Exosome metabolic fingerprint extraction and surface protein quantification integrated detection method based on porous magnetic nano assembly material

Abstract

The invention discloses an exosome metabolic fingerprint extraction and surface protein quantification integrated detection method based on a porous magnetic nano assembly material. The preparation method comprises the following steps: firstly, preparing a porous magnetic nano assembly material Fe3O4 (at) SiO2-Apt-Au, and modifying the surface of a SiO2 shell layer with an exosome surface protein aptamer; au nanoparticles serve as an LDI-MS mass label, the surface of the Au nanoparticles is connected with an antisense strand of the aptamer, and the Au nanoparticles are connected with the Fe3O4 (at) SiO2 magnetic nanoparticles through complementary pairing with a base of the aptamer. In exosome metabolism detection, the nano assembly material can play the roles of exosome capture and LDI-MS auxiliary enhancement of a matrix; a strand displacement reaction site and an LDI-MS mass tag are provided; by combining LDI-MS as a downstream analysis tool, high-sensitivity, high-resolution, low-sample-consumption, high-throughput, rapid and low-cost exosome metabolic fingerprint extraction and surface protein quantitative analysis can be realized.

Description

Technical Field

[0001] The present invention relates to a method for integrated detection of exosome metabolic fingerprint extraction and surface protein quantification based on porous magnetic nano-assembly materials, belonging to the technical field of biomedical detection. Background Art

[0002] Exosomes play unique and important roles in cell communication, antigen presentation, tumor metastasis, etc. In recent years, due to their irreplaceable advantages such as high abundance (up to 10 11 vesicles per milliliter of blood), blood circulation stability, ability to reflect the real-time state of the original cells, and rich content, the metabolic analysis of exosomes has become a research hotspot in the field of in vitro diagnosis. In addition, a variety of exosome surface proteins have been confirmed to be potential tumor markers. For example, glypican-1 (GPC1) has been confirmed to be specifically enriched on exosomes derived from cancer cells, and shows high specificity (100% vs 79.49% vs 82.14%) when distinguishing non-cancer subjects and pancreatic cancer patients compared with CA-199 or serum-free GPC1. Therefore, the combined analysis of serum exosome metabolic fingerprints and exosome surface proteins may provide a new strategy for in vitro diagnosis.

[0003] For exosome metabolic detection, due to the complexity of body fluid components and the huge differences in the expression of disease markers, effective separation and molecular characterization are absolutely necessary. Currently, the methods of exosome separation technology include ultracentrifugation (time-consuming, low yield (~5 - 25%), inconsistent results, and more contamination), sucrose density gradient centrifugation (requiring expensive ultracentrifugation equipment and damaging the exosome membrane), immunoprecipitation (high cost), filtration (limited specificity), size exclusion chromatography (limited specificity), and so on. Functional nanomaterials provide a new low-cost and high-efficiency way for exosome separation by designing strong multiple interactions between the materials and exosomes. Among all the metabolic analysis methods, laser desorption / ionization mass spectrometry (LDI-MS) stands out in practical applications due to its unique advantages such as simple pretreatment, high sensitivity, high resolution, low sample consumption, high detection throughput, and rapidity (used in this method). However, the current nano-material-based methods are limited to single functions and fail to combine efficient exosome separation with downstream metabolic molecular analysis.

[0004] For the characterization of exosomal surface proteins, traditional detection strategies mainly include enzyme-linked immunosorbent assay (ELISA) and fluorescence assay (FL). These methods require expensive labeled antibodies and cumbersome procedures, and consume a large amount of samples to ensure sensitivity and repeatability. As a downstream analysis instrument for exosomal surface proteins, LDI-MS can naturally make up for the above deficiencies. However, the exosomal surface proteins have a large mass-to-charge ratio (beyond the detection range of LDI-MS) and low content. Therefore, signal conversion (such as using mass tags) and amplification strategies are the difficulties in related research. In addition, there is no integrated detection platform for exosomal metabolic profile extraction and surface protein quantification. Summary of the Invention

