Preparation method of reusable Tim4-rMGO magnetic beads and application of reusable Tim4-rMGO magnetic beads in exosome extraction

Abstract

The invention discloses a preparation method of reusable Tim4-rMGO magnetic beads and application of the reusable Tim4-rMGO magnetic beads in exosome extraction.The preparation method comprises the steps that sterile and enzyme-free water is added into Tim4 protein powder, the Tim4 protein powder is dissolved and then added into an activated carboxylated magnetic graphene oxide solution, after an oscillation reaction, pure water magnetic suction washing is conducted, and the Tim4-rMGO magnetic beads are obtained. The Tim4-rMGO magnetic bead provided by the invention has high selectivity, that is, the Tim4-rMGO magnetic bead only depends on Ca < 2 + > chelation, avoids non-specific destruction of interaction of other proteins or lipids, and can be reused. The eluted exosome does not need an additional desalination step, and can be directly used for RNA extraction, proteomics or function research.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome extraction, and specifically relates to a method for preparing reusable Tim4-rMGO magnetic beads and their application in exosome extraction. Background Art

[0002] All cells, both prokaryotes and eukaryotes, release extracellular vesicles (EVs) as part of their normal physiology and during acquired abnormalities. Exosomes (EVs) are extracellular vesicles with a diameter of 40-160 nm (average approximately 100 nm) that can contain many cellular components, including DNA, RNA, lipids, metabolites, and cytoplasmic and cell surface proteins. Consequently, exosomes have been implicated in immune responses, viral pathogenicity, pregnancy, cardiovascular disease, central nervous system-related diseases, and cancer progression. The proteins, metabolites, and nucleic acids delivered by exosomes to recipient cells can effectively alter cellular biological responses. These exosome-mediated responses can promote or suppress disease, and the inherent properties of exosomes in regulating complex intracellular pathways increase their potential utility in the therapeutic control of many diseases, including neurodegenerative diseases and cancer.

[0003] With breakthroughs in single-exosome sequencing and proteomic analysis, exosomes have been identified as "liquid biopsy" biomarkers for disease diagnosis and natural carriers for targeted therapies. Therefore, rapid capture and pure isolation of exosomes is a key research direction in the field of exosome applications. Traditional ultracentrifugation (UC), while considered the gold standard, is time-consuming, equipment-intensive, and prone to vesicle aggregation or rupture. In contrast, kit-based isolation methods have attracted significant attention due to their ease of use and compatibility with routine laboratory conditions. Currently, mainstream techniques include polymer precipitation (e.g., PEG), size exclusion chromatography (SEC), and immunoaffinity capture. However, studies have found that while PEG yields high recovery rates (>80%), it is prone to coprecipitating lipoprotein impurities. SEC, while able to preserve exosome bioactivity, suffers from throughput limitations. While immunoaffinity capture offers high specificity, ease of use, and the preservation of exosome morphology, it is inefficient, hindering downstream experiments. Furthermore, antibodies are expensive and difficult to elute. In addition, the exosome kits developed by patents CN 117095895 A and CN 118258991 A are not reusable, which undoubtedly greatly increases the cost of exosome separation and purification. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem that the exosome kits in the existing exosome extraction and separation methods cannot be reused, and to provide a method for preparing reusable Tim4-rMGO magnetic beads and their application in exosome extraction.

[0005] To achieve the above-mentioned purpose, the technical solution provided by the present invention is:

[0006] A method for preparing reusable Tim4-rMGO magnetic beads comprises the following steps:

[0007] Sterile enzyme-free water was added to the Tim4 protein powder, and after dissolving, the powder was added to the activated carboxylated magnetic graphene oxide solution. After shaking reaction, the powder was magnetically washed with pure water to obtain Tim4-rMGO magnetic beads.

[0008] Furthermore, the mass ratio of Tim4 protein to activated carboxylated magnetic graphene oxide is 1:1000-1500.

[0009] Furthermore, activated carboxylated magnetic graphene oxide is prepared by the following process:

[0010] PBS buffer was added to the carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a clear solution, EDC and NHS powder were added, and the mixture was reacted in an ice bath and then magnetically washed to obtain an activated carboxylated magnetic graphene oxide solution.

