Bowl-shaped nano composite material for capturing exosome as well as preparation method and application of bowl-shaped nano composite material

Abstract

The invention discloses a bowl-shaped nano composite material for capturing exosomes and a preparation method and application thereof.The preparation method comprises the steps that firstly, a mesoporous organic silicon bowl-shaped nano material MONB is prepared through a hard template method and a sol-gel method, then in-situ deposition of platinum nano particles and coupling of streptavidin are carried out, and the bowl-shaped nano composite material for capturing the exosomes is obtained. The nano composite material SA (at) Pt-MONB for capturing the exosome is obtained. The biotin-labeled exosome and SA-Pt-MONB are mixed and co-incubated to capture the exosome, and the captured exosome is further subjected to mass spectrometry, so that the exosome metabolic marker related to diseases can be obtained, and the exosome metabolic marker can be used for cancer diagnosis. The nano composite material provided by the invention has special bowl-shaped morphology, high specific surface area and ultraviolet absorption capacity, can efficiently and conveniently realize continuous exosome capture and metabolite analysis, and has wide clinical application potential, especially in disease diagnosis and prognosis evaluation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mesoporous organosilicon bowl-shaped nanocomposite material and a preparation method thereof, as well as a method for using the material to capture exosomes and exosome metabolites for mass spectrometry analysis for cancer diagnosis. Background Art

[0002] Exosomes are nano-sized vesicles with a diameter of about 30 to 150 nanometers secreted by cells. They are widely present in blood, urine, saliva and other biological fluids. As an important medium for intercellular communication, exosomes play an important role in the occurrence, development and metastasis of diseases. They carry a variety of bioactive molecules, including proteins, lipids, metabolites and RNA, which can reflect the intrinsic state of cells and their microenvironment, and are therefore regarded as potential biomarkers. In various pathological conditions such as cancer, neurodegenerative diseases, and cardiovascular diseases, the composition of exosomes can provide valuable information. Therefore, exosomes have shown great application potential in early diagnosis of diseases, prognosis assessment and personalized treatment. Although exosomes have important biological significance, their separation and purification process still faces many challenges. Traditional separation methods, such as ultracentrifugation, polymer precipitation and immunoaffinity capture, are widely used, but often have defects such as low efficiency, long time and high sample processing requirements. These methods may lead to the loss or low purity of exosomes, thus affecting the subsequent analysis results, especially in metabolomics research, where the purity of the sample is directly related to the quantitative and qualitative analysis of metabolites. In addition, in some complex biological samples, the concentration of exosomes is low, and traditional separation techniques are difficult to meet the needs of high-sensitivity detection. Therefore, it is particularly urgent to develop an efficient, rapid and highly selective exosome capture material.

[0003] In recent years, the application research of nanomaterials in the biomedical field has gradually increased. With its advantages of controllable structure and simple preparation, it can be used as an exosome capturer, and its surface can be chemically modified to connect capture groups to achieve the purpose of highly specific separation of exosomes. In addition, by designing the structure of nanomaterials, more binding sites can be provided, and the fluid dynamics effect can be optimized to promote the adsorption and enrichment of exosomes. Nanomaterial-assisted laser desorption ionization mass spectrometry (LDI-MS) has gradually attracted attention as a highly sensitive and high-throughput analytical technology. LDI-MS can quickly detect and quantify small molecule metabolites in exosomes without the need for complex pretreatment steps. The exosome capture ability of nanomaterials can be combined to build a continuous exosome capture and metabolite analysis platform.

[0004] Mesoporous organosilicon materials have a regular pore size distribution and a high specific surface area, with pore sizes usually ranging from 2 to 50 nanometers. These properties enable them to effectively adsorb and store small molecules. In the field of biomedicine, mesoporous organosilicon materials provide new ideas for the development of drug delivery systems due to their good biocompatibility, adjustable pore structure and relatively flexible skeleton structure. Drugs can be effectively encapsulated in the pores of the material, and the sustained-release properties make the release of drugs in the body more lasting and stable. At the same time, the special bowl-shaped morphology can increase the uptake rate of the material in cells, showing more superior performance. In addition, these materials can be further modified by post-processing methods, carrying specific antibodies, etc., to meet different application requirements. At present, there are relatively few studies and reports on the synthesis of mesoporous organic bowl-shaped morphology nanomaterials, and methods and related processes for large-scale synthesis of nanomaterials with bowl-shaped morphology have yet to be developed. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a method for preparing a mesoporous organosilicon bowl-shaped nanocomposite material for exosome capture and metabolite analysis, as well as to develop its application in cancer diagnosis.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a mesoporous organosilicon bowl-shaped nanomaterial by combining a hard template method and a sol-gel method, comprising the following steps:

