CN112645970B - Fluorescent probe for detecting Abeta oligomer as well as preparation method and application thereof - Google Patents
Fluorescent probe for detecting Abeta oligomer as well as preparation method and application thereof Download PDFInfo
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- CN112645970B CN112645970B CN202011631168.4A CN202011631168A CN112645970B CN 112645970 B CN112645970 B CN 112645970B CN 202011631168 A CN202011631168 A CN 202011631168A CN 112645970 B CN112645970 B CN 112645970B
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
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Abstract
The invention discloses a fluorescent probe for detecting Abeta oligomer and a preparation method and application thereof. The structural general formula of the fluorescent probe is shown as a formula (I). The preparation method comprises the following preparation steps: (1) heating p-hydroxybenzaldehyde and N-acetylglycine in an organic solvent for reaction, and taking sodium acetate as a catalyst to obtain a yellow solid; then heating the yellow solid and a methylamine water solution in an organic solvent for reaction, wherein potassium carbonate is used as a catalyst, and obtaining an intermediate 1; (2) heating the intermediate 1 and boron tribromide dichloromethane in an organic solvent to react to obtain an intermediate 2; (3) and heating the intermediate 2 and p-dimethylaminobenzaldehyde in an organic solvent for reaction, and taking piperidine as a catalyst to obtain the fluorescent probe for detecting the A beta oligomer. The fluorescent probe can be used for beta-amyloid plaque imaging, has high sensitivity, and has wide application prospect in early diagnosis of Alzheimer's disease and disclosure of pathological mechanism.
Description
Technical Field
The invention belongs to the field of specific molecular recognition materials, and particularly relates to a fluorescent probe for detecting Abeta oligomers, and a preparation method and application thereof.
Background
To date, Alzheimer's Disease (AD) is an incurable neurodegenerative disease with progressive cognitive and behavioral disorders. Extracellular misfolded β -amyloid plaques and intracellular tubulin binding (tau) protein tangles are the main pathological features of AD. In recent years, great investment in anti-AD drugs is made in various major pharmaceutical industries all over the world, and due to the complex pathological mechanism, the AD drugs are mostly developed and end up not reaching the main curative effect. At the same time, most AD patients have failed therapy because they have already been diagnosed late. Therefore, increasing the diagnostic level of AD is of great importance for both early treatment of AD and understanding of its pathological mechanisms.
Fluorescent probes are a time and labor-saving method for detecting complex biomarkers. Currently, fluorescent probes on AD are commonly used to detect Α β plaques and tau proteins, such as cyanine derivatives, BODIPY derivatives, curcumin derivatives and indocyanine green derivatives.
A β oligomers are effective targets for early diagnosis and treatment of alzheimer's disease, but due to the unclear strategy of targeting a β oligomer molecules, there are few fluorescent probes that can detect a β effectively today.
Therefore, there is interest in developing molecular probes that specifically bind to a β oligomers. The A beta plaque imaging is carried out by combining an A beta fluorescent probe and a molecular imaging technology, noninvasive and real-time in-vivo tracing and detection of the A beta plaque can be realized, and great convenience is further provided for early diagnosis, curative effect detection, research on therapeutic drugs and the like of patients with diseases such as AD and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a fluorescent probe for detecting A beta oligomer.
The invention also aims to provide a preparation method of the fluorescent probe for detecting the A beta oligomer.
Still another object of the present invention is to provide use of the fluorescent probe for detecting a β oligomers.