[0011] Furthermore, the amount ratio of carboxylated magnetic graphene oxide to EDC is 1:50-100.

[0012] Furthermore, the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:50-100.

[0013] Furthermore, PBS buffer was added to the carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a clear solution. EDC was added and reacted for at least 30 minutes. NHS was then added and reacted for at least 2 hours. Finally, the solution was reacted in an ice bath and then washed by magnetic adsorption to obtain an activated carboxylated magnetic graphene oxide solution.

[0014] A reusable Tim4-rMGO magnetic bead.

[0015] Application of reusable Tim4-rMGO magnetic beads in the extraction of exosomes.

[0016] An exosome extraction kit comprises reusable Tim4-rMGO magnetic beads and an elution solution.

[0017] A method for extracting exosomes, comprising the following steps:

[0018] CaCl2 powder and reusable Tim4-rMGO magnetic beads are added to biological fluid, mixed evenly, and incubated by shaking to obtain a mixed solution. The magnetic beads in the mixed solution are magnetically adsorbed, and the supernatant is discarded to obtain a washed magnetic bead-exosome complex; an eluent is added to the magnetic bead-exosome complex, and the exosomes adsorbed on the magnetic beads are eluted by ultrasonic vibration. The supernatant is then collected by magnetic adsorption. The supernatant is the extracted exosomes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Since the transmembrane protein Tim4 (T cell immunoglobulin and mucin domain-containing protein 4) contains a phosphatidylserine (PS) binding domain at its C-terminus, the PS on the surface of the exosome membrane is a specific ligand for Tim4. The PS binding domain of Tim4 binds to the phosphatidylserine (PS) domain through Ca 2+ The ion stabilizes the conformation and forms a ternary complex of "ion-ligand" (Tim4-Ca2+-PS), thereby specifically anchoring the exosomes. Since EDTA (ethylenediaminetetraacetic acid) is a strong divalent metal ion chelator, it can efficiently bind Ca2+ in the solution. 2+ Mg 2+ Therefore, when EDTA is added in the present invention, its chelating effect will remove the Ca in the system. 2+ , resulting in the collapse of the PS binding domain of Tim4 protein, loss of affinity with PS, and thus release of exosomes. The Tim4-rMGO magnetic beads of the present invention have high selectivity, that is, they only rely on Ca 2+ Chelation avoids nonspecific disruption of other protein or lipid interactions. EDTA acts quickly (usually <10 min), making it suitable for automated processes. Compared to high salt or extreme pH elution, EDTA removes Ca 2+ Achieve lossless dissociation, maximally preserving exosome membrane integrity and content activity. Eluted exosomes require no additional desalting steps and can be directly used for RNA extraction, proteomics, or functional studies.

[0021] The Tim4-rMGO magnetic beads prepared by this method can achieve optimal storage conditions at 4°C in a PBS solution system, and the magnetic beads can be reused at least three times under this storage condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The scanning electron microscope and EDS images of the Tim4-rMGO magnetic beads obtained in Example 1 of the present invention are shown; wherein, (a) is the surface morphology of the Tim4-rMGO magnetic beads, (b) is the C element distribution on the surface of the material analyzed by EDS, (c) is the O element distribution on the surface of the material analyzed by EDS, (d) is the Fe element distribution on the surface of the material analyzed by EDS, (e) is the distribution of all elements on the surface of the material analyzed by EDS, (f) is the N element distribution on the surface of the material analyzed by EDS, (g) is the P element distribution on the surface of the material analyzed by EDS, and (h) is the S element distribution on the surface of the material analyzed by EDS;

[0023] Figure 2 This is a transmission electron micrograph of the exosomes obtained in Example 2 of the present invention;

[0024] Figure 3 This is the DLS & Zeta potential diagram of the exosomes obtained in Example 2 of the present invention;

[0025] Figure 4 The effect of Tim4-rMGO magnetic beads obtained in Example 1 of the present invention on the concentration of isolated exosome proteins was repeated. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in a variety of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0028] 1. A method for preparing Tim4-rMGO magnetic beads, comprising the following steps:

[0029] Step 1-1 Preparation of magnetic graphene oxide: Monolayer graphene oxide (industrial use) is ultrasonically dispersed in ethylene glycol to obtain a uniform graphene oxide solution; ferric chloride powder, sodium acetate powder, and polyethylene glycol 8000 powder are added, magnetically stirred and mixed until uniform, and then sealed and reacted at high temperature and high pressure. After the reaction is completed, the precipitate (i.e., the product) is collected by centrifugation, magnetically washed with anhydrous ethanol and pure water in sequence, and vacuum dried to obtain magnetic graphene oxide powder, which is recorded as rMGO.

[0030] Step 1-2: Preparation of carboxylated magnetic graphene oxide: Pure water is added to the magnetic graphene oxide powder obtained in step 1-1 and ultrasonically dispersed to obtain a uniform clear solution. Then, sodium hydroxide powder and chloroacetic acid powder are added, mixed and reacted, and then vacuum dried to obtain carboxylated magnetic graphene oxide powder, which is recorded as rMGO-COOH.

[0031] Step 1-3: Activating the carboxylated magnetic graphene oxide: PBS buffer was added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonically dispersed to obtain a uniform clear solution. EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide, N-Hydroxysuccinimide) powders were added. After the reaction in an ice bath, the carboxyl groups on the surface of the magnetic graphene were activated to an intermediate form that is more easily reactive, thereby obtaining an activated carboxylated magnetic graphene oxide solution, which was recorded as an activated rMGO-COOH solution.

[0032] Steps 1-4: Prepare Tim4-rMGO magnetic beads: Add sterile, enzyme-free water to Tim4 (Recombinant Biotinylated Human TIM4 Protein) protein powder, vortex to dissolve, then add to the activated rMGO-COOH solution obtained in steps 1-3. After vortexing, wash with magnetic aspiration in pure water to obtain Tim4-rMGO magnetic beads. Long-term storage of the beads is recommended in PBS at 4°C.

[0033] Tim4-rMGO magnetic beads not only maintain good water dispersibility in solutions such as water, PBS buffer and cell culture medium, but also have good magnetic properties and can be quickly (<20s) separated from the solution by magnetic attraction.

[0034] Furthermore, in step 1-1, the mass ratio of the monolayer graphene oxide to ferric chloride is 1:2-10;

[0035] The mass ratio of the monolayer graphene oxide to sodium acetate is 1:20-80;

[0036] The mass ratio of the monolayer graphene oxide to polyethylene glycol 8000 is 1:1-5;

[0037] The reaction temperature is 200-250° C. and the reaction time is 5-8 hours.

[0038] The magnetic graphene oxide prepared within the range of the present invention has better dispersibility and magnetic effect.

[0039] Furthermore, in step 1-2, the mass ratio of magnetic graphene oxide to sodium hydroxide powder is 1:10-50; the mass ratio of magnetic graphene oxide to chloroacetic acid powder is 1:20-30. Under this ratio, the carboxylation effect of the magnetic beads is better.

[0040] Furthermore, in steps 1-3, the mass ratio of carboxylated magnetic graphene oxide to EDC is 1:50-100; the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:50-100. The reaction is carried out for at least 30 minutes after the addition of EDC, and for at least 2 hours after the addition of NHS.

[0041] Furthermore, in steps 1-4, the mass ratio of Tim4 protein to activated carboxylated magnetic graphene oxide is 1:1000-1500;

[0042] The monolayer graphene oxide, ethylene glycol, sodium acetate powder, ferric chloride powder, PEG 8000 powder, EDC powder, NHS powder, and Tim4 protein used in the above preparation process are all reagents that can be prepared by yourself or purchased.

[0043] 2. A method for extracting exosomes using the Tim4-rMGO magnetic beads comprises the following steps:

[0044] Step 2-1 Biological sample processing: The obtained biological fluid (such as serum, cell culture supernatant, other body fluids such as saliva or urine, etc.) is centrifuged at 3000g and 10000g at 4°C to separate larger impurities such as cell debris. The supernatant is obtained to obtain the processed biological sample.

