A modified magnetic nano-molecular sieve material, its preparation method and application in vesicle adsorption

Abstract

The present invention discloses a modified magnetic nano-molecular sieve material, a preparation method thereof, and an application in vesicle adsorption. The preparation method includes the following steps: 1) Dispersing the magnetic nano-molecular sieve material in a strong alkaline solution, performing ultrasonic treatment under ice bath conditions, and then magnetically separating to discard the supernatant to obtain precipitate A; 2) Adding a quaternary ammonium salt aqueous solution to precipitate A, stirring and reacting at 20-40°C, washing, and then drying to obtain a surface-modified magnetic nano-molecular sieve material. The surface-modified magnetic nano-molecular sieve material prepared by the present invention can be used for vesicle adsorption, and the adsorption effect is significantly better than that of the unmodified material and the traditional ultracentrifugation method, and the sample requirement is only 1 / 10 of that of the traditional ultracentrifugation method; it can be applied to almost all sample types, effectively solving the problems of time-consuming and high-abundance protein contamination existing in the traditional vesicle adsorption method.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic nano-molecular sieve materials, and particularly relates to a modified magnetic nano-molecular sieve material, a preparation method thereof, and an application in vesicle adsorption. Background Art

[0002] Extracellular vesicles (EVs) are heterogeneous membrane-bound phospholipid vesicles actively secreted and released by cells. EVs can be further classified into exosomes, microvesicles, and apoptotic bodies according to their biogenesis process, size, and biophysical properties. In recent years, EVs have increasingly been recognized as important carriers for intercellular communication and circulating biomarkers for disease diagnosis and prognosis. However, the large heterogeneity of EVs, their low content in clinically relevant samples, and the difficulty in quantification pose great challenges for the isolation and analysis of EVs. Traditional methods such as density gradient ultracentrifugation, ultracentrifugation, and size exclusion chromatography have problems such as the required sample volume, cumbersome steps, long operation time, low purity of the isolated vesicles, and high-abundance protein contamination, which are not conducive to utilization and application. Summary of the Invention

[0003] Object of the Invention: In view of the problems in vesicle separation, the present invention provides a modified magnetic nano-molecular sieve material, a preparation method thereof, and an application in vesicle adsorption. The modification method of the magnetic nano-molecular sieve proposed by the present invention first creates defects on the surface of the molecular sieve using a strong base to reduce the silicon-aluminum ratio, thereby enhancing the coordination between aluminum and the phosphate groups on the vesicles; then self-assembles positively charged quaternary ammonium cations on the surface of the defective magnetic nano-molecular sieve to endow the magnetic nano-molecular sieve with positive charge, further enhancing the lipophilicity of the magnetic nano-molecular sieve and the electrostatic interaction with the negatively charged vesicle structure. In addition, the alkali treatment method in an ice-water bath ensures that the magnetism and crystal structure of the magnetic nano-molecular sieve are not significantly affected. Therefore, the modification method of the magnetic nano-molecular sieve proposed by the present invention significantly enhances the adsorption of vesicle structures, is applicable to various sample types, and the required sample volume is 1 / 10 of that of traditional ultracentrifugation, providing a simple, rapid, and efficient technical method for vesicle separation.

[0004] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0005] In the first aspect, the present invention provides a preparation method of a modified magnetic nano-molecular sieve material, comprising the following steps:

[0006] A preparation method of a modified magnetic nano-molecular sieve material, comprising the following steps:

[0007] 1) Disperse the magnetic nano-molecular sieve material in a strong alkaline solution, perform ultrasonic treatment under ice bath conditions, and then magnetically separate to discard the supernatant to obtain precipitate A;

[0008] 2) Add an aqueous solution of quaternary ammonium salt to precipitate A, stir and react at 20-40 °C, wash, and then dry to obtain a surface-modified magnetic nano-molecular sieve material.

