Exosome-enriched sheet-shaped magnetic material, and preparation method and application thereof

Abstract

The application discloses a preparation method of a flaky magnetic material for enriching exosomes. The flaky Fe3O4 magnetic material with high specific surface area is taken as a starting point, and a surface of the flaky Fe3O4 magnetic material is coated with silicon dioxide to enhance dispersibility and chemical stability of the flaky Fe3O4 magnetic material, and to provide controllable silicon hydroxyl reaction sites for subsequent surface chemical modification. On this basis, amino active groups are introduced to the surface of the magnetic material through 3-aminopropyl triethoxysilane (APTES), and DSPE-SS-PEG2000-CHO containing a disulfide bond structure is further covalently connected to the surface of the magnetic material. Due to a membrane lipid insertion mechanism, non-antibody-dependent capture of a lipid bilayer exosome can be realized. The flaky magnetic material disclosed by the application shows universality and high affinity advantages, and can realize high affinity capture of a lipid structure of an exosome.

Description

Technical Field

[0001] This invention relates to the field of exosome enrichment and extraction technology, specifically to a sheet-like magnetic material for enriching exosomes, its preparation method, and its application. Background Technology

[0002] Exosomes (EVs) are nanoscale extracellular vesicles, approximately 30–150 nm in diameter, secreted by cells, carrying a variety of cellular macromolecules, including proteins, nucleic acids, and lipids. Exosomes play crucial roles in tumorigenesis and development, immune regulation, and tissue repair. They can transport various bioactive substances between cells, regulate cellular function through signal transduction, and are important mediators of cell communication. Due to their cargo-carrying capacity, immune evasion, and targeting capabilities, EVs have broad application prospects in disease diagnosis, prognostic assessment, and therapeutic molecular delivery. Therefore, efficient and precise isolation and purification of exosomes are key technical steps in promoting their clinical translation and basic research.

[0003] However, isolating high-purity, high-recovery exosomes from complex body fluids remains a challenge, a key bottleneck restricting EV research and clinical translation. Currently used exosome isolation techniques include ultracentrifugation, size exclusion chromatography, polymer precipitation, and immunomagnetic bead capture. Among these, ultracentrifugation equipment is expensive, complex to operate, and time-consuming; precipitation methods easily introduce impurity proteins; and size exclusion methods have limited purity. Immunomagnetic separation, based on its advantages of speed, high selectivity, and automation, has become an important direction for exosome capture research; however, the limited variety of antibodies makes it unsuitable for large-scale sample separation.

[0004] Another strategy is to utilize the properties of EV membranes, where lipid probes such as distearate phosphatidylethanolamine (DSPE), cholesterol, and phosphorylcholine can bind to the lipid bilayer of EVs through non-covalent interactions (as described in patent document CN110036111A). However, existing magnetic bead materials are mostly spherical nanoparticles with limited surface area (Nie Yixin, et al. Accurate Capture and Identification of Exosomes: Nanoarchitecture of the MXene Heterostructure / Engineered Lipid Layer. ACS Sens. 2023 Apr 28;8(4):1850-1857.), resulting in insufficient biomolecular modification density and leaving considerable room for improvement in exosome capture efficiency.

[0005] Furthermore, existing magnetic separation systems, while achieving efficient capture, often lack a controllable and gentle release mechanism for the captured exosomes. Harsh elution conditions (such as low pH or high concentrations of denaturing agents) or drastic physical treatments can damage the membrane structure and internal biomolecular activity of exosomes, affecting downstream functional studies and clinical applications. Although some studies have attempted to introduce breakable linkers (such as disulfide bonds), their cleavage typically relies on high concentrations of exogenous reducing agents (such as dithiothreitol or glutathione). These reagents may remain in the eluent, interfering with subsequent analyses, and the impact of cleavage conditions on the exosomes themselves still needs careful evaluation.

[0006] The core problem facing current exosome separation technology is how to construct a separation material that has high capture efficiency, high selectivity, and can achieve non-destructive and controllable release of exosomes. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing sheet-like magnetic materials enriched with exosomes. Starting with sheet-like Fe3O4 magnetic materials with high specific surface area, the surface is coated with silica. Amino active groups are introduced onto the surface of the magnetic material using 3-aminopropyltriethoxysilane (APTES). Furthermore, DSPE-SS-PEG2000-CHO containing disulfide bonds is covalently linked to the surface of the magnetic material to achieve high affinity capture of exosome membrane lipid structures.

[0008] A method for preparing a sheet-like magnetic material enriched with exosomes includes the following steps: (1) Preparation of sheet-like Fe3O4 magnetic materials; (2) The sheet-like Fe3O4 magnetic material obtained in step (2) is reacted with a mixed solution of tetraethyl silicate and ethanol to obtain sheet-like Fe3O4@SiO2 magnetic material; (2) The sheet-like Fe3O4@SiO2 magnetic material obtained in step (1) is reacted with 3-aminopropyltriethoxysilane to obtain sheet-like Fe3O4@SiO2@NH2 magnetic material; (3) The sheet-like Fe3O4@SiO2@NH2 magnetic material obtained in step (2) is reacted with DSPE-SS-PEG2000-CHO to obtain sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material, that is, sheet-like magnetic material enriched with exosomes; The structure of the DSPE-SS-PEG2000-CHO is shown below: , Where n is 41~50.

[0009] In this invention, starting with a high specific surface area sheet-like Fe3O4 magnetic material, its surface is coated with silica to enhance its dispersibility and chemical stability, and to provide controllable silanol reaction sites for subsequent surface chemical modification. Based on this, amino active groups are introduced onto the surface of the magnetic material using 3-aminopropyltriethoxysilane (APTES), and further, DSPE-SS-PEG2000-CHO containing a disulfide bond structure is covalently linked to the surface of the magnetic material. Due to the lipid intercalation mechanism, non-antibody-dependent capture of lipid bilayer exosomes can be achieved. The sheet-like magnetic material of this invention exhibits advantages of universality and high affinity, enabling high-affinity capture of exosome membrane lipid structures.