[0045] Step 2-2 Exosome extraction: Add CaCl2 powder to the treated biological sample, vortex to dissolve, then add Tim4-rMGO magnetic beads, mix thoroughly, and incubate with shaking to fully capture exosomes to obtain a mixed solution.

[0046] Step 2-3: Washing: Magnetic adsorption of the magnetic beads in the mixed solution of step 2-2, discarding the supernatant, and washing the magnetic beads with PBS buffer to obtain washed magnetic bead-exosome complexes;

[0047] Step 2-4: Exosome elution: Add elution solution to the magnetic bead-exosome complex obtained in step 2-3, elute the exosomes adsorbed on the magnetic beads by ultrasonic vibration, then adsorb the magnetic beads by magnetic force, and collect the supernatant, which is the extracted and separated exosome product.

[0048] Furthermore, in step 2-2, the incubation time of the magnetic beads with the biological sample is 20-60 minutes, and the incubation condition is 4°C; in step 2-2, the concentration of Tim4-rMGO magnetic beads in the treated biological sample is 3-3.5 mg / mL.

[0049] In steps 2-4, the exosome elution solution is an EDTA solution, and the elution time is 10-20 minutes, with the optimal elution condition being an ice bath. This elution method for the magnetic beads only releases the exosomes present on them, so the beads can be reused at least three times after exosome release.

[0050] 3. An exosome extraction kit comprising Tim4-rMGO magnetic beads and an EDTA aqueous solution.

[0051] The Tim4-rMGO magnetic beads of the present invention can be applied to biological fluids such as cell culture supernatant, serum, saliva, urine and plasma.

[0052] The principle of the present invention is as follows: Tim4 (T cell immunoglobulin and mucin domain-containing protein 4) is a transmembrane protein, the C-terminus of which contains a phosphatidylserine (PS) binding domain. The PS on the surface of the exosome membrane is the specific ligand of Tim4, and the PS binding domain of Tim4 binds to the phosphatidylserine (PS) through Ca 2+ Ions stabilize the conformation and form a ternary complex of "ion-ligand" (Tim4-Ca 2+ -PS), thereby specifically anchoring exosomes. EDTA (ethylenediaminetetraacetic acid) is a strong divalent metal ion chelator that can efficiently bind Ca in the solution. 2+ Mg 2+ When EDTA is added, its chelating effect will remove the Ca in the system. 2+ , causing the conformational collapse of the PS-binding domain of Tim4 protein, losing its affinity with PS, and thus releasing exosomes.

[0053] The following are specific examples.

[0054] Example 1

[0055] The preparation method of Tim4-rMGO magnetic beads specifically comprises the following steps:

[0056] Step 1-1: Preparation of magnetic graphene oxide

[0057] Mix the monolayer graphene oxide with ethylene glycol solvent, add it into a Teflon high-temperature and high-pressure reactor, and ultrasonically disperse it for 5 minutes;

[0058] The three compounds were added to the monolayer graphene oxide dispersion in a mass ratio of 1:3 for graphene oxide and ferric chloride, 1:30 for graphene oxide and sodium acetate, and 1:2 for graphene oxide and polyethylene glycol 8000. After magnetic stirring, the reactor was transferred to a forced air drying oven at 200°C for reaction for 6 hours.

[0059] After the reaction is completed, the product is collected by centrifugation at a speed of 10,000 rpm, and magnetically washed three times with anhydrous ethanol and ultrapure water respectively, and then vacuum-dried at 60° C. to obtain magnetic graphene oxide.

[0060] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0061] 20 mL of pure water was added to the magnetic graphene oxide powder obtained in step 1-1 and ultrasonically dispersed to obtain a uniform clear solution. Sodium hydroxide powder and chloroacetic acid powder were added in a mass ratio of 1:30 for the magnetic graphene oxide and sodium hydroxide powder and a mass ratio of 1:25 for the magnetic graphene oxide and chloroacetic acid powder, and the mixture was ultrasonically reacted in an ice-water bath for 2 h. The mixture was vacuum-dried to obtain carboxylated magnetic graphene oxide.

