CN110780076B - Preparation method and application of sensitive beta-amyloid protein nano fluorescent probe - Google Patents

Preparation method and application of sensitive beta-amyloid protein nano fluorescent probe Download PDF

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CN110780076B
CN110780076B CN201911015710.0A CN201911015710A CN110780076B CN 110780076 B CN110780076 B CN 110780076B CN 201911015710 A CN201911015710 A CN 201911015710A CN 110780076 B CN110780076 B CN 110780076B
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abeta
pcl
pnipam
fluorescent probe
beta
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CN110780076A (en
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史林启
杨惠茹
黄帆
马如江
安英丽
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Nankai University
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Abstract

A preparation method and application of a sensitive beta-amyloid (Abeta) nano fluorescent probe. The probe is constructed based on a temperature-responsive composite micelle, GLVFF short peptide capable of being specifically combined with Abeta protein is accessed to a hydrophobic micro-region to improve the selective combination capability of the composite micelle on Abeta, and a fluorescent molecule capable of reacting with amino, namely p-nitrophenyl active ester modified oligo (p-phenylene) derivative (OPV-NP) fluorescent group is accessed to change fluorescent signals before and after the composite micelle captures Abeta, so that the detection of the Abeta protein content in a solution (such as cerebrospinal fluid) is realized. The preparation method is simple and nontoxic, the detection method is simple and easy to implement, easy to operate, short in time consumption and low in cost, and the detection safety and the working efficiency are improved. The invention has sensitive detection effect and specificity to the Abeta, provides a new idea for the in vitro detection of the Abeta protein with low concentration, and has wide application prospect in the early diagnosis of the Alzheimer's disease.

Description

Preparation method and application of sensitive beta-amyloid protein nano fluorescent probe
Technical Field
The invention belongs to the field of nano biomedical materials, and discloses a high-sensitivity low-cost Abeta fluorescent probe which is developed and prepared by chemically modifying GLVFF polypeptide capable of targeting Abeta by using a composite shell micelle and simultaneously combining p-nitrophenyl active ester modified oligo (p-phenylethene) derivative (OPV-NP) fluorescent molecules with amino responsiveness and is used for in-vitro detection of Abeta protein.
Background
Alzheimer's Disease (AD), also known as senile dementia, is one of the most common neurodegenerative diseases in the elderly. The progression of the disease is accompanied by a gradual loss of memory and impairment of cognitive abilities. In the late stages of AD, irreparable pathological lesions have formed, and their therapeutic efficacy is very limited. Therefore, early diagnosis and early treatment are the best approaches to treat AD. However, the methods commonly used at present, such as: diagnosis of brain imaging (PET, CT, etc.), neuropsychology, cognitive and neurological tests, etc., is mainly based on pathological changes occurring in the brain in the middle and late stages of the disease and changes in cognitive ability, and is difficult to adapt to the need of early diagnosis.
Unlike organic pathological changes and cognitive impairment of the brain, the level of the AD biomarker, beta-amyloid (a β), present in body fluids has changed significantly early in AD. Therefore, the detection of the content of the Abeta in the cerebrospinal fluid can timely and effectively carry out early diagnosis of the AD, thereby being beneficial to early treatment of the AD. At present, the detection method for A beta in body fluid is mainly enzyme-linked immunosorbent assay (ELISA). Although ELISA has better specific detection capability, the operation is complex, the detection time is long, the price of enzyme-linked antibody for A beta recognition is very expensive, and the substrate for chemiluminescence detection has carcinogenicity, so that the application of the substrate is limited, and an alternative method is urgently needed to be found. The nano particles have complex and changeable surface structures and modifiability, can meet specific requirements by designing the nano particles with proper functions, and are good choices for developing novel Abeta protein probes.
Disclosure of Invention
In order to improve the sensitivity, convenience and safety of detection on low-concentration Abeta, the invention provides a preparation method and application of a sensitive beta-amyloid nano fluorescent probe, and provides a simple, safe and economic new method for in vitro detection of low-concentration Abeta.