[0008] Step 1: By Stober solution method Preparation of spherical silica cores (SS) as templates;

[0009] Step 2: Using SS as a template, an incompletely wrapped mesoporous organic silicon layer is grown on its surface by a sol-gel method to obtain an intermediate product SS@MOS;

[0010] Step 3: The template SS is etched away by a hydrothermal reaction, and then washed, centrifuged and dried to finally obtain a mesoporous organosilicon bowl-shaped nanomaterial (MONB).

[0011] In the above step 1, preferably, tetraethyl orthosilicate (TEOS) is used as a silicon dioxide precursor, and tetraethyl orthosilicate is hydrolyzed and condensed in a mixed solution of ethanol, water and ammonia to form silicon dioxide particles. The particle size of the silicon dioxide particles used as spherical silicon dioxide cores prepared in step 1 is mainly distributed in 100 to 200 nm.

[0012] In the above step 2, preferably, 1,4-bis(triethoxysilyl)benzene (BTEB) is used as an organosilicon precursor, hexadecyltrimethylammonium bromide (CTAB) is used as a structure directing agent, and ammonia water is used as a catalyst. First, CTAB is dissolved in a mixed solution of water and ethanol as a solvent, and SS prepared in step 1 is added to obtain a mixed solution; BTEB ethanol solution is added to the mixed solution in batches, stirred at room temperature for reaction, and then centrifuged to obtain a precipitate, that is, the intermediate product SS@MOS in which the organosilicon layer does not completely wrap SS. Among them, in the mixed solution of water and ethanol as a solvent, the volume ratio of water: ethanol is preferably in the range of 1:5 to 1:20; in the mixed solution of CTAB and SS, the concentration of CTAB is preferably 2 to 3 mg / mL, and the content of SS is preferably 0.1 to 1 mg / mL; in the BTEB ethanol solution, 3 to 6 μL BTEB is preferably added per milliliter of ethanol.

[0013] In the above step 3, preferably, the hydrothermal reaction uses sodium sulfate as an etchant to etch away the spherical silica core template under high temperature and high pressure conditions. The preferred temperature range of the hydrothermal reaction is 160° C. to 200° C., and the preferred pH range is 1 to 3.

[0014] In the second aspect, based on the mesoporous organosilicon bowl-shaped nanomaterial prepared by the method described in the first aspect, the present invention provides a nanocomposite material for exosome capture, which is a streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite material, and its preparation method comprises the following steps:

[0015] Step I: amino-modify MONB to obtain amino-modified mesoporous organosilicon bowl-shaped nanomaterials (NH 2 -MONB);

[0016] Step II: In-situ deposition in NH 2 - Platinum nanoparticles (Pt NP) were deposited on the surface of MONB, and after centrifugation, washing and drying, platinum-deposited mesoporous organosilicon bowl-shaped nanomaterials (Pt-MONB) were obtained;

[0017] Step III: resuspend Pt-MONB in ​​a buffer solution, and incubate the mixture with a streptavidin solution to obtain a streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite material (SA@Pt-MONB).

[0018] In the above step I, preferably, MONB and an amino coupling agent are refluxed in an organic solvent, wherein the aminosilane coupling agent is, for example, 3-aminopropyltriethoxysilane (APTES) or 3-aminopropyltrimethoxysilane, and the organic solvent is, for example, an alcohol solvent such as methanol or ethanol, or an aromatic solvent such as toluene. In some specific embodiments of the present invention, MONB is dispersed in toluene and 3-aminopropyltriethoxysilane (APTES) is added and heated under reflux to react to obtain NH 2 -MONB.

