A microenvironment-sensitive fluorescent dye for detecting Aβ fibrils and a preparation method thereof

Abstract

The present invention relates to an improvement in the detection technology of Aβ fibrils, in particular to a microenvironment-sensitive fluorescent dye for detecting Aβ fibrils and a preparation method thereof; aiming at the deficiencies of commercially available dyes for detecting Aβ fibrils in terms of poor specificity, low binding affinity, and poor signal-to-noise ratio, a novel class of D-π-A type benzothiazine-based boron fluoride dyes that are sensitive to both the polarity and viscosity of the microenvironment of Aβ fibrils is invented; the boron fluoride fluorescent dye synthesized in the present invention can change parameters such as its fluorescence wavelength and fluorescence quantum yield according to the changes in the polarity and viscosity of Aβ fibrils, and can meet the requirements for detecting Aβ fibrils with high sensitivity and high signal-to-noise ratio. In addition, the boron fluoride fluorescent dye synthesized in the present invention has a good linear configuration and is easy to enter the hydrophobic cavity of Aβ fibrils, thereby improving the binding affinity and selectivity of the dye to Aβ fibrils.

Description

Technical Field

[0001] The present invention relates to an improvement in the detection technology of Aβ fibrils, in particular to a microenvironment-sensitive fluorescent dye for detecting Aβ fibrils and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly population. With the increasing aging of the global population, the incidence rate shows an increasing trend. The World Alzheimer Report 2018 shows that there are approximately 50 million AD patients globally, and it is estimated that by 2050, the number of AD patients will increase to 150 million, which will bring a heavy burden. The pathogenesis of AD is extremely complex, and early identification and diagnosis are still effective measures to prevent the occurrence and development of AD. β-amyloid (Aβ) fibrils formed by protein misfolding and abnormal aggregation in the brain are considered to be the main pathological biomarkers of AD (Villemagne, V.L., Nat. Rev. Neurol., 2018, 14, 446 - 446; Ni, J., Chem 2018, 4, 807 - 820; Riek, R., Nature 2016, 539, 227 - 235.), and the detection of Aβ fibrils is of great significance for the early diagnosis of AD.

[0003] Fluorescent dyes with biorecognition functions have the advantages of high sensitivity, fast response speed, simple technology, non-invasiveness, and non-radioactivity (Li, L., Chem. Sci., 2021, 12, 3308 - 3313; Zhang, K.Y., Chem. Rev., 2018, 118, 1770 - 1839; Chen, H., Acc. Chem. Res., 2017, 50, 1410 - 1422.). Designing and synthesizing fluorescent dyes that can recognize Aβ fibrils in vitro with high selectivity, high specificity, and high signal-to-noise ratio is the primary condition for realizing in vivo imaging of Aβ fibrils and then diagnosing AD patients.

[0004] The formation of Aβ fibrils is often accompanied by changes in the protein microenvironment. On the one hand, the hydrophobic amino acids of the protein are disrupted and exposed in the misfolded protein, resulting in a decrease in polarity. On the other hand, after the misfolded proteins aggregate into insoluble aggregates and further form Aβ fibrils, the viscosity increases (Aliyan, A., Chem. Rev., 2019, 119, 11819 - 11856; Ye, S., Acc. Chem. Res., 2022, 55, 381 - 390; Chiti, F., Annu. Rev. Biochem., 2017, 86, 27 - 68.). Therefore, Aβ fibrils can be detected by designing environment-sensitive fluorescent dyes to sense the changes in protein polarity and viscosity.