The invention utilizes self-assembly of a temperature-responsive diblock polymer and an amphiphilic block polymer to obtain a core-shell-crown structure composite micelle with a surface phase separation structure, and a hydrophobic micro-region part of the core-shell-crown structure composite micelle is chemically modified with LVFF short peptide and p-nitrophenyl active ester functionalized oligo (p-phenylene vinylene) derivative (OPV-NP). A cavity formed by hydrophobic micro-regions and hydrophilic chain segments on the surface of the micelle is utilized to capture A beta protein to the hydrophobic micro-regions, and meanwhile, the entrance and interference of partial large-volume protein are prevented. The fluorescence quenching of the skeleton trimeric phenylacetylene is realized by the electron withdrawing effect of the p-nitrophenyl active ester on the OPV-NP molecule. The introduction of the compound micelle in a hydrophobic micro-region can react with amino of lysine residues of Abeta protein adsorbed by the compound micelle, and the compound micelle has the function of a fluorescent probe by substituting p-nitrophenyl active ester with affinity to cause fluorescence enhancement. The LVFF short peptide is the hydrophobic core of a β protein, and is capable of specifically binding to a β protein and inhibiting a β aggregation. The introduction of the probe can further improve the selective binding capacity of the probe to the A beta protein and prevent the reaction of the small molecular protein and the OPV-NP molecule. The probe avoids the use of expensive enzyme-linked antibodies and toxic substrates in the traditional ELISA method, reduces the cost and improves the safety and the working efficiency. The nanoprobe can realize sensitive and specific detection of low-concentration Abeta protein, and provides a new idea for in vitro detection of Abeta and early diagnosis of Alzheimer's disease.
Technical scheme of the invention
A sensitive beta-amyloid nano fluorescent probe is characterized in that GLVFF short peptide capable of being specifically combined with A beta and a fluorescent molecule with amino responsiveness, namely p-nitrophenyl active ester functionalized oligo (p-phenylene vinylene) derivative (OPV-NP) capable of reacting with an amino group of protein on A beta are introduced into the tail end of a responsive chain segment of a temperature responsive diblock polymer, and then the core-shell-crown structure composite micelle capable of sensitively and specifically detecting the content of the low-concentration A beta is obtained through self-assembly with the amphiphilic block polymer and serves as the A beta nano fluorescent probe.
A preparation method of a sensitive beta-amyloid nano fluorescent probe comprises the following specific steps:
1) Synthesis of amphiphilic block polymer polycyclocaprolactone-b-polyethylene glycol (PCL-b-PEG)
Under the catalysis of stannous octoate, initiating ring-opening polymerization (ROP) of epsilon-caprolactone (epsilon-CL) by hydroxyl-terminated polyethylene glycol (PEG-OH) to obtain the PCL-b-PEG amphiphilic block polymer.
2) Synthesis of polycyclocaprolactone-trithiocarbonate (PCL-TTC) macromolecular chain transfer agent
Initiating epsilon-CL ring-opening polymerization (ROP) by utilizing hydroxyethyl acrylate (HEA) to obtain polycyclocaprolactone (PCL-OH);
and (2) reacting a chain transfer agent 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid (DDMAT) with PCL-OH after acyl chlorination to obtain the macromolecular chain transfer agent PCL-TTC.
3) Synthesis of temperature-responsive diblock polymer polycyclocaprolactone-b-poly-N-isopropylacrylamide (PCL-b-PNIPAM)
Mixing the PCL-TTC obtained in the step 2) with N-isopropylacrylamide (NIPAM) monomers, and synthesizing the PCL-b-PNIPAM through reversible addition-fragmentation chain transfer radical polymerization (RAFT).
4) Synthesis of PCL-b-PNIPAM-APM
Mixing an N- (2-aminoethyl) acrylamide hydrochloride (APM) monomer with the PCL-b-PNIPAM synthesized in the step 3), and synthesizing the PCL-b-PNIPAM-APM through RAFT polymerization.
5) Synthesis of PCL-b-PNIPAM-GLVFF
Reducing trithio ester bond at the end group of the PCL-b-PNIPAM synthesized in the step 3) into sulfydryl by utilizing n-hexylamine and dimethylphenylphosphonium, adding acryloyloxy succinimide (NAS), and carrying out click reaction on sulfydryl-double bonds to obtain the PCL-b-PNIPAM-NHS. Then reacting with amino on GLVFF, dialyzing with pure water, and vacuum freeze-drying to obtain PCL-b-PNIPAM-GLVFF.