[0010] Because disulfide bonds are dynamic covalent bonds that can break under reducing conditions or specific chemical conditions, they exhibit reversible binding characteristics in the biological field. By modifying the surface of sheet-like magnetic materials with lipid molecules containing disulfide bonds (such as DSPE, a derivative of 1,2-stearoyl-sn-glycerol-3-phosphate ethanolamine), efficient capture of exosomes can be achieved, while the exosomes are gently released under the action of reducing agents such as glutathione (GSH), avoiding damage to the structure or function of exosomes.

[0011] Preferably, in step (1), the preparation method of the sheet-like Fe3O4 magnetic material includes the following steps: dissolving ferric chloride hexahydrate in an alcohol solvent, stirring and sonicating, adding anhydrous sodium acetate, mixing evenly, carrying out a solvothermal reaction, and washing to obtain the sheet-like Fe3O4 magnetic material; the alcohol solvent is diethylene glycol.

[0012] In this invention, sheet-like Fe3O4 magnetic materials can be prepared by controlling the type of alcohol solvent. Diethylene glycol has a longer molecular chain, lower hydroxyl density, and significantly weaker reducing power than ethylene glycol. 3+ Reduced to Fe 2+ The rate is extremely slow, and the monomer (Fe) in the system 2+ / Fe 3+ The concentration of hydroxyl complexes remained at a low level for an extended period, failing to meet the conditions for explosive nucleation, resulting in the formation of only a small number of crystal nuclei. Crystal growth occurred within the thermodynamically controlled region, with an extremely slow growth rate, providing ample time for the preferential growth of crystal faces, ultimately leading to the formation of a two-dimensional lamellar structure.

[0013] More preferably, the mass-to-volume ratio of ferric chloride hexahydrate to alcohol solvent is 3~5:60~80 g / mL.

[0014] More preferably, the alcohol solvent further includes ethylene glycol, and the volume ratio of diethylene glycol to ethylene glycol is 6~13:1.

[0015] As the amount of ethylene glycol gradually increases, Fe can be rapidly reduced.3+ Reduced to Fe 2+ Within a short period, the monomer concentration in the system exceeds the nucleation threshold, resulting in explosive nucleation and the instantaneous generation of a large number of nanocrystal nuclei. At this point, the monomers are rapidly consumed, crystal growth enters the kinetic control region, the differences in growth rates among crystal planes are significantly reduced, and the crystals grow isotropically along the three-dimensional direction, ultimately forming spherical particles. Therefore, if ethylene glycol is added, the volume ratio of diethylene glycol to ethylene glycol needs to be controlled within the above-mentioned range to ensure that the obtained Fe3O4 magnetic material is in sheet form.

[0016] More preferably, the mass ratio of anhydrous sodium acetate to ferric chloride hexahydrate is 4~6:3~5 g / g.

[0017] In the Fe3O4 magnetic nucleus preparation system, anhydrous sodium acetate can provide the necessary alkaline environment for crystal nucleus formation; electrostatic stability is achieved through carboxyl coordination adsorption, which inhibits agglomeration and ensures monodispersity; nucleation kinetics are regulated to precisely control the particle size and morphology of magnetic nuclei; the system's reduction ability is enhanced to ensure the stoichiometry and high magnetic properties of Fe3O4, while suppressing the formation of impurity phases and ensuring pure anti-spinel crystal form.

[0018] More preferably, the temperature of the solvothermal reaction is 180~220℃ and the time is 8~16 h.

[0019] Preferably, in step (1), the sheet-like Fe3O4 magnetic material has a two-dimensional sheet structure with a size of 30~120 nm, a thickness of 6~8 nm, and a specific surface area of ​​0.3~0.4 nm. 2 / nm 3 .

[0020] This invention provides a sheet-like Fe3O4 magnetic material with a two-dimensional sheet structure, unlike the spherical magnetic beads in the prior art. Spherical magnetic beads have a limited specific surface area and limited binding capacity, and it is difficult to balance magnetic response efficiency and liquid phase dispersibility. The sheet-like Fe3O4 magnetic material of this invention has a larger effective surface area, better liquid phase suspension stability, and a rapid magnetic response capability within 30 seconds.

[0021] Preferably, in step (2), the mass-to-volume ratio of the sheet-like Fe3O4 magnetic material to tetraethyl silicate is 0.4~0.7:0.5~3 g / mL.

[0022] Preferably, in step (3), the mass-to-volume ratio of the sheet-like Fe3O4@SiO2 magnetic material to 3-aminopropyltriethoxysilane is 0.4~0.7:0.5~3 g / mL.

[0023] Preferably, in step (4), the mass ratio of the sheet-like Fe3O4@SiO2@NH2 magnetic material to DSPE-SS-PEG2000-CHO is 5~10:1~3.

[0024] The present invention also provides sheet-like magnetic materials prepared by the above-described preparation method.

[0025] The present invention also provides the application of the above-mentioned sheet-like magnetic material for enriching exosomes in the enrichment of exosomes.

[0026] Preferably, the specific steps for enriching exosomes are as follows: the above-mentioned sheet-like magnetic material is mixed evenly with the solution rich in exosomes, incubated, and then Tris buffer containing (tris(2-carboxyethyl)phosphine) (TECP) is added for elution. The supernatant is collected to obtain the exosomes.

[0027] More preferably, the concentration of TECP in the Tris buffer solution is 5.0~8 mM.

[0028] More preferably, the mass ratio of the sheet-like magnetic material to TECP is 1:25~40.

[0029] Preferably, the sheet-like magnetic material has an enrichment efficiency of 70%~80% for cell supernatant exosomes and 80%~90% for plasma exosomes.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, starting with a high specific surface area sheet-like Fe3O4 magnetic material, its surface is coated with silica to enhance its dispersibility and chemical stability, and to provide controllable silanol reaction sites for subsequent surface chemical modification. Based on this, amino active groups are introduced onto the surface of the magnetic material using 3-aminopropyltriethoxysilane (APTES), and further, DSPE-SS-PEG2000-CHO containing a disulfide bond structure is covalently linked to the surface of the magnetic material. Due to the lipid intercalation mechanism, non-antibody-dependent capture of lipid bilayer exosomes can be achieved. The sheet-like magnetic material of this invention exhibits advantages of universality and high affinity, enabling high-affinity capture of exosome membrane lipid structures. Attached Figure Description

[0031] Figure 1 This is a TEM image of the sheet-like Fe3O4 magnetic material prepared in Example 1.