[0005] However, most of the environmentally sensitive fluorescent dyes reported currently are designed to be sensitive to either the viscosity or polarity of the protein microenvironment, and there are relatively few fluorescent dyes with both characteristics. For example, the commercially available Thioflavin T (THT, the gold standard dye) is the earliest fluorescent dye specifically for detecting Aβ fibrils (Levine, H., Protein Sci., 1993, 2, 404-410.). Before binding to the protein, the free rotation of the C-C bond within the molecule causes the excited-state energy to dissipate through non-radiative decay, forming a low-intensity fluorescence emission; after interacting with Aβ, the rotation is significantly restricted, preventing non-radiative decay of the excited state and activating fluorescence radiation. This mechanism of action is a typical dye molecule sensitive to viscosity. The research group of Professor Hongbing Fu used pyridinium salt as an electron acceptor and introduced carbazole rings bridged by thiophene and thiadiazole as electron donors. The synthesized dyes showed sensitivity to the polarity of the protein microenvironment (Li, L., Chem. Sci., 2021, 12, 3308-3313; Lv, Z., Nano. Research 2020, 13, 2556-2563.). Although the above fluorescent dyes sensitive to viscosity or polarity have achieved good results in the detection of Aβ fibrils, however, there is still a large room for improvement in their sensitivity and signal-to-noise ratio. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of a microenvironment-sensitive fluorescent dye for detecting Aβ fibrils, and to realize the preparation of a novel D-π-A type benzothiazine fluoroborate dye that is sensitive to both the polarity and viscosity of Aβ fibrils.

[0007] The above technical object of the present invention is achieved through the following technical solutions: A microenvironment-sensitive fluorescent dye for detecting Aβ fibrils, the molecular structural formula is shown in Formula I:

[0008] Wherein:

[0009] The above technical object of the present invention is achieved through the following technical solutions: A preparation method of a microenvironment-sensitive fluorescent dye for detecting Aβ fibrils, the reaction formula is as follows

[0010] Wherein:

[0011] It includes the following steps,

[0012] Step 1: Dissolve 3-methyl-2H-benzo[b][1,4]thiazine in anhydrous THF. Under a nitrogen atmosphere at 0 °C, add NaH to the mixture. After stirring at 0 °C for 0.5 - 1 h, add an anhydrous THF solution of methyl 5-bromo-2-thiophenecarboxylate to the reaction mixture. After the addition is complete, stir the reaction mixture at room temperature for 8 h. After the reaction is complete, quench the excess NaH, then perform separation. The separated organic phase is dried by filtration. Collect the filtrate and remove the solvent. Finally, obtain compound (i) through purification;

[0013] Step 2: Dissolve compound (i), 4-R-phenylboronic acid, Cs2CO3, and Pd(PPh3)4 in a mixture of 1,4-dioxane and H2O; then reflux under a nitrogen atmosphere for 3 - 5 h; subsequently, perform separation again. The separated organic phase is dried by filtration. Collect the filtrate and remove the solvent. Finally, obtain compound (ii) through purification;

[0014] Step 3: At room temperature, add N,N-diisopropylethylamine to a dry CH2Cl2 solution of (ii); after 1 - 2 h, add boron trifluoride diethyl ether complex. Stir the reaction mixture at room temperature for 8 h; quench the reaction mixture with an ice-water mixture, then perform separation. The separated organic phase is dried by filtration. Collect the filtrate and remove the solvent. Finally, obtain compound (I) through purification.

[0015] Preferably, the molar ratio of 3-methyl-2H-benzo[b][1,4]thiazine to NaH to methyl 5-bromo-2-thiophenecarboxylate is 1:3:1 - 1:4:1.5.

[0016] Preferably, the molar ratio of compound (i) to 4-R-phenylboronic acid, Cs2CO3, and Pd(PPh3)4 is 1:2:4:0.2 - 1:2.5:5:0.5.

[0017] Preferably, the volume ratio of 1,4-dioxane to H2O in Step 2 is 8:1 - 11:1.

[0018] Preferably, the reflux temperature in Step 2 is 80 - 120 °C.

[0019] Preferably, the molar ratio of compound (ii) to N,N-diisopropylethylamine and boron trifluoride diethyl ether is 1:5:7 - 1:10:5:14.5.

[0020] Preferably, the drying in Step 1, Step 2, and Step 3 is carried out using anhydrous sodium sulfate for drying and standing for 5 - 10 minutes, and the solvent is removed using a rotary evaporator.

[0021] Preferably, the purification in Step 1, Step 2, and Step 3 is carried out using a chromatographic column.