6) Preparation of Abeta nano fluorescent probe
Dissolving the PCL-b-PEG synthesized in the step 1), the PCL-b-PNIPAM-GLVFF synthesized in the step 5) and the PCL-b-PNIPAM-APM synthesized in the step 4) in DMF according to a molar ratio of 2. Adding OPV-NP, wherein the molar ratio of the OPV-NP to APM in the polymer is more than 1, and shaking and incubating at 37 ℃ for 0.5-2h. Dropwise adding the mixture into pure water at 4 ℃ under electromagnetic stirring, stirring overnight, dialyzing by PBS to obtain composite micelles with the particle sizes of 40-300nm and uniform distribution, and incubating at 37 ℃ to obtain the Abeta nano fluorescent probe.
The A beta nano fluorescent probe can be used for specifically detecting the specificity of the A beta in a solution, namely the A beta nano fluorescent probe for detecting the A beta; the method comprises the following specific steps:
an artificial cerebrospinal fluid (ACSF) is used for preparing a beta protein and other various protein solutions [ such as Bovine Serum Albumin (BSA), ubiquitin (ubiquitin), etc. ]. After the A beta nano fluorescent probe is placed at 37 ℃ for incubation, the protein (such as BSA, ubiquitin and A beta) solutions with equal mass concentration are respectively added, and the constant temperature shaking is continued at 37 ℃ for 30min. And detecting the change of the fluorescence of the system by using a fluorescence spectrophotometer, and recording the corresponding fluorescence spectrum and the fluorescence intensity at the maximum emission wavelength. BSA and ubiquitin were found to have no effect on the system fluorescence, while the system fluorescence was significantly enhanced after addition of A β protein.
The A beta nano fluorescent probe can quantitatively detect the content of A beta in a solution, namely the detection capability of the A beta nano fluorescent probe on the A beta protein; the method comprises the following specific steps:
1) Establishing a standard curve of Abeta concentration-fluorescence intensity;
and (3) incubating the A beta nano fluorescent probe at 37 ℃, mixing the incubated A beta nano fluorescent probe with the A beta protein sample solutions with different concentrations, and continuously oscillating the mixed solution at the constant temperature of 37 ℃ for 30min. And detecting the fluorescence of the mixed solution sample under the same condition, and recording the corresponding fluorescence spectrum and the fluorescence intensity at the maximum emission wavelength to obtain a standard curve of the concentration of Abeta and the fluorescence intensity.
2) Detecting the content of Abeta;
detecting the fluorescence intensity of the mixed liquid of the sample to be detected and the nano fluorescent probe according to the method in the step 1), and obtaining the content of the Abeta through the standard curve of the Abeta concentration-fluorescence intensity established in the step 1).
The application of the A beta nano fluorescent probe can detect the A beta content in the cerebrospinal fluid of a rat, and the specific steps are as follows:
taking SD rat cerebrospinal fluid, and respectively detecting the content of Abeta by using an ELISA kit and the Abeta nano fluorescent probe, wherein the result has no significant difference.
The invention has the advantages and beneficial effects that:
the invention provides a simple and effective nano fluorescent probe for detecting in-vitro Abeta protein. The method has the advantages of simple preparation, low cost and convenient operation, avoids the use of expensive antibodies and toxic carcinogenic luminescent substrates, greatly shortens the detection time of the A beta protein, improves the safety and efficiency of detection, has good detection sensitivity and specificity, and has great value in the in vitro detection of the A beta and the early diagnosis of the Alzheimer's disease.
Drawings
FIG. 1 is a diagram of the particle size distribution of Abeta nano fluorescent probe and the corresponding transmission electron microscope; the figure shows that the nano particles are spherical, have the particle diameter of about 93nm, are uniform in size and have narrow particle size distribution;
FIG. 2 shows the fluorescence intensity change of the Abeta nanophosphoric probe incubated with different proteins; a) Fluorescence spectra of the nanoprobe after incubation with three different proteins, respectively, b) emission intensity at the maximum emission wavelength after incubation of the nanoprobe with three different proteins;
FIG. 3 is the fluorescence emission spectra of the A β nano-fluorescent probe after reaction with different concentrations of A β;
FIG. 4 is a standard curve of the concentration of A β protein relative to the fluorescence intensity of A β nanophosphorescent probes;
FIG. 5 shows the concentration of Abeta in cerebrospinal fluid of rat detected by ELISA kit and Abeta nano fluorescent probe.
