CN110551196A - Abeta 42 aggregation-induced emission fusion and construction and application thereof - Google Patents

Abstract

The invention belongs to the fields of biotechnology and chemical engineering, and in particular relates to an _Aβ42 aggregation-induced luminescence fusion body and its construction method and application. The Aβ42 aggregation-induced light-emitting fusion is specifically EPB- _Aβ42 4F, which is obtained by replacing the phenylalanine _at the fourth position in the amino acid sequence of Aβ42 with _pAZF to obtain the _Aβ42 4F mutant Protein, and then perform bioorthogonal click chemistry reaction between AIE molecule EPB and mutant protein Aβ ₄₂ 4F to obtain EPB‑Aβ ₄₂ 4F, which can be applied to the screening of aggregation inhibitors.

Description

Aggregation-induced luminescent fusion of Aβ42 and its construction and application

Technical field:

The invention belongs to the fields of biotechnology and chemical engineering, and particularly relates to an amyloid beta protein 1-42 (Aβ ₄₂ ) aggregation-induced luminescent system and its construction, and applies the system to the screening of Aβ ₄₂ aggregation inhibitors. Specifically, the verification of the aggregation characteristics of _Aβ42 mutants containing unnatural amino acids, the traceless addition of aggregation-induced luminescent probes, and the application of the system to the screening of aggregation inhibitors.

Background technique:

Aggregation and misfolding of amyloid in cells and tissues to form toxic intermediates and amyloid fibrils may lead to various biological dysfunctions. These aggregation intermediates and fibers are speculated to be related to some neurodegenerative diseases and other conditions, such as Alzheimer's disease (AD), Parkinson's disease (Parkinson's disease, PD), type II diabetes (diabetes type II )Wait. A large number of studies have proved that the more toxic part is the oligomer or fibril intermediate, and the mature fiber may also play an important role. The development of effective amyloid aggregation inhibitors has become one of the effective means for the treatment of these diseases in the field of drug development. Therefore, sensitive and convincing methods for the detection of amyloid fibrils or intermediates, as well as a detailed understanding of the mechanisms of fibril formation at the molecular level, could facilitate the rational design of treatment regimens and prevent or slow the introduction of toxic amyloid fragments into the body. damage. In recent years, breakthroughs have been made in researches on the structure, morphology, and full length of amyloid fibrils. However, the molecular mechanism of amyloid aggregation and misfolding has not yet been ascertained.

The current screening methods for _Aβ42 aggregation inhibitors mainly include the following three aspects: (1) in vitro dye test screening. Amyloid dyes such as Thioflavin T (ThT) and Congo red (Congo red, CR) are sensitive in hydrophobic environments and can fluoresce when bound to the β-sheet structure of amyloid fibrils, so they are widely used in Research on aggregated proteins and high-throughput screening and identification of aggregation inhibitors. However, there are many limitations in the application of these dyes. For example, the morphology of early aggregates cannot be detected with these dyes, and because small molecule drugs may compete with these dyes for binding to amyloid fibrils, they cannot be used to precisely explore small Molecular drug-induced changes in fiber morphology. In addition, the properties of the dye may be affected by other components in the buffer solution, so it is easy to produce some false positives, etc. (2) Fluorescent protein marker screening. To visualize the aggregation process, some fluorescent proteins, such as green fluorescent protein GFP and β-amyloid _1-42 , were expressed as fusion proteins and used to screen for inhibitors of Aβ42 aggregation. However, due to the large molecular weight of GFP, while Aβ42 is only _4.5kDa , it may be difficult for _Aβ42 protein aggregation and folding to cause misfolding of the entire fusion protein, so the aggregation and folding process of GFP- _Aβ42 fusion protein may not be complete Aggregation process of _Aβ42 . In addition, the background fluorescence of dyes and fluorescent proteins may have a greater impact on the detection results. (3) Molecular design and virtual screening. With the rapid development of molecular simulation technology in recent years, molecular simulation technology, such as molecular dynamics simulation, molecular docking and pharmacophore model, has been widely used to analyze the mechanism of amyloid aggregation and misfolding and its inhibition, calculation The type of binding force between the inhibitor and amyloid, the determination of the inhibitor's action site, and the screening and design of the aggregation inhibitor. However, due to the unstable conformation of _Aβ42 aggregates, especially the three-dimensional structures of some of the most toxic oligomers have not yet been resolved, which severely restricts the application of molecular simulation technology in the development of _Aβ42 aggregation inhibitors.

