CN110551196B - Abeta (beta-amyloid peptide)42Aggregation-induced emission fusion and construction and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology and chemical engineering, and particularly relates to Abeta₄₂Aggregation-induced emission fusion and a construction method and application thereof. Said A beta₄₂The aggregation-induced emission fusion body is specifically EPB-Abeta₄₂4F is A.beta.₄₂The phenylalanine at position 4 in the amino acid sequence is replaced by para-azidophenylalanine pAZF to obtain Abeta₄₂4F mutant protein, and then the AIE molecule EPB and the mutant protein Abeta₄₂4F is subjected to bio-orthogonal click chemical reaction to obtain EPB-Abeta₄₂4F, can be applied to screening of aggregation inhibitors.

Description

Abeta (beta-amyloid peptide)42Aggregation-induced emission fusion and construction and application thereof

The technical field is as follows:

the invention belongs to the field of biotechnology and chemical engineering, and particularly relates to amyloid beta protein 1-42 (Abeta)₄₂) Aggregation-induced emission system and construction thereof, and application of the system to Abeta₄₂In the screening of aggregation inhibitors. In particular to A beta containing unnatural amino acid₄₂The method comprises the steps of verifying the aggregation characteristic of the mutant, adding an aggregation-induced emission probe in a traceless manner and applying the system to screening of an aggregation inhibitor.

Background art:

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 presumed to be associated with certain neurodegenerative and other disorders, such as Alzheimer's Disease (AD), Parkinson's Disease (PD), diabetes type II, and the like. Numerous studies have demonstrated that the more toxic moiety is an oligomer or fibril intermediate, and mature fibers may also play an important role. The development of effective amyloid aggregation inhibitors has become one of the effective means for treating these diseases in the field of drug development. Thus, sensitive and convincing methods for detecting amyloid fibrils or intermediates, as well as a detailed understanding of the mechanisms of fibril formation at the molecular level, facilitate rational design of therapeutic regimens and prevent or slow down body damage caused by toxic amyloid fragments. In recent years, some researches on the structure, morphology, full-length amyloid protein and the like of amyloid fibers have been made in a breakthrough manner, but the molecular mechanism of amyloid aggregation and misfolding is still unexplored.

At present, Abeta₄₂The method for screening the aggregation inhibitor mainly comprises the following three aspects: (1) and (4) in-vitro dye experiment screening. Amyloid dyes such as Thioflavin T (ThT) and Congo Red (CR) are sensitive in hydrophobic environments and fluoresce when bound to the β -sheet structure of amyloid fibrils, and are therefore widely used in research and high throughput screening of aggregative proteins and identification of aggregation inhibitors. However, there are many limitations to the use of these dyes, for example, the inability to detect the morphology of early aggregates using these dyes, and the inability to accurately explore the small molecule drug-induced changes in fiber morphology because small molecule drugs may bind competitively to amyloid fibers. In addition, the properties of the dye may be affected by other components in the buffer solution, and thus some false positives and the like are liable to occur. (2) And (4) fluorescent protein marker screening. To visualize the aggregation process, some fluorescent proteins, such as green fluorescent protein GFP, were expressed as fusion proteins with beta-amyloid 1-42 and used to screen for A.beta.₄₂An aggregation inhibitor. However, due to the large molecular weight of GFP, A.beta.₄₂Only 4.5kDa, hence A.beta.₄₂Protein aggregation and folding may hardly lead to ｃA good misfolding of the entire fusion protein, and thus GFP-A β₄₂The aggregation folding process of the fusion protein may not be completely A beta₄₂The aggregation process of (1). In addition, background fluorescence of dyes and fluorescent proteins may have a large influence on the detection result. (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 for analyzing amyloid aggregation and misfolding and inhibition mechanism thereof, calculating binding force type between inhibitor and amyloid, determining inhibitor action site, and screening and designing aggregation inhibitor. However, due to A beta₄₂Conformational instability of aggregates, especially some toxicityThe strongest three-dimensional structure of oligomer is not resolved so far, and the molecular simulation technology is seriously restricted in A beta₄₂Use in the development of an aggregation inhibitor.

