Nanotube based on fluorescently-labeled small peptide and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a nano tube based on fluorescently-labeled small peptide and application of the nano tube, the nano tube is a self-assembled nano tube and is specifically formed by electrostatic adsorption of FAM / heme-KD oligopeptide and a KL-7 polypeptide nano tube; the FAM / heme-KD polypeptide is an FAM / heme-KLVFFAED-NH2 peptide fragment, and the KL-7 polypeptide is an Ac-KLVFFAL-NH2 peptide fragment, and the FAM / heme-KD polypeptide is a KLVFFAED-NH2 peptide fragment. The nanotube based on the fluorescently-labeled small peptide shows excellent sensitivity, rapid response capability, specific recognition and affinity to the Abeta40 aggregate, and can be used for detecting the Abeta40 aggregate, detecting fibrosis of the Abeta40 aggregate, screening an Abeta40 aggregate inhibitor and diagnosing Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a nanotube based on fluorescently labeled small peptides and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease, and the appearance of plaques in the brain of AD patients is one of the signs of the disease. Research shows that the main components of the plaques are Aβ 40 and Aβ 42 . Inhibiting the aggregation of Aβ and the formation of plaques is an indicator for drug screening for AD. Therefore, many studies are devoted to developing efficient methods to detect the aggregation state of Aβ and for rapid drug screening. For example, fluorescence spectroscopy, circular dichroism and Western blotting are commonly used for detection at the molecular level; fluorescence optical microscopy, transmission electron microscopy (TEM) and atomic force microscopy can realize visualization of the aggregation state. However, these methods for detecting Aβ aggregation have many shortcomings, such as the need for high Aβ concentration, special operating skills and expensive instruments.

[0003] The patent CN202310314108.7 discloses a fluorescent probe for detecting Aβ aggregates and a preparation method and application thereof, and the patent CN202410361488.4 discloses a dual-functional fluorescent probe for photo-oxidation and imaging of Aβ 1-42 aggregates and a preparation method and application thereof, but both involve the synthesis of compounds with complex structures, and the preparation process is complex, the cost is high and the yield is low. SUMMARY

[0004] The present application aims at the above technical problems, and provides a nanotube based on fluorescently labeled small peptides and application thereof.

[0005] The technical scheme of the present application is as follows: The present application provides a nanotube based on fluorescently labeled small peptides, which is a self-assembled nanotube and is specifically formed by electrostatic adsorption of FAM / heme-KD short peptides and KL-7 polypeptide nanotubes; the FAM / heme-KD short peptide is a FAM / heme-KLVFFAED-NH2 peptide segment, and the KL-7 polypeptide is an Ac-KLVFFAL-NH2 peptide segment.

[0006] Further, the preparation method of the self-assembled nanotube based on fluorescently labeled small peptides comprises the following steps: (1) Assembly of KL-7 polypeptide nanotube: KL-7 polypeptide is dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (abbreviated as HFIP) and continuously vortexed for dissolution, and then ultrasonic treatment is performed; after incubation in an ice bath, it is dissolved in acetonitrile water and adjusted to neutral pH; finally, it is placed in a 37℃ water bath for 72h to assemble KL-7 polypeptide nanotubes; the KL-7 polypeptide is an Ac-KLVFFAL-NH2 peptide segment.

[0007] (2) Synthesis of FAM / heme-KD: First, the carboxyl group in ferriheme (abbreviated as Heme) is activated to obtain activated Heme, and it is mixed with FAM-KD short peptide and triethylamine and stirred in an ice bath, then sequentially stirred at room temperature, dialyzed, and dried to obtain FAM / heme-KD short peptide; the FAM-KD short peptide is FAM-KLVFFAED-NH2 peptide segment; (3) Self-assembly of nanotube based on fluorescently labeled small peptide: KL-7 polypeptide nanotubes are dissolved in acetonitrile water to obtain a KL-7 nanotube solution; FAM / heme-KD short peptide is dissolved in dimethyl sulfoxide (abbreviated as DMSO) and adjusted to neutral pH to obtain a FAM / heme-KD solution; the KL-7 nanotube solution, FAM / heme-KD solution, and Tris buffer are mixed uniformly and reacted at room temperature to obtain a nanotube based on fluorescently labeled small peptide.

[0008] In step (1) and step (3), the acetonitrile water consists of 0.1% trifluoroacetic acid, 40% acetonitrile, and the balance being water by weight percentage.