[0005] The object of the present invention is: aiming at the deficiencies of the prior art, the present invention provides an integrated detection method for exosomal metabolic fingerprint extraction and surface protein quantification based on a porous magnetic nano-assembly material; the present invention designs an integrated exosomal metabolism and protein detection platform combining a porous magnetic nano-assembly material (Fe3O4@SiO2-Apt-Au) with LDI-MS; on the one hand, in exosomal metabolism detection, the Fe3O4@SiO2-Apt-Au assembly can play the role of exosome capture and LDI-MS-assisted enhanced matrix; on the other hand, in exosomal surface protein quantification, the Fe3O4@SiO2-Apt-Au assembly provides a strand displacement reaction site (targeting the exosomal surface protein) and an LDI-MS mass tag; in addition, combined with LDI-MS as a downstream analysis tool, high-sensitivity, high-resolution, low-sample consumption, high-detection throughput, fast, and low-cost exosomal metabolic fingerprint extraction and surface protein quantitative analysis can be achieved.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a preparation method of a porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au, comprising the following steps:

[0008] Step 1: Coupling reaction is carried out between Fe3O4@SiO2 nanoparticles and a silane coupling agent containing amino groups to prepare amino-modified Fe3O4@SiO2 nanoparticles;

[0009] Step 2: The amino-modified Fe3O4@SiO2 nanoparticles are dispersed in a solvent, and a carboxyl-modified aptamer Apt solution and a carboxyl activator are added for coupling reaction to prepare a nano-material Fe3O4@SiO2-Apt modified with an aptamer; the aptamer is an aptamer targeting exosomal surface proteins;

[0010] Step 3: After incubating the thiol-modified corresponding aptamer antisense strand Apt’ and the PBS solution of tricarboxyethylphosphine, disperse them into the Au colloid suspension and stir overnight at room temperature to obtain aptamer antisense strand-modified gold nanoparticles Au-Apt’.

[0011] Step 4: Mix the Fe3O4@SiO2-Apt obtained in Step 2 and the Au-Apt’ obtained in Step 3 and stir for reaction. After the reaction is completed, wash to obtain the porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au.

[0012] Preferably, the silane coupling agent containing amino groups in Step 1 is selected from 3-aminopropyltriethoxysilane (APTES) and / or 3-aminopropyltrimethoxysilane (APTMS).

[0013] Preferably, the Fe3O4@SiO2 nanoparticles in Step 1 are prepared by the following steps:

[0014] Step S1, preparation of Fe3O4 nanoparticles: Add ferric salt to the mixed solution of ethylene glycol and diethylene glycol, and magnetically stir the reaction at room temperature; then add polyvinylpyrrolidone, magnetically stir the reaction under heating conditions, add sodium acetate and stir, transfer the obtained mixture to a high-pressure reaction vessel, and heat the reaction; after the reaction is completed, perform magnetic separation and wash and dry to obtain Fe3O4 nanoparticles;

[0015] Step S2, preparation of Fe3O4@SiO2 nanoparticles: Disperse the Fe3O4 nanoparticles prepared in Step 1 in a solution containing triethanolamine, cetyltrimethylammonium bromide, sodium salicylate and ultrapure water, and stir the reaction under heating conditions; then add tetraethyl orthosilicate and continue to stir the reaction under heating; after the reaction is completed, collect the nanoparticles by magnetic separation and wash; finally, dry and calcine to remove the residual surfactant in the nanoparticles to obtain Fe3O4@SiO2 nanoparticles.

[0016] Preferably, the carboxyl activator in Step 2 is EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide).