[0009] Preferably, in step 1), the alkali source of the strong alkaline solution is one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, and calcium hydroxide; the concentration of the strong alkaline solution is 0.1 M to 1 M; when the solid dosage is in grams and the liquid dosage is in ml, the ratio of the magnetic nano-molecular sieve material to the strong alkali solution is 1:50 to 500; the time of ice bath ultrasonic treatment is 10 min to 6 h, and the power is 40-80 KHZ.

[0010] Preferably, in step 2), the quaternary ammonium salt in the aqueous solution of quaternary ammonium salt is one or more of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, benzyltriethylammonium chloride, and benzyltriethylammonium bromide; the concentration of the aqueous solution of quaternary ammonium salt is 0.12 M to 1 M; the molar ratio of the quaternary ammonium salt in the aqueous solution of quaternary ammonium salt to the strong base in the strong alkaline solution in step 1) is 1 to 1.2; the time of the stirring reaction is 2 h to 6 h; the washing is carried out by washing with water, washing with alcohol, and washing with water in sequence, and repeating several times; among them, the alcohol source for washing with alcohol is ethanol or methanol, and washing with water, washing with alcohol, and washing with water are regarded as one cycle, and the cycle washing is carried out 3 to 6 times.

[0011] Preferably, in step 1), the magnetic nano-molecular sieve material is mainly prepared by the following method:

[0012] Take citric acid-sodium modified Fe3O4, add it to deionized water, disperse it evenly, then add the alkali source and dissolve it completely; continue to add the template agent, stabilizer and surfactant, and stir to dissolve; continue to add the silicon source and alkali metal source, crystallize at room temperature after dissolution, and then carry out hydrothermal crystallization. After the hydrothermal crystallization is completed, filter, wash, dry, and calcine to obtain the magnetic nano-molecular sieve.

[0013] More preferably, the particle size of the citric acid-sodium modified Fe3O4 is 10-500 nm; the alkali source is one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, di-isopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; the surfactant is one or more of sodium dodecyl sulfate, cetyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride; the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetra-n-propyl orthosilicate, tetra-n-butyl orthosilicate, silica sol, water glass, and diatomite; the alkali metal source is one or more of sodium aluminate, aluminum chloride, copper sulfate, copper chloride, zinc sulfate, and zinc chloride.

[0014] More preferably, the mass ratio of Fe3O4, alkali source, template agent, stabilizer, surfactant, silicon source, and alkali metal source is 1:(2-20):(0.1-10):(0.05-5):(0.01-3):(20-100):(1-20); the room temperature crystallization duration is 1-6 h, the temperature of hydrothermal crystallization is 100-200 °C, and the duration is 24-120 h.

[0015] In a second aspect, the present invention provides a modified magnetic nanozeolite material prepared by the above preparation method.

[0016] In a third aspect, the present invention provides the application of the modified magnetic nanozeolite in vesicle adsorption.

[0017] In a fourth aspect, the present invention provides a method for adsorbing vesicles using the modified magnetic nanozeolite, comprising the following steps:

[0018] 1) Adding a buffer solution and surface-modified magnetic nanozeolite to a sample to be processed to obtain a suspension;

[0019] 2) After shaking and incubating the suspension, performing magnetic separation, removing the supernatant and retaining the precipitate;

[0020] 3) Adding a washing buffer solution to wash the precipitate, and the obtained precipitate is a mixture of the surface-modified magnetic nanozeolite and the vesicle structure adsorbed thereon;

[0021] 4) Detecting the target proteome or target protein.

[0022] Preferably, in step 1), the sample type is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, egg white or cell supernatant; when the solid dosage is measured in mg and the liquid dosage is measured in ml, the ratio of the surface-modified magnetic nanozeolite material to the sample to be detected is 1:(0.005-10);

[0023] Preferably, in step 1), the components of the binding buffer solution include one or any combination of Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium chloride, sodium chloride, citric acid, sodium citrate, barbituric acid, sodium barbital, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, methanol, and preferably a combined buffer solution of Tris and EDTA.