[0009] In step (2), the activation is mixing Heme, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (abbreviated as EDCI), and N-hydroxysuccinimide (abbreviated as NHS) in a ratio of 1:1.2:1.2 and stirring at room temperature for two hours.

[0010] In step (3), the concentration of the KL-7 nanotube solution is 0-1750 μM and not zero.

[0011] In step (3), the concentration of the FAM / heme-KD solution is 0-10 µg / mL and not zero.

[0012] In step (3), the volume ratio of the KL-7 nanotube solution to the FAM / heme-KD solution is 1:1.

[0013] The application also provides the use of the aforementioned nanotube based on fluorescently labeled small peptide in the preparation of products with at least one of the following uses: (1) for quantitative detection of Aβ 40 aggregates; (2) for monitoring the fibrillation of Aβ 40 aggregates; (3) for evaluating the drug efficacy of Aβ 40 aggregate inhibitors; (4) for identifying and screening Aβ 40 aggregate inhibitors; (5) early diagnosis of Alzheimer's disease.

[0014] Advantages of the present application: (1) The fluorescently labeled small peptide-based nanotube of the present application is formed by electrostatic adsorption and has self-assembly ability. The FAM / heme-KD short peptide is adsorbed to the surface of the KL-7 nanotube by electrostatic interaction, and the local aggregation of FAM / heme-KD causes quenching of FAM fluorescence. When Aβ 40 is added to the fluorescence-quenched system, Aβ 40 competes with FAM / heme-KD from the surface of the nanotube by hydrogen bonding, hydrophobic interaction, and interaction with Heme, thereby restoring fluorescence and enabling rapid fluorescence imaging.

[0015] (2) The fluorescently labeled small peptide-based nanotube of the present application exhibits excellent sensitivity, rapid response ability, specific recognition, and affinity for Aβ 40 aggregates, and can be used for detecting Aβ 40 aggregates, monitoring the fibrillation of Aβ 40 aggregates, identifying Aβ 40 aggregate inhibitors, and diagnosing Alzheimer's disease.

[0016] (3) The FAM / heme-KD short peptide in the present application has different binding abilities for Aβ 40 in different aggregation states, and thus can selectively recognize Aβ 40 in different aggregation states.

[0017] (4) The method of the present application has the characteristics of simple synthesis operation, high synthesis efficiency, and excellent synthesis success rate. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1are characterization figures of KL-7 nanotube; wherein, A is a transmission electron microscope figure of KL-7 nanotube; B is a scanning electron microscope figure of KL-7 nanotube; C is zeta potential of KL-7 nanotube, FAM / heme-KD, FAM / heme-KD@KPNT; D is an infrared spectrum of KL-7 nanotube; E is a circular dichroism spectrum of KL-7 nanotube; F is a fluorescence imaging figure of KL-7 nanotube adsorbing fluorescent dye Nile Red; Figure 2 are characterization figures of FAM / heme-KD short peptide: wherein, A is a nuclear magnetic resonance hydrogen spectrum figure of Heme and FAM / heme-KD; B is an ultraviolet spectrum figure of Heme, FAM-KD and FAM / heme-KD; C is a fluorescence figure of Heme and FAM / heme-KD; Figure 3 are fluorescence spectra of Example 1; wherein, A is a fluorescence spectrum of 5 µg / mL FAM / heme-KD adding different concentrations of KL-7 nanotube; B is a fluorescence spectrum of 5 µg / mL FAM / heme-KD and 35 µM KL-7 nanotube adding different concentrations of Aβ 40 monomer; C is a fluorescence spectrum of 5 µg / mL FAM / heme-KD and 35 µM KL-7 nanotube adding different concentrations of Aβ 40 fiber; D is a linear graph of normalized signal intensity at 525 nm and Aβ 40 monomer and Aβ 40 fiber; Figure 4 , wherein A is a molecular docking figure of KD short peptide and Aβ 40 monomer; B is a multiple change of fluorescence signal intensity at 525 nm and different concentrations of Aβ 40 , BSA, BLG and α-syn; Figure 5 , wherein A is the inhibitory effect of Aβ 40 aggregation detected by KL-7 nanotube and FAM / heme-KD system; B is the inhibitory effect of Aβ 40 aggregation detected by KL-7 nanotube and FAM / heme-KD system; C is the inhibitory effect of Aβ 40 aggregation detected by KL-7 nanotube and FAM / heme-KD system; Figure 6 are determination of Aβ 40 in simulated cerebrospinal fluid: A is the determination of Aβ 40fluorescence spectra under the action of KL-7 nanotubes; B is the relationship diagram of the multiple change of fluorescence signal intensity at 525 nm and different concentrations of Aβ in simulated cerebrospinal fluid; C is the calibration curve of the system composed of KL-7 nanotubes and FAM / heme-KD for detecting Aβ in simulated cerebrospinal fluid; D is the calibration curve of the commercial ELISA kit for detecting Aβ in simulated cerebrospinal fluid. 40 40 40