Detailed Description
Example 1:
a preparation method of an A beta nanometer fluorescent probe comprises the following implementation steps:
1) Synthesis of amphiphilic block polymer PCL-b-PEG
3.0g of dried CH 3 O-PEG 114 -OH and 6.3g of vacuum distilled epsilon-CL were mixed in a 50mL dry eggplant-shaped bottle, dissolved in 15mL redistilled anhydrous toluene, and one drop of stannous octoate (Sn (Oct) was added 2 ). Then, after three cycles of freezing by liquid nitrogen, vacuum pumping, nitrogen filling and thawing, the reaction was carried out for 12 hours in an oil bath at 110 ℃ under the protection of nitrogen. After the reaction, the reaction mixture was diluted with an appropriate amount of dichloromethane and then precipitated in ten times the volume of ethyl acetate. After the precipitation is completed, the amphiphilic block polymer PCL-b-PEG is obtained after suction filtration, washing and vacuum drying.
2) Synthesis of macromolecular chain transfer agent PCL-TTC
Adding 0.1g hydroxyethyl acrylate (HEA) and 7.0g reduced epsilon-CL into a bottle shaped like a eggplant, adding 12mL redistilled toluene to dissolve, adding one drop of Sn (Oct) 2 . Then, freezing with liquid nitrogen, vacuumizing, introducing nitrogen, thawing and repeating for three times. Then the reaction is carried out for 12h in an oil bath at 110 ℃ under the protection of nitrogen. After the reaction is finished, adding a proper amount of dichloromethane for dilution, precipitating with ethyl glacial ether with ten times of volume, carrying out suction filtration, washing, and carrying out vacuum drying to obtain the polycaprolactone PCL-OH.
0.6g of the chain transfer agent DDMAT was added to a 50mL round bottom flask and dissolved in 10mL dichloromethane. 0.6mL of oxalyl chloride was dissolved in 5mL of dichloromethane and added dropwise to the round bottom flask over 10 minutes under ice water bath and magnetic stirring. After the dropwise addition, the temperature is raised to 25 ℃ for reaction for 2h. Roto-steaming at 30 ℃ to remove dichloro and excess oxalyl chloride, adding 2mL of dichloromethane, and roto-steaming again, repeated three times. 5g of PCL-OH was added to a round-bottomed flask, dissolved in 20mL of dichloromethane, and 0.17g of triethylamine was added to the solution to react at 25 ℃ for 24 hours. And concentrating the reaction solution to 5mL, precipitating in ethyl acetate, filtering, washing, and drying in vacuum to obtain the PCL-TTC macromolecular chain transfer agent.
3) Synthesis of temperature-responsive block polymer PCL-b-PNIPAM
Firstly, carrying out recrystallization on NIPAM by using n-hexane as a solvent to remove a polymerization inhibitor, and carrying out vacuum drying to obtain the NIPAM monomer. Mixing 1.2g of recrystallized NIPAM and 3.0g of PCL-TTC, dissolving in DMF, adding 15mg of Azobisisobutyronitrile (AIBN) as an initiator, freezing by liquid nitrogen, vacuumizing, filling nitrogen and unfreezing, circulating for three times, reacting in an oil bath at 70 ℃ for 24h under the protection of nitrogen, synthesizing PCL-b-PNIPAM by RAFT polymerization, precipitating by glacial ethyl ether, carrying out suction filtration, washing, and carrying out vacuum drying to obtain PCL-b-PNIPAM powder.
4) Synthesis of PCL-b-PNIPAM-APM
Mixing 25.5mg of APM monomer and 1.0g of PCL-b-PNIPAM, dissolving in DMF, adding 5mg of AIBN serving as an initiator, and carrying out RAFT reaction to obtain PCL-b-PNIPAM-APM, wherein the specific reaction conditions are the same as the PCL-b-PNIPAM synthesis conditions in the step 3).