[0032] Figure 2 The image shows a TEM image of the sheet-like Fe3O4 magnetic material prepared in Example 1, as labeled.

[0033] Figure 3This is a TEM image of the spherical Fe3O4 magnetic material prepared in Comparative Example 1.

[0034] Figure 4 This is a TEM image of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Example 1.

[0035] Figure 5 The image shows a TEM image of the spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Comparative Example 1.

[0036] Figure 6 This is a TEM image of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Example 2.

[0037] Figure 7 This is a TEM image of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Example 3.

[0038] Figure 8 This is a TEM image of the magnetic material enriched with exosomes in Example 1.

[0039] Figure 9 This is a TEM image of exosomes in the cell supernatant after enrichment and elution with the sheet-like magnetic material of Example 1.

[0040] Figure 10 This is a TEM image of plasma exosomes enriched with the sheet-like magnetic material of Example 1.

[0041] Figure 11 This is a TEM image of plasma exosomes enriched in the spherical magnetic material of Comparative Example 1.

[0042] Figure 12 This is a TEM image of exosomes in the plasma supernatant after enrichment and elution with the sheet-like magnetic material of Example 1.

[0043] Figure 13 The images show NTA test results for cells and plasma exosomes after enrichment and elution of the sheet-like magnetic material in Example 1. In Example 1, A and C are NTA test results for cells or plasma exosomes enriched with the sheet-like magnetic material, while B and D are NTA test results for cells or plasma exosomes after elution of the sheet-like magnetic material. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0045] All raw materials used in this invention are commercially available.

[0046] Example 1 (1) Preparation of sheet-like Fe3O4 magnetic materials Dissolve 3.62 g of ferric chloride hexahydrate (purchased from Aladdin, F102739-2.5 kg, 99%, AR) in 70 mL of diethylene glycol (purchased from Aladdin, D110833-100 mL, >99%, GC). Place the solvent in a 500 mL round-bottom three-necked flask, install a mechanical stirrer, and place it in an ultrasonic bath. Set the stirring speed to 200 rpm and sonicate for 10 min to ensure homogeneous mixing. Then, while maintaining sonication and stirring, add 5.42 g of anhydrous sodium acetate (purchased from Sigma, S8750-5KG, ≥99.0%), continue ultrasonic stirring for 30 min, then stop sonication and maintain stirring for another 30 min to ensure homogeneous mixing. Transfer the reaction mixture to a 100 mL polytetrafluoroethylene-lined reactor, seal it, and place it in a forced-air drying oven for a solvothermal reaction: heat to 200 °C and react for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the product was removed. It was then magnetically washed six times with anhydrous ethanol to remove unreacted substances and impurities. A dispersion of flake-shaped Fe3O4 magnetic material was obtained. The concentration of the flake-shaped Fe3O4 magnetic material dispersion was adjusted to 10 mg / mL and stored at 4℃ for later use.

[0047] (2) Preparation of sheet-like Fe3O4@SiO2 magnetic materials Based on the concentration of the magnetic material dispersion, 0.5 g of flake-shaped Fe3O4 magnetic material was placed in a 500 mL round-bottom three-necked flask. The supernatant was discarded by magnetic suction. 21.5 mL of ultrapure water and 117.5 mL of ethanol (purchased from Titan, G73537F, AR, 99.7%) were added to the flask. A mechanical stirrer was installed and placed in an ultrasonic instrument. The stirring speed was set to 200 rpm, the ultrasonic time to 30 min, and the ultrasonic temperature to 45 ℃ to ensure thorough dispersion of the magnetic material. 2.5 mL of ammonia (purchased from Aladdin, A112079-500mL, GR, 25~28%) was added to a constant-pressure dropping funnel. The ammonia was added dropwise to the reaction system under ultrasonic stirring. After the addition was complete, stirring continued for 5 min. Take 12.75 mL of a pre-prepared tetraethyl silicate ethanol solution (specifically, a mixture of 11.25 mL ethanol and 1.5 mL tetraethyl silicate (purchased from Aladdin, T110593-500 mL, GC, 99%)), add it to a constant pressure dropping funnel, and add it dropwise to the reaction system at 45 °C. Continue stirring at this constant temperature overnight. After the reaction is complete, remove the apparatus, magnetically collect the magnetic material, discard the supernatant, and magnetically wash the magnetic material five times with ultrapure water to obtain a dispersion of sheet-like Fe3O4@SiO2 magnetic material. Adjust the concentration of the sheet-like Fe3O4@SiO2 magnetic material to 10 mg / mL and store it at 4 °C for later use.

[0048] (3) Preparation of sheet-like Fe3O4@SiO2@NH2 magnetic materials Based on the concentration of the magnetic material dispersion, 0.5 g of the flake-shaped Fe3O4@SiO2 magnetic material dispersion was placed in a 500 mL round-bottom flask. The supernatant was discarded, and 200 mL of ethanol was added to the flask. A mechanical stirrer was installed on an ultrasonic apparatus, and the ultrasonic time was set to 40 min. The round-bottom flask was installed, and the reaction system was ultrasonically treated while stirring at a speed of 200 rpm. 1.5 mL of 3-aminopropyltriethoxysilane (APTES) was taken and diluted with 10 mL of anhydrous ethanol, and then added to a constant-pressure dropping funnel. After ultrasonic reaction for 40 min, a constant-pressure dropping funnel was installed on the flask, and the APTES / ethanol mixture was added dropwise to the reaction system. Ultrasonication was stopped, and the reaction was stirred for 15 min. 1.25 mL of ammonia water was then placed in a constant-pressure separatory funnel and added dropwise to a three-necked flask. The reaction was stirred at 25 °C for 17 h. After the reaction is complete, the reaction apparatus is removed. The magnetic material is first collected using a magnetic separator and then washed multiple times with ultrapure water until neutral. Finally, a flake-shaped Fe3O4@SiO2@NH2 magnetic material dispersion is obtained and stored at 4°C for later use.

