Alzheimer disease early diagnosis kit, fluorescent probe and application thereof

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly provides an Alzheimer's disease early diagnosis kit, a fluorescent probe and application thereof. The kit comprises a reagent A, a reagent B, a reagent C and a reagent D, wherein the reagent A is a boric acid modified metal organic framework material, boric acid modified graphene oxide or boric acid modified silicon dioxide microspheres, the reagent B is a PBS buffer solution with the pH value of 7.8-8.5, the reagent C is a PBS buffer solution containing fructose or glucose and with the pH value of 6.6-7.2, and the reagent D is a fluorescent probe of a specific targeting extracellular vesicle A beta 1-42 oligomer. The kit disclosed by the invention can be used for accurately and sensitively detecting the content of the extracellular vesicle (at) A beta 1-42 oligomer so as to judge the risk of suffering from the Alzheimer's disease. The detection method is simple and easy to operate, has high sensitivity and specificity, is suitable for large-scale population screening, and has important practical value for detection and early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and in particular to an early diagnosis kit for Alzheimer's disease, a fluorescent probe and its application. Background Art

[0002] Alzheimer's disease is a common neurodegenerative disease, and the role of β-amyloid (Aβ) in its disease progression cannot be ignored. The over-secretion of Aβ will lead to the deposition of a large amount of Aβ in the brain tissue, especially Aβ 1-42 is more likely to aggregate and form plaques. Among them, Aβ 1-42 oligomers have stronger neurotoxicity, can change the neuronal signaling pathway, lead to abnormal synaptic function and cause neuronal death, and ultimately lead to dementia. It is worth noting that the imbalance of Aβ metabolism may occur decades before the clinical symptoms appear, which provides an important possibility for the diagnosis of the early asymptomatic stage of Alzheimer's disease.

[0003] At present, the detection of Alzheimer's disease biomarkers is mainly brain imaging and cerebrospinal fluid biomarker analysis. However, brain imaging examinations such as magnetic resonance imaging and positron emission tomography (PET) are expensive; while the biomarker detection based on cerebrospinal fluid is invasive, and the acceptance of patients is low, and its clinical application is limited. In contrast, the detection of biomarkers from peripheral body fluids such as blood and urine is relatively simple, inexpensive and non-invasive, which can improve the accessibility of diagnosis and has gradually attracted attention. However, due to the too low concentration of biomarkers for diagnosis in peripheral body fluids, it often faces problems such as difficult separation from body fluids, low sensitivity and poor specificity, and is difficult to be popularized and applied. Summary of the Invention

[0004] The purpose of the present invention is to provide an early diagnosis kit for Alzheimer's disease, a fluorescent probe and its application. The use method of the kit is simple and easy to operate, and at the same time has high sensitivity and specificity. Compared with the currently commonly used cerebrospinal fluid and PET biomarker detection technologies in clinics, this method is non-invasive and low-cost, suitable for large-scale population screening, and has good prospects for popularization and application.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] First of all, the present invention provides an early diagnosis kit for Alzheimer's disease, and the detection target of the kit is extracellular vesicle surface-bound Aβ 1-42 oligomers (extracellular vesicle@Aβ 1-42 oligomers).

[0007] Furthermore, the kit includes reagent A, reagent B, reagent C and reagent D; wherein,

[0008] The reagent A is a boric acid-modified metal-organic framework material, boric acid-modified graphene oxide, or boric acid-modified silica microspheres;

[0009] The reagent B is a PBS buffer solution with a pH of 7.8 - 8.5;

[0010] The reagent C is a PBS buffer solution with a pH of 6.6 - 7.2 containing fructose or glucose;

[0011] The reagent D is a fluorescent probe specifically targeting extracellular vesicle @Aβ 1-42 oligomers; The structural formula of the fluorescent probe specifically targeting extracellular vesicle @Aβ 1-42 oligomers is as follows:

[0012]

[0013] Furthermore, the concentration of the reagent A is 1 - 6 mg / mL; the concentration of the reagent B is 0.01 M; the concentration of the reagent C is 0.01 M; the concentration of the reagent D is 2.5 - 20 μM.

[0014] Furthermore, in the reagent C, the concentration of the fructose or glucose is 20 - 100 mM.