Aggregation-Induced Emission (AIE) molecules do not fluoresce in the free state, but can emit strong fluorescence when forming aggregates or constrained conformation, so they can be used as the best biosensors for analyzing environmental and conformational changes . The principle of aggregation-induced luminescence may be that the internal rotation of the probe molecule is restricted by the environment, and a local excited state appears, which produces an abnormal photophysical effect. This conformational switch-dependent luminescence is not interfered by background fluorescence, making it applicable to the study of amyloid biokinetics. In recent years, several AIE molecules have been reported to detect and identify amyloid fibrils and explore the relationship between proteins, including TPE, TPE-TPP, BSPOTPE, EPB, etc. However, it is not perfect that these AIE molecules have disadvantages such as low sensitivity and low specificity in the application process.

In order to solve the current problems in the screening of _Aβ42 aggregation inhibitors, the present invention combines aggregation-induced luminescent technology to introduce unnatural amino acid ( _Unnatural Amino Acid, UAA) into Aβ42, and then use the azido group on its side chain to combine with the AIE molecule Through bio-orthogonal reaction coupling, AIE molecules are labeled on _Aβ42 at fixed points, so as to improve the specificity and sensitivity of AIE in detecting protein conformational transitions, obtain AIE- _Aβ42 fusion system and apply it to the inhibition of amyloid aggregation agent screening.

Invention content:

In order to achieve the above purpose, the present invention combines the screening of _Aβ42 non-natural amino acid mutants and bio-orthogonal reaction technology to construct an _Aβ42 aggregation-induced luminescent fusion, and apply it to the screening of _Aβ42 aggregation inhibitors.

One of the technical solutions provided by the present invention is an _Aβ42 aggregation-induced light-emitting fusion body. The _Aβ42 aggregation-induced light-emitting fusion body is specifically EPB- _Aβ42 4F, which is the fourth phenylpropanoid in the _Aβ42 amino acid sequence. Amino acid was replaced by p-Azido-L-phenylalanine (p-Azido-L-phenylalanine, pAZF) to obtain Aβ ₄₂ 4F mutant protein, and then the AIE molecule EPB and mutant protein Aβ ₄₂ 4F were subjected to bioorthogonal click chemistry reaction to obtain EPB-Aβ ₄₂ 4F, the chemical formula of the AIE molecule EPB is: C ₃₈ H ₄₄ N ₂ O ₂ Br ₂ , and the structural formula is shown in Figure 1-a.

The present invention also provides a construction method of EPB-Aβ ₄₂ 4F, specifically as follows:

(1) Using a solid-phase chemical synthesis method, _pAZF is used to replace the 4-phenylalanine in Aβ42 to obtain the _Aβ42 4F mutant protein;

(2) EPB and UAA mutant protein Aβ ₄₂ 4F were subjected to bioorthogonal click chemistry reaction by copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) to obtain EPB-Aβ ₄₂ 4F.

The present invention also provides the application of EPB-Aβ ₄₂ 4F in the screening of Aβ ₄₂ aggregation inhibitors, the method is as follows: add potential inhibitors to the EPB-Aβ ₄₂ 4F solution (dissolved in PBS), the excitation wavelength is 350nm, and the emission wavelength is in the range of 380nm-600nm If the fluorescence intensity is weaker than that of the EPB- _Aβ42 4F system without potential inhibitor, it is determined that the potential inhibitor is an _Aβ42 aggregation inhibitor.

When inhibitors are present in EPB- _Aβ424F solution (dissolved in PBS), the aggregation of _Aβ424F can be inhibited, resulting in no luminescence or weak luminescence in the screening system; and when there is no inhibitor in the solution, _Aβ424F aggregates rapidly , so that the screening system can emit strong fluorescence;

Preferably, the fluorescence intensity is detected in the range of EPB-Aβ ₄₂ 4F concentration of 20 μM, excitation wavelength of 350 nm, and emission wavelength of 380 nm-600 nm, preferably emission wavelength of 480 nm.