[0062] Steps 1-3: Preparation of Tim4-rMGO magnetic beads

[0063] PBS buffer was added to the carboxylated magnetic graphene oxide powder obtained in steps 1-2 and ultrasonically dispersed to obtain a homogeneous, clear solution. EDC and NHS powders were then added at a mass ratio of 1:50 for carboxylated magnetic graphene oxide and 1:50 for NHS. After an ice bath reaction, the mixture was magnetically washed with pure water. This activated the carboxyl groups on the surface of the magnetic graphene into a more reactive intermediate. The mixture was magnetically washed three times with PBS buffer. The Tim4 protein solution was then added at a mass ratio of 1:1000 for the activated carboxylated magnetic graphene oxide. After shaking at 4°C for 24 hours, the mixture was magnetically washed three times with PBS buffer to obtain Tim4-rMGO magnetic beads.

[0064] See also Figure 1 In (a)-(h), it can be seen that this material exhibits the lamellar structure unique to graphene, and there are also microspheres attached to its surface, which are Fe3O4 attached to the surface. In addition, the distribution of its N and P elements is mainly concentrated on the surface of the material, proving the successful grafting of Tim4 protein.

[0065] Example 2

[0066] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0067] Step 2-1: Construction of Tim4-rMGO magnetic beads

[0068] In this embodiment, step 2-1 adopts Example 1 to obtain magnetic beads;

[0069] Step 2-2 Biological sample processing

[0070] The obtained animal mesenchymal stem cell culture supernatant was centrifuged at 3000g and 10000g at 4°C for at least 30 minutes to separate larger impurities such as cell debris, and the supernatant was obtained to obtain the processed biological sample.

[0071] Steps 2-3: Exosome extraction

[0072] CaCl2 powder was added to the treated biological sample at a concentration of 1 mmol / L. After vortexing to dissolve, Tim4-rMGO magnetic beads were added. After thorough mixing, the mixture was incubated at 4°C with shaking to fully capture exosomes, thereby obtaining immunomagnetic bead-exosome complexes.

[0073] Steps 2-4: Exosome elution

[0074] The immunomagnetic beads-exosome complex in step 2-3 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. This was repeated twice, and EDTA solution was added at a concentration of 1 mmol / L. Ice bath ultrasonication was performed for 15 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0075] Identification of exosomes:

[0076] (1) Exosomes obtained by electron microscopy:

[0077] 10 μL of the exosome extract obtained in Example 2 was added dropwise to the copper mesh and adsorbed at room temperature for 10 minutes. The excess liquid was removed with filter paper. Then 10 μL of 2% phosphotungstic acid solution was added dropwise to the copper mesh and stained at room temperature for 2 minutes. The excess liquid was removed with filter paper and the copper mesh was air-dried at room temperature. The observation voltage was set to 120 kV and the transmission electron microscope was used for observation. Figure 2 , it was observed that the obtained extracellular vesicles had a uniform particle size and a saucer-shaped double-layer membrane structure unique to exosomes, which was consistent with the morphology of exosomes.

[0078] (2) Exosome particle size and potential determination:

[0079] The exosome solution obtained in Example 2 was added to the Malvern potential cell to measure its Zeta potential. The potential value obtained was between -10mV and -70mV, which is consistent with the traditional potential value of exosomes. The particle size of the obtained exosome solution was then measured, and the particle size was found to be consistent with the exosome particle size of 50-150nm, see Figure 3 , it can be seen that the particle size is about 55.2nm, the Zeta potential is about -11.9mV, and the PDI is 0.184. The data are credible and consistent with the exosome particle size.