[0022] Preferably, in the first, second, and third steps, silica gel filler is used as the stationary phase and dichloromethane - petroleum ether eluent is used as the mobile phase in the column chromatography for purification, where the volume ratios of dichloromethane to petroleum ether are 1:4, 1:1, and 1:4 respectively.

[0023] In summary, the present invention has the following beneficial effects: In view of the deficiencies of commercially available dyes for detecting Aβ fibrils, such as poor specificity, low binding affinity, and poor signal-to-noise ratio, the present invention has invented a novel class of D-π-A type benzothiazine fluoroboron dyes that are sensitive to both the polarity and viscosity of the microenvironment of Aβ fibrils; the synthesized fluoroboron fluorescent dyes of the present invention can change parameters such as their fluorescence wavelength and fluorescence quantum yield according to the changes in the polarity and viscosity of Aβ fibrils, and can meet the requirements for highly sensitive and high signal-to-noise ratio detection of Aβ fibrils. In addition, the synthesized fluoroboron fluorescent dyes of the present invention have a good linear configuration and can easily enter the hydrophobic cavity of Aβ fibrils, thereby improving the binding affinity and selectivity between the dye and Aβ fibrils. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 1H NMR spectrum of the prepared compound (I-1);

[0025] Figure 2 19F NMR spectrum of the prepared compound (I-1);

[0026] Figure 3 Fluorescence emission spectra of the prepared compound (I-1) in different polar solvents;

[0027] Figure 4 Fluorescence emission spectra of the prepared compound (I-1) in different viscous solvents;

[0028] Figure 5 TEM image of Aβ42 fibrils;

[0029] Figure 6 Fluorescence response performance test chart of the prepared compound (I-1) to Aβ42 fibrils;

[0030] Figure 7 Concentration-dependent saturation curve fitting chart of the prepared compound (I-1) to Aβ42 fibrils;

[0031] Figure 8 Signal-to-noise ratio evaluation chart of the prepared compound (I-1) to Aβ42 fibrils;

[0032] Figure 9 Selectivity evaluation chart of the prepared compound (I-1) to Aβ42 fibrils;

[0033] Figure 10 It is the molecular structural formula diagram of the present invention. Specific embodiments

[0034] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0035] When the present invention is specifically implemented, the following steps can be adopted.

[0036] (1) Weigh 3.34 mmol of 3-methyl-2H-benzo[b][1,4]thiazine and dissolve it in 15 mL of anhydrous THF under a nitrogen atmosphere at 0 °C. Then quickly add 13.36 mmol of NaH to the mixture. After stirring at 0 °C for 0.5 hour, add an anhydrous tetrahydrofuran solution of 4.00 mmol of methyl 5-bromo-2-thiophenecarboxylate to the reaction mixture. After the addition is completed, the reaction mixture is stirred overnight at room temperature. Subsequently, quench the excess NaH with an ice-water mixture, extract it three times with 45 mL of CH2Cl2, wash the organic layer three times with 30 mL of water and dry it with anhydrous Na2SO4. After the solvent is concentrated, purify it with a chromatographic column (CH2Cl2:PE = 1:4) to obtain a yellow product (i-1) with a yield of 15%;

[0037] (2) Weigh 0.40 mmol of (i-1), 1.00 mmol of 4-(N,N-dimethylamino)phenylboronic acid, 2.00 mmol of Cs2CO3 and 0.02 mmol of Pd(PPh3)4 and dissolve them in a mixture of 1,4-dioxane and H2O Then reflux for 5 h under a nitrogen atmosphere. After the reaction is completed, extract it three times with 45 mL of CH2Cl2, wash the organic layer three times with 30 mL of water and dry it with anhydrous Na2SO4. After the solvent is concentrated, purify it with a chromatographic column (CH2Cl2:PE = 1:1) to obtain a product (ii-1) with a yield of 71%;

[0038] (3) 0.20 mmol of (ii-1) was weighed and dissolved in dry CH2Cl2 (10 mL), and 2.00 mmol of N,N-diisopropylethylamine was added. After 2 hours, 2.90 mmol of boron trifluoride diethyl ether was added. The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was quenched with an ice-water mixture and extracted three times with 30 mL of CH2Cl2. The organic layer was washed three times with 30 mL of water and dried over anhydrous Na2SO4. After concentration of the solvent, purification was carried out by column chromatography (CH2Cl2:PE = 1:4) to obtain the final product (I-1) with a yield of 64%.