[0024] Preferably, in step 2), the conditions for shaking and incubating are: 18-37 °C, 500-2000 rpm, incubating for 1-120 min; placing it on a magnetic rack, and the placement time is 1-5 min;

[0025] Preferably, in step 3), the washing buffer is selected from the binding buffer or the corresponding dilution used in step 1); the precipitate is washed 3 times, and the process is as follows: add the washing buffer, shake at room temperature for 3 min, place the sample on a magnetic rack for magnetic separation for 2 min, discard the supernatant, and retain the precipitate; repeat the above process 3 times.

[0026] Preferably, in step 4), the means for detecting the target protein include one or more of mass spectrometry, IHC, Elisa, Western blot, and chemiluminescence, preferably mass spectrometry.

[0027] Beneficial effects:

[0028] 1. The modification method of the magnetic nano-molecular sieve proposed by the present invention first creates defects on the surface of the molecular sieve by using a strong base to reduce the silicon-aluminum ratio, thereby enhancing the coordination between aluminum and the phosphate groups on the vesicles; then, positively charged quaternary ammonium cations are self-assembled on the surface of the defective magnetic nano-molecular sieve to endow the magnetic nano-molecular sieve with positive charge, further enhancing the lipophilicity of the magnetic nano-molecular sieve and the electrostatic interaction with the negatively charged vesicle structure.

[0029] 2. The modification method of the magnetic nano-molecular sieve proposed by the present invention uses alkali sonication under ice bath conditions to reduce the silicon-aluminum ratio of the molecular sieve, and does not cause any impact on the crystal structure and magnetism of the magnetic nano-molecular sieve.

[0030] 3. The magnetic nano-molecular sieve material modified by the present invention significantly enhances the adsorption of the vesicle structure, effectively solving the problems of separation and analysis of the vesicle structure.

[0031] 4. The method of the present invention has a wide range of applications and is applicable to various samples containing high-abundance proteins such as blood, urine, cerebrospinal fluid, saliva, emulsion, egg white, and cell supernatant.

[0032] 5. Compared with the traditional density gradient ultracentrifugation, ultracentrifugation method, and molecular exclusion method, the sample volume required by the method of the present invention is only 1 / 10 of the traditional method, significantly reducing the sample processing steps and operation time, and effectively solving the problems of low vesicle purity and interference of high-abundance proteins in the traditional method.

[0033] Description of the attached drawings

[0034] Figure 1 Scanning electron microscope pictures of the magnetic nano-molecular sieve and the modified magnetic nano-molecular sieve (both obtained in Example 3), where a. magnetic nano-molecular sieve; b. surface-modified magnetic nano-molecular sieve.

[0035] Figure 2Results of X-ray diffraction spectra of magnetic nanozeolites and modified magnetic nanozeolites (both obtained in Example 3).

[0036] Figure 3 Identification of some typical vesicle structure specific markers in magnetic nanozeolites and modified magnetic nanozeolites after incubation with plasma samples. Detailed implementation mode

[0037] The following gives a comprehensive description of the solution of the present invention. The described implementation cases are the most preferred implementation modes in the present invention, but the present invention is not limited to the following examples.

[0038] Example 1

[0039] A surface-modified magnetic nanozeolite is prepared by the following steps:

[0040] 1) Add 10 nm Fe3O4 to a sodium citrate solution with a concentration of 0.01 mol / L. The mass ratio of Fe3O4 to the sodium citrate solution is 1:1. After ultrasonic dispersion, stir in an oil bath at 40 °C for 6 h, then perform magnetic separation and wash with water 3 times, and dry in a vacuum oven to obtain sodium citrate-modified Fe3O4.

[0041] 2) Take the sodium citrate-modified Fe3O4 and add it to deionized water, ultrasonic disperse it evenly, and then add an appropriate amount of ammonia water. The mass ratio of Fe3O4 to ammonia water is Fe3O4:ammonia water = 1:2.