Beneficial effect:

EPB cannot produce fluorescence along with the aggregation of _Aβ42 , that is, EPB cannot be used alone for the study of the aggregation characteristics of _Aβ42 . The Aβ ₄₂ mutated by pAzF cannot cause EPB to aggregate to produce fluorescence during the aggregation process, while the EPB-Aβ ₄₂ 4F constructed by the present invention can induce the system to emit strong fluorescence when forming aggregates, thus providing a strong anti-inflammatory effect for Aβ ₄₂ aggregation inhibitors. Screening provides new methodological ideas.

Description of drawings:

Figure 1 EPB molecular structure and verification of its aggregation-induced luminescent properties

Among them, a. EPB molecular structure; b. EPB fluorescence value changes in different concentrations of glycerol;

Fig. 2 Flow chart of construction of _Aβ42 aggregation-induced luminescent screening system;

Fig. 3 Schematic diagram of protein synthesis of Aβ ₄₂ UAA mutant

Among them, a. unnatural amino acid pAzF structure and replacement principle; b. _pAzF replacement site in Aβ42 protein;

Figure 4 MALDI TOF mass spectrometry identification of Aβ ₄₂ 4F (a) and Aβ ₄₂ 10Y (b) mutant proteins;

Figure 5 ThT fluorescence staining to detect the aggregation properties of two mutant proteins, Aβ ₄₂ 4F and Aβ ₄₂ 10Y;

Fig. 6 Construction and verification of EPB-Aβ ₄₂ 4F screening system

Among them, a. Use CuAAC to connect EPB to Aβ ₄₂ 4F to construct EPB-Aβ ₄₂ 4F; b. UV-visible spectroscopic full-wavelength scanning to verify whether EPB-Aβ ₄₂ 4F is successfully constructed, and the scanning wavelength range is 200-600nm;

Figure 7 Application of EPB- _Aβ42 4F aggregation-induced luminescent screening system

Among them, a. Fluorescence scanning spectrum detection of fluorescence intensity of different concentrations of EPB-Aβ ₄₂ 4F; b. Fluorescence scanning spectrum detection of fluorescence intensity of 20 μM EPB-Aβ ₄₂ 4F samples added with different concentrations of EGCG;

Figure 8 Aggregation characteristics of different luminescent systems;

Figure 9 Validation results of small molecule inhibitors.

Detailed ways:

In order to make the purpose, technical solutions and advantages of this patent more clear, the following will further describe this patent in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the patent, not to limit the present invention.

The unnatural amino acid pAzF used in the present invention was purchased from MCE Company, and other reagents whose sources were not specified were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Aggregation-induced luminescent molecule EPB entrusts chemical synthesis formula for synthesis. All experimental operations that were not described in detail were performed according to laboratory manuals or existing techniques.

Tetraphenylethylene and its derivatives are the most studied AIE molecules due to their simple synthesis and easy modification. EPB molecule is a derivative of tetraphenylethylene.

The AIE molecule selected in the present invention is EPB, full name 2,20-(((2-(4-ethylphenyl)-2-phenylethene-1,1-diyl)bis(4,1-phenylene))bis(oxy))bis -(N,N,N-trimethylethanaminium)bromide, the molecule is a derivative of tetraphenylethylene, which has good aggregation-induced luminescent properties, and has been published in Hu, R.; Yap, H.K.; Fung, Y.H.; Wang, Y .; Cheong, W.L.; So, L.Y.; Tsang, C.S.; Lee, L.Y.; -protein interaction through bioorthogonal incorporation of a turn-onfluorescent probe into beta-lactamase. Mol. Biosyst. 2016, 12(12), 3544-3549. Those skilled in the art can synthesize EPB according to the relevant information such as the above literature or structural formula and chemical formula.