[0019] In the above step II, preferably, NH 2 -MONB was ultrasonically dispersed in water and the platinum precursor chloroplatinic acid (H 2 PtCl 4 6H 2 O), an organic polymer stabilizer such as polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA) or polyacrylic acid (PAA) is mixed, and then a reducing agent such as sodium borohydride (NaBH 4 ), citric acid or ascorbic acid for reduction reaction, so that platinum nanoparticles are deposited on the surface of mesoporous organosilicon bowl-shaped nanomaterials.

[0020] In the above step III, the buffer can be phosphate buffer (PBS), ammonium acetate buffer, Tris hydrochloric acid buffer, etc. In some specific embodiments of the present invention, Pt-MONB is resuspended in phosphate buffer, mixed with a solution of streptavidin, and incubated under stirring conditions to obtain a streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite (SA@Pt-MONB). Furthermore, bovine serum albumin (BSA) is used to block the streptavidin coupling product to reduce nonspecific adsorption.

[0021] In a third aspect, based on the SA@Pt-MONB prepared by the method described in the second aspect, the present invention provides an exosome capture method, comprising the following steps:

[0022] Step 1, biotin labeling of exosomes;

[0023] Step 2: The biotin-labeled exosomes are mixed with SA@Pt-MONB and co-incubated, and then centrifuged and washed to obtain a nanocomposite-exosome complex to achieve exosome capture.

[0024] In the above step 1, a biotin-labeled probe such as a distearoylphosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-Biotin)-labeled probe is used and incubated with the exosome suspension to obtain biotin-labeled exosomes.

[0025] In the above step 2, preferably, the biotin-labeled exosomes are mixed evenly with SA@Pt-MONB, co-incubated and centrifuged, and the precipitate is washed with a buffer solution for multiple times to complete the capture of the exosomes. In some embodiments of the present invention, for a concentration of 1×10 8 particles / mL of exosome solution, preferably 1 to 5 mg of capture material SA@Pt-MONB is added per mL of biotin-labeled exosome solution, and the incubation time is 15 to 35 minutes.

[0026] In a fourth aspect, the present invention provides a method for mass spectrometry analysis of serum exosome metabolites, comprising the following steps:

[0027] Step A, capturing serum exosomes using the method described in the third aspect above, adding the obtained nanocomposite material-exosome complex to deionized water to obtain a suspension, and performing laser desorption ionization mass spectrometry (LDI MS) analysis on the suspension to obtain a mass spectrum of serum exosomes;

[0028] Step B: Use data processing software to process mass spectrometry data, and then use an orthogonal partial least squares discriminant analysis model to screen exosome metabolic markers that reflect metabolic differences.

[0029] Preferably, the sample analyzed in step A is from human serum.

[0030] Preferably, the mass spectrometry conditions in step A are: using an UltrafleXtreme MALDI-TOF / TOF mass spectrometer from Bruker, Germany, using a 355nm Nd:YAG laser light source, a laser frequency of 2000Hz, and an acceleration voltage of 20kV; the acquisition mode is a cation reflector mode, and the collection mass-to-charge ratio range is 100-1000Da; the exosome mass spectrometry data is obtained by the built-in flexControl 3.4, and the data is exported in flexAnalysis 3.4.

[0031] Preferably, in step B, the mass spectrometry data is preprocessed using R language, and the orthogonal partial least squares discriminant analysis model is used to screen metabolic markers using SIMCA14.1 software.

[0032] The above-mentioned serum exosome metabolite mass spectrometry analysis method can be applied to the diagnosis of diseases. In one embodiment of the present invention, the exosome metabolite markers of liver cancer obtained by the above-mentioned serum exosome metabolite mass spectrometry analysis method are α-ketoisovalerate, 2-ketobutyrate, 3-hydroxybutyrate, and succinylacetone.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention proposes a method for preparing a controllable bowl-shaped nanocomposite material. The synthesized material has a special bowl-shaped morphology, a high specific surface area and an easily modifiable surface, and can bind to the labeled exosomes through the high affinity between streptavidin and biotin. The bowl-shaped morphology and large surface area provide more abundant binding sites. Therefore, the method of the present invention can achieve exosome capture simply and efficiently.