[0042] 3) Add an appropriate amount of template agent dipropylamine, stabilizer ethanol, and surfactant cetyltrimethylammonium bromide to 2) and stir or ultrasonically dissolve them; taking the dosage of Fe3O4 as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4:template agent:stabilizer:surfactant = 1:0.1:0.05:0.01.

[0043] 4) Add an appropriate amount of silicon source tetramethyl orthosilicate and alkali metal source aluminum chloride to 3). Taking the dosage of Fe3O4 as a comparison, the mass ratio of the silicon source and alkali metal source is Fe3O4:silicon source:alkali metal source = 1:20:1. After stirring and dissolving, crystallize at room temperature for 1 h, place it in a polytetrafluoroethylene inner liner, put it into a hydrothermal autoclave, and hydrothermally crystallize at 100 °C for 120 h. After the hydrothermal treatment, obtain the magnetic nanozeolite through filtration, washing, drying, and calcination.

[0044] 5) Take 3 g of the magnetic nanozeolite material obtained in 4) and disperse it in 500 mL of 0.1 M potassium hydroxide solution. Under ice bath conditions, ultrasonically treat it at 40 KHZ for 6 h, perform magnetic separation and discard the supernatant to obtain precipitate A;

[0045] 6) Add 500 mL of an aqueous solution of cetyltrimethylammonium bromide with a concentration of 0.12 M to precipitate A; stir and react at 20 °C for 6 h, wash with water, wash with methanol, and then wash with water again. After repeating the cycle 3 times, dry to obtain the surface-modified magnetic nanozeolite material.

[0046] Example 2

[0047] A surface-modified magnetic nanozeolite is prepared by the following steps:

[0048] 1) Add Fe3O4 with a size of 500 nm to a sodium citrate solution with a concentration of 1 mol / L. The mass ratio of Fe3O4 to the sodium citrate solution is 1:100. After ultrasonic dispersion until homogeneous, stir in an oil bath at 100 °C for 0.5 h, then perform magnetic separation and wash 3 times with water, and dry in a vacuum oven to obtain sodium citrate-modified Fe3O4.

[0049] 2) Take the sodium citrate-modified Fe3O4 and add it to deionized water, ultrasonic disperse until homogeneous, then add an appropriate amount of urea and ultrasonic until the alkali source is completely dissolved. The mass ratio of Fe3O4 to the alkali source is Fe3O4:alkali source = 1:20.

[0050] 3) Add an appropriate amount of template agent diisopropylamine, stabilizer glycerol, and surfactant octadecyldimethylbenzylammonium chloride to step 2), and stir or ultrasonic dissolve; taking the amount of Fe3O4 as a reference, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4:template agent:stabilizer:surfactant = 1:10:5:3.

[0051] 4) Add an appropriate amount of silicon source tetraethyl orthosilicate and alkali metal source copper sulfate to step 3). Taking the amount of Fe3O4 as a reference, the mass ratio of the silicon source to the alkali metal source is Fe3O4:silicon source:alkali metal source = 1:100:20. After stirring and dissolving, crystallize at room temperature for 6 h, place it in a polytetrafluoroethylene inner liner, put it into a hydrothermal autoclave, and hydrothermally crystallize at 200 °C for 24 h. After the hydrothermal reaction is completed, obtain the magnetic nanozeolite through filtration, washing, drying, and calcination.

[0052] 5) Take 3 g of the magnetic nanozeolite material obtained in step 4), disperse it in 150 mL of 1 M sodium hydroxide solution, ultrasonic at 80 KHZ for 10 min under ice bath conditions, perform magnetic separation and discard the supernatant to obtain precipitate A;

[0053] 6) Add 150 mL of an aqueous solution of benzyltriethylammonium chloride with a concentration of 1 M to precipitate A; stir and react at 40 °C for 30 min, wash with water, wash with ethanol, and then wash with water again. After repeating the cycle 3 times, dry to obtain the surface-modified magnetic nanozeolite material.