Aggregation-Induced Emission (AIE) molecules have the property of not fluorescing in the free state, and of fluorescing strongly when aggregates are formed or when conformation is restricted, and thus can be used as optimal biosensors for analyzing environmental and conformational changes. The principle of aggregation-induced emission may be that the rotation of the probe molecule is limited by the environment, and a local excited state appears, thereby generating an abnormal photophysical effect. The conformation transformation dependent luminescence phenomenon is not interfered by background fluorescence, so that the conformation transformation dependent luminescence phenomenon can be applied to the biological dynamics research of amyloid protein. Several AIE molecules have been reported in recent years to detect and identify amyloid fibrils and to explore the relationship of proteins to proteins, including TPE, TPE-TPP, BSPOTPE, EPB, etc. Unfortunately, these AIE molecules suffer from low sensitivity and poor specificity during application.

To solve the problem of the existing Abeta₄₂The invention combines aggregation-induced emission technology to screen A beta₄₂Introducing Unnatural Amino Acid (UAA), coupling with AIE molecule by using azido group on its side chain through bioorthogonal reaction, and fixing at A beta₄₂The AIE molecule is marked, thereby improving the specificity and the sensitivity of the AIE applied to detecting the protein conformation transition and obtaining the AIE-Abeta₄₂The fusion system is applied to screening of amyloid aggregation inhibitors.

The invention content is as follows:

to achieve the above object, the present invention binds to Abeta₄₂Screening of unnatural amino acid mutant and bio-orthogonal reaction technology, and A beta is constructed₄₂Aggregation-induced emission fusions and their application to Abeta₄₂In the screening of aggregation inhibitors.

One of the technical schemes provided by the invention is Abeta₄₂Aggregation-induced emission fusion of A β₄₂The aggregation-induced emission fusion body is specifically EPB-Abeta₄₂4F is A.beta.₄₂The phenylalanine at position 4 in the amino acid sequence is replaced by para-azidophenylalanine (pAZF), and Abeta is obtained₄₂4F mutant protein, and then the AIE molecule EPB and the mutant protein Abeta₄₂4F is subjected to bio-orthogonal click chemical reaction to obtain EPB-Abeta₄₂4F, the chemical formula of the AIE molecule EPB is: c₃₈H₄₄N₂O₂Br₂The structural formula is shown in figure 1-a.

The invention also provides EPB-A beta₄₂The construction method of 4F specifically comprises the following steps:

(1) replacing pAZF with A beta by using a solid-phase chemical synthesis method₄₂Phenylalanine at position 4 to obtain Abeta₄₂4F mutant proteins;

(2) EPB and UAA mutant protein Abeta by using copper-catalyzed azide terminal alkyne cycloaddition reaction (CuAAC)₄₂4F is subjected to bio-orthogonal click chemical reaction to obtain EPB-Abeta₄₂4F。

The invention also provides EPB-A beta₄₂4F at A.beta.₄₂The application of aggregation inhibitor screening comprises the following steps: in EPB-A beta₄₂Adding potential inhibitor into 4F solution (PBS solution), detecting fluorescence intensity in the range of excitation wavelength 350nm and emission wavelength 380nm-600nm, and detecting if fluorescence intensity is weaker than EPB-A beta without potential inhibitor₄₂4F system, judging the potential inhibitor to be Abeta₄₂An aggregation inhibitor.

When EPB-A beta₄₂Inhibition of A beta in the presence of inhibitors in 4F solution (PBS dissolution)₄₂4F aggregates, resulting in the screening system not emitting or emitting less light; when the solution does not contain the inhibitor, A beta₄₂4F is rapidly gathered, so that a screening system can emit stronger fluorescence;

preferably, in EPB-A beta₄₂The 4F concentration is 20 μ M, the excitation wavelength is 350nm, the emission wavelength is 380nm-600nm, and the fluorescence intensity is preferably 480 nm.