[0049] (4) Preparation of sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic materials Based on the concentration of the magnetic material dispersion, 10 mg of the sheet-like Fe3O4@SiO2@NH2 magnetic material dispersion was placed in a 50 mL centrifuge tube, and the supernatant was magnetically removed. A PBS mixture was prepared by adding 2 mL of pH 7.4 PBS buffer (1×PBS, pH 7.2-7.4, P1020, Solarbio) to a 50 mL centrifuge tube, along with 38 mL of ultrapure water, and mixing thoroughly. 8 mL of the PBS mixture was then used to rinse the sheet-like Fe3O4@SiO2@NH2 magnetic material three times, and the supernatant was magnetically removed. 2 mg of DSPE-SS-PEG2000-CHO (TYK0607-01, 95%, Shanghai Tuoyang) was weighed and placed in a 5 mL centrifuge tube, and 1 mL of the PBS mixture was added to dissolve it thoroughly. All of the above solutions were transferred to centrifuge tubes containing the washed sheet-like Fe3O4@SiO2@NH2 magnetic material and incubated at 4°C for 16 h. After the reaction was complete, the sample was washed three times with 8 mL of PBS solution, and then three times with pure water. Finally, a dispersion of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material was obtained and stored at 4°C for later use.

[0050] Two mL of ultrapure water was placed in a cuvette, and 20 μL of a 10 mg / mL magnetic nanoparticle dispersion was added. After sonication to ensure uniform dispersion, the outer surface of the cuvette was wiped with lint-free paper. Subsequently, the electrode was inserted into the cuvette and connected to a NanoBrook 90 Plus PALS instrument (Brookhaven Instruments, USA) to measure the zeta potential. The results are shown in Table 1.

[0051] Table 1: Zeta potential (mV) measurement results of magnetic materials in each step of Example 1

[0052] As shown in Table 1, the surface potential of the unmodified sheet-like Fe3O4 magnetic material is 4.43 mV, which is close to neutral. After being coated with silica, the surface potential of the sheet-like Fe3O4@SiO2 magnetic material drops to -25.63 mV due to the introduction of easily deprotonated silanol groups on the surface. After further aminosilanization modification, amino functional groups with positive charges are introduced on the surface, changing the surface potential of the sheet-like Fe3O4@SiO2@NH2 magnetic material from negative to 22.95 mV. On this basis, after introducing an SS-DSPE modification layer containing polyethylene glycol segments and phospholipid structures, the outer organic shell layer covers and shields the surface charge and changes the ion distribution at the interface, adjusting the surface potential of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material to -18.61 mV, indicating that the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material was successfully prepared.

[0053] Example 2: Preparation of sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material The preparation method is the same as the preparation scheme of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material in step 4 of Example 1. The only difference is that the amount of sheet-like Fe3O4@SiO2@NH2 magnetic material used is replaced with 5 mg, and the amount of DSPE-SS-PEG2000-CHO used is replaced with 1 mg.

[0054] 2 mL of ultrapure water was placed in a cuvette, and 20 μL of a 10 mg / mL magnetic nanoparticle dispersion was added. After sonication to ensure uniform dispersion, the outer surface of the cuvette was wiped with lint-free paper. Subsequently, the electrode was inserted into the cuvette and connected to a NanoBrook 90 Plus PALS instrument (Brookhaven Instruments, USA) to measure the zeta potential. The results are shown in Table 2.

[0055] Table 2: Zeta potential (mV) measurement results of sheet-like Fe3O4@SiO2@NH2@SS-DSPE in Example 2

[0056] As shown in Table 2, when the amount of sheet-like Fe3O4@SiO2@NH2 magnetic material was replaced with 5 mg and the amount of DSPE-SS-PEG2000-CHO was replaced with 1 mg, the outer organic shell layer covered and shielded the surface charge and changed the ion distribution at the interface. The surface potential was adjusted from 22.95 mV to -16.25 mV, indicating that the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material was successfully prepared.

[0057] Example 3: Preparation of sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material The preparation method is the same as the preparation scheme of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material in step 4 of Example 1. The only difference is that the amount of sheet-like Fe3O4@SiO2@NH2 magnetic material used is replaced with 10 mg, and the amount of DSPE-SS-PEG2000-CHO used is replaced with 3 mg.

[0058] 2 mL of ultrapure water was placed in a cuvette, and 20 μL of a 10 mg / mL magnetic nanoparticle dispersion was added. After sonication to ensure uniform dispersion, the outer surface of the cuvette was wiped with lint-free paper. Subsequently, the electrode was inserted into the cuvette and connected to a NanoBrook 90 Plus PALS instrument (Brookhaven Instruments, USA) to measure the zeta potential. The results are shown in Table 3.

[0059] Table 3: Zeta potential (mV) measurement results of sheet-like Fe3O4@SiO2@NH2@SS-DSPE in Example 3

[0060] As shown in Table 3, when the amount of sheet-like Fe3O4@SiO2@NH2 magnetic material was replaced with 10 mg and the amount of DSPE-SS-PEG2000-CHO was replaced with 3 mg, the outer organic shell layer covered and shielded the surface charge and changed the ion distribution at the interface. The surface potential was adjusted from 22.95 mV to -20.96 mV, indicating that the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material was successfully prepared.