[0054] Example 3

[0055] Material Preparation

[0056] 1) Weigh 0.5 g of 20-nm Fe3O4 and add it to 200 mL of 0.1 mol / L sodium citrate solution. After ultrasonic dispersion until uniform, stir in an 80 °C oil bath for 1.5 h. Then, perform magnetic separation, wash with deionized water 3 times, and dry in a 60 °C vacuum oven for 6 h to obtain sodium citrate-modified Fe3O4.

[0057] 2) Take 0.5 g of sodium citrate-modified Fe3O4 and add it to 30 mL of deionized water. After ultrasonic dispersion until uniform, add 2 g of NaOH and ultrasonicate until the NaOH is completely dissolved.

[0058] 3) Add 0.25 g of triethylamine, 0.1 g of isopropanol, and 0.15 g of sodium dodecyl sulfate to 2). Ultrasonicate until the solids are completely dissolved.

[0059] 4) Add 3 g of sodium aluminate to 3), stir to dissolve, add 30 g of silica sol, stir to dissolve, crystallize at room temperature for 1 h, perform hydrothermal treatment at 150 °C for 72 h, and obtain the magnetic nanomolecular sieve material after filtration, washing, drying, and calcination.

[0060] 5) Take 3 g of the magnetic nanomolecular sieve material obtained in 4), disperse it in 300 mL of 0.1 M sodium hydroxide solution, ultrasonicate at 80 KHZ for 60 min under ice bath conditions, perform magnetic separation and discard the supernatant to obtain precipitate A;

[0061] 6) Add 300 mL of an aqueous solution of cetyltrimethylammonium chloride with a concentration of 0.12 M to precipitate A; stir and react at 25 °C for 4 h. After washing with water, ethanol, and water, repeat the cycle 3 times and then dry to obtain the surface-modified magnetic nanomolecular sieve material.

[0062] 7) As Comparative Example 1: Prepare a magnetic nanomolecular sieve material treated only with strong base: Take 3 g of the magnetic nanomolecular sieve material obtained in 4), disperse it in 300 mL of 0.1 M sodium hydroxide solution, ultrasonicate at 80 KHZ for 60 min under ice bath conditions, perform magnetic separation and discard the supernatant to obtain precipitate A; and after washing with water, ethanol, and water, repeat the cycle 3 times and then dry to obtain the magnetic nanomolecular sieve material treated only with strong base, denoted as magnetic nanomolecular sieve material - strong base.

[0063] 8) As Comparative Example 2: Prepare a magnetic nanomolecular sieve material treated only with quaternary ammonium salt: Take 3 g of the magnetic nanomolecular sieve material obtained in 4), disperse it in 300 mL of an aqueous solution of cetyltrimethylammonium chloride with a concentration of 0.12 M; stir and react at 25 °C for 4 h. After washing with water, ethanol, and water, repeat the cycle 3 times and then dry to obtain the magnetic nanomolecular sieve material treated only with quaternary ammonium salt, denoted as magnetic nanomolecular sieve material - quaternary ammonium salt.

[0064] Vesicle Adsorption and Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) Detection of Plasma Samples

[0065] 1) Take 4 portions of 100 μL plasma samples, and add 0.5 mg of magnetic nano-molecular sieve (obtained in Example 3), 0.5 mg of modified magnetic nano-molecular sieve (obtained in Example 3), 0.5 mg of magnetic nano-molecular sieve-strong base (obtained in Example 3), and 0.5 mg of magnetic nano-molecular sieve-quaternary ammonium base (obtained in Example 3) respectively; then add 300 μL of buffer to each of them to obtain a suspension;

[0066] 2) Incubate the suspension with shaking at 1000 rpm at room temperature for 15 min, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate;

[0067] 3) Add 500 μL of washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate;

[0068] 4) Resuspend the precipitate with a certain volume of buffer containing DTT, react at 95 °C for 1 h; then add a certain volume of IAM and react in the dark at room temperature for 45 min.