[0015] Furthermore, the preparation method of the reagent A is as follows:

[0016] (1) Ultrasonically dissolve carboxylphenylboronic acid in dimethyl sulfoxide, then add EDC·HCl and NHS, and stir and react at room temperature to obtain an activated carboxylphenylboronic acid solution;

[0017] (2) After activating the amino-functionalized metal-organic framework material, amino-functionalized graphene oxide, or amino-modified silica microspheres at high temperature, ultrasonically disperse them in dimethyl sulfoxide to obtain an activated amino-functionalized material;

[0018] (3) Drop the activated amino-functionalized material into the activated carboxylphenylboronic acid solution, stir and react at room temperature, centrifuge to collect the precipitate, wash it successively with dimethyl sulfoxide and ethanol, and then dry it under vacuum to obtain the boric acid-modified metal-organic framework material, boric acid-modified graphene oxide, or boric acid-modified silica microspheres.

[0019] Even further, in the preparation of the reagent A, the concentration of the amino-functionalized metal-organic framework material, amino-functionalized graphene oxide, or amino-modified silica microspheres is 0.25 - 2.5 mg / mL, and the mass ratio of it to carboxylphenylboronic acid is 1:2 - 8;

[0020] The concentration of the carboxylphenylboronic acid is 2 - 5 mg / mL;

[0021] The molar ratio of the carboxylphenylboronic acid to EDC·HCl is 1:1 - 10;

[0022] The molar ratio of the EDC·HCl to NHS is 1:2 - 5.

[0023] Furthermore, the kit further includes a sample diluent and an Aβ 1-42 oligomeric protein standard;

[0024] wherein, the 1-42 molecular weight of the Aβ oligomeric protein is 32.4 kDa; the concentrations of the Aβ 1-42 oligomeric protein standards are 1.0 pM, 2.0 pM, 3.0 pM, 4.0 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, and 10.0 pM respectively.

[0025] Furthermore, the detection method of the above-mentioned kit includes the following steps:

[0026] (1) Mix the biological sample with Reagent A, adjust the pH to 7.8 - 8.5 with Tris-HCl, incubate at 2 - 8 °C for 5 - 60 min, centrifuge to discard the supernatant, and wash three times with Reagent B; add Reagent C and gently shake for 5 - 30 min, centrifuge to collect the supernatant as the sample to be tested;

[0027] (2) Take the sample to be tested and mix it with Reagent D, incubate at room temperature for 1 - 5 min, measure the fluorescence intensity value of the mixed solution, substitute it into the standard curve, and calculate the level of extracellular vesicle @Aβ 1-42 oligomer in the biological sample to be tested.

[0028] wherein, the biological sample includes plasma and serum;

[0029] The detection wavelength of the fluorescence intensity value of the mixed solution is 560 - 590 nm, and the excitation wavelength is 446 - 460 nm;

[0030] The standard curve is constructed by curve fitting with the concentration of Aβ 1-42 oligomer as the abscissa and the change in the fluorescence intensity value of the corresponding mixed solution as the ordinate.

[0031] Second, the present invention also provides a fluorescence probe for quantitatively detecting extracellular vesicle @Aβ 1-42 oligomer, and the structural formula of the fluorescence probe is as follows:

[0032]

[0033] The preparation method of the above fluorescence probe includes the following steps:

[0034] S1: Dissolve carbazole-3-bromo, 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate and potassium carbonate in tetrahydrofuran (THF), reflux at 75 °C for 10 - 16 h. After the reaction, cool to room temperature, rotary evaporate to remove THF, and purify by column chromatography to obtain Intermediate 1:

[0035]

[0036] S2: Add Intermediate 1 and (1,3-dioxolan-2-yl)triphenylphosphonium to a mixed solution of saturated potassium carbonate solution and dichloromethane, reflux at 100 °C for 22 - 26 h. After the reaction, cool to room temperature, add THF containing 10% hydrochloric acid, stir for 1 - 3 h, adjust the pH to 6.8 - 7.2, and purify by extraction, rotary evaporation and column chromatography to obtain Intermediate 2:

[0037]

[0038] S3: Dissolve Intermediate 2 and 1-butyl-4-methylpyridinium iodide in toluene, add piperidine, heat to 110 °C and reflux for 2 - 4 h. After the reaction, cool to room temperature, rotary evaporate to remove toluene, and purify by column chromatography to obtain a red solid, which is the fluorescent probe for quantitatively detecting extracellular vesicle @Aβ 1-42 oligomers.

[0039] Furthermore, the molar ratio of carbazole-3-bromo, 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate to potassium carbonate described in step S1 is 1:3 - 5:15 - 25;

[0040] The volume ratio of the saturated potassium carbonate solution to dichloromethane described in step S2 is 1:1;

[0041] The molar ratio of Intermediate 1 to (1,3-dioxolan-2-yl)triphenylphosphonium described in step S2 is 1:1 - 2;

[0042] The molar ratio of Intermediate 2, 1-butyl-4-methylpyridinium iodide to piperidine described in step S3 is 1 - 2:1:0.4 - 0.8.