[0061] Comparative Example 1: Preparation of spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic materials (1) Preparation of spherical Fe3O4 magnetic materials 6.28 g of ferric chloride hexahydrate was dissolved in 70 mL of ethylene glycol. The solvent was placed in a 250 mL round-bottom three-necked flask, a mechanical stirrer was installed, and the flask was placed in an ultrasonic apparatus. The stirring speed was set to 200 rpm, and the mixture was ultrasonicated for 10 min to ensure homogeneous mixing. Then, while maintaining ultrasonication and stirring, 10.22 g of anhydrous sodium acetate was added, and ultrasonication and stirring were continued for 30 min. After stopping ultrasonication, stirring was maintained for another 30 min to ensure homogeneous mixing. The reaction mixture was transferred to a 100 mL polytetrafluoroethylene-lined reactor, sealed, and placed in a forced-air drying oven for a solvothermal reaction: the temperature was raised to 200 °C and the reaction was carried out for 24 h. After the reaction was completed, the mixture was cooled to room temperature, and the product was removed. It was magnetically washed 6 times with anhydrous ethanol to remove unreacted substances and impurities, yielding spherical Fe3O4 magnetic material. The concentration of the spherical Fe3O4 magnetic material dispersion was adjusted to 10 mg / mL and stored at 4 °C.

[0062] (2) Preparation of spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic materials The preparation method is the same as in Example 1, except that the sheet-like Fe3O4 magnetic material is replaced with spherical Fe3O4 magnetic material, and the amount of magnetic beads and other modifying reagents is the same.

[0063] 2 mL of ultrapure water was placed in a cuvette, and 20 μL of a 10 mg / mL magnetic nanoparticle dispersion was added. After sonication to ensure uniform dispersion, the outer surface of the cuvette was wiped with lint-free paper. Subsequently, the electrode was inserted into the cuvette and connected to a NanoBrook 90 Plus PALS instrument (Brookhaven Instruments, USA) to measure the zeta potential. The results are shown in Table 4.

[0064] Table 4: Zeta potential (mV) measurement results of magnetic materials in each step of Comparative Example 1

[0065] As shown in Table 4, the average surface potential of spherical Fe3O4 is 19.82 mV, indicating a positive charge. After coating with silica, the surface potential of spherical Fe3O4@SiO2 decreases to -39.56 mV. This change is mainly attributed to the deprotonation of silanol groups on the SiO2 surface in the aqueous phase, resulting in a negative charge on the particle surface. After further aminosilanization modification, the surface potential of spherical Fe3O4@SiO2@NH2 increases to 11.77 mV, due to the formation of positive charges by amino groups on the surface. Based on this, the introduction of an SS-DSPE modification layer containing polyethylene glycol segments and phospholipid structures allows the outer organic shell to cover and shield the surface charge and change the ion distribution at the interface, adjusting the surface potential of the spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic material to -21.55 mV. This indicates that the spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic material was successfully prepared.

[0066] Sample Analysis I. TEM Testing of Sheet-like and Spherical Fe3O4 Magnetic Materials (1) The TEM of the sample was determined by the Testing Center of Yangzhou University. First, the sample to be tested was made ultrathin and fixed on the transmission electron microscope grid. Then, the grid was installed into the sample rod and inserted into the transmission electron microscope under vacuum conditions. The electron beam was centered and the beam spot was adjusted under the set acceleration voltage. The thin area of ​​the sample was located in low magnification mode and adjusted to the isofocal height. Clear imaging was obtained by focusing the objective lens and correcting astigmatism. Finally, the transmission electron microscopy image or selected area electron diffraction pattern of the sample was acquired under the set magnification and exposure parameters, and the obtained data was stored and analyzed.

[0067] Figure 1 This is a TEM image of the sheet-like Fe3O4 magnetic material prepared in Example 1. Figure 1 As shown, the sheet-like Fe3O4 magnetic material exhibits a distinct two-dimensional sheet structure with an overall size in the hundreds of nanometers range (corresponding to the 100 nm scale). The sheet outlines are clear and the shapes are irregular, displaying typical characteristics of two-dimensional nanosheets. There is a certain degree of stacking and overlap between the sheet structures, mainly due to the self-assembly of the nanosheets during the drying process under the influence of van der Waals forces and magnetic interactions. No significant large-scale aggregation was observed, indicating that this sheet-like magnetic material has good dispersibility and structural integrity.

[0068] (2) Using ImageJ software, measure and calculate the values ​​of each sheet-like Fe3O4 magnetic material (e.g., according to the actual scale of the TEM image) based on the actual scale of the image. Figure 2 The dimensions of the spherical Fe3O4 magnetic material (shown in the figure) and Comparative Example 1 are calculated, retaining one decimal place. The surface area, volume and specific surface area are also calculated. The measurement and calculation results are shown in Table 5.

[0069] Table 5: Statistical data of the sheet-like Fe3O4 magnetic material prepared in Example 1

[0070] The average surface area of ​​the sheet-like magnetic beads in the table is approximately 12634.65 nm. 2 Its volume is approximately 37563.75 nm. 3 Its specific surface area was calculated to be 0.340 nm. 2 / nm 3 .

[0071] Figure 3 The image shows a TEM image of the spherical Fe3O4 magnetic material prepared in Comparative Example 1. The diameter of the tested magnetic beads is 510 nm, and the calculated surface area is approximately 817128.25 nm. 2 Its volume is approximately 69455741.10 nm. 3 Its specific surface area was calculated to be 0.012 nm. 2 / nm 3 .

[0072] In summary, the unit is nm. 3 Under these conditions, the specific surface area of ​​the sheet-like magnetic beads is 0.340 nm. 2 / nm 3 This is much larger than the 0.012 nm of spherical magnetic beads. 2 / nm 3 This provides a larger contact surface for the enrichment of exosomes by sheet-like materials, thereby increasing the enrichment amount and efficiency of exosomes by sheet-like magnetic beads.

[0073] II. TEM Testing of Flake and Spherical Fe3O4@SiO2@NH2@SS-DSPE Magnetic Materials The testing method is the same as above. Figure 4 The image shows a TEM image of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Example 1. As shown, the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material exhibits a distinct two-dimensional sheet-like structure, with its lateral dimensions mainly distributed in the range of hundreds of nanometers, consistent with the scale bar (100 nm). The material outline is relatively clear, but there are certain differences in the thickness of the sheets, indicating that the sheet-like Fe3O4 has polyhedral or irregular lamellar characteristics. Figure 4The darker regions within the nanosheets exhibit strong absorption of the electron beam, indicating a high electron density and presumably corresponding to a Fe3O4 magnetic core. The surrounding lighter, semi-transparent regions are attributable to the SiO2 coating and the organic functional layers (NH2 and SS-DSPE) introduced to the surface, demonstrating the successful construction of the magnetic core-shell structure. Slight stacking and contact exist between some nanosheets, likely due to magnetic interactions and the minimization of surface energy during drying, but the overall structure remains well-dispersed.