[0069] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and enzymatically digest at 37 °C for 4 h.

[0070] 6) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the enzymatically digested peptides to the SDB column.

[0071] 7) Wash the SDB column several times and desorb to obtain a purified peptide solution.

[0072] 8) Lyophilize the purified peptide solution and redissolve the peptides with an on-machine buffer.

[0073] 9) The peptides are analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0074] 10) Use DIA-NN software (version 1.8.1) for extraction to obtain protein qualitative and quantitative results; perform the same treatment on 3 plasma samples from different sources, with 3 parallel experiments for each sample. The number of identified proteins is shown in Table 1, Figure 3Shows the mass spectrometry quantitative values of typical markers of partial vesicle structures in each group.

[0075] Ultracentrifugation for extracting vesicles (including exosomes) from plasma samples and LC-MS / MS detection

[0076] To better compare with the modified magnetic nano-molecular sieve, the present invention extracted vesicle structures (including exosomes) by the traditional ultracentrifugation method and performed LC-MS / MS detection. The specific operations are as follows:

[0077] 1) Take 5 mL of plasma sample and centrifuge it at 2000 g for 30 min to remove large cell debris and organelles, and collect the supernatant; then centrifuge the supernatant at 10000 g for 30 min. The precipitate obtained by centrifugation is used as the large vesicle fraction. The supernatant is centrifuged at 100000 g for 90 min. Discard the supernatant, resuspend the precipitate with PBS and centrifuge it again at 100000 g for 90 min. Discard the supernatant, and resuspend the precipitate with 100 μL of PBS as the exosome fraction.

[0078] 2) Take 20 μL of the large vesicle fraction and exosome fraction in 1) respectively, resuspend the precipitate with a certain volume of buffer containing DTT, and react at 95 °C for 1 h; then add a certain volume of IAM and react in the dark at room temperature for 45 min. Then perform steps 5) to 10) in "Vesicle adsorption and liquid chromatography tandem mass spectrometry (LC-MS / MS) detection of plasma samples", and the protein identification results of the large vesicle fraction and exosome fraction extracted by the ultracentrifugation method can be obtained.

[0079] 3) Perform the same treatment on 3 plasma samples from different sources. The number of identified proteins is shown as "Ultracentrifugation - Large Vesicles" and "Ultracentrifugation - Exosomes" in Table 1. Figure 3 Shows the mass spectrometry quantitative values of typical markers of partial vesicle structures in 2 groups.

[0080] Table 1: Number of protein identifications in plasma samples

[0081]

[0082] Vesicle adsorption and liquid chromatography tandem mass spectrometry (LC-MS / MS) detection of urine samples

[0083] 1) Take 1 mL of urine sample, add 0.5 mg of modified magnetic nano-molecular sieve (obtained in Example 3) thereto; then add 200 μL of buffer to obtain a suspension.

[0084] 2) Shake and incubate the suspension at 1000 rpm at room temperature for 15 min, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate.

[0085] 3) Add 500 μL of washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place it on a magnetic stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate; repeat this process 3 times;

[0086] 4) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, react at 95 °C for 1 h; then add a certain volume of IAM and react at room temperature in the dark for 45 min.

[0087] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and digest at 37 °C for 4 h.

[0088] 6) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the digested peptide segments to the SDB column.

[0089] 7) Wash the SDB column several times and desorb to obtain a purified peptide solution.

[0090] 8) Freeze-dry the purified peptide solution and redissolve the peptide segments with the loading buffer.