[0043] Thirdly, the present invention also provides the application of the above fluorescent probe for quantitatively detecting extracellular vesicle @Aβ 1-42 oligomers in the preparation of products for diagnosing Alzheimer's disease.

[0044] Furthermore, the products include fluorescent probes, detection reagents, diagnostic reagents or kits.

[0045] The beneficial effects of the present invention are as follows:

[0046] The present invention provides a kit for early diagnosis of Alzheimer's disease, which can efficiently isolate and enrich extracellular vesicles and accurately and sensitively detect extracellular vesicle @Aβ 1-42 oligomer content. It is based on the content of extracellular vesicle @Aβ 1-42 oligomers to achieve early diagnosis of Alzheimer's disease, greatly improving the detection specificity and accuracy rate. The sensitivity can reach 100%, and the specificity is 96.3%. Compared with the existing detection methods, the kit of the present invention is simple to operate, only requires a small amount of blood to achieve detection, has a low detection cost and is relatively non-invasive. At the same time, it does not rely on antibodies, has higher specificity, sensitivity and accuracy, is particularly suitable for large-scale population screening, has broad application prospects, and is suitable for popularization and application.

[0047] In addition, the fluorescence probe for quantitatively detecting extracellular vesicle @Aβ 1-42 oligomers provided by the present invention can meet the detection and precise quantification requirements of extracellular vesicle @Aβ 1-42 oligomers at a lower concentration, providing the possibility for earlier and more accurate identification of Alzheimer's disease and delaying the progression of the disease. Brief Description of the Drawings

[0048] Figure 1 is the standard curve graph of the detection of extracellular vesicle @Aβ 1-42 oligomers by the kit for early diagnosis of Alzheimer's disease of the present invention.

[0049] Figure 2 is the specific detection result of the detection of extracellular vesicle @Aβ 1-42 oligomers by the kit for early diagnosis of Alzheimer's disease of the present invention.

[0050] Figure 3 is the result of the quantitative detection of extracellular vesicle @Aβ 1-42 oligomers in the plasma of healthy mice and mice with Alzheimer's disease at different degrees.

[0051] Figure 4 is the receiver operating characteristic curve analysis graph of the detection results of Alzheimer's disease patients and healthy control groups. Detailed Embodiments

[0052] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0053] Example 1

[0054] This embodiment provides a kit for early diagnosis of Alzheimer's disease, which includes: reagent A, reagent B, reagent C, reagent D, sample diluent, and a series of Aβ oligomeric protein standards with different concentrations. 1-42 The oligomeric protein standards.

[0055] Among them, reagent A is a boric acid-modified metal-organic framework material with a concentration of 1 mg / mL; reagent B is a PBS buffer solution with pH 7.8 and a concentration of 0.01 M; reagent C is a PBS eluent containing 80 mM fructose (pH = 6.8) with a concentration of 0.01 M; reagent D is a fluorescent probe (with a concentration of 10 μM) that specifically targets extracellular vesicles @Aβ oligomers. 1-42 The fluorescent probe for the oligomeric protein standards.

[0056] The preparation method of the boric acid-modified metal-organic framework material is as follows:

[0057] Weigh 223 mg (1.0 mmol) of zirconium chloride and 181 mg (1.0 mmol) of 2-aminoterephthalic acid and add them to 30 mL of N,N-dimethylformamide (DMF). Ultrasonic for 20 min, then add 4.3 mL of acetic acid and 0.3 mL of water to the above mixed solution, continue ultrasonic for 10 min, and then pour it into a high-pressure reaction kettle with a 100 mL polytetrafluoroethylene liner and place it in an oven at 120 °C for heating for 24 h. After the reaction is completed, it is naturally cooled to room temperature, and the precipitate is collected by centrifugation at 12,000 rpm for 10 min, washed 3 times with DMF and methanol, and vacuum dried at 60 °C to obtain an amino-functionalized metal-organic framework material.

[0058] Weigh 40 mg (0.2 mmol) of 4-carboxyphenylboronic acid and dissolve it in 10 mL of dimethyl sulfoxide. Ultrasonic for 20 min. Add 77 mg (0.4 mmol) of EDC·HCl and 115 mg (1.0 mmol) of NHS to the above solution, and stir at room temperature to activate the carboxyl group for 40 min. Add 10 mg of the prepared amino-functionalized metal-organic framework material, activate it at 120 °C for 6 h, then add it to 10 mL of dimethyl sulfoxide, ultrasonic for 30 min until completely dispersed, and then drop it into the activated carboxyphenylboronic acid solution, and stir at 150 rpm at room temperature for 24 h. Centrifuge at 12,000 rpm for 10 min, wash 3 times with dimethyl sulfoxide and ethanol in turn, and vacuum dry at 60 °C to obtain the boric acid-modified metal-organic framework material.