[0019] Figure 7 Figure 6 is a statistical chart of the results of the system composed of KL-7 nanotubes and FAM / heme-KD for detecting 52 control serum samples and 50 AD serum samples. DETAILED DESCRIPTION

[0020] The materials used in the following examples are as follows: 1.1 Reagents and materials FAM-KLVFFAED-NH2 peptide and Ac-KLVFFAL-NH2 peptide were purchased from Genview Biotech Co., Ltd. (Shanghai, China), and the purity of the polypeptides was >98% and was characterized by mass spectrometry. Aβ 40 was purchased from Abeam, USA. Rhodamine, ThT and Heme were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Rifampicin, rutin, glutathione, EGCG and curcumin, quercetin, paeoniflorin and myricetin were purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China). All reagents were of analytical grade.

[0021] 1.2 Instruments and equipment Fluorescence spectrophotometer (Santa Clara, CA, USA), ZS 90 Malvern particle size analyzer (Malvern, UK), Nicolet 380 Fourier transform infrared spectrometer (Thermo Nicolet Co., USA), HT7700 transmission electron microscope (Hitachi, Ltd, Japan), upright fluorescence microscope (Nikon, Japan) 1.3 Preparation of Aβ 40 monomers and fibers About 1 mg of Aβ 40 monomers was weighed into 250 μL DMSO. After complete dissolution, it was aliquoted in EP tubes (20 μL per tube) and stored in a -20°C refrigerator. For Aβ 40 monomers, 100 μL of 10 mM Tris buffer (pH 7.4) was added in the EP tube for dissolution, and then the absorbance at 278 nm was detected. The concentration of Aβ 40 monomers was determined according to the molar absorption coefficient at 278 nm (ε278nm=1490 M -1 ·cm​​​-1 ), and used immediately. The fibers were prepared by dissolving Aβ 40 monomer solution was incubated at 37 °C and then matured by shaking at 400 rpm for 48 h.

[0022] Example 1 Self-assembly of KL-7 polypeptide Assembly of KL-7 polypeptide: 3.5 mM KL-7 polypeptide was first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) with continuous vortexing, followed by sonication. After incubation in ice bath for 30 min, the peptide was dissolved in 333 μL of aqueous acetonitrile solvent, and the pH was adjusted to 7.0 with NaOH. Finally, it was allowed to assemble in a 37 °C water bath for 72 h to obtain KL-7 nanotubes. The aqueous acetonitrile consisted of 0.1% trifluoroacetic acid, 40% acetonitrile, and the balance water by mass percentage.

[0023] Results: KL-7 monomer was found to self-assemble into KL-7 peptide nanotubes (KPNT) after incubation in 40% acetonitrile / water (0.1% TFA, pH 7.0) for 72 h. The Figure 1 A and Figure 1 B show that the resulting KL-7 peptide nanotubes have a uniform morphology with a well-defined tubular structure, a diameter of about 50 ± 10 nm, and a length of several microns. Figure 1 D shows that the Fourier transform infrared (FT-IR) spectrum of the KL-7 peptide nanotubes has two absorption bands at 1625 cm -1 and 1693 cm -1 , which indicates a stable antiparallel β-sheet secondary structure. Since the KL-7 nanotubes have many amine groups on their surface, their zeta potential was measured to be 35.2 mV (C), while the FAM / heme-KD has many carboxyl groups, and its zeta potential was measured to be -28.5 mV. Thus, the FAM / heme-KD can bind to the KL-7 nanotubes through electrostatic interactions. F shows that the KL-7 nanotubes have a uniform morphology and can adsorb fluorescent small molecules. Figure 1 Figure 1 Figure 1 F shows that the KL-7 nanotubes have a uniform morphology and can adsorb fluorescent small molecules.