The purpose of the invention is realized by the following technical scheme:
a fluorescent probe for detecting Abeta oligomers has a structural general formula shown in formula (I):
the preparation method of the fluorescent probe for detecting the A beta oligomer comprises the following preparation steps:
(1) preparation of intermediate 1
Adding p-hydroxybenzaldehyde, N-acetylglycine and sodium acetate (serving as catalysts) into an organic solvent, uniformly mixing, heating to 110 +/-5 ℃ for reflux reaction, adding ethanol after the reaction is finished, cooling to room temperature to precipitate crystals, filtering, and washing to obtain a yellow solid;
adding the yellow solid, a methylamine aqueous solution and potassium carbonate (serving as a catalyst) into an organic solvent, heating to 80 +/-5 ℃ for reflux reaction, cooling to room temperature after the reaction is finished, adjusting the pH value to be neutral, cooling to separate out a solid, filtering, and drying to obtain an intermediate 1; the structural formula is shown as a formula (II):
(2) preparation of intermediate 2
Dissolving the intermediate 1 in an organic solvent, adding a boron tribromide dichloromethane solution, heating to 110 +/-5 ℃ in a protective gas atmosphere to perform reflux reaction, cooling, filtering and washing after the reaction is finished, then adding a Hydrogen Fluoride (HF) solution, uniformly stirring, sequentially extracting with ethyl acetate, a potassium carbonate solution and water, concentrating and purifying to obtain an intermediate 2; the structural formula is shown as formula (III):
(3) preparation of fluorescent Probe
And adding the intermediate 2, p-dimethylaminobenzaldehyde and piperidine (serving as a catalyst) into an organic solvent, reacting at 100 +/-5 ℃, and concentrating, washing and purifying after the reaction is finished to obtain the fluorescent probe for detecting the Abeta oligomer.
The molar ratio of the p-hydroxybenzaldehyde, the N-acetylglycine and the sodium acetate in the step (1) is 1: 0.9-1.1: 0.7-1.3; preferably 1:1: 0.9.
The organic solvent in the first step (1) is preferably acetic anhydride.
The organic solvent in the step (1) is calculated according to the proportion of 1L of organic solvent to each mole of p-hydroxybenzaldehyde.
The reflux reaction time in the step (1) is 3-5 h; preferably 4 hours.
The temperature of the added ethanol in the step (1) is 50-60 ℃; preferably 50 deg.c.
The time for precipitating crystals in the step (1) is 3-5 hours; preferably 4 hours.
The washing in the step (1) is washing by using glacial ethanol and hot water in sequence.
The molar ratio of the yellow solid, the potassium carbonate and the methylamine in the step (1) and the step (2) is 1: 1.5: 4.
the concentration of the methylamine water solution in the step (1) and the step (II) is 33 percent by volume.
The organic solvent in the step (1) and the step (2) is preferably ethanol.
The volume ratio of the methylamine water solution to the organic solvent in the step (1) and the step (1) is 1:1.
The reflux reaction time in the step (1) is 2-3 h; preferably 2.5 h.
Adjusting the pH value in the step (1) and the step (2) by adopting an HCl ethanol solution; preferably, the adjustment is carried out by using 10% by volume of ethanol HCl solution.
And (2) cooling to 0 ℃.
The molar ratio of the intermediate 1 to the boron tribromide dichloromethane in the step (2) is 1: 1.1-2; preferably 1: 2.
The organic solvent in the step (2) is dichloroethane.
The dosage of the organic solvent in the step (2) is calculated according to the proportion of 1-2 mL of organic solvent to 1 millimole (mmol) of intermediate; preferably 1.33mL of organic solvent per mmol of intermediate 1.
The protective gas in the step (2) is nitrogen.
The reflux reaction time in the step (2) is 6-8 h; preferably 7 h.
The concentration of the Hydrogen Fluoride (HF) solution in the step (2) is 48-55% by mass.
The dosage of the hydrogen fluoride solution in the step (2) is calculated according to the proportion of 1 mmol of intermediate to 3-5 mL of hydrogen fluoride solution; preferably 3.33mL of hydrogen fluoride solution per mmol of intermediate 1.
The stirring time in the step (2) is 20-30 min; preferably 30 min.
The concentration of the potassium carbonate solution in the step (2) is 5% by mass.
The purification in the steps (2) and (3) is performed by adopting column chromatography; the column chromatography conditions are as follows: volume ratio of chloroform to ethanol (CHCl)3EtOH) is 50: 1; silica gel 200-300 meshes.
The molar ratio of the intermediate 2, the p-dimethylaminobenzaldehyde and the piperidine in the step (3) is 1:5: 1.2-1.5; preferably 1:5: 1.25.
The organic solvent in the step (3) is pyridine.
The organic solvent in the step (3) is calculated by 25mL of organic solvent in a ratio of 2mL per mol of intermediate.
The reaction time in the step (3) is 2-5 min; preferably for 3 min.
Washing in the step (3) is washing by adopting ethyl acetate; preferably, ethyl acetate is adopted for washing for 3-5 times; more preferably 4 washes with ethyl acetate.