[0059] The fluorescent probe that specifically targets extracellular vesicles @Aβ oligomers 1-42 The structural formula is as follows:

[0060]

[0061] Its preparation method includes the following steps:

[0062] S1: Dissolve carbazole-3-bromo (1 mmol, 195.2 mg), 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate (4 mmol, 1.10 g) and potassium carbonate (20 mmol, 2.76 g) in 20 mL of THF, and reflux the reaction at 75 °C for 12 h. After the reaction is cooled to room temperature, remove THF by rotary evaporation and purify by column chromatography to obtain intermediate 1; its structural formula is as follows:

[0063]

[0064] S2: Add intermediate 1 (1 mmol, 297.4 mg) and (1,3-dioxolan-2-yl)triphenylphosphonium (1 mmol, 336.4 mg) to a mixed solution of 40 mL of saturated potassium carbonate solution: dichloromethane with a volume ratio of 1:1, and reflux at 100 °C for 22 h. After cooling to room temperature, add 20 mL of THF containing 10% hydrochloric acid and stir for 2 h, then neutralize to pH 7.2 with sodium hydroxide. Extract with ethyl acetate, concentrate by rotary evaporation, and purify by column chromatography to obtain intermediate 2; its structural formula is as follows:

[0065]

[0066] S3: Dissolve intermediate 2 (1.5 mmol, 485.1 mg) and 1-butyl-4-methylpyridinium iodide (1 mmol, 277.2 mg) in 40 mL of toluene, then add piperidine (0.66 mmol, 65.2 μL), heat to 110 °C and reflux for 2 h. After the reaction is completed, cool to room temperature, remove toluene by rotary evaporation, and purify by column chromatography to obtain a red solid, which is the fluorescent probe of the specific targeting extracellular vesicle @Aβ 1-42 oligomer.

[0067] Example 2

[0068] This example provides a kit for early diagnosis of Alzheimer's disease, which includes: reagent A, reagent B, reagent C, reagent D, sample diluent and a series of concentrations of Aβ 1-42 oligomer protein standards. Aβ 1-42 The concentrations of the Aβ

[0069] oligomer protein standards are 1.0 pM, 2.0 pM, 3.0 pM, 4.0 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, 10.0 pM respectively. Among them, reagent A is boric acid-modified graphene oxide with a concentration of 3 mg / mL; reagent B is a PBS buffer solution with pH 8.5 and a concentration of 0.01 M; reagent C is a PBS eluent (pH = 7.2) containing 20 mM fructose with a concentration of 0.01 M; reagent D is the specific targeting extracellular vesicle @Aβ1-42 Fluorescent probe for oligomers (concentration: 5 μM).

[0070] The preparation of the boric acid-modified graphene oxide is as follows:

[0071] Weigh 0.2 g of graphene oxide and add it to 40 mL of DMF. Ultrasonically disperse for 3 h to obtain a uniform suspension. Slowly add 9.0 g of ethylenediamine under vigorous stirring and react at room temperature for 24 h. After the reaction is completed, add 100 mL of absolute ethanol and let it stand overnight. Remove the supernatant, filter by vacuum filtration, wash repeatedly with ethanol and deionized water, and dry in vacuum at 60 °C for 24 h to obtain amino-functionalized graphene oxide.

[0072] Activate 2.5 mg of amino-functionalized graphene oxide at 120 °C for 6 h, add it to 10 mL of dimethyl sulfoxide solution, and ultrasonically disperse for 1 h. Weigh 20 mg (0.1 mmol) of 4-carboxyphenylboronic acid and dissolve it in 10 mL of dimethyl sulfoxide. Add 192 mg (1.0 mmol) of EDC·HCl and 230 mg (2.0 mmol) of NHS, activate for 40 min, and dropwise add the above-mentioned amino-functionalized graphene oxide under stirring, and continue to stir and react for 24 h. Centrifuge to collect the precipitate, wash 3 times with dimethyl sulfoxide and ethanol respectively, and dry in vacuum at 60 °C to obtain boric acid-modified graphene oxide.