[0024] Example 2 Synthesis and characterization of FAM / heme-KD 1) Activation of the carboxyl group in Heme: Heme, EDCI, and NHS were mixed in a ratio of 1:1.2:1.2 and stirred at room temperature for two hours to obtain activated Heme; ​​FAM-KD, activated Heme, triethylamine were mixed in the ratio of 1.5:1:1 and stirred in an ice bath for one hour, and then stirred at room temperature for 24 hours. Subsequently, the unreacted Heme was removed with a 1000 Da dialysis bag. Finally, drying was performed with a rotary evaporator, and the sample was collected.

[0025] It was found that the FAM / heme-KD dry sample was characterized. As shown in Figure 2 A shows that FAM / heme-KD has an overlapping peak with Heme in the nuclear magnetic resonance spectrum, indicating that FAM / heme-KD synthesis is successful. As shown in Figure 2 B shows that FAM / heme-KD has a peak of Heme in the absorption spectrum, further indicating that FAM-KD and Heme are successfully coupled. As shown in Figure 2 C shows that the fluorescence emission peak of FAM / heme-KD is consistent with that of FAM-KD, indicating that FAM-KD and Heme are successfully coupled.

[0026] Example 3 Construction of a system based on fluorescently labeled small peptide nanotubes The KL-7 polypeptide nanotube was dissolved in acetonitrile water to obtain a KL-7 nanotube solution of 35 µM; FAM / heme-KD was dissolved in dimethyl sulfoxide (abbreviated as DMSO) and adjusted to neutral pH to obtain a FAM / heme-KD solution of 5 µg / mL; 20 μL of KL-7 nanotube solution, 20 μL of FAM / heme-KD solution, 60 μL of 10 mM Tris buffer were mixed uniformly and reacted at room temperature for 10 min to obtain a fluorescently labeled small peptide nanotube (abbreviated as FAM / heme-KD@KPNT). The acetonitrile water consists of the following components by mass percentage: 0.1% trifluoroacetic acid, 40% acetonitrile, and the balance being water.

[0027] Example 4 KL-7 nanotube concentration optimization experiment The concentration of FAM / heme-KD was fixed at 5 µg / mL. To optimize the binding concentration of KL-7 nanotubes with FAM / heme-KD, KL-7 nanotubes were first dissolved in aqueous acetonitrile solvent at the desired concentrations (350, 175, 88, 44, 35, 22, 11, 0 µM). KL-7 nanotube solutions (20 µL 350 µM, 20 µL 175 µM, 20 µL 88 µM, 20 µL 44 µM, 20 µL 35 µM, 20 µL 22 µM, 20 µL 11 µM, 20 µL 0 µM) were added to 20 µL FAM / heme-KD solution (50 mg / mL DMSO pH 7.00) and 60 µL 10 mM Tris buffer (pH 8.0) was added to each and vortexed to mix well. The fluorescence intensity was measured after 10 min of reaction at room temperature (462 nm excitation). According to the measured fluorescence intensity data, the concentration with the most complete quenching was selected. The aqueous acetonitrile consisted of 0.1% trifluoroacetic acid, 40% acetonitrile, and the remainder water by mass percentage.

[0028] From the potential detection, it was found that FAM / heme-KD could bind to KL-7 nanotubes through electrostatic interaction. We then detected the fluorescence change of FAM when the same concentration of FAM / heme-KD was added with different concentrations of KL-7 nanotubes. Figure 3 As can be seen, the fluorescence quenching effect was the best when 35 µM KL-7 nanotubes were added. This is because aggregation of FAM / heme-KD occurs when it is adsorbed on the KL-7 nanotubes, causing aggregation-caused quenching (ACQ). When the concentration of KL-7 nanotubes is further increased, the fluorescence recovers. This is because as the concentration of KL-7 nanotubes increases, the relative aggregation of FAM / heme-KD on the surface of the nanotubes decreases, the ACQ effect weakens, and the fluorescence recovers. Therefore, in all subsequent experiments, the concentration of KL-7 nanotubes was 35 µM.