[0035] (2) The benzene ring structure in the skeleton of the nanocomposite prepared by the present invention and the platinum nanoparticles deposited on the surface work together to determine its high efficiency in photothermal conversion and ion transfer. The organosilicon and platinum nanoparticles have stable structures, which can greatly reduce the background interference in laser desorption ionization mass spectrometry analysis and achieve effective desorption of metabolites. The surface of the material is rough, has strong ultraviolet absorption ability, and has a surface plasmon resonance effect, which can achieve selective adsorption of small molecular metabolites in complex samples, and transfer the laser energy of the mass spectrometry ion source to the metabolites, promoting the ionization of the metabolites. Therefore, the present invention can achieve rapid mass spectrometry detection of exosome metabolites.

[0036] (3) The present invention can analyze the expression differences of serum exosome metabolites between healthy controls and liver cancer patients through a machine learning model, and screen exosome metabolic markers to achieve accurate identification of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the SEM image of the mesoporous organosilicon bowl-shaped nanomaterial (MONB) prepared in Example 1.

[0038] Figure 2 TEM image of MONB prepared in Example 1.

[0039] Figure 3 This is the SEM image of the platinum-deposited mesoporous organosilicon bowl-shaped nanomaterial (Pt-MONB) prepared in Example 2.

[0040] Figure 4 TEM image of Pt-MONB prepared in Example 2.

[0041] Figure 5 These are the nitrogen adsorption-desorption curves of the MONB prepared in Example 1 and the Pt-MONB prepared in Example 2.

[0042] Figure 6 Zeta potential for the stepwise synthesis of mesoporous organosilica bowl-shaped nanocomposites.

[0043] Figure 7 This is a graph showing the effects of the dosage and co-incubation time of SA@Pt-MONB on the capture efficiency during exosome capture in Example 5.

[0044] Figure 8 This is the mass spectra of using SA@Pt-MONB to capture serum exosomes and analyze the serum exosome metabolites of liver cancer patients and healthy controls in Example 6.

[0045] Fig. 9 This is a violin plot of the four marker metabolites screened out in Example 6 for identifying liver cancer.

[0046] Fig.10 Receiver characteristic curves for the training set and test set in Example 6. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples.

[0048] Example 1: Preparation of a Mesoporous Organosilicon Bowl-Shaped Nanomaterial (MONB)

[0049] In this example, the following method is used to prepare mesoporous organosilicon bowl-shaped nanomaterials (MONB):

[0050] Step 1: By Stober solution method Preparation of spherical silica core (SS). Take 20mL of anhydrous ethanol and 1mL of deionized water, mix and add 1.4mL of 25% ammonia solution to make the pH value of the system finally 9.5, add 1mL of tetraethyl orthosilicate (TEOS) under stirring in an oil bath at 40℃, and then continue stirring and reacting at 40℃ for 8 hours. After the reaction is completed, the reaction liquid does not need to be centrifuged and is directly used for the next step of synthesis. The diameter of the synthesized SS in the solution is about 160nm, and the concentration is 13.5mg / mL.

[0051] Step 2: Weigh 150 mg of hexadecyltrimethylammonium bromide (CTAB), dissolve it in 50 mL of deionized water, add 5 mL of ethanol and 1 mL of 25% ammonia water. Next, take 2 mL of the reaction solution containing SS in step 1 and add it to the above solution, and stir vigorously at room temperature for 30 minutes. Dissolve 15 μL of 1,4-bis(triethoxysilyl)benzene (BTEB) in 3 mL of ethanol, and add the prepared BTEB ethanol solution to the above mixture three times, once every 10 minutes, then stir at room temperature for 24 hours, and then centrifuge to obtain a precipitate to form an intermediate product SS@MOS that is not completely wrapped by the silicone layer. The precipitate was washed three times with deionized water and anhydrous ethanol respectively. Finally, the precipitate was redissolved in 20 mL of deionized water, 80 mg of sodium sulfate was added, and ultrasonic treatment was performed for 20 minutes to fully disperse the precipitate in the aqueous solution. Then, the pH value of the mixed solution was adjusted to 3 with 0.1 M sulfuric acid, and then stirred at room temperature for 24 hours. After that, the reaction solution was transferred to a reactor and reacted at 180°C for 10 hours. After the reaction solution was cooled to room temperature, it was centrifuged, and the precipitate was washed three times with water, and then placed in an oven for drying to obtain a mesoporous organosilicon bowl-shaped nanomaterial (MONB). Figure 1 and Figure 2 Showing its bowl-shaped morphology.