[0073] The specific extracellular vesicle-targeted @Aβ 1-42 The structural formula of the fluorescent probe for oligomers is as follows:

[0074]

[0075] Its preparation method includes the following steps:

[0076] S1: Dissolve carbazole-3-bromo (1 mmol, 195.2 mg), 2-(2-methoxyethoxy)ethyl 4-methylbenzenesulfonate (3 mmol, 823.0 mg) and potassium carbonate (24 mmol, 3.32 g) in 15 mL of THF, and reflux and react at 75 °C for 16 h. After the reaction is cooled to room temperature, remove THF by rotary evaporation and purify by column chromatography to obtain intermediate 1; its structural formula is as follows:

[0077]

[0078] S2: Add intermediate 1 (2 mmol, 594.8 mg) and (1,3-dioxolan-2-yl) triphenylphosphonium (4 mmol, 1.35 g) to a mixed solution of 60 mL saturated potassium carbonate solution and dichloromethane with a volume ratio of 1:1, and reflux at 100 °C for 26 h. After cooling to room temperature, add 25 mL of THF containing 10% hydrochloric acid and stir for 1.5 h, then neutralize to pH 7.0 with sodium hydroxide. Extract with ethyl acetate, concentrate by rotary evaporation, and purify by column chromatography to obtain intermediate 2; its structural formula is as follows:

[0079]

[0080] S3: Dissolve intermediate 2 (2 mmol, 646.8 mg) and 1-butyl-4-methylpyridinium iodide (1 mmol, 277.2 mg) in 50 mL of toluene, then add piperidine (0.75 mmol, 74.1 μL), heat to 110 °C and reflux for 4 h. After the reaction is completed, cool to room temperature, rotary evaporate to remove toluene, and purify by column chromatography to obtain a red solid, which is the fluorescent probe of specific targeting extracellular vesicles @Aβ 1-42 oligomer.

[0081] Example 3

[0082] This example provides a kit for early diagnosis of Alzheimer's disease, which includes: reagent A, reagent B, reagent C, reagent D, sample diluent, and a series of concentrations of Aβ 1-42 oligomer protein standards.

[0083] Among them, reagent A is boric acid-modified silica microspheres with a concentration of 6 mg / mL; reagent B is a PBS washing buffer with pH 8.2 and a concentration of 0.01 M; reagent C is a PBS eluent containing 100 mM fructose (pH = 6.6) with a concentration of 0.01 M; reagent D is a fluorescent probe of specific targeting extracellular vesicles @Aβ 1-42 oligomer (concentration of 20 μM).

[0084] The preparation of the boric acid-modified silica microspheres is as follows:

[0085] Weigh 0.1 g of spherical silica and activate it at 110 °C for 4 h, resuspend it in 20 mL of anhydrous toluene, ultrasonically disperse it evenly, then add 0.15 mL of 3-aminopropyltriethoxysilane, and carry out condensation reflux at 80 °C, stir and react for 24 h. After the reaction is completed, wait until it cools to room temperature, centrifuge at 6,000 rpm for 5 min, wash the precipitate with anhydrous ethanol 5 times, and dry it at 60 °C to constant weight to obtain amino-modified silica microspheres.

[0086] Weigh 50 mg (0.3 mmol) of 4-carboxyphenylboronic acid and ultrasonically dissolve it in 10 mL of dimethyl sulfoxide. Add 345 mg (1.8 mmol) of EDC·HCl and 978 mg (8.5 mmol) of NHS, and stir for activation for 40 min. Activate 25 mg of amino-modified silica microspheres at 120 °C for 9 h, then add them to 10 mL of dimethyl sulfoxide. After ultrasonic dispersion, dropwise add them to the above-activated carboxyphenylboronic acid solution, and stir at 120 rpm at room temperature for 24 h. Centrifuge to collect the precipitate, wash it three times with dimethyl sulfoxide and ethanol successively, and dry it under vacuum at 25 °C to obtain boronic acid-modified silica microspheres.

[0087] The specific extracellular vesicle-targeting @Aβ 1-42 The structural formula of the fluorescent probe for oligomers is as follows:

[0088]

[0089] Its preparation method includes the following steps:

[0090] S1: Dissolve carbazole-3-bromo (1 mmol, 195.2 mg), 2-(2-methoxyethoxy)ethyl-4-methylbenzenesulfonate (5 mmol, 1.37 g) and potassium carbonate (16 mmol, 2.21 g) in 25 mL of THF, and reflux at 75 °C for 12 h. After the reaction is cooled to room temperature, remove THF by rotary evaporation and purify by column chromatography to obtain intermediate 1; its structural formula is as follows:

[0091]