[0091] 9) The peptide segments are analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) in tandem with mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0092] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0093] 11) When 3 personnel operate simultaneously, process urine samples from 3 different sources, with three parallel replicate experiments for each sample. The number of identified proteins is shown in Table 2:

[0094] Table 2 Number of urine protein identifications

[0095] Person 1 Person 2 Person 3 Sample1-1 4517 4604 4496 Sample1-2 4519 4527 4533 Sample1-3 4437 4514 4603 Sample2-1 4308 4466 4412 Sample2-2 4465 4300 4468 Sample2-3 4402 4423 4398 Sample3-1 4793 4701 4668 Sample3-2 4749 4611 4592 Sample3-3 4701 4629 4605

[0096] Protein adsorption of cell supernatant and LC-MS / MS detection

[0097] 1) Take 1 mL of cerebrospinal fluid sample, add 0.5 mg of modified magnetic nano-molecular sieve (obtained in Example 3) to it; then add 200 μL of buffer to obtain a suspension;

[0098] 2) Incubate the suspension with shaking at 1000 rpm for 15 min at room temperature, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate;

[0099] 3) Add 500 μL of washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; Repeat this process 3 times;

[0100] 4) Resuspend the precipitate by adding a certain volume of buffer containing DTT, and react at 95 °C for 1 h; Then add a certain volume of IAM and react in the dark at room temperature for 45 min.

[0101] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and digest at 37 °C for 4 h.

[0102] 6) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the digested peptides to the SDB column.

[0103] 7) Wash the SDB column several times and desorb to obtain a purified peptide solution.

[0104] 8) Lyophilize the purified peptide solution and redissolve the peptides with the loading buffer.

[0105] 9) The peptides were analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) coupled with tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0106] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0107] 11) Three personnel operated simultaneously to process three urine samples from different sources, with three parallel replicates for each sample. The number of identified proteins is shown in Table 3:

[0108] Table 3 Protein identification numbers in cell supernatant

[0109] Person 1 Person 2 Person 3 Sample1-1 5155 5090 5087 Sample1-2 4917 4985 5076 Sample1-3 4952 5027 4983 Sample2-1 5087 5002 5078 Sample2-2 5015 5108 4901 Sample2-3 4984 4972 5026 Sample3-1 4969 4948 4892 Sample3-2 4892 5076 5003 Sample3-3 5031 4997 4903

[0110] Conclusion:

[0111] 1. Through Figure 1 It can be seen that the surface of the modified magnetic nano-molecular sieve is rougher;

[0112] 2. Through Figure 2 It can be seen that the crystal structure of the modified magnetic nano-molecular sieve is not damaged and still has good crystallinity;

[0113] 3. As can be seen from Table 1-3, the modified magnetic nano-molecular sieve has excellent vesicle adsorption effects on different types of biological samples; compared with the magnetic nano-molecular sieve (unmodified), the number of protein identifications has been significantly improved;

[0114] 4. Through Table 1 and Figure 3 It can be seen that compared with the magnetic nano-molecular sieve (unmodified), the magnetic nano-molecular sieve treated only with strong base (magnetic nano-molecular sieve - strong base), and the magnetic nano-molecular sieve treated only with quaternary ammonium salt (magnetic nano-molecular sieve - quaternary ammonium salt), the number of plasma protein identifications of the magnetic nano-molecular sieve modified by our process has been improved to a certain extent, and the quantitative intensity values of some typical vesicle structure specific markers have increased significantly. The number of protein identifications and the intensity of vesicle markers of the magnetic nano-molecular sieve - strong base and the magnetic nano-molecular sieve - quaternary ammonium salt are significantly lower than those of the magnetic nano-molecular sieve without any treatment.

[0115] 5. Through Table 1 and Figure 3 It can be seen that compared with the method of extracting vesicle structures by the traditional ultracentrifugation method, the number of plasma protein identifications of the magnetic nano-molecular sieve modified by our process has increased significantly, and the quantitative intensity values of some typical vesicle structure specific markers have also increased significantly. Moreover, the sample amount of the modified magnetic nano-molecular sieve is only 1 / 10 of that of the ultracentrifugation method.