[0074] Figure 5 The image shows a TEM image of the spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Comparative Example 1. As shown in the figure, the surface of the Fe3O4@SiO2@NH2@SS-DSPE magnetic material becomes smoother and flatter. Even after functionalization modification, this magnetic material still exhibits good dispersibility and structural integrity.

[0075] Figure 6 and Figure 7 The figures show TEM images of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic materials prepared by substituting the amounts of sheet-like Fe3O4@SiO2@NH2 magnetic material and DSPE-SS-PEG2000-CHO used in Examples 2 and 3, respectively. Figure 6 and Figure 7 The plate-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material exhibits a distinct two-dimensional plate-like structure, with lateral dimensions mainly distributed in the range of hundreds of nanometers, consistent with the scale bar (100 nm). The material outline is relatively clear, but there are certain differences in the thickness of the plates, indicating that the plate-like Fe3O4 has polyhedral or irregular lamellar characteristics. Figure 6 and Figure 7 The darker regions within the nanosheets exhibit strong absorption of the electron beam, indicating a high electron density and presumably corresponding to a Fe3O4 magnetic core. The surrounding lighter, semi-transparent regions are attributable to the SiO2 coating and the organic functional layers (NH2 and SS-DSPE) introduced to the surface, demonstrating the successful construction of the magnetic core-shell structure. Slight stacking and contact exist between some nanosheets, likely due to magnetic interactions and the minimization of surface energy during drying, but the overall structure remains well-dispersed.

[0076] III. Exosome enrichment test NTA (Nanoparticle Size Analyzer) Operation Procedure: Turn on the Nanoparticle Tracking Analyzer instrument (manufacturer: Particle Metrix, model: ZetaView) main unit and control terminal, inject purified water into the sample cell to complete system initialization and quality testing; prepare standard nanoparticle calibration solution and inject it into the sample cell to perform automatic alignment and calibration operations. After calibration, confirm that the system is in a testable state; then rinse the sample cell and tubing with purified water and sample buffer solution in sequence, inject the sample to be tested into the sample cell, and adjust the detection sensitivity according to the particle density to ensure that the number of particles in the field of view is within the preset range; after imaging and drift verification of the sample at different detection positions, acquire particle motion video data in the scattered light mode according to the preset test program, and obtain the particle size distribution and particle concentration results of the sample based on multi-site analysis, and finally generate and save the test report.

[0077] (1) Enrichment and extraction of cell exosomes S1. Extraction of exosomes Transfer 15 mL of cell supernatant using a pipette into a 15 mL centrifuge tube. Centrifuge the tube containing the cell supernatant at 4 °C and 500 × g for 5 min. After centrifugation, gently and slowly aspirate the supernatant into a new centrifuge tube for later use; this is designated as Supernatant 1. Centrifuge Supernatant 1 at 4 °C and 10000 × g for 30 min. After centrifugation, gently and slowly aspirate the supernatant into a new centrifuge tube for later use; this is designated as Supernatant 2. Filter Supernatant 2 using a 0.22 µm syringe filter to further remove larger cell vesicles and apoptotic bodies. Transfer the filtered supernatant into a new centrifuge tube for later use; this is designated as Supernatant 3. Add supernatant 3 to the cell supernatant exosome extraction reagent (cell supernatant exosome extraction kit, Beyotime, C3620S), and pipette the solution. After pipetting, place the solution at 4 ℃ and let it stand overnight, which is recorded as mixture 1. Centrifuge mixture 1 at 4 ℃ and 10000×g for 30 min. After centrifugation, discard the supernatant and collect the precipitate using 10 mM PBS (pH 7.4). This precipitate is the exosome, and the particle size and number of the extracted exosomes are determined using NTA.

[0078] S2, enriched exosomes Preparation of HEPES binding buffer: Take 1.6 mL of HEPES buffer (0.5 M, pH 7.4, H885790-100 mL, Maclean) and place it in a 50 mL centrifuge tube. Add 38.4 mL of ultrapure water and mix well to obtain HEPES binding buffer.

[0079] Based on the concentration of the magnetic material dispersion, 2 mg of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material prepared in Example 1 was placed in a 5 mL centrifuge tube. The supernatant was discarded by magnetic aspiration, and the magnetic material was washed with HEPES binding buffer by magnetic aspiration. 0.5 mL of the extracted cell supernatant exosomes was added to the centrifuge tube containing the magnetic material using a pipette, followed by 0.5 mL of HEPES binding buffer. The centrifuge tubes were placed in a centrifuge mixer and incubated at 37 °C for 1 h. After incubation, the magnetic material was washed with HEPES binding buffer by magnetic aspiration, and the supernatant was collected. The number of exosomes in the supernatant was determined using NTA.

[0080] Figure 8 The image shows a TEM image of the magnetic material enriched with exosomes in Example 1. As shown in the figure, a large number of typical exosome structures are present in the TEM image of the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material enriched with exosomes. Figure 8 The central region shows dark clumps with high electron density, corresponding to the magnetic material itself, surrounded by numerous, relatively uniform nanoscale vesicle structures. These vesicles are approximately circular or elliptical, with clear boundaries and a distinct membrane-like outline. Their particle size is mainly concentrated in the range of tens to approximately 150 nm, consistent with the size distribution characteristics of exosomes. Simultaneously, the overall electron density of the vesicles is lower than that of the magnetic material core, exhibiting a typical "cup-shaped / vesicle-shaped" morphology, a common TEM feature of exosomes under negative staining conditions. These results demonstrate that the sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material can effectively capture and enrich cell-extracted exosomes, while maintaining the integrity of the exosome structure after elution.