[0092] S2: Add intermediate 1 (1 mmol, 297.4 mg) and (1,3-dioxolan-2-yl)triphenylphosphonium (1.5 mmol, 504.6 mg) to a mixed solution of 40 mL of saturated potassium carbonate solution: dichloromethane with a volume ratio of 1:1, and reflux at 100 °C for 24 h. After cooling to room temperature, add 20 mL of THF containing 10% hydrochloric acid and stir for 3 h, then neutralize to pH 6.8 with sodium hydroxide. Extract with ethyl acetate, concentrate by rotary evaporation, and purify by column chromatography to obtain intermediate 2; its structural formula is as follows:

[0093]

[0094] S3: Dissolve intermediate 2 (1 mmol, 323.4 mg) and 1-butyl-4-methylpyridinium iodide (1 mmol, 277.2 mg) in 45 mL of toluene, then add piperidine (0.45 mmol, 44.5 μL), heat to 110 °C and reflux for 3 h. After the reaction is completed, cool to room temperature, remove toluene by rotary evaporation, and purify by column chromatography to obtain the red solid, which is the specific extracellular vesicle-targeting @Aβ1-42 Fluorescent probes for oligomers.

[0095] Example 4

[0096] This example provides a detection method for the Alzheimer's disease early diagnosis kit prepared in Example 1 and detects its sensitivity and accuracy, and the steps are as follows:

[0097] (1) Collect plasma or serum from patients with Alzheimer's disease and healthy subjects, mix 500 μL of plasma or 300 μL of serum with reagent A, adjust the pH to 7.8-8.2 with Tris-HCl, incubate at 4°C for 20-60 min, centrifuge and discard the supernatant, and wash three times with reagent B. Add reagent C and shake gently for 10-30 min, centrifuge and collect the supernatant as the sample to be tested;

[0098] (2) Set up blank wells, standard wells, and test sample wells. Add 50 μL of blank sample diluent to the blank wells; add 50 μL of different concentrations of Aβ to the standard wells. 1-42 Oligomer protein standard (Aβ 1-42 The concentrations of the oligomeric protein standards were 1.0 pM, 2.0 pM, 3.0 pM, 4.0 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, and 10.0 pM, respectively); 50 μL of the supernatant collected by centrifugation in step (1) was added to the sample well to be tested;

[0099] (3) Add 50 μL of reagent D to each well and incubate at room temperature for 1 to 4 min. Detect the fluorescence intensity of the mixed solution at a wavelength of 560 to 576 nm under an excitation wavelength of 446 to 460 nm.

[0100] Aβ 1-42 The concentration of oligomers is used as the horizontal axis, and the corresponding change in fluorescence intensity value is used as the vertical axis, and the standard curve is obtained by fitting. Figure 1 As shown, Aβ 1-42 The fluorescence intensity changes with Aβ in the oligomer concentration range of 0 to 10 pM 1-42 The oligomer concentration has a good linear relationship (R 2 =0.997), indicating that the kit of the present invention has high sensitivity and high accuracy within this concentration range.

[0101] Example 4

[0102] This example investigates the specificity of the kit prepared in Example 2. The detection method is the same as that in Example 3, except that different concentrations of Aβ 1-42 The oligomeric protein standards were replaced with Aβ 1-42 Monomer or Aβ 1-42 Fiber, Mg 2+, potential interfering substances such as CD63 protein, serine, and human serum albumin.

[0103] The results are as Figure 2 shown. It can be seen that when the kits prepared in Example 2 are used to detect Aβ 1-42 monomers, Aβ 1-42 oligomers, and Aβ 1-42 fibers, only Aβ 1-42 oligomers can cause a strong fluorescence enhancement, indicating that the kit of the present invention has high specificity for Aβ 1-42 oligomers.

[0104] In addition, when the kit interacts with potential interfering substances such as Mg 2+ , CD63 protein, serine, and human serum albumin, the fluorescence change is negligible and can be ignored, further confirming that the kit of the present invention has excellent specificity.

[0105] Example 5

[0106] Plasma from 5XFAD-transgenic Alzheimer's disease model mice (5XFAD mice) and healthy control mice (C57BL / 6J) at 3 weeks old, 2 months old, 5 months old, and 11 months old was collected respectively. The extracellular vesicle @Aβ 1-42 oligomers in their plasma were quantitatively detected using the kit prepared in Example 1. Since the older the 5XFAD mice are, the more severe the degree of Alzheimer's disease is. 2-month-old 5XFAD mice represent the mild early stage of Alzheimer's disease, and 11-month-old 5XFAD mice represent severe Alzheimer's disease. If the kit of the present invention can effectively distinguish 5XFAD mice at different age stages, it indicates that it can achieve early diagnosis of Alzheimer's disease.

