figure 16 The figure shows the interference resistance of the compound III complex with acetylcholinesterase in the detection of butyrylcholinesterase in different biological components. The fluorescence intensity of sample vial 1 is represented as I0, and the fluorescence intensity of the other sample vials is represented as I. As can be seen from the figure, the fluorescence intensity of the sample vial containing butyrylcholinesterase is significantly enhanced compared to sample vial 1, while the fluorescence intensity of the sample vial without butyrylcholinesterase is not significantly enhanced compared to sample vial 1. This result indicates that the compound III complex with acetylcholinesterase can achieve a specific response to butyrylcholinesterase and is not affected by interference from other biological components.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A water-soluble AIE compound, characterized in that, It has the following general structural formula: ； Where R is , and At least one of them.

2. The water-soluble AIE compound according to claim 1, characterized in that, The water-soluble AIE compound includes at least one of the following structures: （Ⅰ）、 （Ⅱ）、 （Ⅲ）。 3. The method for preparing the water-soluble AIE compound according to claim 1 or 2, characterized in that, The steps include the following: (a) Methyl 4-aminobenzoate or 4-aminobenzoic acid reacts with aldehydes in a first solvent; then 2,3-butanedione, an organic acid and a catalyst are added to carry out a second reaction to obtain an intermediate product; (b) The intermediate product is refluxed in a second solvent containing sodium hydroxide to obtain the water-soluble AIE compound; The aldehyde compounds include , and At least one of them.

4. The preparation method according to claim 3, characterized in that, It has at least one of the following characteristics: (1) The first solvent includes at least one of benzene, toluene, and xylene; (2) The organic acid includes at least one of acetic acid, oxalic acid and propionic acid; (3) The catalyst includes at least one of p-toluenesulfonic acid and ferric perchlorate.

5. The preparation method according to claim 3, characterized in that, It has at least one of the following characteristics: (1) The molar ratio of the methyl 4-aminobenzoate or 4-aminobenzoic acid to the aldehyde compound is (0.8~1.2):1; (2) The molar ratio of the aldehyde compound to the 2,3-butanedione is 1:(0.4~0.6). (3) The molar ratio of the aldehyde compound to the catalyst is 1:(0.05~0.15); (4) In step (a), the temperature of the first reaction is 85~95℃ and the reaction time is 2~4 h; (5) In step (a), the temperature of the second reaction is 55~65℃ and the time of the second reaction is 10~14 h.

6. The preparation method according to claim 3, characterized in that, It has at least one of the following characteristics: (1) The second solvent comprises water and tetrahydrofuran; (2) The molar ratio of sodium hydroxide to the intermediate product is not less than 5:1; (3) In step (b), the reaction time is 10-14 h.

7. The use of the water-soluble AIE compound of claim 1 or 2 in the preparation of fluorescent probes for the detection of butyrylcholinesterase or for the detection of organophosphorus compounds.

8. A fluorescent probe for detecting butyrylcholinesterase, characterized in that, Includes the water-soluble AIE compound and solvent as described in claim 1 or 2; The solvent includes either water or PBS buffer; Preferably, the concentration of the water-soluble AIE compound in the fluorescent probe is 10. -5 ~10 -7 mol / L.

9. The fluorescent probe for detecting butyrylcholinesterase according to claim 8, characterized in that, The fluorescent probe also includes a carrier protein; the carrier protein includes acetylcholinesterase. Preferably, in the fluorescent probe, the molar ratio of the water-soluble AIE compound to the carrier protein is 1:

1.

10. A fluorescent probe for detecting organophosphorus compounds, characterized in that, Includes the water-soluble AIE compound, butyrylcholinesterase, and solvent as described in claim 1 or 2; The solvent includes either water or PBS buffer; Preferably, the concentration of the water-soluble AIE compound in the fluorescent probe is 10. -5 ~10 -7 mol / L; Preferably, the molar ratio of the water-soluble AIE compound to the butyrylcholinesterase is 1:1; Preferably, the organophosphorus compound includes at least one of dichlorophen and methyl parathion.

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