The unnatural amino acid selected in the present invention is an azido-containing phenylalanine structural analogue, p-Azido-L-phenylalanine (p-Azido-L-phenylalanine, pAZF). In order to reduce the influence of the replacement of UAA on the aggregation properties of the protein itself, according to the principle of structural similarity, the 4-phenylalanine in Aβ42 was selected as the replacement site of _pAzF . The pAzF mutant Aβ ₄₂ isoform protein, Aβ ₄₂ 4F, was obtained by solid-phase chemical synthesis. The aggregation properties of the mutant were analyzed by ThT fluorescent staining, and the results showed that the aggregation of Aβ ₄₂ 4F was less affected by the introduction of pAzF, so the Aβ ₄₂ 4F mutant protein was selected for subsequent experiments.

The present invention will be further explained through specific examples below, wherein the construction flow of the _Aβ42 aggregation-induced luminescence screening system is shown in FIG. 2 .

Example 1: Selection of aggregation-induced luminescence molecules and identification of luminescence characteristics

The aggregation-induced luminescent molecule EPB is a derivative of tetraphenylethylene, and its structural formula is shown in Figure 1-a. In order to verify the aggregation-inducing properties of the AIE molecule, the same concentration of EPB (2 μM) was used to detect the change of its fluorescence value in different concentrations of glycerol. The glycerol concentrations were 0%, 20%, 40%, 60%, 70%, buffer as PBS, pH 7.4. After fully mixing, detect the fluorescence under the conditions of excitation wavelength 350nm and emission wavelength 460nm.

The experimental results are shown in Figure 1-b. The fluorescence intensity of EPB solution gradually increases with the increase of glycerol concentration, which proves that it does have the characteristics of aggregation-induced luminescence.

Example 2: Study on the Synthesis and Aggregation Properties of _Aβ42 UAA Mutants

(1) Selection of UAA replacement sites in UAA and _Aβ42 sequences

The unnatural amino acid selected in the present invention is pAzF, and its structure is similar to phenylalanine and tyrosine, so according to the principle of structural similarity, the 4-position phenylalanine and the 10-position tyrosine in Aβ42 are selected as _pAzF . Replacement sites, as shown in Figure 3-a, 3-b.

(2) Synthesis of _Aβ42 UAA mutant protein

Two Aβ ₄₂ mutant proteins (Aβ ₄₂ 4F and Aβ ₄₂ 10Y) in which the 4th and 10th amino acids of Aβ ₄₂ were replaced by pAzF were synthesized by solid-phase chemical synthesis, and the synthesis was entrusted to Jill Biochemical Shanghai Co., Ltd.

The amino acid sequence of the wild-type _Aβ42 protein is as follows:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID No. 1).

The molecular weight of the mutant protein was further confirmed by MALDI TOF mass spectrometry, and the mass spectrogram is shown in Figure 4. The results of mass spectrometry showed that the molecular weight of Aβ ₄₂ 4F was 4555.15, and the molecular weight of Aβ ₄₂ 10Y was 4539.15, which were consistent with the theoretical values, thus proving that the obtained protein was the pAzF mutant protein.

In situ culture was used for ThT fluorescent staining experiments. The above two proteins were prepared into protein solutions, mixed with ThT solution at a concentration ratio of 1:1, and placed at 37°C for in situ culture. Fluorescence intensity was detected at fixed time, and the detection conditions were excitation wavelength 440nm and emission wavelength 480nm. The test results are shown in Figure 5. It can be seen from the figure that the fluorescence intensity of wild-type Aβ ₄₂ increases gradually with the increase of culture time, and basically enters a stable phase after 24 hours; among the two mutant proteins, the fluorescence intensity of Aβ ₄₂ 4F also increases with After 24 hours, it basically entered a stable period, and then the fluorescence intensity decreased to a certain extent; the fluorescence intensity of Aβ ₄₂ 10Y was always low. The above results prove that when _pAzF replaces the 4-phenylalanine, it has little effect on the aggregation of the protein itself, and when the 10-position tyrosine is replaced, the mutant protein basically loses the aggregation. experiment of.

The above protein and ThT buffers use PBS buffer, pH 7.4.

Example 3: Construction of EPB-Aβ ₄₂ 4F aggregation-induced luminescent screening system

In the present invention, copper-catalyzed azide-terminated alkyne cycloaddition reaction (CuAAC) is used to link EPB with Aβ ₄₂ 4F to construct an EPB-Aβ ₄₂ 4F screening system, as shown in Figure 6-a. 1mL reaction system, the reaction conditions and the amount of reagents used are as follows:

last joined

Then make up 1 mL with PBS. Mix and shake gently, and react at 4°C for 8 hours (the above buffers and reagents are all prepared with PBS). After the reaction is completed, use a dialysis bag or a desalting column to desalt the reaction system to remove excess ions, etc., and keep the dialysis or desalting process at a low temperature as much as possible.