[0029] Example 5 Detection ability of the system based on fluorescently labeled small peptide nanotubes for Aβ 40 1. ThT fluorescence detection experiment Aβ 40 was added to 10 mM Tris buffer (pH 8.0) with different incubation times (0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 6, 12, 24, 48, 72, 96 h), then ThT solution was added to a final concentration of 20 µM, vortexed to mix well, and then reacted for half an hour at room temperature in the dark before measuring the fluorescence intensity (446 nm excitation). ​

[0030] Different concentrations of Aβ were added to the fluorescently labeled peptide-based nanotubes prepared in Example 3. 40 Monomers and Aβ 40 The fibers were subjected to fluorescence detection, with BSA (bovine serum albumin), BLG (β-lactoglobulin), and α-synuclein serving as control groups. Figure 3 As shown in Figure B, 0.2 µM–9 µM Aβ were added respectively. 40 After monomerization, the fluorescence of FAM recovered to varying degrees. Adding Aβ... 40 Fibers can also undergo fluorescence recovery, but compared to monomers, the fluorescence recovery ability of fibers is much smaller. Figure 3 (C and 3D). This is because FAM / heme-KD affects Aβ. 40 This is due to the difference in the binding ability between monomers and fibers.

[0031] In addition, we have a different understanding of this system regarding Aβ. 40 The selectivity of Aβ was detected. Different concentrations of Aβ were added to the above-mentioned [product / method / treatment]. 40 Replace with BSA, BLG, and α-syn, by Figure 4 B shows that this system is effective against Aβ. 40 It exhibits good selectivity. This is because the Heme in FAM / heme-KD can interact with Aβ. 40 In addition, the KLVFFAED sequence in FAM / heme-KD is Aβ. 40 A segment ( Figure 4 A), therefore FAM / heme-KD affects Aβ. 40 It is selective.

[0032] From the above experiments, we found that adding Aβ to the fluorescence quenching system... 40 The fluorescence was stronger after monomerization, but stronger after the addition of Aβ. 40 The fibers do not provide significant reinforcement, therefore the FAM / heme-KD and KL-7 nanotube system can be used for Aβ. 40 Fibrosis is monitored. For example... Figure 5 As shown in A, with Aβ 40 With increasing incubation time, fluorescence intensity decreased exponentially, while traditional ThT detection showed an S-shaped curve. Similar to ThT detection, Aβ was also observed in the FAM / heme-KD and KL-7 nanotube systems. 40 The aggregation reached a plateau after approximately 10 hours. Therefore, the FAM / heme-KD and KL-7 nanotube system can be applied to the aggregation of Aβ. 40 Monitoring of fibrosis.

[0033] Example 6: A system based on fluorescently labeled small peptide nanotubes for Aβ40 The ability to identify aggregation inhibitors Aβ was added to the fluorescently labeled peptide-based nanotubes prepared in Example 3. 40 After incubating the monomers individually for 48 hours, rifampin, rutin, glutathione, EGCG, curcumin, quercetin, paeoniflorin, and myricetin were added separately to detect the effects of rifampin, rutin, glutathione, EGCG, curcumin, quercetin, paeoniflorin, and myricetin on Aβ. 40 Detection of aggregation inhibition effect. When Aβ can be inhibited... 40 When FAM aggregates, its fluorescence increases; however, when its fiber growth cannot be suppressed, the fluorescence remains quenched. For example... Figure 5 As shown in B, after adding Aβ 40 The addition of rifampin, rutin, glutathione, EGCG, and curcumin during incubation all induced fluorescence enhancement, indicating that these inhibitors all produce an inhibitory effect, consistent with the results obtained from the ThT experiment. Figure 5 C).

[0034] Example 7: A system based on fluorescently labeled small peptide nanotubes for the treatment of Aβ in simulated cerebrospinal fluid. 40 recognition ability Adding different concentrations of Aβ to the fluorescently labeled peptide-based nanotubes prepared in Example 3 40 Simulated cerebrospinal fluid was used to detect the effect of a fluorescently labeled small peptide-based nanotube system on Aβ in biological samples. 40 The ability to recognize. For example... Figure 6 A and 6B show that, with Aβ 40 As the concentration of FAM increases, its fluorescence gradually intensifies, and the fluorescence intensity is related to the concentration of Aβ. 40 By fitting curves to the concentrations, it was found that the system based on fluorescently labeled small peptide nanotubes can be used to simulate Aβ in cerebrospinal fluid. 40 Detection ( Figure 6 C), sensitivity is higher than that of commercially available kits ( Figure 6 D).