[0039] The above preparation process is as follows:

[0040]

[0041] The nuclear magnetic resonance (NMR) test was performed on the compound prepared in this example. Figure 1 1H-NMR spectrum of the prepared compound (I-1). It can be proved from the 1H-NMR spectrum of the compound (I-1) that the final product was obtained.

[0042] 1 H-NMR (400 MHz, DMSO-d6, ppm): δ = 8.06 (d, J = 4.0 Hz, 1H, Ph-H), 7.75 (d, J = 8.1 Hz, 1H, Ph-H), 7.66 (d, J = 8.7 Hz, 2H, Ph-H), 7.58 (d, J = 4.0 Hz, 1H, Ph-H), 7.49 (d, J = 7.5 Hz, 1H, Ph-H), 7.33 (t, J = 7.7 Hz, 1H, Ph-H), 7.23 (t, J = 7.4 Hz, 1H, Ph-H), 6.78 (d, J = 8.7 Hz, 2H, Ph-H), 6.65 (s, 1H, alkene-H), 3.81 (s, 2H, CH2), 3.00 (s, 6H, 2CH3).

[0043] The nuclear magnetic resonance (NMR) test was performed on the compound prepared in this example. Figure 2 19F-NMR spectrum of the compound (I-1) prepared in Example 1. It can be further proved from the 19F-NMR spectrum of the compound (I-1) that the final product was obtained.

[0044] 19 19F NMR (376 MHz, DMSO-d6, ppm): δ = -130.21 (s).

[0045] Polarity sensitivity test of the fluorescent dye (I-1):

[0046] Prepare a dimethyl sulfoxide stock solution of fluorescent dye (I-1) with a concentration of 1 mM. Pipette 20 μL of the (I-1) stock solution into 2-mL volumetric flasks numbered ①, ②, ③, ④, and ⑤, and dilute to the mark with 1,4-dioxane, ethyl acetate (EtOAc), ethanol (EtOH), dimethyl sulfoxide (DMSO), and water, respectively, to obtain test solutions with a final concentration of 1×10 -5 M. Measure their fluorescence emission spectra as shown in Figure 3 a, and the normalized graph as shown in Figure 3 b. As can be seen from Figure 3 , as the polarity of the solvent increases, the maximum emission wavelength of fluorescent dye (I-1) redshifts from 600 nm in 1,4-dioxane to 720 nm in water, and the fluorescence intensity gradually weakens due to the TICT effect, indicating that the dye has strong polarity sensitivity. The weak initial fluorescence in water helps to achieve the "off-on" mode detection of biomarker Aβ fibrils by the dye.

[0047] Viscosity sensitivity test of fluorescent dye (I-1):

[0048] Prepare a dimethyl sulfoxide stock solution of fluorescent dye (I-1) with a concentration of 1 mM. Pipette 20 μL of the (I-1) stock solution into 2-mL volumetric flasks numbered ①, ②, ③, ④, ⑤, ⑥, and ⑦, and dilute to the mark with ethylene glycol, V 甘油 :V 乙二醇 =1:9, V 甘油 :V 乙二醇 =2:8, V 甘油 :V 乙二醇 =3:7, V 甘油 :V 乙二醇 =4:6, V 甘油 :V 乙二醇 =5:5, and V 甘油 :V 乙 two 醇 =6:4 mixed solvents to obtain test solutions with a final concentration of 1×10 -5 M, and the volume fractions of glycerol are 0%, 10%, 20%, 30%, 40%, 50%, and 60%, respectively. Measure their fluorescence emission spectra as shown in Figure 4 a, and plot the fluorescence intensity at the maximum emission wavelength as shown in Figure 4 b. As can be seen from Figure 4 , as the viscosity of the solvent increases, the emission intensity of fluorescent dye (I-1) gradually increases, indicating that the dye has strong viscosity sensitivity. The weak initial fluorescence helps to achieve the "off-on" mode detection of biomarker Aβ fibrils by the dye.