The fluorescent probe for detecting the A beta oligomer is applied to the preparation of products for diagnosing Alzheimer's Disease (AD).
The product comprises a fluorescent probe, a detection (diagnosis) reagent, a kit and the like.
The fluorescent probe for detecting the A beta oligomer is applied to the preparation of medicaments for treating Alzheimer's Disease (AD).
The fluorescent probe for detecting the A beta oligomer is applied to the preparation of products for detecting beta-amyloid.
The product comprises a fluorescent probe, a detection (diagnosis) reagent, a kit and the like.
The beta-amyloid protein is an A beta oligomer.
The detecting comprises quantitatively detecting the A beta oligomer.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a fluorescent probe for detecting Abeta oligomer, which only needs three steps for synthesis, has simple post-treatment process, easy operation and easily obtained product, and has wide application prospect in the aspects of early diagnosis of Alzheimer's disease and disclosure of pathological mechanism.
(2) The fluorescent probe can sensitively detect the A beta oligomer and image the A beta plaque.
(3) The invention relates to a switch-type probe designed based on a green fluorescent protein chromophore, which can be used for beta-amyloid plaque imaging and has high sensitivity.
Drawings
FIG. 1 is a synthesis scheme of the fluorescent probe preparation method of the present invention (in the figure, 1 and 2 respectively represent intermediate 1 and intermediate 2; a: p-hydroxybenzaldehyde, N-acetylglycine, sodium acetate, acetic anhydride, refluxing at 110 ℃ for 4 h; potassium carbonate, methylamine/water, ethanol, refluxing for 2.5 h; b: dichloroethane, boron tribromide/dichloromethane, refluxing at 110 ℃ for 7 h; c: 4-dimethylaminobenzaldehyde, pyridine, piperidine, 100 ℃).
FIG. 2 shows a probe (I) obtained in example 1 of the present invention1H-NMR spectrum.
FIG. 3 shows a probe (I) obtained in example 1 of the present invention13C-NMR spectrum.
FIG. 4 is a HRMS spectrum of probe (I) obtained in example 1 of the present invention.
FIG. 5 is a graph showing the results of evaluating the binding ability of the probe (I) obtained in example 1 of the present invention to A.beta.protein; wherein A is an ultraviolet-fluorescence spectrum of the probe (I) in DMSO; b is the specificity result of the probe (I) on the A beta protein; c and D are the selectivity results for probe (I); e is the titration result; f is the binding constant.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available. The following examples are carried out with reference to conventional techniques for parameters not specifically mentioned.
In the embodiment of the invention, nuclear magnetic spectrum is measured by adopting an Avance III 400MHz (600MHz) nuclear magnetic resonance instrument of Bruker company in Germany, and deuterated chloroform, deuterated methanol or deuterated DMSO is used as a solvent. The fluorescence spectrum was measured by using FL-4500 fluorescence spectrometer of Hitachi, Japan.
EXAMPLE 1 Synthesis of organic Small molecule fluorescent Probe (I)
In this example, a synthesis scheme of the organic small molecule fluorescent probe (I) is shown in fig. 1, and the specific synthesis steps are as follows:
(1) synthesis of intermediate 1
Uniformly mixing reactants of p-hydroxybenzaldehyde (0.02mol,2.44g), N-acetylglycine (0.02mol, 2.34g), anhydrous sodium acetate (0.018mmol,1.47g) and 20mL of acetic anhydride, heating and stirring at 110 ℃, refluxing for 4h, slowly adding ethanol heated at 50 ℃ after the reaction is finished, cooling the reaction liquid to room temperature, cooling for 4h, precipitating crystals, filtering, washing with ice ethanol, washing with hot water to obtain yellow solid, and drying to obtain 3.64 g.
② mixing yellow solid (0.01mol,2.03g), potassium carbonate (0.015mol,2.07g), 33% (v/v) methylamine aqueous solution 4mL and 4mL ethanol in a 75mL volumetric flask, heating the reaction solution (80 ℃) for reflux for 2.5h, cooling to room temperature after the reaction is finished, adjusting the pH of the reaction solution to be neutral by using 10% (v/v) HCl ethanol solution, cooling to crystallize for 4h at 0 ℃, filtering the solid to obtain light yellow solid, drying to obtain 1.43g of intermediate 1 (the structural formula is shown as formula (II)), wherein the yield is 66.20%.