Has the advantages that:

EPB does not follow A beta₄₂To produce fluorescence, i.e. EPB cannot be used alone for A beta₄₂Investigation of aggregation properties. pAzF mutated Abeta₄₂The EPB cannot be polymerized to generate fluorescence in the aggregation process, and the EPB-A beta constructed by the invention₄₂4F can induce the system to emit stronger fluorescence when forming the aggregate, thereby being Abeta₄₂Screening for aggregation inhibitors provides a new approach.

Description of the drawings:

FIG. 1EPB molecular Structure and verification of aggregation-induced emission characteristics thereof

Wherein, a.epb molecular structure; change in fluorescence of epb in different concentrations of glycerol;

FIG. 2A β₄₂Constructing a flow chart of an aggregation-induced emission screening system;

FIG. 3A β₄₂Schematic diagram of UAA mutant protein synthesis

Wherein, a, the structure and the substitution principle of the unnatural amino acid pAzF; b.A beta₄₂A pAzF substitution site in the protein;

FIG. 4MALDI TOF mass spectrometry for identification of Abeta₄₂4F (a) and Abeta₄₂10y (b) a mutant protein;

FIG. 5 detection of Abeta by ThT fluorescent staining₄₂4F and A β₄₂10Y two mutant protein aggregation properties;

FIG. 6EPB-A β₄₂4F screening system construction and verification

Wherein, a. connecting EPB to a β using CuAAC₄₂Construction of EPB-A β on 4F₄₂4F; UV-visible Spectroscopy full wavelength scanning verification of EPB-Abeta₄₂Whether the 4F is successfully constructed or not, wherein the scanning wavelength range is 200-600 nm;

FIG. 7EPB-A β₄₂Application of 4F aggregation-induced emission screening system

Wherein, a, fluorescence scanning spectrum detection of EPB-A beta with different concentrations₄₂4F fluorescence intensity; b. fluorescence scanning spectrum detection of 20 mu M EPB-Abeta₄₂Adding EGCG (epigallocatechin gallate) with different concentrations into a 4F sample;

FIG. 8 aggregation characteristics of different light emitting systems;

figure 9 small molecule inhibitor validation results.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The unnatural amino acid pAzF used in the present invention was purchased from MCE, and other reagents, the sources of which are not specifically indicated, were purchased from Shanghai-derived leaf Biotech, Inc. The aggregation-induced emission molecule EPB is synthesized by using a chemical synthesis formula. The experimental procedures, which are not described in detail, were performed according to the laboratory manual or the prior art.

Tetraphenyl ethylene and derivatives thereof are the most deeply studied AIE molecules at present due to the characteristics of simple synthesis, easy modification and the like. The EPB molecule is a tetraphenylethylene derivative.

The AIE molecule selected by the invention is EPB, which is named as 2,20- (((2- (4-ethylphenyl) -2-phenylethene-1,1-diyl) bis (4,1-phenylene)) bis (oxy)) bis- (N, N, N-trimethyl thietaneaminium) bromide, is a derivative of tetraphenylethylene, has good aggregation-induced emission characteristics, and is disclosed in Hu, R.; yap, h.k.; fung, y.h.; wang, y.; cheong, w.l.; so, l.y.; tsang, c.s.; lee, l.y.; lo, w.k.; yuan, j.; sun, n.; leung, y.c.; yang, G.; wong, K.Y., 'Light up' protein-protein interaction through bioorganic interaction of a turn-on fluorescent interaction. mol.biosystem.2016, 12, (12), 3544-. The EPB can be synthesized by the skilled person according to the above documents or the related information of structural formula, chemical formula and the like.

The unnatural amino acid selected by the invention is a phenylalanine structural analogue containing Azido, namely p-Azido phenylalanine (pAZF). To reduce the effect of UAA substitution on the aggregation properties of the protein itself, A.beta.was chosen according to the principle of structural similarity₄₂Phenylalanine at position 4 as a replacement site for pAzF. pAzF mutant Abeta obtained by solid-phase chemical synthesis method₄₂Isoform protein, Abeta₄₂4F, and (5). The aggregation characteristics of the mutant are analyzed by ThT fluorescent staining, and the result shows that the Abeta is₄₂The aggregability of 4F was less affected by the introduction of pAzF, and therefore A.beta.was selected₄₂4F mutant eggThe subsequent experiments were carried out on white.