[0017] Preferably, the exosome surface proteins in Step 3 include CD63, LZH8, HER2, PSA, CA125, CEA or A33; the aptamer Apt targeting the exosome surface protein and the corresponding aptamer antisense strand Apt’ are as follows:

[0018] Apt CD63 As shown in SEQ ID NO: 1, its antisense strand Apt’ CD63 As shown in SEQ ID NO: 2;

[0019] Apt LZH8 As shown in SEQ ID NO: 3, its antisense strand Apt’ LZH8 As shown in SEQ ID NO: 4;

[0020] Apt HER2 As shown in SEQ ID NO: 5, its antisense strand Apt’ HER2 As shown in SEQ ID NO: 6;

[0021] Apt PSA As shown in SEQ ID NO: 7, its antisense strand Apt’ PSA As shown in SEQ ID NO: 8;

[0022] Apt CA125 As shown in SEQ ID NO: 9, its antisense strand Apt’ CA125 As shown in SEQ ID NO: 10;

[0023] Apt CEA As shown in SEQ ID NO: 11, its antisense strand Apt’ CEA As shown in SEQ ID NO: 12;

[0024] Apt A33 As shown in SEQ ID NO: 13, its antisense strand Apt’ A33 As shown in SEQ ID NO: 14.

[0025] Preferably, the Au colloid suspension in step 3 is prepared by the following method: dissolving gold trichloride or its hydrate in water, stirring and heating to boiling or refluxing; then adding a trisodium citrate solution and stirring and reacting under boiling or refluxing conditions to obtain the Au colloid suspension.

[0026] In a second aspect, the present invention provides the porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au prepared by the above preparation method, including Fe3O4@SiO2-Apt CD63 -Au, Fe3O4@SiO2-Apt LZH8 -Au, Fe3O4@SiO2-Apt HER2 -Au, Fe3O4@SiO2-Apt PSA -Au, Fe3O4@SiO2-Apt CA125 -Au, Fe3O4@SiO2-Apt CEA -Au and Fe3O4@SiO2-Apt A33 -Au.

[0027] In a third aspect, the present invention provides the use of the porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au prepared by the above preparation method in the detection of exosomes.

[0028] In some embodiments of the present invention, the detection of exosomes includes extracting the exosome metabolic fingerprint and quantitatively detecting the exosome surface proteins based on LDI-MS.

[0029] In a fourth aspect, the present invention provides an integrated detection method for exosome metabolic fingerprint extraction and surface protein quantification, comprising the following steps:

[0030] Step A1: Mix the exosome sample solution and the porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au as claimed in claim 7 at room temperature and incubate for 3.5 h. After magnetic separation, the exosome precipitate and the supernatant are collected separately. After washing, the exosome precipitate is dispersed in a solution to obtain a precipitate dispersion.

[0031] Step A2: Take the supernatant and the precipitate dispersion obtained in Step A1 and drop them on the target plate respectively. After drying at room temperature, LDI-MS detection is carried out to obtain the exosome metabolic fingerprint and the surface protein quantitative detection map.

[0032] In some embodiments of the present invention, the quantitative detection of the surface proteins further includes the step of fitting a standard curve: fitting the Au signal intensity (m / z 196.967) with exosome standards of different concentrations to obtain the exosome surface protein quantitative standard curve.

[0033] The technical principle of the present invention is as follows:

[0034] The Fe3O4@SiO2-Apt-Au assembled nano-material provided by the present invention has a porous SiO2 shell layer coated on the surface of Fe3O4 magnetic nanoparticles, and the exosome surface protein aptamer is modified on the surface of the SiO2 shell layer; the Au nanoparticles are used as LDI-MS mass tags, with the antisense strand of the aptamer connected to the surface, and are connected to the Fe3O4@SiO2 magnetic nanoparticles through base complementary pairing with the aptamer. When the test solution is mixed and incubated with the Fe3O4@SiO2-Apt-Au assembly, when there are exosome surface proteins in the test solution, the Fe3O4@SiO2 magnetic nanoparticles are tightly connected to the exosomes through the induced fit effect of the aptamer with the exosome surface proteins, and at the same time, the Au nanoparticles are detached from the Fe3O4@SiO2 magnetic nanoparticles and free in the mixed solution. Through the magnetic separation procedure, the precipitate (Fe3O4@SiO2-Apt-exosomes) and the supernatant (containing the mass tag - Au nanoparticles) are collected, and through further LDI-MS detection, exosome metabolic analysis and surface protein quantification are realized respectively.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention provides a porous magnetic nano-assembly material (Fe3O4@SiO2-Apt-Au), which can be combined with LDI-MS to serve as an integrated detection platform for exosome metabolism and proteins; in the detection of exosome metabolism, the specific surface protein aptamer binding sites of the Fe3O4@SiO2-Apt-Au assembly can target the target exosomes in complex biological samples, and its strong magnetism enables the efficient sorting and enrichment of exosomes with only one magnet, simplifying the sample processing steps, and can solve the problems of long time consumption, low yield, exosome damage, poor specificity, and more pollution in traditional exosome enrichment; as an LDI-MS matrix, its porous structure can improve the LDI efficiency through the pore-guided electromagnetic field enhancement effect, thereby realizing the rapid, sensitive and high-throughput detection of exosome metabolites; in the quantification of exosome surface proteins, the Fe3O4@SiO2-Apt-Au assembly uses gold nanoparticles as LDI-MS mass tags, without the expensive antibodies in traditional methods; and chain displacement can be completed through simple incubation, and impurities can be further removed through the magnetic separation step, solving the problems of cumbersome procedures, low sensitivity and poor repeatability in traditional ELISA and FL.

[0037] (2) The method of the present invention only needs simple incubation, magnetic separation and sample loading detection operations to complete the integrated detection of exosome metabolism fingerprint extraction and surface protein quantification. The cost of materials for detecting one sample (extracting the metabolic fingerprint map + quantifying one surface protein) is only 3 yuan, the consumption of original serum is only 20 nL, and the total time consumption is less than 4 h, which can meet the needs of clinical blood detection and has clinical application potential and transformation value. Description of the Drawings

[0038] Figure 1 Characterization results of the Fe3O4@SiO2-Apt-Au nano-assembly material prepared in Example 1 and its components; among them, a is the scanning electron microscope image of Fe3O4 nanoparticles; b and c are the scanning electron microscope image and transmission electron microscope image of Fe3O4@SiO2 nanoparticles respectively; d is the transmission electron microscope image of Au nanoparticles; e and f are the scanning transmission electron microscope and elemental mapping images of the Fe3O4@SiO2-Apt-Au assembly respectively;

[0039] Figure 2 Standard curves fitted by the Fe3O4@SiO2-Apt-Au assisted LDI-MS analysis and detection platform for the surface protein quantification detection of standard exosomes in Example 2; among them, a is HER2 protein, b is A33 protein; c is CA125 protein; d is CEA protein; e is LZH8 protein; f is PSA protein; g is CD63 protein;

[0040] Figure 3 For the integrated detection results of metabolite fingerprint extraction and CD63 quantification of serum exosomes by the Fe3O4@SiO2-Apt-Au assisted LDI-MS analysis and detection platform in Example 3, where a is the metabolite fingerprint map of serum exosomes; b is the CD63 quantitative detection map. Detailed implementation mode

[0041] To make the present invention more obvious and understandable, preferred embodiments are described in detail below in conjunction with the accompanying drawings.

[0042] Example 1: Preparation and characterization of Fe3O4@SiO2-Apt CD63 -Au nanoassemblies

[0043] (1) Preparation of Fe3O4 nanoparticles: Add 2 mmol of FeCl3·6H2O to a mixed solution of 6 mL of ethylene glycol and 14 mL of diethylene glycol, and stir magnetically at room temperature for 30 min. Subsequently, add 2 g of polyvinylpyrrolidone to the above mixture, and stir magnetically at 125 °C for 1 h. Add 1.5 g of sodium acetate and stir for 0.5 h. Then pour the mixture into a 50 mL stainless steel autoclave and heat at 200 °C for 12 h. Obtain Fe3O4 NPs by magnetic separation, wash three times with ethanol and water respectively, and dry at 50 °C for standby;