[0107] The specific detection steps are as follows:

[0108] (1) Mix 500 μL of plasma with reagent A, adjust the pH to 7.8 - 8.2 with Tris-HCl, incubate at 4°C for 20 min, centrifuge to discard the supernatant, and wash three times with reagent B. Add reagent C and gently shake for 10 min, centrifuge to collect the supernatant as the test sample;

[0109] (2) Set up blank wells, standard wells, and test sample wells respectively. Among them, add 50 μL of blank sample diluent to the blank well; add 50 μL of Aβ 1-42 oligomer protein standards with different concentrations to the standard wells (Aβ 1-42The concentrations of the oligomeric protein standards were 1.0 pM, 2.0 pM, 3.0 pM, 4.0 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, and 10.0 pM respectively; 50 μL of the supernatant collected by the above centrifugation was added to the wells of the sample to be tested;

[0110] (3) Add 50 μL of Reagent D to each well and incubate at room temperature for 3 min; detect the fluorescence intensity of the mixed solution at a wavelength of 585 nm under an excitation wavelength of 450 nm.

[0111] According to the detected fluorescence intensity values, the concentration of extracellular vesicle @Aβ oligomers in the plasma to be tested was calculated using the standard curve of Example 3. 1-42 The concentration of the oligomers.

[0112] As Figure 3 shown, with the increase of age, the concentration of extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice showed a gradually increasing trend. Extracellular vesicle @Aβ oligomers could be detected in the plasma of 2-month-old 5XFAD mice. 1-42 The fluorescence signal of the oligomers increased in a dependent manner with age. After 5 months of age, this trend of increasing concentration became more significant. Compared with 5-month-old mice, the content of extracellular vesicle @Aβ oligomers in 11-month-old 5XFAD mice increased significantly (P < 0.0001). 1-42 The above results indicate that the kit of the present invention can accurately quantify extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice of different months of age, and the present invention will provide important support for distinguishing 5XFAD mice at different age stages and the early diagnosis of Alzheimer's disease. 1-42 The above results show that the kit of the present invention can accurately quantify extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice of different months of age, and the present invention will provide important support for distinguishing 5XFAD mice at different age stages and the early diagnosis of Alzheimer's disease.

[0113] The above results indicate that the kit of the present invention can accurately quantify extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice of different months of age, and the present invention will provide important support for distinguishing 5XFAD mice at different age stages and the early diagnosis of Alzheimer's disease. 1-42 The above results indicate that the kit of the present invention can accurately quantify extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice of different months of age, and the present invention will provide important support for distinguishing 5XFAD mice at different age stages and the early diagnosis of Alzheimer's disease.

[0114] Example 6

[0115] Plasma from 27 healthy donors and 24 Alzheimer's disease patients was collected respectively, and the extracellular vesicle @Aβ oligomers in their plasma were quantitatively detected using the kit prepared in Example 1 to effectively distinguish healthy individuals and Alzheimer's disease patients. 1-42 The above results indicate that the kit of the present invention can accurately quantify extracellular vesicle @Aβ oligomers in the plasma of 5XFAD mice of different months of age, and the present invention will provide important support for distinguishing 5XFAD mice at different age stages and the early diagnosis of Alzheimer's disease.

[0116] The specific detection steps were as follows:

[0117] (1) Mix 500 μL of plasma with Reagent A, adjust the pH to 7.8 - 8.2 with Tris-HCl, incubate at 4°C for 20 min, centrifuge to discard the supernatant, and wash three times with Reagent B. Add Reagent C and gently shake for 10 min, centrifuge to collect the supernatant as the sample to be tested;

[0118] (2) Set up blank wells, standard wells, and test sample wells. Add 50 μL of blank sample diluent to the blank wells; add 50 μL of different concentrations of Aβ to the standard wells. 1-42 Oligomer protein standard (Aβ 1-42 The concentrations of the oligomeric protein standards were 1.0 pM, 2.0 pM, 3.0 pM, 4.0 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, and 10.0 pM, respectively); 50 μL of the supernatant collected by centrifugation was added to the sample well to be tested;

[0119] (3) Add 50 μL of reagent D to each well and incubate at room temperature for 5 min; under an excitation wavelength of 446 nm, detect the fluorescence intensity at 562 nm.

[0120] According to the detected fluorescence intensity value, it is converted into Aβ using the standard curve 1-42 The concentration of oligomers is calculated to determine the extracellular vesicles@Aβ in the plasma to be tested. 1-42 Concentration of oligomers.

