An AIE fluorescent probe molecular material, its preparation method and application

Abstract

The present invention discloses an AIE fluorescent probe molecular material, its preparation method and application. The preparation method includes the following steps: (1) Synthesis of compound 3: Using 4-ethynylphthalic anhydride, Ac2O, Et3N and tert-butyl acetoacetate as raw materials to synthesize compound 3; (2) Synthesis of compound 4: Using compound A, Pd(PPh)3, a basic substance, tetrahydrofuran hydrate and compound B as raw materials to synthesize compound 4; (3) Synthesis of the AIE fluorescent probe molecular material: Using compound 4, acetic anhydride anhydrous, compound 3 and piperidine as raw materials to obtain the AIE fluorescent probe molecular material. The AIE fluorescent probe molecular material prepared by the present invention has high sensitivity, high selectivity and non-toxicity, can perform real-time monitoring of heavy metal ions, and can achieve rapid, accurate and low-concentration labeling of heavy metal ions in plants.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular probe synthesis, and particularly relates to an AIE fluorescent probe molecular material, a preparation method thereof and an application thereof. Background Art

[0002] China is the largest rice producer and consumer in the world. However, the preference of rice for absorbing cadmium (Cd) causes a large accumulation of Cd in rice grains, seriously threatening food security and human health. At present, a large number of studies focus on the absorption and transport of Cd in rice plants. Therefore, it is necessary to deeply locate the occurrence state of heavy metals in plant tissues. Currently, the commonly used in-situ micro-region Cd localization techniques include laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray fluorescence spectrometry. The former not only has a high analysis cost but also is interfered by the matrix, making it difficult to accurately locate heavy metals in plants. The latter determines the distribution of cadmium through the characteristic X-ray fluorescence signal generated after the excitation of high-energy X-rays in plants, and it is difficult to locate trace amounts of Cd, severely restricting researchers from exploring the absorption and transport of Cd in plants.

[0003] AIE (Aggregation-Induced Emission) molecules are a special type of fluorescent molecules that do not emit light in the monomer state but emit strong fluorescence in the aggregated state. Based on the luminescence characteristics of AIE molecules, an AIE molecular detection and localization technique can be developed to achieve high-sensitivity and high-resolution detection and localization of molecules in living organisms.

[0004] The AIE molecular detection and localization technique has the following advantages:

[0005] 1. High sensitivity: The fluorescence emission intensity of AIE molecules is proportional to the degree of aggregation. Therefore, high-sensitivity detection of biomolecules can be achieved.

[0006] 2. High resolution: The fluorescence emission of AIE molecules has a high spatial resolution, and high-resolution localization of biomolecules can be achieved.

[0007] 3. Non-toxicity: AIE molecules are a type of non-toxic fluorescent molecules, and non-destructive detection of biomolecules can be achieved in living organisms.

[0008] 4. Multiplex labeling: AIE molecules can be chemically modified to achieve multiplex labeling, and multiple biomolecules can be detected and localized simultaneously.

[0009] The AIE molecular detection and localization technique has currently been widely applied in the field of biomedical research, such as cell imaging, molecular diagnosis, drug screening, etc.

[0010] AIE (Aggregation-Induced Emission) probes are a type of molecular materials with aggregation-induced emission properties. They emit very weakly in the monomer state, and their emission intensity increases significantly when aggregated or dissolved in non-polar solvents. This aggregation-induced emission property enables AIE probes to have broad application potential in the fields of bioimaging, chemical sensing, materials science, etc. In the past decade, many AIE fluorescent probes for detecting heavy metal ions have been developed, but they generally have the following disadvantages in practical applications:

[0011] 1. Small tissue penetration: The emission wavelengths of general fluorescent probes are relatively short, in the blue or green light range, and their penetration ability is relatively weak. They can only perform co-localization of cadmium in relatively shallow tissue layers or on the cell surface, and cannot penetrate the plant cell wall, making it difficult to image inside plants.