[0044] (2) Preparation of Fe3O4@SiO2 nanoparticles: Disperse 80 mg of Fe3O4 NPs in a solution containing 0.17 g of triethanolamine, 0.95 g of cetyltrimethylammonium bromide, 0.315 g of sodium salicylate and 73.5 mL of ultrapure water. The mixture is vigorously stirred at 80 °C for 3 h. Then add 1.8 mL of tetraethyl orthosilicate and continue to stir at 80 °C for 45 min. The prepared particles are collected by magnetic separation, and washed three times with ethanol and deionized water respectively. Dry the product in air at 50 °C for 12 h, and then calcine in air at 550 °C for 5 h to remove the residual surfactant in the nanoparticles;

[0045] (3) Preparation of Fe3O4@SiO2-Apt nanoparticles: Ultrasonically disperse 10 mg of Fe3O4@SiO2 in 20 mL of ethanol. Add 2 mL of aminopropyltrimethoxysilane, stir mechanically at room temperature for 5 h, and then reflux at 80 °C for 2 h. Wash three times by magnetic separation with ethanol and deionized water. Redisperse the amino-modified Fe3O4@SiO2 NPs into 1 ml of PBS and store at 4 °C for use. 6 mL of aptamer solution (3.0 nM; Apt CD63 : 5’-COOH-TTTTTCACCCCACCTCGCTCCCGTGACACTAATGCTA-3’, SEQ ID NO: 1; Apt LZH8: 5’-COOH-ATCCAGAGTGACGCAGCATATTAGTACGGCTTAACCCCATGGTGGACACGGTGGCTTAGT-3’, SEQ ID NO: 3; Apt HER2 : 5’-COOH-GGGCCGTCGAACACGAGCATGGTGCGTGGACCTAGGATGACCTG AGTACTGTCC-3’, SEQ ID NO: 5; Apt PSA : 5’-COOH-AATTAAAGCTCGCCATCAAATAGC-3’, SEQ ID NO: 7; Apt CA125 : 5’-COOH-TATCAATTACTTACCCTAGTGGTGTGATGTCGTATGGATG-3’, SEQ ID NO: 9; Apt CEA : 5’-COOH-ATACCAGCTTATTCAATT-3’, SEQ IDNO: 11; Apt A33 : 5’-COOH-GCTGTGTGACTCCTGCAAGGCGGTACGCGTGTGGACAGAAGTGA CCGCCAAATAGCGCCTGGCAGCTGTATCTTGTCTCC-3’, SEQ ID NO: 13) was added to a mixture of 600 μL EDC (3×10 -2 mg / mL) and 600 μL NHS (3×10 -2 mg / mL), and activated at room temperature for 30 min. The activated aptamer solution was mixed with the above amino-modified Fe3O4@SiO2 NPs (1 ml of the stock solution was dispersed in 50 ml of PBS), and stirred overnight at room temperature. The final product was purified by magnetic separation and washing three times, and then redispersed in 50 ml of PBS and stored at 4 °C for use;

[0046] (4) Preparation of Au-Apt’ nanoparticles: First, Au NPs were synthesized. 240 mg of gold(III) chloride trihydrate was dissolved in 500 mL of ultrapure water, stirred vigorously and heated to boiling. 50 ml of 1% sodium citrate was added all at once, and the resulting wine-red solution was stirred vigorously for 45 minutes while boiling. The resulting Au colloidal suspension was diluted to 1000 mL and stored in the dark. To obtain Au-Apt’, first, Apt’ (the thiol-modified antisense strand of the aptamer; Apt’ CD63 : 5’-SH-TTTTAGCATTAGTGTCACGGG-3’, SEQ ID NO: 2; Apt’ LZH8: 5’-SH-ACTAAGCCACCGTGTCCACCATGGGGTT-3’, SEQ ID NO: 4; Apt’ HER2 : 5’-SH-GGACAGTACTCAGGTCATCCTAGGT-3’, SEQ ID NO: 6; Apt’ PSA : 5’-SH-GCTATTTGATGG-3’, SEQ IDNO: 8; Apt’ CA125 : 5’-SH-CATCCATACGACATCACAC-3’, SEQ ID NO: 10; Apt’ CEA : 5’-SH-AATTGAATAA-3’, SEQ ID NO: 12; Apt’ A33 : 5’-SH-GGAGACAAGATACAGCTGCCAGGCGCTATTTGGCG-3’, SEQ ID NO: 14) Dilute to 3 nmol with 6 ml of PBS solution of tricarboxyethylphosphine (25 mM; adjust the pH to 8.0 with 10% sodium hydroxide), incubate for 1 h; Disperse the incubated Apt’ solution into 70 mL of Au colloid suspension and stir overnight at room temperature. The obtained Au-Apt’ is stored at 4 °C;