[0052] Among them, in step 1, ammonia water is used as a catalyst, and its dosage can be 0.6mL, 1mL, 1.4mL, 1.8mL, and the optimized value is 1.4mL.

[0053] In step 2, the alcohol-water mixture used to dissolve the structure-directing agent CTAB, the preferred volume ratio of water:ethanol can be: 1:5, 1:10, 1:15, 1:20, and the final optimized value is 1:10. The amount of the organosilicon precursor 1,4-bis(triethoxysilyl)benzene can be 10μL, 15μL, 20μL, and the final optimized value is 15μL. The reaction temperature during the etching process will affect the etching effect. The preferred reaction temperatures are: 160℃, 180℃, 200℃, and the final optimized value is 180℃.

[0054] Example 2: Preparation of platinum-deposited mesoporous organosilicon bowl-shaped nanomaterials (Pt-MONB)

[0055] Step 1: 50 mg of MONB synthesized in Example 1 was ultrasonically dispersed in 20 mL of toluene, and then 50 μL of 3-aminopropyltriethoxysilane was added. The mixture was refluxed at 90°C for 12 hours. After the reaction, the mixture was centrifuged, and the precipitate was washed twice with toluene and ethanol, and then dried at 80°C to obtain product 1: amino-modified mesoporous organosilicon bowl-shaped nanomaterial NH 2 -MONB.

[0056] Step 2: 5 mg NH 2 -MONB was ultrasonically dispersed in 20 mL of deionized water, and then 1.54 mL of 3.05 mM H 2 PtCl 4 6H 2 O solution and 10 mg polyvinylpyrrolidone (PVP); the resulting reaction mixture was magnetically stirred at room temperature for 2 hours; finally, 1% NaBH 4 0.37 mL of the solution was used as a reducing agent, and the reaction was continued for 10 hours; then the product precipitate was separated from the reaction solution by centrifugation, and the precipitate was subsequently washed 4 times with toluene and ethanol to obtain product 2: platinum-deposited mesoporous organosilicon bowl-shaped nanomaterial (Pt-MONB), which was then dried in vacuum at 60°C.

[0057] Furthermore, the specific surface area and pore size distribution of MONB and Pt-MONB were determined by nitrogen adsorption-desorption test, such as Figure 5 As shown in Figure 2, after the deposition of platinum nanoparticles, the specific surface area decreased, but Figure 3 , Figure 4The morphology of the platinum nanoparticles after deposition is shown. It can be seen that the bowl-shaped morphology can still be maintained, and the deposition of platinum nanoparticles provides the material with a nanoscale rough structure.

[0058] Example 3. Preparation of Streptavidin-coupled Platinum-deposited Mesoporous Organic Silicon Bowl-shaped Nanocomposite Material (SA@Pt-MONB)

[0059] 1 mg of the platinum-deposited mesoporous organosilicon bowl-shaped nanomaterial prepared in Example 2 was ultrasonically dispersed in 1 mL of PBS, washed several times by centrifugation with PBS, and then resuspended again, and 100 μL of 1 mg / mL streptavidin (SA) solution was added. The reaction was shaken at room temperature for 24 hours, and the precipitate obtained by centrifugation was washed three times with PBS, and then 1 mL of 1% bovine serum albumin (BSA) PBS solution was added to block for 30 minutes, and then washed three times with PBS to obtain the product streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite (SA@Pt-MONB), which was stored at 4°C.

[0060] The surface modification can be verified by Zeta potential test, such as Figure 6 As shown, the surface of the material is still negatively charged after coupling with streptavidin.