[0081] S3, Eluting exosomes By adding the reducing agent TCEP to the Tris buffer system, the disulfide bonds in SS-DSPE on the surface of the magnetic material are broken, disrupting the connection between the magnetic material and exosomes, and exosomes are selectively released under mild conditions. Preparation of the exosome elution buffer: Measure 32 mL of ultrapure water into a 50 mL centrifuge tube, weigh 0.06 g of TCEP and add it to the centrifuge tube, then add 8 mL of Tris-HCl (0.1 M, pH=7.5) to the centrifuge tube, mix well, and store at 4℃ for later use.

[0082] Figure 9 This is a TEM image of exosomes in the cell supernatant after enrichment and elution with the sheet-like magnetic material of Example 1. Figure 9 As shown, exosomes were successfully separated after elution. Figure 9The background was relatively uniform, and no obvious large-sized, high-electron-density clusters were observed, indicating that the magnetic material had been largely removed and the elution process was relatively thorough. Numerous dispersed nanoscale vesicle particles were visible in the field of view, generally spherical or elliptical in shape with relatively clear boundaries. Some particles exhibited typical "cup-shaped" or concave morphologies, a common structural feature of exosomes under negative staining conditions. Their particle size was mainly concentrated in the range of tens to approximately 150 nm, consistent with the size range of exosomes. Furthermore, no significant aggregation of exosomes was observed, indicating that the elution conditions were relatively mild, which was conducive to maintaining the structural integrity and good dispersibility of the exosomes. These results further verify that this magnetic material system can not only effectively capture exosomes but also achieve effective release of exosomes during the elution step.

[0083] (2) Enrichment and extraction of plasma exosomes S1. Extraction of plasma exosomes Plasma samples were centrifuged at 4 °C and 1200 × g for 20 min. After centrifugation, cell debris was separated by precipitation, and the supernatant was transferred to a new centrifuge tube, designated as Supernatant 1. Supernatant 1 was then centrifuged at 4 °C and 10000 × g for 30 min to separate large extracellular vesicles by precipitation, and the supernatant was transferred to a new centrifuge tube, designated as Supernatant 2. Supernatant 2 contained extracted plasma exosomes, and the particle size and number of the extracted exosomes were determined using NTA.

[0084] S2, enriched plasma exosomes and eluted plasma exosomes The method follows the steps of enriching and eluting exosomes.

[0085] Figure 10 This is a TEM image of plasma exosomes enriched with the sheet-like magnetic material of Example 1. Compared with cell-derived exosomes, the morphology of plasma exosomes exhibits certain heterogeneity. Figure 10 Nanoscale vesicles with a relatively dispersed size distribution were observed, with particle sizes mainly ranging from tens to approximately 200 nm, consistent with the complex origin and diverse composition of plasma exosomes. Some vesicles exhibited typical cup-shaped or near-circular structures with clear outlines and intact membrane structures, indicating that they still retain the lipid bilayer membrane characteristics of exosomes; at the same time, a small number of irregularly shaped or larger particles were also observed, which may be related to lipoproteins or microvesicles coexisting in plasma. Overall, a large number of nanovesicle structures were enriched around the magnetic material, indicating that the material still has good exosome capture ability in complex biofluid environments.

[0086] Figure 11The image shown is a TEM image of plasma exosomes enriched by spherical magnetic materials in Comparative Example 1. The spherical Fe3O4@SiO2@NH2@SS-DSPE magnetic beads did not exhibit the typical cup-shaped or vesicle-like nanostructure features (usually approximately 30–150 nm in diameter) characteristic of exosomes on their surface, indicating that no exosome enrichment behavior clearly identifiable by TEM occurred on the surface of the spherical magnetic beads. No further NTA testing was performed.

[0087] Figure 12 This is a TEM image of exosomes from the plasma supernatant after enrichment and elution with sheet-like magnetic material in Example 1. As shown in the figure, the plasma exosomes after elution and further elution / washing exhibit a relatively pure and dispersed nanovesicle morphology. The image background is uniform, and no obvious high electron density impurities or magnetic material residues were observed, indicating that the purity of the exosome sample was further improved after elution. A large number of nanoscale particles are visible in the field of view, with an overall spherical or typical cup-shaped structure, clear edge contours, and the common lipid bilayer membrane morphology characteristics of exosomes. Their particle size is mainly distributed in the range of tens to approximately 150 nm, consistent with the size characteristics of plasma exosomes. Compared with the uneluted sample, Figure 12 The particle aggregation phenomenon was significantly reduced, indicating that the elution and washing steps effectively removed non-specific adsorbed components, while maintaining the structural integrity and dispersibility of exosomes well, further verifying the effectiveness of this magnetic material system in the purification process of plasma exosomes.

[0088] (3) NTA analysis of enriched and extracted cell exosomes and plasma exosomes After enriching the cell supernatant using a coagulation method, ZetaView nanoparticle tracking analysis (NTA) was used to detect the particle size and concentration of the obtained samples. Figure 13 As shown in Figure A, the particle size distribution of the extracellular vesicle (EV) preparation is mainly concentrated in the range of approximately 20–400 nm, with a main peak particle size of approximately 101.0 nm, indicating that the obtained particle population conforms to the typical particle size characteristics of exosome-like extracellular vesicles. Simultaneously, the particle concentration measured at a 100-fold dilution was 1.7 × 10⁻⁶. 8 particles / mL, which translates to an initial concentration of approximately 1.7 × 10⁻⁶. 10 The particle size distribution (particles / mL) indicates that this method can achieve efficient enrichment and significant concentration of extracellular vesicles in cell supernatants. These results demonstrate that the described sedimentation enrichment process can stably obtain extracellular vesicle concentrates with reasonable particle size and high yield, providing a foundation for subsequent enrichment of exosomes using magnetic materials.