[0012] 2. Limited detection sensitivity: Although AIE molecules have high sensitivity, for some heavy metal ions, such as chromium and cadmium, at low concentrations, the fluorescent probes cannot produce obvious fluorescent signal responses.

[0013] 3. Restricted in practical applications: Although AIE molecules have good performance as heavy metal probes, some problems still need to be solved in their practical applications, such as detection in complex sample matrices and applications in real environments. Summary of the Invention

[0014] To solve the above technical problems, the purpose of the present invention is to provide an AIE fluorescent probe molecular material, its preparation method and application, in order to solve the problems of small tissue penetration, limited detection sensitivity, restricted practical application and high cost of existing AIE fluorescent probes.

[0015] The technical solution of the present invention to solve the above technical problems is as follows:

[0016] An AIE fluorescent probe molecular material, the structural formula is:

[0017]

[0018] The preparation method of the above AIE fluorescent probe molecular material is characterized by including the following steps:

[0019] (1) Synthesis of compound 3: Mix 4-ethynylphthalic anhydride with Ac2O, add Et3N and tert-butyl acetoacetate, stir and react at 20 - 25 °C, then cool to -10 - 10 °C and concentrated hydrochloric acid, and stir and react at 75 - 80 °C to obtain it;

[0020] (2) Synthesis of Compound 4: Mix Compound A, Pd(PPh)₃ and a basic substance, introduce an inert gas to expel oxygen, and then add tetrahydrofuran hydrate and Compound B, and carry out a reflux reaction at 78 - 80 °C to obtain it;

[0021] (3) Synthesis of the AIE fluorescent probe molecular material: Dissolve Compound 4 in acetic anhydride anhydrous, then add Compound 3 and piperidine, and carry out a mixing reaction at 58 - 62 °C to obtain it;

[0022] The reaction formula of the above preparation method is:

[0023]

[0024] Furthermore, in step (1), the mass - volume ratio of 4 - ethynylphthalic anhydride, Ac₂O, Et₃N, tert - butyl acetoacetate and concentrated hydrochloric acid is (1.23 - 1.26) g : (4.2 - 4.5) g : (2 - 3) mL : (1.0 - 1.3) g : (3 - 5) mL.

[0025] Furthermore, in step (1), the stirring time is 18 - 20 h; the re - stirring time is 0.5 - 2 h.

[0026] Furthermore, in step (2), the mass - volume ratio of Compound A, Pd(PPh)₃, the basic substance, tetrahydrofuran hydrate and Compound B is (1.4 - 1.5) g : (0.08 - 0.1) g : (1 - 2) g : (20 - 25) mL : (0.9 - 1.0) g; the basic substance includes K₂CO₃; the volume ratio of tetrahydrofuran to water in tetrahydrofuran hydrate is (8 - 10) : (1 - 2).

[0027] Furthermore, in step (2), the reflux time is 10 - 15 h.

[0028] Furthermore, in step (3), the mass - volume ratio of Compound 4, acetic anhydride anhydrous, Compound 3 and piperidine is (300 - 400) mg : (10 - 12) mL : (200 - 300) mg : (0.85 - 0.88) mg.

[0029] Furthermore, in step (3), the mixing reaction time is 5 - 8 h.

[0030] The above AIE fluorescent probe molecular material is used in the detection of cadmium.

[0031] Furthermore, the purification process of the AIE fluorescent probe molecular material includes: distilling off the solvent under reduced pressure, dissolving the sample in dichloromethane, loading the sample wet onto a short silica gel column with 100 - 200 mesh, carrying out column chromatography with petroleum ether / dichloromethane with a volume ratio of 1:1, and when the orange mono - condensation product is eluted, eluting with pure dichloromethane with a greater polarity.