[0047] (5) Preparation of Fe3O4@SiO2-Apt-Au assembly (Fe3O4@SiO2-Apt CD63 -Au, Fe3O4@SiO2-Apt LZH8 -Au, Fe3O4@SiO2-Apt HER2 -Au, Fe3O4@SiO2-Apt PSA -Au, Fe3O4@SiO2-Apt CA125 -Au, Fe3O4@SiO2-Apt CEA -Au, Fe3O4@SiO2-Apt A33 -Au): Take 50 mL of Fe3O4@SiO2-Apt stock solution and mix it with 76 mL of the corresponding Au-Apt’ stock solution obtained in step (4), and gently mechanically stir at room temperature for 4 h. The obtained nano-assembly is washed three times with PBS and then dispersed in 5 ml of PBS and stored at 4 °C for use.

[0048] (6) Material characterization: Obtain scanning electron microscope results using Hitachi S-4800, and obtain transmission electron microscope, scanning transmission electron microscope and elemental mapping results using JEOL JEM-2100F. Scanning electron microscope results of Fe3O4 nanoparticles ( Figure 1a) It shows uniform spherical particles with a particle size of about 190 nm. Scanning electron microscopy results of Fe3O4@SiO2 nanoparticles ( Figure 1 b) and transmission electron microscopy results ( Figure 1 c) show that it is a homogeneous macroporous spherical nanostructure with a size of about 320 nm. Transmission electron microscopy image of Au nanoparticles ( Figure 1 d) shows a uniform spherical morphology with an average diameter of 13 nm. Fe3O4@SiO2-Apt CD63 -Au assembly scanning transmission electron microscopy ( Figure 1 e) and elemental mapping ( Figure 1 f) results show the successful assembly of each component.

[0049] Example 2: Quantitative detection of surface proteins of standard exosomes

[0050] (1) Preparation of instruments and reagents: Matrix-assisted laser desorption / ionization mass spectrometry, using positive ion reflection mode;

[0051] (2) Mix 50 μL of standard exosome solution (0 - 8.0×10 10 particles / mL) and 50 μL of Fe3O4@SiO2-Apt-Au nanoassembly (Fe3O4@SiO2-Apt CD63 -Au, Fe3O4@SiO2-Apt LZH8 -Au, Fe3O4@SiO2-Apt HER2 -Au, Fe3O4@SiO2-Apt PSA -Au, Fe3O4@SiO2-Apt CA125 -Au, Fe3O4@SiO2-Apt CEA -Au, Fe3O4@SiO2-Apt A33 -Au) stock solution, and gently shake and incubate at room temperature for 3.5 h. Magnetically separate and collect the supernatant; among them, the standard exosome solution is obtained by diluting the exosomes extracted from serum with PBS solution at different multiples;

[0052] (3) Take 2 μL of the supernatant and drop it on the target plate, dry it at room temperature and then perform mass spectrometry detection;

[0053] (4) Fit the Au signal intensity (m / z 196.967) with the exosome concentration to obtain the quantitative standard curve of exosome surface proteins, as Figure 2 shown. The detection results show that the linear detection range of HER2 is 9.0×10 5 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.933( Figure 2a); The linear detection range of A33 is 1.9×10 6 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.989( Figure 2 b); The linear detection range of CA125 is 1.9×10 6 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.992( Figure 2 c); The linear detection range of CEA is 1.9×10 6 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.971( Figure 2 d); The detection range of LZH8 is 1.9×10 6 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.965( Figure 2 e); The detection range of PSA is 4.8×10 7 -3×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.996( Figure 2 f); The detection range of CD63 is 1.0×10 8 -8.0×10 10 particles / mL, and the linear correlation coefficient R 2 = 0.994( Figure 2 g).