[0089] The concentration of the original exosomes derived from the cell supernatant was 1.7 × 10⁻⁶. 10The particles / mL were collected and exosomes were enriched using magnetic materials. The samples enriched with magnetic materials were then analyzed using ZetaView nanoparticle tracking analysis (NTA). Figure 13 As shown in Figure B, the main peak particle size of the exosomes in this sample is approximately 133.4 nm, which falls within the typical particle size range of exosome-like extracellular vesicles, indicating that the magnetic enrichment process did not significantly alter the particle size characteristics of the exosomes. Under a 100-fold sample dilution, the concentration of enriched exosome particles was measured to be 1.3 × 10⁻⁶. 8 The particle / mL value translates to an exosome concentration in the original solution obtained through magnetic enrichment of approximately 1.3 × 10⁻⁶ particles / mL. 10 The results show that the magnetic material can enrich exosomes from the extracted cell supernatant with an efficiency of 76.5%, achieving highly efficient enrichment of exosomes.

[0090] The statistical analysis and calculation process of the above experimental data is shown in Table 6.

[0091] Table 6: Summary and Analysis of NTA Data from Enriched Extraction of Exosomes

[0092] After extracting exosomes from plasma using centrifugation, ZetaView nanoparticle tracking analysis (NTA) was employed to detect the particle size distribution and concentration of the obtained samples. Figure 13 As shown in C, the particle size distribution of this exosome-like extracellular vesicle is mainly concentrated in the range of approximately 20–250 nm, with a main peak particle size of approximately 97.5 nm, consistent with the typical particle size characteristics of plasma-derived exosome-like extracellular vesicles. Under a 100-fold sample dilution, the particle concentration was measured to be 8.2 × 10⁻⁶. 7 particles / mL, which translates to an initial particle concentration of approximately 8.2 × 10⁻⁶. 9 The particle size distribution (particles / mL) indicates that this centrifugal extraction method can effectively enrich and significantly concentrate exosomes in plasma. These results demonstrate that the obtained extracellular vesicles have a reasonable particle size distribution and high concentration, laying a solid foundation for subsequent research on exosome enrichment based on magnetic materials.

[0093] For plasma-derived exosomes (initial concentration 8.2 × 10⁻⁶), 9 After enriching and eluting the magnetic material (particles / mL), the eluted exosomes were characterized by ZetaView-NTA. Figure 13As shown in Figure D, the particle size distribution of the enriched products is mainly in the hundreds of nanometers, with a main peak particle size of approximately 150.8 nm. This falls within the typical size range of exosome-like extracellular vesicles, indicating that the magnetic enrichment and recovery process effectively maintains the particle size characteristics of exosomes. Furthermore, the particle concentration of the recovered sample was measured to be 6.9 × 10⁻⁶ after a 100-fold dilution. 7 The particle / mL concentration translates to approximately 6.9 × 10⁻⁶ exosomes enriched from magnetic materials in the original solution. 9 The results show that the magnetic material can enrich the extracted plasma exosomes with an efficiency of 84.1%, achieving highly efficient enrichment of exosomes.

[0094] The statistical analysis and calculation process of the above experimental data is shown in Table 7.

[0095] Table 7: Summary and Analysis of NTA Data from Enriched Plasma Secretions

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a sheet-like magnetic material enriched with exosomes, characterized in that, Includes the following steps: (1) Preparation of sheet-like Fe3O4 magnetic materials; (2) The sheet-like Fe3O4 magnetic material obtained in step (1) is reacted with a mixed solution of tetraethyl silicate and ethanol to obtain sheet-like Fe3O4@SiO2 magnetic material; (3) The sheet-like Fe3O4@SiO2 magnetic material obtained in step (2) is reacted with 3-aminopropyltriethoxysilane to obtain sheet-like Fe3O4@SiO2@NH2 magnetic material; (4) The sheet-like Fe3O4@SiO2@NH2 magnetic material obtained in step (3) is reacted with DSPE-SS-PEG2000-CHO to obtain sheet-like Fe3O4@SiO2@NH2@SS-DSPE magnetic material, that is, sheet-like magnetic material enriched with exosomes; The structure of the DSPE-SS-PEG2000-CHO is shown below: ， Where n is 41~50.

2. The method for preparing the sheet-like magnetic material enriched with exosomes according to claim 1, characterized in that, In step (1), the preparation method of the sheet-like Fe3O4 magnetic material includes the following steps: dissolving ferric chloride hexahydrate in an alcohol solvent, stirring and sonicating, adding anhydrous sodium acetate, mixing evenly, carrying out a solvothermal reaction, and washing to obtain the sheet-like Fe3O4 magnetic material; the alcohol solvent is diethylene glycol.

3. The method for preparing the sheet-like magnetic material enriched with exosomes according to claim 2, characterized in that, The alcohol solvent also includes ethylene glycol, and the volume ratio of diethylene glycol to ethylene glycol is 6~13:

1.

4. The method for preparing the sheet-like magnetic material enriched with exosomes according to claim 1, characterized in that, In step (1), the sheet-like Fe3O4 magnetic material has a two-dimensional sheet structure with a size of 30~120 nm, a thickness of 6~8 nm, and a specific surface area of ​​0.3~0.4 nm. 2 / nm 3 .

5. The method for preparing the sheet-like magnetic material enriched with exosomes according to claim 1, characterized in that, In step (4), the mass ratio of the sheet-like Fe3O4@SiO2@NH2 magnetic material to DSPE-SS-PEG2000-CHO is 5~10:1~3.

6. A sheet-like magnetic material enriched with exosomes is prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the sheet-like magnetic material for enriching exosomes according to claim 6 in the enrichment of exosomes.

8. The application according to claim 7, characterized in that, The specific steps for enriching exosomes are as follows: the sheet-like magnetic material described in claim 6 is mixed evenly with a solution rich in exosomes, incubated, and then eluted with Tris buffer containing (tris(2-carboxyethyl)phosphine). The supernatant is collected to obtain the exosomes.

9. The application according to claim 8, characterized in that, The mass ratio of the sheet-like magnetic material to (tris(2-carboxyethyl)phosphine) is 1:25~40.

10. The application according to claim 8, characterized in that, The sheet-like magnetic material described above has an enrichment efficiency of 70%~80% for cell supernatant exosomes and 80%~90% for plasma exosomes.

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