[0032] Furthermore, the purification method of the AIE fluorescent probe molecular material further includes: rotary evaporating the reaction mixture at a pressure of 70 mbar and a temperature of 50 °C until no obvious liquid distillate appears, loading the reaction solution onto a short silica gel column with 100 - 200 mesh by wet method, eluting with pure dichloromethane, and quickly collecting the eluate containing the target product; combining and concentrating the eluate containing the target product, then loading it onto another short silica gel column with 100 - 200 mesh by wet method, performing column chromatography with a volume ratio of petroleum ether:dichloromethane of 1:1. After eluting the aforementioned orange mono - condensation product, elute with pure dichloromethane with a greater polarity.

[0033] The present invention has the following beneficial effects:

[0034] (1) The AIE fluorescent probe molecular material synthesized by the present invention has high sensitivity. The AIE fluorescent probe molecular material is highly sensitive to cadmium. The content of cadmium in plants is low, and the fluorescent probe cannot produce an obvious fluorescent signal response at low concentrations. The synthesized product of this method, through appropriate molecular design and modification strategies, enables the thiophene group to highly complex with cadmium, and can highly sensitively detect the target molecule at low concentrations, generating an obvious fluorescent signal response. The AIE molecule can undergo a fluorescent change by binding to cadmium ions, thereby realizing the highly sensitive detection of cadmium ions.

[0035] (2) The AIE fluorescent probe molecular material synthesized by the present invention has high selectivity. The recognition of cadmium ions by the AIE fluorescent probe molecular material is highly selective, which can avoid interference from other ions.

[0036] (3) The AIE fluorescent probe molecular material synthesized by the present invention can be monitored in real - time. Since the fluorescent change of the AIE fluorescent probe molecular material prepared by the present invention is real - time, the real - time monitoring of cadmium ions can be realized, which helps to study the real - time migration and transport of Cd in rice plants.

[0037] (4) The AIE fluorescent probe molecular material synthesized by the present invention is non - toxic. Compared with traditional organic fluorescent probes, the AIE fluorescent probe molecular material prepared by the present invention has lower toxicity and biotoxicity, so it can be applied to the monitoring of cadmium ions in living organisms.

[0038] (5) The AIE fluorescent probe molecular material synthesized in the present invention has strong penetrability. Hydroxyl groups are introduced into the synthesized product molecules, and long fluorescence emission is obtained by enhancing the conjugation degree, enhancing the cell penetration ability, and achieving a breakthrough in the positioning range from cells to tissues. It can make heavy metal cadmium emit light in plant tissues. When general fluorescent probes are used for in-situ co-localization, the emission wavelength is only 400 - 500 nm, and observations can only be carried out on relatively shallow tissue layers or cell surfaces. An alkyne group is introduced into the synthesized product molecules of this method, and long fluorescence emission is obtained by enhancing the conjugation degree, and the maximum emission wavelength reaches 1000 nm, entering the second near-infrared region. At this time, the penetrability is stronger when excited and detected using a laser confocal microscope, and it can penetrate tissue or cell structures, thereby achieving high-sensitivity and real-time monitoring in-situ co-localization on plant tissues. Description of the Drawings

[0039] Figure 1 It is the experimental result of thin-layer chromatography monitoring in Example 2;

[0040] Figure 2 It is the schematic diagram of thin-layer chromatography in Example 2;

[0041] Figure 3 It is the mass spectrometry analysis chart of Compound 3 in Example 3;

[0042] Figure 4 It is the mass spectrometry analysis chart of Compound 4 in Example 3;

[0043] Figure 5 It is the mass spectrometry analysis chart of the AIE fluorescent probe molecular material in Example 3;

[0044] Figure 6 It is the comparison chart of the emission wavelengths of the commercial AIE probe in Example 4 and the AIE fluorescent probe molecular material prepared in Example 1;

[0045] Figure 7 It is the imaging diagram of plant leaves by a laser confocal microscope in Example 5, where (a) is the commercial AIE probe and (b) is the AIE fluorescent probe molecular material prepared in Example 1;