The invention will be further illustrated by the following specific examples, in which A.beta.₄₂The construction process of aggregation-induced emission screening system is shown in FIG. 2.

Example 1: selection of aggregation-induced emission molecules and characterization of emission characteristics

The aggregation-induced emission molecule EPB is a tetraphenylethylene derivative, and the structural formula is shown in figure 1-a. To verify the aggregation-inducing properties of the AIE molecules, the changes in fluorescence values of the AIE molecules in different concentrations of glycerol were measured using the same concentration of EPB (2. mu.M), the concentrations of glycerol being 0%, 20%, 40%, 60%, 70%, the buffer being PBS, pH 7.4. After being fully and uniformly mixed, the fluorescence is detected under the conditions of the excitation wavelength of 350nm and the emission wavelength of 460 nm.

The experimental result is shown in figure 1-b, the EPB solution fluorescence intensity is gradually increased along with the increase of the glycerol concentration, and the EPB solution is proved to have the characteristic of aggregation-induced luminescence.

Example 2: abeta (beta)₄₂UAA mutant synthesis and aggregation property study

(1) UAA and Abeta₄₂Selection of UAA substitution sites in the sequence

The unnatural amino acid of choice in the invention is pAzF, which has a structure similar to that of phenylalanine and tyrosine, and therefore, according to the principle of structural similarity, A.beta.is selected₄₂Phenylalanine at position 4 and tyrosine at position 10 in the sequence serve as substitution sites for pAzF, as shown in FIGS. 3-a, 3-b.

(2)Aβ₄₂Synthesis of UAA mutant proteins

Synthesis of Abeta by solid phase chemical Synthesis₄₂Two A beta-amino acids respectively substituted by pAzF at positions 4 and 10₄₂Mutant proteins (A beta)₄₂4F and A β₄₂10Y), entrusted to gill biochemical shanghai ltd.

Wild type Abeta₄₂The protein amino acid sequence is shown as follows:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID No.1)。

the molecular weight of the mutant protein is further confirmed by MALDI TOF mass spectrometry detection, and the mass spectrogram is shown in the figure4, respectively. The mass spectrum detection result shows that A beta₄₂4F has a molecular weight of 4555.15, Abeta₄₂The molecular weight of 10Y is 4539.15, which is consistent with the theoretical value, therefore, the obtained protein is proved to be pAzF mutant protein.

Performing ThT fluorescent staining experiment by in-situ culture, preparing the two proteins into protein solution, and mixing the protein solution with the ThT solution according to the concentration ratio of 1: 1, mixing well, standing at 37 ℃ for in-situ culture. The fluorescence intensity is detected in fixed time under the conditions of 440nm excitation wavelength and 480nm emission wavelength. The results are shown in FIG. 5, which shows that wild-type A.beta.increases with the culture time₄₂The fluorescence intensity is gradually enhanced, and basically enters a stable period after 24 hours; of the 2 mutant proteins, Abeta₄₂The 4F fluorescence intensity is also enhanced along with the increase of time, and basically enters a stable period after 24h, and then the fluorescence intensity is reduced to a certain degree; abeta (beta)₄₂The 10Y fluorescence intensity was consistently low. The above results demonstrate that: pAzF, when substituted for phenylalanine at position 4, had a small effect on the aggregation of the protein itself, and when substituted for tyrosine at position 10, the mutant protein had substantially lost aggregation, and thus A.beta.was selected₄₂The 4F mutant protein was subjected to subsequent experiments.

The protein and the ThT buffer solution adopt PBS buffer solution, and the pH value is 7.4.

Example 3: EPB-A beta₄₂Construction of 4F aggregation-induced emission screening system

The invention adopts copper-catalyzed azide terminal alkyne cycloaddition reaction (CuAAC) to react EPB and Abeta₄₂4F ligation to construct EPB-A beta₄₂4F screening system, as shown in FIG. 6-a. 1mL of reaction system, the reaction conditions and the added amounts of the reagents used were as follows:

then 1mL was made up with PBS. Mix and shake gently, react at 4 ℃ for 8h (both buffer and reagent are prepared in PBS). After the reaction is finished, desalting the reaction system by using a dialysis bag or a desalting column, removing redundant ions and the like, and keeping the dialysis or desalting process at low temperature as much as possible.