[0054] Example 3: Integrated detection of metabolic fingerprint extraction and surface protein quantification of human serum exosomes

[0055] (1) Preparation of instruments and reagents: Matrix-assisted laser desorption / ionization mass spectrometry, using positive ion reflection mode;

[0056] (2) Dilute 100 μL of serum to 500 μL with PBS and filter it with a 0.22 μm filter membrane to remove other interfering substances. The treated serum sample is stored at -80 °C;

[0057] (3) Mix 50 μL of the treated serum sample and 50 μL of the Fe3O4@SiO2-Apt CD63 -Au nano-assembly stock solution and gently shake and incubate at room temperature for 3.5 h. At the same time, exosome standard solutions with different concentrations (1.0×10 8 particles / mL, 6.5×10 8particles / mL, 3.2×10 9 particles / mL, 1.6×10 10 particles / mL, 8.0×10 10 particles / mL) were also treated under the same conditions as the standard control. The precipitates of the serum samples and the supernatants of all samples were collected separately with the help of a magnet. Among them, the precipitates were washed 3 times by magnetic separation with PBS and then dispersed into 50 μL of PBS;

[0058] (4) 2 μL of the precipitate dispersion and the supernatant were respectively dropped on the target plate, dried at room temperature, and then subjected to mass spectrometry detection;

[0059] (5) Analyze the results of the mass spectrometry detection to draw a conclusion. The exosome fingerprint map and the surface protein quantitative detection map are shown in Figure 3 Figures a and 3b respectively.

[0060] As mentioned above, it is only a preferred embodiment of the present invention, and there is no limitation in any form and substance to the present invention. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au, characterized in that, It includes the following steps: Step 1: Carry out a coupling reaction between Fe3O4@SiO2 nanoparticles and a silane coupling agent containing an amino group to prepare amino-modified Fe3O4@SiO2 nanoparticles; Step 2: Disperse the amino-modified Fe3O4@SiO2 nanoparticles in a solvent, add a carboxyl-modified aptamer Apt solution and a carboxyl activator, and carry out a coupling reaction to prepare a nanomaterial Fe3O4@SiO2-Apt modified with an aptamer; the aptamer is an aptamer targeting the surface protein of exosomes; Step 3: Incubate a PBS solution of a thiol-modified corresponding aptamer antisense strand Apt’ and tris(carboxyethyl)phosphine, and then disperse it into an Au colloid suspension, and stir overnight at room temperature to obtain aptamer antisense strand-modified gold nanoparticles Au-Apt’; Step 4: Mix the Fe3O4@SiO2-Apt obtained in Step 2 and the Au-Apt’ obtained in Step 3 and stir to react. After the reaction is completed, wash to obtain a porous magnetic nano-assembly material Fe3O4@SiO2-Apt-Au.

2. The preparation method according to claim 1, characterized in that, The silane coupling agent containing an amino group in Step 1 is selected from 3-aminopropyltriethoxysilane (APTES) and / or 3-aminopropyltrimethoxysilane (APTMS).