[0046] Figure 8 It is the experimental result of the sensitivity experiment in Example 6, where from left to right are aqueous solutions containing Cd ions with Cd mass fractions of 10, 20, 30, 40, and 50 mg·kg -1 ;

[0047] Figure 9 It is the experimental result of the selectivity experiment in Example 7;

[0048] Figure 10 It is the test result of biomass and the photo of potted rice in the biotoxicity experiment in Example 8;

[0049] Figure 11 The SPAD value test results of rice leaves in the biotoxicity experiment in Example 8. Specific implementation mode

[0050] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] Example 1:

[0052] A preparation method of an AIE fluorescent probe molecular material, comprising the following steps:

[0053] (1) Synthesis of compound 3

[0054]

[0055] A. Add 1.25 g of 4-ethynylphthalic anhydride to 4.32 g of Ac2O, and add 2.5 mL of Et3N to the suspension;

[0056] B. Add 1.2 g of tert-butyl acetoacetate to the mixture obtained in step A, and stir and react at room temperature for 20 hours;

[0057] C. After the reaction is completed, add 5 g of crushed ice, and dropwise add 4 mL of concentrated hydrochloric acid with a mass fraction of 37%, and stir and react at 80 °C for 1 hour;

[0058] D. After the reaction mixture is cooled to room temperature, extract it 3 times with 30 mL of CH2Cl2. Take the organic phase, dry it over anhydrous sodium sulfate, filter it, and concentrate the filtrate under reduced pressure to obtain a dark brown solid, that is, the crude product compound 3, with a yield of 85%.

[0059] (2) Synthesis of compound 4

[0060]

[0061] A. Add 1.45 g of compound A, 90 mg of Pd(PPh)3, and 1.38 g of K2CO3 to a 100 mL two-necked flask, and perform evacuation and argon replacement through Schlenk operation to ensure that there is no oxygen in the system;

[0062] B. Add 25 ml of a furan hydrate with a volume ratio of THF (tetrahydrofuran):H2O of 9:1 to the Schlenk container, and add 955 mg of compound B for reaction, and reflux at 80 °C for 12 h;

[0063] C. The reaction mixture was successively extracted with ethyl acetate and H2O. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by suction filtration, and the solvent was rotary evaporated. Column chromatography was first performed using petroleum ether / dichloromethane with a volume ratio of 3:1, and then column chromatography was performed using petroleum ether / dichloromethane with a volume ratio of 1:1. A yellow solid compound 4 was obtained with a yield of 85%.

[0064] (3) Synthesis of AIE fluorescent probe molecular materials

[0065] A. Acetic anhydride was dehydrated with phosphorus pentoxide. 355 mg of compound 4 was dissolved in 12 mL of anhydrous acetic anhydride, and 266 mg of compound 3 and 0.88 mg of piperidine were added. The reaction was carried out at 60 °C for 6 h;

[0066] B. The reaction mixture was rotary evaporated at 70 mbar pressure and 50 °C until no obvious liquid distillate was obtained. The reaction solution was wet-loaded onto a short column of 200-mesh silica gel and eluted with pure dichloromethane. The eluate containing the target product was quickly collected;

[0067] C. The eluates containing the target product were combined and concentrated, and then wet-loaded onto another short column of 200-mesh silica gel. Column chromatography was carried out using petroleum ether:dichloromethane with a volume ratio of 1:1. When the orange mono-condensation product was eluted, it was eluted with more polar pure dichloromethane, and the dark blue-green product 6, namely the AIE fluorescent probe molecular material, was collected. The yield was about 12%.

[0068]

[0069] Example 2: Thin-layer chromatography detection experiment

[0070] The reaction solution finally obtained in step A of the synthesis of the AIE fluorescent probe molecular material (product 6) in Example 1 was aspirated, diluted with ethyl acetate, added with a small amount of water, and separated in a centrifuge tube. The organic phase was taken with a capillary tube and monitored by thin-layer chromatography (TLC) using petroleum ether:ethyl acetate with a volume ratio of 4:1 as the developing agent.