In order to verify whether the above system was successfully constructed, a UV-visible spectrophotometer was used to scan in the wavelength range of 200-600nm to compare the changes in the absorbance of the target protein before and after EPB labeling. The results are shown in Figure 6-b. It is obvious from the figure that the absorbance of EPB-Aβ ₄₂ 4F complex after EPB labeling in the wavelength range of 250–350nm is significantly larger than the fluorescence value of unlabeled Aβ ₄₂ 4F, which is caused by the π-π bond on EPB The increase in absorbance proves that EPB is successfully linked to Aβ ₄₂ 4F.

Example 4: Application of EPB-Aβ ₄₂ 4F aggregation-induced luminescence screening system

In order to identify the stability of the screening system and whether it can really be applied to the screening of Aβ ₄₂ aggregation inhibitors, different concentrations of EPB-Aβ ₄₂ 4F were detected by fluorescence scanning, and small molecules known to have Aβ ₄₂ aggregation inhibitory effects were selected. The inhibitor EGCG was verified, and the function evaluation of the screening system was carried out.

The EPB-Aβ ₄₂ 4F was prepared into different concentrations with PBS buffer solution, and the fluorescence was detected in the range of excitation wavelength 350nm and emission wavelength 380nm-600nm. The results are shown in Figure 7-a, when the emission wavelength is 480nm, EPB-Aβ ₄₂ 4F has the maximum fluorescence intensity, and the fluorescence intensity gradually increases with the increase of the sample concentration. Based on the comprehensive analysis of cost and luminescence, 20 μM is the best experimental concentration.

EGCG powder was prepared in PBS buffer to a stock solution with a concentration of 100 μM. The final concentration of EPB-Aβ ₄₂ 4F solution was 20 μM. EGCG and EPB-Aβ ₄₂ 4F solution with final concentrations of 5, 10, 15 and 20 μM were placed in a 96-well cell culture plate to prepare a 200 μL system. After fully mixing, scan and detect with a fluorescence spectrophotometer, the detection conditions are: excitation wavelength 350nm, emission wavelength 380nm-600nm. And the sample with the same final concentration of EPB-Aβ ₄₂ 4F without adding EGCG was used as the control group.

The experimental results are shown in Figure 7-b, the fluorescence intensity of the EPB-Aβ ₄₂ 4F sample without EGCG was significantly higher than that of the sample with EGCG added. And with the increase of EGCG concentration, the fluorescence intensity of EPB-Aβ ₄₂ 4F samples decreased gradually. It is proved that it is because EGCG inhibits the aggregation of the target protein that the aggregation-induced luminescent system is in a free state, so the fluorescence value is low. When EGCG is not added, the target protein forms aggregates, and finally induces the system to emit strong fluorescence. In addition, the analysis results show that within this scanning range, the fluorescence value is the highest when the emission wavelength is about 480nm, which can be used as a detection condition for rapid screening of small molecule drugs in the later stage.

In addition, the present invention also uses other small molecule inhibitors fast green, congo red, caffeic acid and braziliann which have _Aβ42 aggregation inhibitory effect for verification, and the results are shown in FIG. 9 . When the EPB-Aβ ₄₂ 4F with a final concentration of 25 μM was added with the above four small molecule inhibitors at a final concentration of 25 μM, the fluorescence intensity of the system decreased to varying degrees, and the blank control group (25 μM EPB-Aβ ₄₂ 4F ) 11.28%, 52.12%, 56.5% and 74.99% of the fluorescence intensity. Therefore, it can be proved that the EPB-Aβ ₄₂ 4F system can be used to screen Aβ ₄₂ aggregation inhibitors.