3. The preparation method according to claim 1, characterized in that, The Fe3O4@SiO2 nanoparticles in Step 1 are prepared by the following steps: Step S1, Preparation of Fe3O4 nanoparticles: Add a ferric salt to a mixed solution of ethylene glycol and diethylene glycol, and carry out a magnetic stirring reaction at room temperature; then add polyvinylpyrrolidone, carry out a magnetic stirring reaction under heating conditions, and then add sodium acetate and stir. Transfer the obtained mixture to a high-pressure reaction vessel and heat to react; after the reaction is completed, carry out magnetic separation and wash and dry to obtain Fe3O4 nanoparticles; Step S2, Preparation of Fe3O4@SiO2 nanoparticles: Disperse the Fe3O4 nanoparticles prepared in Step 1 in a solution containing triethanolamine, cetyltrimethylammonium bromide, sodium salicylate and ultrapure water, and carry out a stirring reaction under heating conditions; then add tetraethyl orthosilicate and continue to carry out a heating and stirring reaction; after the reaction is completed, collect the nanoparticles by magnetic separation and wash; finally, dry and calcine to remove the residual surfactant in the nanoparticles to obtain Fe3O4@SiO2 nanoparticles.

4. The preparation method according to claim 1, characterized in that, The carboxyl activator in Step 2 is EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide).

5. The preparation method according to claim 1, characterized in that, The exosome surface proteins in Step 3 include CD63, LZH8, HER2, PSA, CA125, CEA or A33; the aptamer Apt targeting the exosome surface protein and the corresponding aptamer antisense strand Apt’ are as follows: Apt CD63 As shown in SEQ ID NO: 1, its antisense strand Apt’ CD63 As shown in SEQ ID NO: 2; Apt LZH8 As shown in SEQ ID NO: 3, its antisense strand Apt' LZH8 As shown in SEQ ID NO: 4; Apt HER2 As shown in SEQ ID NO: 5, its antisense strand Apt' HER2 As shown in SEQ ID NO: 6; Apt PSA As shown in SEQ ID NO: 7, its antisense strand Apt’ PSA As shown in SEQ ID NO: 8; Apt CA125 As shown in SEQ ID NO: 9, its antisense strand Apt' CA125 As shown in SEQ ID NO: 10; Apt CEA As shown in SEQ ID NO: 11, its antisense strand Apt' CEA As shown in SEQ ID NO: 12; Apt A33 As shown in SEQ ID NO: 13, its antisense strand Apt’ A33 As shown in SEQ ID NO:

14.

6. The preparation method according to claim 1, wherein The Au colloid suspension in Step 3 is prepared by the following method: Dissolve gold trichloride or its hydrate in water, stir and heat to boiling or reflux; then add a trisodium citrate solution and stir to react under boiling or reflux conditions to obtain an Au colloid suspension.

7. The porous magnetic nano-assembled material Fe3O4@SiO2-Apt-Au prepared by the preparation method according to any one of claims 1 to 6, including Fe3O4@SiO2-Apt CD63 -Au, Fe3O4@SiO2-Apt LZH8 -Au, Fe3O4@SiO2-Apt HER2 -Au, Fe3O4@SiO2-Apt PSA -Au, Fe3O4@SiO2-Apt CA125 -Au, Fe3O4@SiO2-Apt CEA -Au and Fe3O4@SiO2-Apt A33 -Au.

8. Use of the porous magnetic nano-assembled material Fe3O4@SiO2-Apt-Au prepared by the preparation method according to any one of claims 1 to 6 in the detection of exosomes.

9. The application according to claim 8, wherein The detection of exosomes includes extracting the exosome metabolic fingerprint and quantitatively detecting the exosome surface protein based on LDI-MS.

10. An integrated detection method for extracting exosomal metabolic fingerprint and quantifying surface proteins, characterized in that, It includes the following steps: Step A1: Mix the exosome sample solution and the porous magnetic nano-assembled material Fe3O4@SiO2-Apt-Au according to claim 7 at room temperature and incubate for 3.5 h. After magnetic separation, the exosome precipitate and the supernatant are collected respectively. After washing the exosome precipitate, it is dispersed in a solution to obtain a precipitate dispersion; Step A2: Drop the supernatant and the precipitate dispersion obtained in Step A1 onto the target plate respectively. After drying at room temperature, LDI-MS detection is carried out to obtain the exosome metabolic fingerprint and the surface protein quantitative detection map.

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