1H NMR(400MHz,DMSO)δ10.11(s,1H),8.07(d,J＝8.8Hz,2H),6.88(s,1H),6.82(d,J＝8.8Hz,2H),3.07(s,3H),2.31(s,3H).
(2) Synthesis of intermediate 2
Dissolving the intermediate 1(3mmol,648mg) in 4mL of dry dichloroethane, adding 6mL of 1mol/L boron tribromide dichloromethane solution, heating (110 ℃) in nitrogen for refluxing for 7h, cooling after the reaction is finished, filtering by a molecular sieve, washing by ethanol and dichloroethane in sequence, adding 10mL of Hydrogen Fluoride (HF) aqueous solution (48-55%, w/w), stirring for 30min, extracting by ethyl acetate, 5% (w/v) potassium carbonate solution and water for 2 times respectively, concentrating and passing through a column (column chromatography: CHCl)3EtOH 50: 1; silica gel 200-300 mesh) to obtain 121.68mg of orange solid, namely the intermediate 2 (the structural formula is shown as the formula (III), and the yield is 15.36%.
1H NMR(400MHz,DMSO)10.2(s,1H),7.56(s,1H),7.48(d,J＝8.31Hz,1H),7.00(d,J＝2.20Hz,1H,),6.74(dd,J＝8.3Hz,2.4Hz,1H,),3.22(s,3H),2.71(s,3H).
(3) Synthesis of organic Small molecule fluorescent Probe (I)
Intermediate 2(0.4mmol,105mg), p-dimethylaminobenzaldehyde (2mmol,300mg) and piperidine (0.05mL) were dissolved in 10mL of pyridine, and the reaction was reacted at 100 ℃ for 3min, followed by concentration of the reaction solution. The crude product was washed with ethyl acetate (4X 10mL) and then subjected to column chromatography (CHCl)3EtOH 50: 1; silica gel 200-300 mesh), and the final green solid 54mg with 35.68% yield.
1H NMR(600MHz,DMSO)δ10.16(s,1H),8.20(d,J＝16.4Hz,1H),7.66(d,J＝8.9Hz,2H),7.46(d,J＝8.3Hz,1H),7.42(s,1H),7.11(d,J＝16.4Hz,1H),7.03(d,J＝1.7Hz,1H),6.84(d,J＝8.8Hz,2H),6.73(dd,J＝8.3,2.4Hz,1H),3.51(s,3H),3.08(s,6H).13C NMR(151MHz,DMSO)δ163.22,160.87,157.64,152.90,149.04,133.31,131.18,126.18,125.26,121.86,118.41,115.06,111.94,103.16,28.50.HRMS(ESI):calcd for(M-F)+(C21H20BFN3O2 +)376.1633,found 376.1637.
Of synthetic organic small-molecule fluorescent probes1The H-NMR spectrum is shown in FIG. 2,13the C-NMR spectrum is shown in FIG. 3, the HRMS spectrum is shown in FIG. 4, and the chemical structure is shown in formula (I).
Example 2 UV-fluorescence assay of organic Small molecule fluorescent Probe (I)
The probe (I) prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10mM stock solution; then, 3. mu.L of 10mM stock solution was dissolved in 3mL of DMSO to obtain a 10. mu.M probe solution. The uv absorption spectrum was tested under a uv spectrophotometer and the maximum absorption wavelength was recorded. The fluorescence spectra were tested under a fluorescence spectrophotometer and the maximum emission wavelength was recorded.
The results are shown in FIG. 5A: 10 μ M of probe (I) in DMSO solution has a maximum absorption wavelength of 546nm, a maximum emission wavelength of 650nm, and a Stokes shift of 104 nm.
EXAMPLE 3 evaluation of the binding Capacity of Small organic molecule fluorescent Probe (I) to A.beta.protein
Fluorescence titration spectrometry: an appropriate amount of 100 μ M a β protein (a β monomer, a β oligomer and a β aggregate, available from lakeda gawarrior organisms) solution was added to PBS buffer (pH 7.4,10mM) in a total volume of 600 μ L, with the concentration of probe (I) (prepared in example 1) kept constant at 1 μ M and the protein concentration at 10 μ M, and the fluorescence spectrum thereof was measured under a fluorescence spectrophotometer to perform data processing. The parameters of the fluorescence spectrophotometer were: the working voltage is 700V, the outer slit is 10nm, and the electromagnetic slit is 10 nm.