[0071] The results of the thin-layer chromatography monitoring experiment are as Figure 1 , and the thin-layer chromatography schematic diagram is as Figure 2 . The yellow spot (relative migration rate Rf = 0.6) at the top of the thin-layer chromatography plate is compound 4, the middle blue-green spot (Rf = 0.4) is the AIE fluorescent probe molecular material (product 6), and there are more dark blue or dark brown spots above the origin (Rf = 0.05) for compound 3 and other impurities.

[0072] Example 3: Mass spectrometry detection experiment

[0073] To further determine the product synthesis results, mass spectrometry (MS) analysis was performed on Compound 3, Compound 4, and the AIE fluorescent probe molecular material (Product 6).

[0074] (1) The molecules of Compound 3 were negatively ionized in the mass spectrometer to form the negative ion [M-H] - , which represents the ion formed by the loss of a proton (hydrogen ion) in the molecule, causing the mass of the negative ion to decrease by the mass of one proton compared to the original molecule. The theoretical molecular weight of Compound 3 is 170.0368, and the mass spectrometry analysis results are shown as Figure 3 . The highest peak in the mass spectrum [M-H] - has a mass-to-charge ratio of 160.0329 and a relative abundance of 70%, and the mass spectrometry data is consistent with the theoretical value.

[0075] (2) The theoretical molecular weight of Compound 4 is 355.10, and [M+H] + represents the positive ion formed by the molecule gaining an additional proton during the ionization process of mass spectrometry analysis, causing the mass of the positive ion to increase by the mass of one proton compared to the original molecule. The experimental results are shown as Figure 4 . Compound 4 is charged positively in the positive mode by adding H + , and the mass-to-charge ratio m / z = 336.1094, indicating the successful synthesis of Product 4.

[0076] (3) The theoretical molecular weight of the AIE fluorescent probe molecular material is 507.1293, and the mass spectrometry analysis results are shown as Figure 5 . The highest peak in the mass spectrum [M+H] + has a mass-to-charge ratio of 508.1229 and a relative abundance of 60%, and the mass spectrometry data is consistent with the theoretical value; although separation and purification were carried out using high-performance liquid chromatography, there are still some isomers, and the yield of the AIE fluorescent probe molecular material exceeds 90%. This indicates the successful synthesis of the AIE fluorescent probe molecular material.

[0077] Example 4: Emission Wavelength Comparison Experiment

[0078] A commercial AIE probe (purchased from Xi'an Ruixi Biotechnology Co., Ltd., customized AIE probe: CAS: 2566678-02-0) and the AIE fluorescent probe molecular material introduced with alkynyl prepared in Example 1 of the present invention were used for in-situ co-localization experiments to detect their maximum emission wavelengths.

[0079] Experimental instruments: Fluorescence spectrophotometer (PerkinElmer UV650, USA), ultraviolet-visible spectrophotometer (UV2450, Shanghai Yuanxi Instruments).

[0080] Experimental method: The commercial AIE probe and the AIE fluorescent probe molecular material prepared in Example 1 of the present invention were respectively dissolved in dichloromethane and irradiated with an excitation wavelength of 405 nm.

[0081] The experimental results are as Figure 6 shown. When the commercial AIE probe was used for in-situ co-localization, the emission wavelength was relatively short, only 400 - 500 nm, within the blue or green light range. Its penetration ability was relatively weak, and it could only perform co-localization of cadmium in relatively shallow tissue layers or on the cell surface. An alkyne group was introduced into the synthetic material molecule of this method, and long fluorescence emission was obtained by enhancing the conjugation degree. The maximum emission wavelength reached 1000 nm, entering the second near-infrared region. Therefore, when detected by laser confocal microscopy, it had stronger penetration ability and could penetrate tissue or cell structures.