The research of embodiment 5 aggregation characteristics

(1) Construct the luminescent system ① Aβ ₄₂ and EPB solution are mixed at equal concentrations, and the final concentration is 25 μM; ② Aβ ₄₂ 4F is mixed with EPB at equal concentrations, and the final concentration is 25 μM; ③ 25 μM EPB-Aβ ₄₂ 4F

(2) Using a fluorescence spectrophotometer to scan and detect the above systems respectively, the detection conditions are: the excitation wavelength is 350nm, and the emission wavelength is 380nm-600nm.

The results are shown in Figure 8. From Figure 8, it can be known that EPB cannot produce fluorescence along with the aggregation of _Aβ42 , that is, EPB alone cannot be used for the study of the aggregation characteristics of _Aβ42 . The Aβ ₄₂ mutated by pAzF cannot cause the EPB to aggregate to produce fluorescence during the aggregation process, while the EPB-Aβ ₄₂ 4F constructed by the present invention can induce the system to emit strong fluorescence when forming aggregates.

To sum up, the aggregation-induced luminescence screening system constructed in the present invention can be applied to the screening of _Aβ42 aggregation inhibitors. When the inhibitor exists in the solution, the aggregation of _Aβ42 4F can be inhibited, resulting in the screening system not emitting light; and When there is no inhibitor in the solution, Aβ ₄₂ 4F aggregates rapidly, so that the screening system can emit strong fluorescence.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, without departing from the concept of the patent, several modifications, combinations and improvements can be made to the above-mentioned embodiments, all of which belong to the protection scope of the patent. Therefore, the scope of protection of this patent should be determined by the claims.

sequence listing

<110> Tianjin University of Science and Technology

<120> Aggregation-induced luminescent fusion of Aβ42 and its construction and application

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<141> 2019-08-01

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Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

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Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

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Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

Claims (6)

1. An _Aβ42 aggregation-induced light-emitting fusion body, characterized in that, the _Aβ42 aggregation-induced light-emitting fusion body is specifically EPB- _Aβ42 4F, which replaces the 4th phenylalanine in the _Aβ42 amino acid sequence with Azidophenylalanine pAZF was used to obtain the Aβ ₄₂ 4F mutant protein, and then the AIE molecule EPB was subjected to bioorthogonal click chemistry reaction with the mutant protein Aβ ₄₂ 4F to obtain EPB-Aβ ₄₂ 4F; The amino acid sequence of _Aβ42 is shown in the sequence listing SEQ ID No.1; The chemical formula of the EPB is: C ₃₈ H ₄₄ N ₂ O ₂ Br ₂ . 2. The construction method of _Aβ42 aggregation-induced luminescent fusion body according to claim 1, characterized in that, it is as follows: (1) Using a solid-phase chemical synthesis method, _pAZF is used to replace the 4-phenylalanine in Aβ42 to obtain the _Aβ42 4F mutant protein; (2) EPB was subjected to bioorthogonal click chemistry reaction with mutant protein Aβ ₄₂ 4F by copper-catalyzed azide-terminal alkyne cycloaddition reaction to obtain EPB-Aβ ₄₂ 4F. 3. The method for constructing _Aβ42 aggregation-induced luminescence fusion body as claimed in claim 2, characterized in that EPB is connected to Aβ42 4F by copper-catalyzed azide-terminal alkyne cycloaddition reaction to construct EPB- _{Aβ42 4F} screening The method of the system is as follows: 1mL reaction system, the reaction conditions and the amount of reagents used are as follows: Then make up 1mL with PBS; react at 4°C for 8h, after the reaction is completed, use a dialysis bag or a desalting column to desalt the reaction system. 4. The application of the _Aβ42 aggregation-induced light-emitting fusion body according to claim 1. 5. The application according to claim 4, characterized in that, it is specifically applied to screening _Aβ42 aggregation inhibition, the method is as follows: add potential inhibitors to the EPB- _Aβ42 4F solution, the excitation wavelength is 350nm, and the emission wavelength is in the range of 380nm-600nm If the fluorescence intensity is weaker than that of the EPB- _Aβ42 4F system without potential inhibitor, it is determined that the potential inhibitor is an _Aβ42 aggregation inhibitor. 6 . The application according to claim 5 , wherein the detection is performed under the conditions of EPB-Aβ ₄₂ 4F concentration of 20 μM, excitation wavelength of 350 nm, and emission wavelength of 480 nm.