5) Synthesis of PCL-b-PNIPAM-GLVFF
0.5g of PCL-b-PNIPAM was mixed with 30mg of acryloxysuccinimide (NAS), dissolved in 10mL of dichloromethane, 35. Mu.L of Dimethylphenylphosphonium (DMPP) was added as a catalyst, and N was introduced 2 Deoxygenation by bubbling for 20min. Then 2.5mL of N-hexylamine is added, and N is introduced into the reaction system again 2 Deoxygenated by bubbling for 15min, then stirred overnight at room temperature in the dark. After the reaction is finished, diluting with dichloromethane, precipitating with glacial ethyl ether, filtering, washing, and drying in vacuum to obtain PCL-b-PNIPAM-NHS. And mixing the product with excessive GLVFF, dissolving in 5mL of DMF, adding 100 mu L of triethylamine, electromagnetically stirring overnight under the condition of water bath at 30 ℃, dialyzing with pure water, and performing vacuum freeze drying to obtain the PCL-b-PNIPAM-GLVFF.
6) Preparation of Abeta nano fluorescent probe
Dissolving 2mg of PCL-b-PEG,1mg of PCL-b-PNIPAM-GLVFF and 1mg of PCL-b-PNIPAM-APM in DMF, adding 0.5mg of OPV-NP to obtain a stock solution with the polymer concentration of 5mg/ml, and performing shake incubation at 37 ℃ for 30min. Dropwise adding the mixture into pure water at 4 ℃ under electromagnetic stirring, stirring overnight, dialyzing by PBS to obtain 0.5mg/ml micelle, and incubating for 30min at 37 ℃ to obtain the Abeta nano fluorescent probe.
The micelle particle size is characterized by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), the micelle is spherical in shape, has an average particle size of 93nm, is uniform in size and has narrow particle size distribution, as shown in figure 1.
7) Specificity of A beta nano fluorescent probe for A beta detection
An artificial cerebrospinal fluid (ACSF) is used for preparing solutions of Bovine Serum Albumin (BSA), ubiquitin protein (ubiquitin) and Abeta protein with equal mass concentration. And (3) placing the Abeta nano fluorescent probe at the constant temperature of 37 ℃ for 30min, respectively adding three protein solutions with equal mass concentration, and continuously oscillating at the constant temperature of 37 ℃ for 30min. The change in fluorescence of the system (Ex =376 nm) was detected using a fluorescence spectrophotometer, and the corresponding fluorescence spectrum and fluorescence intensity at the maximum emission wavelength (Em =458 nm) were recorded. BSA and ubiquitin were found to have no effect on system fluorescence, whereas system fluorescence was significantly enhanced after addition of A β protein, as shown in FIG. 2.
8) Detection capability of Abeta nano fluorescent probe on Abeta protein
(1) Establishing a standard curve of Abeta concentration-fluorescence intensity;
artificial cerebrospinal fluid (ACSF) was used to formulate a range of concentrations of Α β protein solutions. Keeping the temperature of the A beta fluorescent probe constant at 37 ℃ for 30min, mixing the A beta fluorescent probe with A beta solutions with different concentrations in an equal volume, and continuing to shake and incubate at 37 ℃ for 30min; detecting the fluorescence (Ex =376 nm) of the mixed solution by using a fluorescence spectrophotometer, recording the corresponding fluorescence spectrum (fig. 3) and the fluorescence intensity at the maximum emission wavelength (Em =458 nm), and establishing a standard curve of the concentration of the A beta to the fluorescence intensity, such as fig. 3 and fig. 4;
(2) Detecting the content of Abeta;
detecting the fluorescence intensity of the mixed solution according to the method in the step 1), and obtaining the content of the Abeta through the standard curve of the Abeta concentration-fluorescence intensity established in the step 1).
9) Method for detecting content of Abeta in cerebrospinal fluid of rat by Abeta nano fluorescent probe
Taking the cerebrospinal fluid of a female SD rat, respectively detecting the content of Abeta by using an ELISA kit and the Abeta nano fluorescent probe, wherein the result has no significant difference, and the reliability of the Abeta nano fluorescent probe is proved, as shown in figure 5.