[0082] Example 5: Practical application experiment on rice leaves

[0083] A commercial AIE probe (purchased from Xi'an Ruixi Biotechnology Co., Ltd., customized AIE probe: CAS: 2566678 - 02 - 0) and the AIE fluorescent probe molecular material prepared in Example 1 were used to detect heavy metal Cd in rice leaves by laser confocal microscopy.

[0084] When the rice was at the flowering stage (the rice mainly has 4 growth stages: the returning green stage, the tillering stage, the flowering stage, and the filling stage), at 5 pm, 10 mL of the commercial AIE probe with a concentration of 10 μmol / L and the AIE fluorescent probe molecular material prepared in Example 1 were respectively sprayed on the leaves of potted rice (the total Cd content in the potted soil was 5 mg·kg -1 ) (just covering the leaf surface), and gently pasted with tin foil to increase the contact time of the product and improve the absorption efficiency of the plant. After 30 minutes, the leaves were collected and stored in a -48 °C refrigerator for later observation. After the collected leaves were washed with ethanol, the leaves were laid flat in a Corning confocal culture dish with an area of 20×30 mm, fixed solution was added, and detected and observed with a Leica SP8 laser confocal microscope. With 488 nm as the excitation wavelength, orange and red fluorescence in the wavelength range of 800 - 900 nm was collected.

[0085] The experimental results are as Figure 7 shown, among which, Figure 7 a Fluorescence image of a commercial AIE probe used to locate heavy metal Cd in plant leaves. Since the fluorescence wavelength belongs to the short wavelength range, it is difficult to penetrate plant tissues. Figure 7 b Cd content distribution map using the AIE probe with an introduced alkyne group. Since the wavelength increases to 800 - 900 nm, it can easily penetrate plant tissues for the localization and imaging of heavy metal - Cd.

[0086] Example 6: Sensitivity Experiment

[0087] Take the AIE fluorescent probe molecular material prepared in Example 1 of the present invention for the sensitivity experiment, which specifically includes the following steps: Prepare 20 mL aqueous solutions containing Cd ions with Cd mass fractions of 10, 20, 30, 40, and 50 mg·kg -1 respectively, add 0.5 mmol of the AIE fluorescent probe molecular material to each, and stir for 5 min to observe the fluorescence change.

[0088] The experimental results are as Figure 8 shown. Obvious fluorescence signals were generated in all five experimental groups. The results indicate that the AIE fluorescent probe molecular material prepared by the present invention can achieve highly sensitive detection of Cd content from 10 mg·kg -1 to 50 mg·kg -1 .

[0089] Example 7: Selectivity Experiment

[0090] Take the AIE fluorescent probe molecular material prepared in Example 1 of the present invention for the selectivity experiment, which specifically includes the following steps: Prepare 50 mg·kg -1 ion solutions of Ca 2+ , Mg 2+ , Zn 2+ , Cd 2+ , Ni 2+ , Cu 2+ , Co 2+ , Pb 2+ , and Fe 2+ with a volume of 50 mL each. Add 0.5 mmol of the AIE fluorescent probe molecular material to each, stir for 5 min, then measure the fluorescence spectrum and calculate the change in fluorescence intensity.

[0091] The experimental results are as Figure 9 shown. Fluorescence was generated only in the Cd 2+ solution and the intensity was more than 3 times that of other elements, while other ions did not have an obvious effect on the fluorescence intensity of the alkynyl-AIE probe. The results indicate that the AIE fluorescent probe molecular material prepared by the present invention has high selectivity for Cd and can avoid interference from other ions.