[0116] 6. As can be seen from Table 4, the silicon-aluminum ratio of the modified magnetic nano-molecular sieve has decreased significantly;

[0117] Table 4 Results of ICP-OES of magnetic nano-molecular sieve and modified magnetic nano-molecular sieve (both obtained from Example 3)

[0118] <![CDATA[SiO2]]> <![CDATA[Al2O3]]> <![CDATA[Na2O]]> Silica-alumina ratio Magnetic nanomolecular sieve 74.416％ 24.328％ 1.256％ 5.2 Modified magnetic nanomolecular sieve 63.190％ 35.808％ 1.003％ 3

Claims

1. A preparation method of a modified magnetic nano-molecular sieve material, characterized in that, It includes the following steps: 1) Disperse the magnetic nano-molecular sieve material in a strong alkaline solution, perform ultrasonic treatment under ice bath conditions, and then magnetically separate to discard the supernatant to obtain precipitate A; the magnetic nano-molecular sieve material is mainly prepared by the following method: Take citric acid-sodium modified Fe3O4 and add it to deionized water, disperse it evenly, then add an alkali source and dissolve it completely; continue to add a template agent, a stabilizer and a surfactant, and stir to dissolve; continue to add a silicon source and an aluminum source, and after dissolution, crystallize at room temperature, and then perform hydrothermal crystallization. After the hydrothermal crystallization is completed, through filtration, washing, drying, and calcination, the magnetic nano-molecular sieve can be obtained; the aluminum source is one of sodium aluminate and aluminum chloride; 2) Add a quaternary ammonium salt aqueous solution to precipitate A, stir and react at 20~40 °C, after washing, dry to obtain a surface-modified magnetic nano-molecular sieve material; The quaternary ammonium salt in the quaternary ammonium salt aqueous solution is one or more of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, benzyltriethylammonium chloride, and benzyltriethylammonium bromide.

2. The preparation method of the modified magnetic nano-molecular sieve material according to claim 1, characterized in that, In step 1), the alkali source of the strong alkaline solution is one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, and calcium hydroxide; the concentration of the strong alkaline solution is 0.1 M~1 M; when the solid dosage is in g and the liquid dosage is in ml, the ratio of the magnetic nano-molecular sieve material to the strong alkali solution is 1:50~500; the time of ice bath ultrasonic treatment is 10 min~6 h, and the power is 40-80 KHZ.

3. The preparation method of the modified magnetic nano-molecular sieve material according to claim 1, wherein, In step 2), the concentration of the quaternary ammonium salt aqueous solution is 0.12 M~1 M; the molar ratio of the quaternary ammonium salt in the quaternary ammonium salt aqueous solution to the strong alkali in the strong alkaline solution in step 1) is 1~1.2; the time of the stirring reaction is 2 h~6 h; the washing is successively washing with water, washing with alcohol, and washing with water, and cycling several times; among them, the alcohol source for washing with alcohol is ethanol or methanol, and the washing with water, washing with alcohol, and washing with water are taken as one cycle, and the cycle washing is 3~6 times.

4. The preparation method of the modified magnetic nano molecular sieve material according to claim 1, characterized in that, In step 1), the particle size of the citric acid-sodium modified Fe3O4 is 10~500 nm; the alkali source is one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, di-isopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; the surfactant is one or more of sodium dodecyl sulfate, cetyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride; the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetra-n-propyl orthosilicate, tetra-n-butyl orthosilicate, silica sol, water glass, and diatomite.

5. The preparation method of the modified magnetic nano molecular sieve material according to claim 1, characterized in that, In step 1), the mass ratio of Fe3O4, alkali source, template agent, stabilizer, surfactant, silicon source, and alkali metal source is 1:(2~20):(0.1~10):(0.05~5):(0.01~3):(20~100):(1~20); the duration of room temperature crystallization is 1~6 h, and the temperature of hydrothermal crystallization is 100~200 °C, and the duration is 24~120 h.

6. A modified magnetic nano-molecular sieve material prepared by the preparation method according to any one of claims 1-5.

7. Use of the modified magnetic nano-molecular sieve material according to claim 6 in the preparation of a vesicle adsorbent.

Images