[0035] Example 8: The ability of a fluorescently labeled small peptide-based nanotube system to identify AD patients. Serum from 52 healthy controls or 50 clinically diagnosed Alzheimer's disease (AD) patients was added to the fluorescently labeled peptide-based nanotubes prepared in Example 3. The ability of the system to recognize AD patients in human plasma using fluorescently labeled peptide-based nanotubes was then tested. Figure 7 The results showed that the fluorescence intensity leading to FAM in the serum of AD patients was significantly higher than that in healthy individuals. This is because the serum of AD patients contains more Aβ. 40 The concentration was higher than that in healthy human serum, confirming that the system based on fluorescently labeled small peptide nanotubes can detect Aβ in the serum of AD patients. 40, and then realize the diagnosis of AD.

Claims

1. A fluorescently labeled nanotube based on a small peptide, characterized in that, The nanotube is a self-assembled nanotube, which is specifically formed by electrostatic adsorption of FAM / heme-KD short peptide and KL-7 polypeptide nanotube; the FAM / heme-KD short peptide is FAM / heme-KLVFFAED-NH2 peptide segment, and the KL-7 polypeptide is Ac-KLVFFAL-NH2 peptide segment.

2. The nanotube based on fluorescently labeled small peptides as described in claim 1, characterized in that, The preparation method of the self-assembled nanotube based on the fluorescently labeled small peptide comprises the following steps: (1) Assembly of KL-7 polypeptide nanotube: dissolve KL-7 polypeptide in 1,1,1,3,3,3-hexafluoro-2-propanol and continuously vortex to dissolve, and then perform ultrasonic treatment; after incubation in an ice bath, dissolve in acetonitrile water and adjust the pH to neutral; finally, assemble in a 37°C water bath for 72 hours to obtain the KL-7 polypeptide nanotube; the KL-7 polypeptide is Ac-KLVFFAL-NH2 peptide segment. (2) Synthesis of FAM / heme-KD short peptide: First, activate the carboxyl group in Heme to obtain activated Heme, and mix it with FAM-KD short peptide and triethylamine and stir in an ice bath, then sequentially stir at room temperature, dialysis, and drying to obtain FAM / heme-KD short peptide; the FAM-KD short peptide is FAM-KLVFFAED-NH2 peptide segment; (3) Self-assembly of nanotube based on fluorescently labeled small peptide: Dissolve the KL-7 polypeptide nanotube in acetonitrile water to obtain a KL-7 nanotube solution; dissolve FAM / heme-KD in dimethyl sulfoxide and adjust the pH to neutral to obtain a FAM / heme-KD solution; mix the KL-7 nanotube solution, FAM / heme-KD solution, and Tris buffer uniformly and react at room temperature to obtain the nanotube based on the fluorescently labeled small peptide.

3. The nanotube based on fluorescently labeled small peptides as described in claim 2, characterized in that, In steps (1) and (3), the acetonitrile water consists of 0.1% trifluoroacetic acid, 40% acetonitrile, and the balance being water by weight percentage.

4. The fluorescently labeled nanotube based on a small peptide according to claim 2, wherein, In step (2), the activation is mixing Heme, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and N-hydroxysuccinimide in a ratio of 1:1.2:1.2 and stirring at room temperature for two hours.

5. The nanotube based on fluorescently labeled small peptides as described in claim 2, characterized in that, In step (3), the concentration of the KL-7 nanotube solution is 0-350 µM and does not take zero.

6. The nanotube based on fluorescently labeled small peptides as described in claim 2, characterized in that, In step (3), the concentration of the FAM / heme-KD solution is 0-10 µg / mL and does not take zero.

7. The nanotube based on fluorescently labeled small peptides as described in claim 2, characterized in that, In step (3), the volume ratio of the KL-7 nanotube solution to the FAM / heme-KD solution is 1:

1.

8. Use of the nanotube based on the fluorescently labeled small peptide according to any one of claims 1-7 in the preparation of a product for at least one of the following uses: (1) for quantitative detection of Αβ 40 aggregates; (2) for monitoring Abeta 40 fibrillation of aggregates; (3) for assessing the efficacy of a drug on the aggregation of Aβ 40 a pharmaceutical effect of an aggregate inhibitor (4) for identifying, screening, and inhibiting Aβ 40 aggregates (5) Early diagnosis of Alzheimer's disease.

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