In order to verify whether the system is successfully constructed, the change of the absorbance of the target protein before and after the EPB labeling is detected by comparing the scanning method of the UV-visible spectrophotometer in the wavelength range of 200-600nm, and the result is shown in FIG. 6-b. As is evident from the figure, EPB-A.beta.after EPB labeling₄₂The absorbance of the 4F complex in the wavelength range of 250-350 nm is obviously higher than that of the unlabeled Abeta₄₂4F has a large fluorescence value, which is a phenomenon that the absorbance is enhanced due to the pi-pi bond on EPB, and proves that EPB is successfully connected with Abeta₄₂4F.

Example 4: EPB-A beta₄₂Application of 4F aggregation-induced emission screening system

To determine the stability of the screening system and whether it can be really applied to Abeta₄₂In the screening of aggregation inhibitors, different concentrations of EPB-A beta were measured₄₂4F for fluorescence scanning detection and selection of known A beta₄₂And (3) verifying a small molecule inhibitor EGCG with the aggregation inhibition effect, and evaluating the function of the screening system.

Separately using PBS buffer to separate EPB-A beta₄₂4F is prepared into different concentrations, and the fluorescence condition is detected within the range of excitation wavelength of 350nm and emission wavelength of 380nm-600 nm. As a result, EPB-A β at an emission wavelength of 480nm was shown in FIG. 7-a₄₂4F has the greatest fluorescence intensity and the fluorescence intensity increases gradually as the sample concentration increases. And (3) analyzing the comprehensive cost and the luminescence condition, wherein 20 mu M is the optimal experimental concentration.

EGCG powder was formulated in 100. mu.M stock solution with PBS buffer. EPB-A beta₄₂The final concentration of the 4F solution is 20 mu M, and EGCG and EPB-Abeta with the final concentrations of 5, 10, 15 and 20 mu M respectively are taken₄₂The 4F solution was placed in a 96-well cell culture plate to make a 200. mu.L system. After fully mixing, scanning and detecting by using a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 350nm, and the emission wavelength is 380nm-600 nm. And using the same final concentration of EPB-A beta₄₂4F and no EGCG was used as a control.

The results are shown in FIG. 7-b, and EPB-A β without EGCG₄₂The fluorescence intensity of the 4F sample is obviously higher than that of the EGCG added sample. And as the concentration of EGCG increases, EPB-Aβ₄₂The fluorescence intensity of the 4F sample gradually decreased. The result proves that the EGCG inhibits the aggregation of the target protein, so that the aggregation-induced emission system is in a free state, the fluorescence value is low, and when the EGCG is not added, the target protein forms an aggregate, and finally the induction system emits strong fluorescence. In addition, analysis results show that the fluorescence value is highest when the emission wavelength is about 480nm in the scanning range, and the fluorescent material can be used as a detection condition for rapidly screening small-molecule drugs at the later stage.

In addition, other compounds having A beta are also useful in the present invention₄₂The small molecule inhibitors of aggregation inhibition, fast green, congo red, caffeic acid and brazilin, were validated and the results are shown in fig. 9. EPB-A beta at a final concentration of 25. mu.M₄₂After 4F was added with the 4 small molecule inhibitors at a final concentration of 25. mu.M, the fluorescence intensity of the system showed a different decrease, which was a blank control (25. mu.M EPB-A. beta. of EPB-A. beta. respectively)₄₂4F) 11.28%, 52.12%, 56.5% and 74.99% of the fluorescence intensity. Thus, it was confirmed that EPB-A.beta.₄₂The 4F system can be used for screening A beta₄₂An aggregation inhibitor.