[0061] Example 4: Biotin labeling of exosomes

[0062] First, DSPE-PEG-Biotin was used as a labeling probe and inserted into the exosome membrane through the hydrophobic interaction between the two alkyl chains on DSPE and the exosome phospholipid bilayer. This process involved mixing 100 μL of 10 nM labeling probe with 1 mL of the sample to be tested (exosome suspension with a concentration of 1×10 8 The cells were incubated at room temperature for 15 min to allow the exosomes to be labeled with biotin.

[0063] Example 5. Exosome capture and optimization of conditions

[0064] Take 1 mL of the exosome sample labeled with biotin in Example 4 and co-incubate it with streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite (SA@Pt-MONB) at room temperature to achieve the capture and separation of exosomes on the material, and then collect the material and exosomes by simple centrifugation. The amount of capture material SA@Pt-MONB is selected to be 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg, and centrifuged after co-incubation for 15, 20, 25, 30, and 35 minutes, and the exosome protein concentration in the supernatant is tested by BCA protein quantification method to determine the capture efficiency and screen out the optimal amount and optimal incubation time. Figure 7As shown in the figure, when the amount of capture material was 4 mg and the total incubation time was 30 minutes, the capture efficiency reached a peak of 92.56±1.85%, and the capture efficiency decreased with the increase of the amount of capture material, which was attributed to the increase of steric hindrance. The captured substances were verified to be exosomes by Western blotting experiments.

[0065] Example 6. Serum exosome capture and mass spectrometry analysis

[0066] Step 1. Similar to Examples 4 and 5, for serum exosome capture, 50 μL serum was taken and diluted 10 times with 450 μL PBS; then 100 μL 10 nM DSPE-PES-Biotin was added, incubated at room temperature for 15 minutes, and finally 100 μL 1 mg / mL SA@Pt-MONB suspension was added. Incubate for 20 minutes to capture serum exosomes. Finally, rinse with PBS three times and disperse in water to form a suspension with a final concentration of 1 mg / mL for laser desorption ionization mass spectrometry (LDI MS) analysis.

[0067] Step 2: Take 1 μL of the suspension in step 1 and drop it onto the polished stainless steel target plate of laser desorption ionization mass spectrometry, wait for natural drying and then perform mass spectrometry analysis. The instrument used is UltrafleXtreme MALDI-TOF / TOF mass spectrometer from Bruker, Germany, which adopts 355nm Nd:YAG laser light source, laser frequency of 2000Hz, acceleration voltage of 20kV; acquisition mode is cation reflector mode, and the acquisition mass-to-charge ratio range is 100-1000Da; laser intensity is optimized to 80%; and the mass spectrum is obtained by the data processing software flexControl 3.4 provided by the instrument as shown in the figure. Figure 8 As shown, the raw data is exported through the built-in flexAnalysis software.

[0068] Step 3: The raw data obtained in step 2 were preprocessed using the R language MALDIquant data package for peak alignment, smoothing, normalization based on total ion intensity, and peak extraction to reduce the data dimension and output it in the form of a matrix.

[0069] Step 4: Based on the matrix containing multiple groups of data obtained in step 3, the clinical samples were divided into training set and test set, and the orthogonal partial least squares discriminant analysis model (OPLS-DA) was constructed using SIMCA-P 14.1 to screen exosome metabolite markers that can be used to distinguish liver cancer from healthy controls. The four metabolites finally screened were α-ketoisovalerate, 2-ketobutyrate, 3-hydroxybutyrate, and succinylacetone. Fig. 9 As shown, through Fig.10The receiver characteristic curves of the training set and the test set are used to evaluate the differentiation effect. It can be seen from the figure that this method can well distinguish liver cancer patients from healthy controls and has great potential for clinical diagnosis of liver cancer.

[0070] The analysis method mentioned in the above Example 6 is also applicable to other diseases. The present invention only takes liver cancer as an example.

[0071] Analysis results: The nanocomposite material of the present invention can not only efficiently capture exosomes through its special bowl-shaped morphology, high specific surface area and high specificity between streptavidin and biotin, but also can be used as an LDI MS matrix to quickly analyze serum exosome metabolites. Thus, the present invention constructs an efficient platform for continuous exosome capture and analysis, showing its potential for application in precision medicine.