[0092] Example 8: Biotoxicity Experiment

[0093] Take the commercial AIE probe (purchased from Xi'an Ruixi Biotechnology Co., Ltd., customized AIE probe: CAS: 2566678-02-0) and the AIE fluorescent probe molecular material prepared in Example 1 for the biotoxicity experiment, which specifically includes the following steps: Select three groups of potted rice at the flowering stage (rice is mainly divided into 4 periods, namely: the green-recovery period, the tillering period, the flowering stage, and the filling period). At 5 pm, spray 10 mL of 10 μmol / L commercial AIE probe on the leaves of one group of potted rice, spray 10 mL of 10 μmol / L AIE fluorescent probe molecular material prepared in Example 1 on another group, and spray the same volume of water on one group as the control group CK. After culturing at room temperature for 7 days, detect its biomass and the SPAD value of rice leaves.

[0094] The experimental results are as Figure 10 and Figure 11 shown. Compared with the control group CK, the use of the commercial AIE and the AIE fluorescent probe molecular material prepared in the present invention has almost no effect on the growth of plants, and there is no obvious difference in the biomass of rice during the growth period. In addition, by comparing the SPAD values of rice leaves, there is no obvious decrease, indicating that the AIE fluorescent probe molecular material prepared in the present invention has no obvious change in the chlorophyll synthesis of rice. The results show that the AIE fluorescent probe molecular material prepared in the present invention has almost no biotoxicity and does not affect the photosynthesis of rice.

[0095] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An AIE fluorescent probe molecular material, characterized in that, The structural formula is as follows:

2. The preparation method of the AIE fluorescent probe molecular material according to claim 1, characterized in that, It includes the following steps: (1) Synthesis of compound 3: Mix 4-ethynylphthalic anhydride with Ac2O, add Et3N and tert-butyl acetoacetate, stir and react at 20 - 25 °C, then cool down to -10 - 10 °C and add concentrated hydrochloric acid, and stir and react again at 75 - 80 °C to obtain it; (2) Synthesis of compound 4: Mix compound A, Pd(PPh)3 and a basic substance, introduce an inert gas to expel oxygen, then add tetrahydrofuran hydrate and compound B and reflux at 78 - 80 °C to obtain it; (3) Synthesis of the AIE fluorescent probe molecular material: Dissolve compound 4 in acetic anhydride anhydride, then add compound 3 and piperidine, and carry out a mixing reaction at 58 - 62 °C to obtain it; The reaction formula of the above preparation method is:

3. The preparation method of the AIE fluorescent probe molecular material according to claim 2, characterized in that In the step (1), the mass-volume ratio of 4-ethynylphthalic anhydride, Ac2O, Et3N, tert-butyl acetoacetate and concentrated hydrochloric acid is (1.23 - 1.26) g : (4.2 - 4.5) g : (2 - 3) mL : (1.0 - 1.3) g : (3 - 5) mL.

4. The preparation method of the AIE fluorescent probe molecular material according to claim 2, wherein In the step (1), the stirring time is 18 - 20 h; the re-stirring time is 0.5 - 2 h.

5. The preparation method of the AIE fluorescent probe molecular material according to claim 2, wherein, In the step (2), the mass-volume ratio of compound A, Pd(PPh)3, the basic substance, tetrahydrofuran hydrate and compound B is (1.4 - 1.5) g : (0.08 - 0.1) g : (1 - 2) g : (20 - 25) mL : (0.9 - 1.0) g; the basic substance includes K2CO3; the volume ratio of tetrahydrofuran to water in the tetrahydrofuran hydrate is (8 - 10) : (1 - 2).

6. The preparation method of the AIE fluorescent probe molecular material according to claim 2, characterized in that, In the step (2), the reflux time is 10 - 15 h.

7. The preparation method of the AIE fluorescent probe molecular material according to claim 2, characterized in that, In the step (3), the mass-volume ratio of compound 4, acetic anhydride anhydride, compound 3 and piperidine is (300 - 400) mg : (10 - 12) mL : (200 - 300) mg : (0.85 - 0.88) mg.

8. The preparation method of the AIE fluorescent probe molecular material according to claim 2, characterized in that, In the step (3), the mixing reaction time is 5 - 8 h.

9. Use of the AIE fluorescent probe molecular material according to claim 1 in the preparation of a cadmium detection preparation.

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