[0049] Response performance test of fluorescent dye (I-1) to Aβ42 fibrils:

[0050] Configuration method of Aβ42 fibrils: Weigh 1.0 mg of Aβ protein and dissolve it in 2215 μL of Tris buffer solution (pH = 7.4). Incubate it in a constant temperature environment at 37 °C for 7 days. The final concentration of Aβ42 is 100 μM. Use transmission electron microscopy (TEM) to confirm the morphology of Aβ42 as Figure 5 shown. It can be seen from the figure that the Aβ protein has formed a fibrous morphology after incubation.

[0051] Prepare a dimethyl sulfoxide stock solution of fluorescent dye (I-1) with a concentration of 1 mM. Respectively transfer 2 μL of the (I-1) stock solution into 2 mL volumetric flasks numbered ①, ②, ③, ④, ⑤, ⑥, ⑦, ⑧, ⑨, and ⑩. Then add 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 μL of the above Aβ42 stock solution to each of them, and make up the volume with PBS buffer solution (pH = 7.4). The final concentrations of Aβ42 fibrils are 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 μM respectively. Test their fluorescence emission spectra as Figure 6 shown in a. The ratio of the fluorescence intensity at the maximum emission wavelength of 620 nm to the fluorescence intensity at 720 nm in water is as Figure 6 shown in b. It can be seen from Figure 6 that the dye (I-1) has good response performance to Aβ42 fibrils. As the concentration of Aβ42 fibrils increases, the fluorescence of the dye gradually increases and has a good linear relationship, indicating that the dye (I-1) can achieve quantitative detection of Aβ42 fibrils within a certain range.

[0052] Binding affinity test of fluorescent dye (I-1) to Aβ42 fibrils:

[0053] Prepare a dimethyl sulfoxide test solution of fluorescent dye (I-1) with a concentration of 100 μM. Respectively transfer 60 μL of the Aβ42 test solution (100 μM) into 2 mL volumetric flasks numbered ①, ②, ③, ④, ⑤, ⑥, ⑦, ⑧, ⑨, and ⑩. Then add 0, 1, 2, 3, 4, 8, 12, 14, 16, and 20 μL of the above (I-1) test solution (100 μM) to each of them, and make up the volume with PBS buffer solution (pH = 7.4). The final concentrations of (I-1) are 0, 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, 0.7, 0.8, and 1.0 μM respectively. Test their fluorescence emission spectra, and fit the concentration-dependent saturation curve with the formula Y = B max *X / (K d +X) as Figure 7 shown. The dissociation constant K of the fluorescent dye (I-1)d It is 25 nM, far lower than the dissociation constant of 270 nM of the commercially available dye ThT, indicating that the dye ((I-1)) of the present invention has a strong binding affinity for Aβ42 fibrils.

[0054] Evaluation of the signal-to-noise ratio of the fluorescent dye (I-1) for Aβ42 fibrils:

[0055] Prepare dimethyl sulfoxide test solutions of the fluorescent dye (I-1) with a concentration of 100 μM and the commercially available dye THT respectively. Pipette 20 μL of the fluorescent dye (I-1) and the commercially available dye THT into 2-mL volumetric flasks numbered ① and ② respectively, add 60 μL of the Aβ42 fibril test solution with a concentration of 100 μM to each of them, make up the volume with PBS buffer solution (pH = 7.4), and use the blank solution without adding the Aβ42 fibril test solution as a control to measure their fluorescence emission spectra as Figure 8 shown in a, and the ratio of the maximum emission wavelength intensities corresponding to the fluorescent dye (I-1) and the commercially available dye THT before and after adding Aβ42 fibrils is as Figure 8 shown in b. It can be seen from the figure that compared with the commercially available dye THT, the dye (I-1) of the present invention shows a higher signal-to-noise ratio and can achieve highly sensitive detection of Aβ42 fibrils.