Example 5 investigation of aggregation Properties

(1) Construction of luminescent System (a beta)₄₂Mixing with EPB solution at equal concentration to 25 μ M; ② Abeta₄₂4F and EPB are fully mixed with equal concentration, and the final concentration is 25 mu M; ③ 25 μ M EPB-Abeta₄₂4F

(2) Respectively carrying out scanning detection on the system by using a fluorescence spectrophotometer under the following detection conditions: the excitation wavelength is 350nm, and the emission wavelength is 380nm-600 nm.

As shown in FIG. 8, it is clear from FIG. 8 that EPB does not follow A.beta.₄₂To produce fluorescence, i.e. EPB cannot be used alone for A beta₄₂Investigation of aggregation properties. pAzF mutated Abeta₄₂The EPB cannot be polymerized to generate fluorescence in the aggregation process, and the EPB-A beta constructed by the invention₄₂4F can induce the system to emit stronger fluorescence when forming the aggregate.

In conclusion, the aggregation-induced emission screening system constructed by the invention can be applied to Abeta₄₂In the screening of the aggregation inhibitor, the aggregation inhibitor is,when an inhibitor is present in the solution, A beta can be inhibited₄₂4F aggregated, resulting in non-luminescence of the screening system; when the solution does not contain the inhibitor, A beta₄₂4F rapidly aggregates, enabling the screening system to emit stronger fluorescence.

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

Sequence listing

<110> Tianjin science and technology university

<120> Abeta 42 aggregation-induced emission fusion and construction and application thereof

<130> 1

<141> 2019-08-01

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 42

<212> PRT

<213> person ()

<400> 1

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

1 5 10 15

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

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

Claims (6)

1. Abeta (beta-amyloid peptide)₄₂An aggregation-induced emission fusion comprising A β₄₂Aggregation-induced emission fusion toolThe form is EPB-Abeta₄₂4F is A.beta.₄₂The phenylalanine at position 4 in the amino acid sequence is replaced by para-azidophenylalanine pAZF to obtain Abeta₄₂4F mutant protein, and then the AIE molecule EPB and the mutant protein Abeta₄₂4F is subjected to bio-orthogonal click chemical reaction to obtain EPB-Abeta₄₂4F；

Said A beta₄₂The amino acid sequence is shown as a sequence table SEQ ID No. 1;

the EPB has the chemical formula: c₃₈H₄₄N₂O₂Br₂。

2. A β as defined in claim 1₄₂The construction method of the aggregation-induced emission fusion body is characterized by comprising the following steps:

(1) replacing pAZF with A beta by using a solid-phase chemical synthesis method₄₂Phenylalanine at position 4 to obtain Abeta₄₂4F mutant proteins;

(2) EPB and mutant protein Abeta by copper-catalyzed azide-terminal alkyne cycloaddition reaction₄₂4F is subjected to bio-orthogonal click chemical reaction to obtain EPB-Abeta₄₂4F。

3. The A β of claim 2₄₂The construction method of aggregation-induced emission fusion is characterized in that the copper-catalyzed azide-terminated alkyne cycloaddition reaction is adopted to carry out the EPB and Abeta₄₂The method for performing the bio-orthogonal click chemistry reaction of 4F is as follows: 1mL of reaction system, the reaction conditions and the added amounts of the reagents used were as follows:

then make up 1mL with PBS; reacting for 8 hours at 4 ℃, and desalting the reaction system by using a dialysis bag or a desalting column after the reaction is finished.

4. A β as defined in claim 1₄₂Screening of Abeta by aggregation-induced emission fusions₄₂Use in an aggregation inhibitor.

5. The use of claim 4, wherein screening for A β is performed₄₂The method of aggregation inhibitors is as follows: in EPB-A beta₄₂Adding potential inhibitor into 4F solution, detecting fluorescence intensity in the range of excitation wavelength 350nm and emission wavelength 380nm-600nm, and detecting if the fluorescence intensity is weaker than that of EPB-Abeta without potential inhibitor₄₂4F system, judging the potential inhibitor to be Abeta₄₂An aggregation inhibitor.

6. Use according to claim 5, characterized in that in EPB-A β₄₂The concentration of 4F is 20 mu M, the excitation wavelength is 350nm, and the emission wavelength is 480 nm.

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