Claims (4)

1. A sensitive beta-amyloid protein A beta nano fluorescent probe is characterized in that: GLVFF short peptide capable of being specifically combined with Abeta and fluorescent molecule with amino responsiveness, namely p-nitrophenyl active ester functionalized oligomeric p-phenylene vinylene derivative OPV-NP capable of reacting with Abeta protein amino are introduced into the tail end of a responsive chain segment of a temperature responsive diblock polymer, namely polycyclocaprolactone-b-poly N-isopropyl acrylamide (PCL-b-PNIPAM), and then the core-shell-crown structure composite micelle capable of sensitively and specifically detecting the content of Abeta is obtained to serve as an Abeta nano fluorescent probe through self-assembly with an amphiphilic block polymer, namely polycyclocaprolactone-b-polyethylene glycol (PCL-b-PEG).
2. The preparation method of the A beta nano fluorescent probe of claim 1, which is characterized by comprising the following steps:
1) Synthesis of amphiphilic block polymer polycyclocaprolactone-b-polyethylene glycol PCL-b-PEG
Under the catalysis of stannous octoate, the PCL-b-PEG amphiphilic block polymer is obtained by ring-opening polymerization of epsilon-caprolactone epsilon-CL initiated by hydroxyl-terminated polyethylene glycol PEG-OH;
2) Synthesis of polycyclohexyllactone-trithiocarbonate PCL-TTC macromolecular chain transfer agent
Initiating epsilon-CL ring-opening polymerization by hydroxyethyl acrylate HEA to obtain polycaprolactone PCL-OH; performing acyl chlorination on a chain transfer agent 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid DDMAT, and then reacting with PCL-OH to obtain a macromolecular chain transfer agent PCL-TTC;
3) Synthesis of temperature-responsive diblock polymer polycyclocaprolactone-b-poly (N-isopropylacrylamide) PCL-b-PNIPAM
Mixing the PCL-TTC obtained in the step 2) with an N-isopropyl acrylamide NIPAM monomer, and performing reversible addition-fragmentation chain transfer free radical polymerization to obtain PCL-b-PNIPAM;
4) Synthesis of PCL-b-PNIPAM-APM
Mixing an N- (2-aminoethyl) acrylamide hydrochloride APM monomer with the PCL-b-PNIPAM synthesized in the step 3), and performing reversible addition-fragmentation chain transfer free radical polymerization to obtain the PCL-b-PNIPAM-APM;
5) Synthesis of PCL-b-PNIPAM-GLVFF
Reducing trithiocarbonate bonds at the end group of the PCL-b-PNIPAM synthesized in the step 3) into sulfydryl by utilizing n-hexylamine and dimethylphenylphosphonium, adding acryloyloxy succinimide NAS, obtaining PCL-b-PNIPAM-NHS through a sulfydryl-double bond click reaction, then reacting with amino on short peptide GLVFF, dialyzing with pure water, and performing vacuum freeze drying to obtain PCL-b-PNIPAM-GLVFF;
6) Preparation of Abeta nano fluorescent probe
Dissolving the PCL-b-PEG synthesized in the step 1), the PCL-b-PNIPAM-GLVFF synthesized in the step 5) and the PCL-b-PNIPAM-APM synthesized in the step 4) in DMF according to the molar ratio of 2; dropwise adding the mixture into pure water at 4 ℃ under electromagnetic stirring, stirring overnight, dialyzing by PBS to obtain composite micelles with the particle sizes of 40-300nm and uniform distribution, and incubating at 37 ℃ to obtain the Abeta nano fluorescent probe.
3. The application of the A beta nano fluorescent probe of claim 1, which is characterized in that the application can quantitatively detect the content of A beta in a solution, and the specific steps are as follows:
1) Establishing a standard curve of Abeta concentration-fluorescence intensity;
incubating the Abeta fluorescent probe at 37 ℃, mixing the Abeta fluorescent probe with Abeta sample solutions with different concentrations in equal volume, and continuing to perform oscillation incubation at 37 ℃ for more than 30min; detecting the fluorescence of each sample under the same condition, recording the corresponding fluorescence spectrum and the fluorescence intensity at the maximum emission wavelength, and establishing a standard curve of Abeta concentration-fluorescence intensity;
2) Detecting the content of Abeta;
detecting the fluorescence intensity of the sample to be detected according to the method in the step 1), and obtaining the content of the Abeta in the sample through the Abeta concentration-fluorescence intensity standard curve established in the step 1).
4. The use of the A beta nano fluorescent probe according to claim 3, characterized in that the probe is capable of specifically detecting A beta protein in solution.
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