[0072] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a nanocomposite material for capturing exosomes, wherein the nanocomposite material is a streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite material, and the preparation method comprises the following steps: Step 1: Prepare mesoporous organosilicon bowl-shaped nanomaterials, referred to as MONB, by template method and sol-gel method; Step 2, amino-modifying the mesoporous organosilicon bowl-shaped nanomaterial to obtain an amino-modified mesoporous organosilicon bowl-shaped nanomaterial, referred to as NH2-MONB; Step 3: Platinum nanoparticles are deposited on the surface of NH2-MONB by an in-situ deposition method, and after centrifugation, washing and drying, platinum-deposited mesoporous organosilicon bowl-shaped nanomaterials, referred to as Pt-MONB, are obtained; Step 4: resuspend Pt-MONB in ​​a buffer solution, and incubate it with a solution of streptavidin to obtain a streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite material, referred to as SA@Pt-MONB.

2. The preparation method according to claim 1, characterized in that In step one, 1,4-bis(triethoxysilyl)benzene is used as an organosilicon precursor, hexadecyltrimethylammonium bromide is used as a structure directing agent, and ammonia water is used as a catalyst to grow an incompletely wrapped mesoporous organosilicon layer on the surface of a spherical silica core; then sodium sulfate is used as an etchant to remove the spherical silica core under hydrothermal reaction conditions of pH 1 to 3 and 160°C to 200°C to obtain a mesoporous organosilicon bowl-shaped nanomaterial.

3. The preparation method according to claim 1, characterized in that: In step 2, the mesoporous organosilicon bowl-shaped nanomaterial and the amino coupling agent are refluxed in an organic solvent, wherein the aminosilane coupling agent is selected from 3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane, and the organic solvent is an alcohol solvent or an aromatic solvent.

4. The preparation method according to claim 1, characterized in that, In step three, NH2-MONB is ultrasonically dispersed in water, platinum precursor chloroplatinic acid and an organic polymer stabilizer are added and mixed, and then a reducing agent is added to carry out a reduction reaction, so that platinum nanoparticles are deposited on the surface of the mesoporous organosilicon bowl-shaped nanomaterial.

5. The preparation method according to claim 1, characterized in that: In step 4, the buffer is phosphate buffer, ammonium acetate buffer or Tris hydrochloric acid buffer; after Pt-MONB and streptavidin are co-incubated, bovine serum albumin is used to block the streptavidin coupling product to reduce nonspecific adsorption. 6 . The streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped nanocomposite material obtained according to the preparation method of any one of claims 1 to 5 .

7. A method for capturing exosomes, comprising the following steps: 1) Using the biotin-labeled probe DSPE-PEG-Biotin to co-incubate with the exosome suspension to obtain biotin-labeled exosomes; 2) The biotin-labeled exosomes are mixed with the streptavidin-coupled platinum-deposited mesoporous organosilicon bowl-shaped composite nanomaterial according to claim 6, and co-incubated, and then centrifuged and washed to obtain a nanocomposite material-exosome complex to achieve exosome capture.

8. A method for mass spectrometry analysis of serum exosome metabolites, comprising the following steps: A. Capturing serum exosomes according to the exosome capture method of claim 7, adding the obtained nanocomposite material-exosome complex to deionized water to obtain a suspension, taking the suspension for laser desorption ionization mass spectrometry analysis, and obtaining a mass spectrum of serum exosomes; B. Use data processing software to process mass spectrometry data, and then use an orthogonal partial least squares discriminant analysis model to screen exosome metabolic markers that reflect metabolic differences.

9. Use of the serum exosome metabolite mass spectrometry analysis method according to claim 8 in obtaining exosome metabolite markers for disease diagnosis.

10. The use according to claim 9, characterized in that The serum exosome metabolite mass spectrometry analysis method was used to compare the serum exosome metabolite mass spectrometry data of liver cancer patients and healthy individuals, and the exosome metabolic markers of liver cancer were obtained as α-ketoisovalerate, 2-ketobutyrate, 3-hydroxybutyrate, and succinylacetone.

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