The results are shown in FIG. 5B: the fluorescence intensity after binding of probe (I) to a β oligomers was significantly increased compared to a β monomers and a β aggregates.
Example 4 evaluation of Selectivity of organic Small molecule fluorescent Probe (I)
Fluorescence titration spectrometry: a beta oligomer (Haematococcus Shanghai) was added to PBS buffer (pH 7.4,10mM), and then different metal ions (K) were added thereto+,Ni+,Na+,Cu+,Cd2+,Pd2+,Fe2+,Mg2+,Al3+And Fe3+) (stock solutions of the corresponding metal ions were obtained by diluting standard solutions of the corresponding chloride salts, respectively) and various amino acid (alanine, arginine, aspartic acid, cysteine, glutamine, isoleucine, lysine, methionine, serine and glutathione) solutions in a total volume of 600. mu.L, maintaining the concentration of probe (I) (prepared in example 1)The degree is 1 μ M, the concentration of A β oligomers, different metal ions and various amino acids is 10 μ M, and the fluorescence spectra are measured in a fluorescence spectrophotometer without adding metal ions and amino acids as blank controls for data processing. The parameters of the fluorescence spectrophotometer were: the working voltage is 700V, the outer slit is 10nm, and the electromagnetic slit is 10 nm.
The results are shown in FIGS. 5C and 5D: compared with the A beta oligomer, the fluorescence of the probe (I) is hardly changed slightly before and after the probe (I) is combined with different metal ions and various amino acids, and the probe (I) shows good selectivity.
Example 5 titration assay of organic Small molecule fluorescent Probe (I) with A beta oligomer
Fluorescence titration spectrometry: in PBS buffer (pH 7.4,10mM), different volumes of 100 μ M a β oligomer protein (shanghai qiao organism) solutions were added, the total volume was 600 μ L, the concentration of the probe (I) (prepared in example 1) was kept constant at 1 μ M, the protein concentration was increased from 0 μ M to 5 μ M, and the fluorescence spectrum was measured under a fluorescence spectrophotometer to perform data processing. The parameters of the fluorescence spectrophotometer were: the working voltage is 700V, the outer slit is 10nm, and the electromagnetic slit is 10 nm.
The results are shown in FIG. 5E: the fluorescence intensity gradually increased with increasing concentration of a β oligomers. When the concentration of the A beta oligomer is in the range of 0-5 mu M, the fluorescence intensity of the probe (I) and the concentration of the A beta oligomer are in a linear relation, and the linear correlation coefficient (R) is 0.9906, which indicates that the probe (I) can be used for quantitative detection of the A beta oligomer.
EXAMPLE 6 in vitro saturation binding assay for Small organic molecule fluorescent probes (I)
In vitro binding constant assay saturated binding method was used: after adding a PBS buffer solution (pH 7.4,10mM) to the luciferase assay plate, 4. mu.L of a 100. mu.M solution of A.beta.oligomer protein (Shanghai Qiaozhibio; final protein concentration 2. mu.M) was added to each well, organic small molecule fluorescent probe (I) was further added at various concentrations ranging from 0. mu.M to 4. mu.M (prepared in example 1), and the final volume of each well solution was determined to be 200. mu.L. Scanning the mixed solution prepared by the method in multifunctional micropore detection equipment, and recording the fluorescence value at the maximum emission wavelength. The binding constant Kd value was calculated using GraphPad Prism 8.0.1(GraphPad Software, Inc, USA).