[0121] like Figure 4 As shown, the test results of Alzheimer's disease patients and healthy control samples were evaluated by the receiver operating characteristic curve, and the area under the curve (AUC) value obtained was 0.995. This shows that the kit of the present invention has excellent diagnostic performance, with a sensitivity of 100% and a specificity of 96.3%, and has good diagnostic accuracy and the ability to distinguish Alzheimer's disease.

[0122] The protection scope of the present invention is not limited to the above-mentioned specific embodiments. Any equivalent substitution or modification made by any technician familiar with the art after understanding the technical solution of the present invention shall be deemed to be within the protection scope of the present invention as long as it complies with the claims.

Claims

1. An Alzheimer's disease early diagnosis kit, characterized in that: The detection target of the kit is extracellular vesicles@Aβ 1-42 Oligomers.

2. The Alzheimer's disease early diagnosis kit according to claim 1, characterized in that: The kit comprises reagent A, reagent B, reagent C and reagent D; wherein, The reagent A is a boric acid-modified metal organic framework material, a boric acid-modified graphene oxide or a boric acid-modified silica microsphere; The reagent B is a PBS buffer with a pH of 7.8 to 8.5; The reagent C is a PBS buffer with a pH of 6.6 to 7.2 containing fructose or glucose; The reagent D specifically targets extracellular vesicles@Aβ 1-42 Fluorescent probes of oligomers that specifically target extracellular vesicles@Aβ 1-42 The structural formula of the oligomeric fluorescent probe is as follows:

3. The kit according to claim 2, characterized in that: The concentration of the reagent A is 1-6 mg / mL; the concentration of the reagent B is 0.01 M; the concentration of the reagent C is 0.01 M; and the concentration of the reagent D is 2.5-20 μM.

4. The kit according to claim 2, characterized in that In reagent C, the concentration of fructose or glucose is 20-100 mM.

5. The kit according to claim 2, characterized in that The kit also includes a sample diluent and Aβ 1-42 Oligomeric protein standards.

6. A quantitative detection method for extracellular vesicles@Aβ 1-42 An oligomeric fluorescent probe, characterized in that The structural formula of the fluorescent probe is as follows:

7. The method for preparing the fluorescent probe according to claim 6, characterized in that: The following steps are involved: S1: Dissolve carbazole-3-bromobenzene, 2-(2-methoxyethoxy)ethyl-4-methylbenzenesulfonate and potassium carbonate in tetrahydrofuran, and reflux at 75°C for 10-16 hours. After the reaction is completed, cool to room temperature, remove tetrahydrofuran by rotary evaporation, and purify by column chromatography to obtain intermediate 1: S2: Add intermediate 1 and (1,3-dioxolane-2-yl)triphenylphosphorane to a mixed solution of saturated potassium carbonate solution and dichloromethane, reflux at 100°C for 22 to 26 hours. After the reaction is completed, cool to room temperature, add tetrahydrofuran containing 10% hydrochloric acid, stir for 1 to 3 hours, adjust the pH to 6.8 to 7.2, extract, concentrate by rotary evaporation and purify by column chromatography to obtain intermediate 2: S3: Dissolve intermediate 2 and 1-butyl-4-methylpyridinium iodide in toluene, add piperidine, heat to 110°C and reflux for 2-4 hours. After the reaction is completed, cool to room temperature, remove toluene by rotary evaporation, and purify by column chromatography to obtain a red solid, which is the extracellular vesicle @Aβ for quantitative detection. 1-42 Fluorescent probes for oligomers.

8. The method for preparing a fluorescent probe according to claim 7, characterized in that: In step S1, the molar ratio of carbazole-3-bromo, 2-(2-methoxyethoxy)ethyl-4-methylbenzenesulfonate and potassium carbonate is 1:3-5:15-25; The volume ratio of the saturated potassium carbonate solution to dichloromethane in step S2 is 1:1; In step S2, the molar ratio of the intermediate 1 to (1,3-dioxolane-2-yl)triphenylphosphorane is 1:1-2; In step S3, the molar ratio of the intermediate 2, 1-butyl-4-methylpyridinium iodide and piperidine is 1-2:1:0.4-0.

8.

9. The method for quantitatively detecting extracellular vesicles@Aβ according to claim 6 1-42 Application of oligomeric fluorescent probes in the preparation of products for diagnosing Alzheimer's disease.

10. The use according to claim 9, characterized in that: The products include fluorescent probes, detection reagents, diagnostic reagents or kits.

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