[0056] Selective test of the fluorescent dye (I-1) for Aβ42:

[0057] Select potential competing substances for Aβ42 fibrils (including amino acids, enzymes, serum markers, and certain metabolites), and prepare their test concentration to be 100 μM. Pipette 20 μL of the fluorescent dye (I-1) with a concentration of 100 μM into 2-mL volumetric flasks numbered ①, ②, ③, ④,⑤,⑥,⑦,⑧,⑨,⑩, and respectively, add 60 μL of the Aβ42 fibril test solution with a concentration of 100 μM and the potential competing substance test solution to each of them, make up the volume with PBS buffer solution (pH = 7.4), measure their fluorescence emission spectra and plot the graph as Figure 9 shown. It can be seen from the figure that the dye (I-1) of the present invention does not show any obvious fluorescence response to the above potential competing substances, indicating that the dye (I-1) has high selectivity for Aβ42 fibrils.

[0058] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microenvironment-sensitive fluorescent dye for detecting Aβ fibrils, characterized in that, The molecular structural formula is as shown in Formula I: Wherein:

2. A preparation method of a microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 1, characterized in that: Step 1. Dissolve 3-methyl-2H-1,4-benzothiazine in anhydrous THF. Under a nitrogen atmosphere at 0 °C, add NaH to the mixture. After stirring at 0 °C for 0.5 - 1 h, add an anhydrous tetrahydrofuran solution of methyl 5-bromo-2-thiophenecarboxylate to the reaction mixture. After the addition is complete, stir the reaction mixture at room temperature for 8 h. After the reaction is completed, quench the excess NaH, then carry out separation. The separated organic phase is dried and filtered. Collect the filtrate and remove the solvent. Finally, obtain compound (i) through purification. The structure is Step 2: Dissolve compound (i), 4-R-phenylboronic acid, Cs2CO3 and Pd(PPh3)4 in a mixture of 1,4-dioxane and H2O; then reflux for 3-5 h under a nitrogen atmosphere; Subsequently, separation is carried out. The separated organic phase is dried and filtered. The filtrate is collected and the solvent is removed. Finally, compound (ii) with the structure of Step 3: At room temperature, add N,N-diisopropylethylamine to the dry CH2Cl2 solution of (ii); after 1-2 hours, add boron trifluoride diethyl ether complex, and stir the reaction mixture at room temperature for 8 hours; Quench the reaction mixture with an ice-water mixture, then separate it. The separated organic phase is dried and filtered, the filtrate is collected and the solvent is removed, and finally compound (I) is obtained through purification.

3. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The molar ratio of the 3-methyl-2H-1,4-benzothiazine, NaH and methyl 5-bromo-2-thiophenecarboxylate is 1:3:1 to 1:4:1.

5.

4. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The molar ratio of compound (i), 4-R-phenylboronic acid, Cs2CO3 and Pd(PPh3)4 is 1:2:4:0.2 to 1:2.5:5:0.

5.

5. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The volume ratio of 1,4-dioxane and H2O in Step 2 is 8:1 to 11:

1.

6. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The reflux temperature in Step 2 is 80-120 °C.

7. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The drying in Step 1, Step 2 and Step 3 is carried out by drying with anhydrous sodium sulfate and standing for 5-10 minutes, and the solvent is removed by a rotary evaporator.

8. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, The purification in Step 1, Step 2 and Step 3 is carried out by using a chromatographic column.

9. The preparation method of the microenvironment-sensitive fluorescent dye for detecting Aβ fibrils according to claim 2, characterized in that, In the column chromatography method in Step 1, Step 2 and Step 3, silica gel filler is used as the stationary phase, and dichloromethane-petroleum ether eluent is used as the mobile phase for column chromatography purification, wherein the volume ratios of dichloromethane to petroleum ether are 1:4, 1:1 and 1:4 respectively.

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