The results are shown in FIG. 5F: the in vitro binding constant (Kd) of probe (I) was 88.16 nM.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A fluorescent probe for detecting Abeta oligomers is characterized in that the structural general formula is shown as the formula (I):
2. the method for preparing a fluorescent probe for detecting a β oligomers of claim 1, comprising the steps of:
(1) preparation of intermediate 1
Adding p-hydroxybenzaldehyde, N-acetylglycine and sodium acetate into an organic solvent, uniformly mixing, heating to 110 +/-5 ℃ for reflux reaction, adding ethanol after the reaction is finished, cooling to room temperature to precipitate crystals, filtering, and washing to obtain a yellow solid;
adding the yellow solid, methylamine water solution and potassium carbonate into an organic solvent, heating to 80 +/-5 ℃ for reflux reaction, cooling to room temperature after the reaction is finished, adjusting the pH value to be neutral, cooling to separate out a solid, filtering, and drying to obtain an intermediate 1;
(2) preparation of intermediate 2
Dissolving the intermediate 1 in an organic solvent, adding a boron tribromide dichloromethane solution, heating to 110 +/-5 ℃ in a protective gas atmosphere to perform reflux reaction, cooling, filtering and washing after the reaction is finished, then adding a hydrogen fluoride solution, uniformly stirring, sequentially extracting with ethyl acetate, a potassium carbonate solution and water, concentrating and purifying to obtain an intermediate 2;
(3) preparation of fluorescent Probe
And adding the intermediate 2, p-dimethylaminobenzaldehyde and piperidine into an organic solvent, reacting at 100 +/-5 ℃, and concentrating, washing and purifying after the reaction is finished to obtain the fluorescent probe for detecting the A beta oligomer.
3. The method for producing a fluorescent probe for detecting a β oligomers as claimed in claim 1, wherein:
the molar ratio of the p-hydroxybenzaldehyde, the N-acetylglycine and the sodium acetate in the step (1) is 1: 0.9-1.1: 0.7-1.3;
the molar ratio of the yellow solid, the potassium carbonate and the methylamine in the step (1) and the step (2) is 1: 1.5: 4;
the molar ratio of the intermediate 1 to the boron tribromide dichloromethane in the step (2) is 1: 1.1-2;
the molar ratio of the intermediate 2, the p-dimethylaminobenzaldehyde and the piperidine in the step (3) is 1:5: 1.2-1.5.
4. The method for producing a fluorescent probe for detecting a β oligomers as claimed in claim 1, wherein:
the organic solvent in the step (1) is acetic anhydride;
the organic solvent in the step (1) and the step (II) is ethanol;
the organic solvent in the step (2) is dichloroethane;
the organic solvent in the step (3) is pyridine.
5. The method for producing a fluorescent probe for detecting a β oligomers as claimed in claim 1, wherein:
the concentration of the methylamine water solution in the step (1) and the step (II) is 33 percent by volume;
the concentration of the hydrogen fluoride solution in the step (2) is 48-55% by mass;
the concentration of the potassium carbonate solution in the step (2) is 5% by mass.
6. The method for producing a fluorescent probe for detecting a β oligomers as claimed in claim 1, wherein:
the reflux reaction time in the step (1) is 3-5 h;
the temperature of the added ethanol in the step (1) is 50-60 ℃;
the time for precipitating crystals in the step (1) is 3-5 hours;
washing in the step (1) is washing by using glacial ethanol and hot water in sequence;
the reflux reaction time in the step (1) is 2-3 h;
adjusting the pH value in the step (1) and the step (2) by adopting an HCl ethanol solution;
the temperature reduction in the step (1) and the step (II) is to 0 ℃;
the protective gas in the step (2) is nitrogen;
the reflux reaction time in the step (2) is 6-8 h;
the stirring time in the step (2) is 20-30 min;
the purification in the steps (2) and (3) is performed by adopting column chromatography; wherein the column chromatography conditions are as follows: the volume ratio of the trichloromethane to the ethanol is 50: 1; 200-300 meshes of silica gel;
the reaction time in the step (3) is 2-5 min;
the washing in the step (3) is washing by using ethyl acetate.
7. Use of the fluorescent probe for detecting A β oligomers of claim 1 for the preparation of a product for the diagnosis of Alzheimer's disease.
8. Use of the fluorescent probe for detecting A β oligomers as claimed in claim 1 for the preparation of a medicament for the treatment of Alzheimer's disease.
9. Use of the fluorescent probe for detecting a β oligomers of claim 1 for the preparation of a product for detecting β -amyloid, characterized in that:
the beta-amyloid protein is an A beta oligomer.
10. Use according to claim 7 or 9, characterized in that:
the product comprises a fluorescent probe, a detection or diagnosis reagent and a kit.
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