[0080] (3) Determination of the concentration of extracted exosome protein by BCA kit:

[0081] An appropriate amount of the exosome solution prepared in Example 2 was added to RIPA lysis buffer at a ratio of 1:1 and ultrasonically disrupted for 30 seconds; centrifuged at 12000g and 4°C for 10 minutes, and the supernatant was taken as the sample; the sample was added to a 96-well plate, 20 μL per well, and each sample was repeated in 3 wells; 200 μL of working solution was added, and the plate was allowed to stand at 37°C for 20 minutes; the OD value at 562 nm was measured with a microplate reader, and the protein concentration was calculated according to the standard curve, see Figure 4 , it can be seen that the extraction efficiency of the magnetic beads after one recovery is 86% of the previous one, and after two recovery it is 69%.

[0082] It can be seen that the exosome capture immunomagnetic beads prepared by the present invention can effectively extract exosomes with high purity, and the extraction and separation methods adopted will not cause damage to the exosomes. The extracted exosomes are complete in morphology and can be used for downstream applications.

[0083] Example 3

[0084] The preparation method of Tim4-rMGO magnetic beads specifically comprises the following steps:

[0085] Step 1-1: Preparation of magnetic graphene oxide

[0086] Mix the monolayer graphene oxide with ethylene glycol solvent, add it into a Teflon high-temperature and high-pressure reactor, and ultrasonically disperse it for 5 minutes;

[0087] The three compounds were added to the monolayer graphene oxide dispersion at a mass ratio of 1:10 for graphene oxide and ferric chloride, 1:80 for graphene oxide and sodium acetate, and 1:1 for graphene oxide and polyethylene glycol 8000. After magnetic stirring, the reactor was transferred to a forced air drying oven at 200°C for reaction for 8 hours.

[0088] After the reaction is completed, the product is collected by centrifugation at a speed of 10,000 rpm, and magnetically washed three times with anhydrous ethanol and ultrapure water respectively, and then vacuum-dried at 60° C. to obtain magnetic graphene oxide.

[0089] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0090] 20 mL of pure water was added to the magnetic graphene oxide powder obtained in step 1-1 and ultrasonically dispersed to obtain a uniform clear solution. Sodium hydroxide powder and chloroacetic acid powder were added in a mass ratio of 1:10 for the magnetic graphene oxide and sodium hydroxide powder and a mass ratio of 1:20 for the magnetic graphene oxide and chloroacetic acid powder. The mixture was ultrasonically reacted in an ice-water bath for 2 h and vacuum dried to obtain carboxylated magnetic graphene oxide.

[0091] Steps 1-3: Preparation of Tim4-rMGO magnetic beads

[0092] PBS buffer was added to the carboxylated magnetic graphene oxide powder obtained in steps 1-2 and ultrasonically dispersed to obtain a homogeneous, clear solution. EDC and NHS powders were then added at a mass ratio of 1:70 for carboxylated magnetic graphene oxide and 1:70 for NHS. After an ice bath reaction, the mixture was magnetically rinsed with pure water to activate the carboxyl groups on the surface of the magnetic graphene into a more reactive intermediate. The mixture was magnetically rinsed three times with PBS buffer. The Tim4 protein solution was then added at a mass ratio of 1:1300 for the activated carboxylated magnetic graphene oxide. The mixture was shaken at 4°C for 24 hours and then magnetically rinsed three times with PBS buffer to obtain Tim4-rMGO magnetic beads.

[0093] Example 4

[0094] The preparation method of Tim4-rMGO magnetic beads specifically comprises the following steps:

[0095] Step 1-1: Preparation of magnetic graphene oxide

[0096] Mix the monolayer graphene oxide with ethylene glycol solvent, add it into a Teflon high-temperature and high-pressure reactor, and ultrasonically disperse it for 5 minutes;

[0097] The three compounds were added to the monolayer graphene oxide dispersion at a mass ratio of 1:7 for graphene oxide and ferric chloride, 1:20 for graphene oxide and sodium acetate, and 1:2 for graphene oxide and polyethylene glycol 8000. After magnetic stirring, the reactor was transferred to a forced air drying oven at 250°C for reaction for 5 hours.

[0098] After the reaction is completed, the product is collected by centrifugation at a speed of 10,000 rpm, and magnetically washed three times with anhydrous ethanol and ultrapure water respectively, and then vacuum-dried at 60° C. to obtain magnetic graphene oxide.

[0099] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0100] 20 mL of pure water was added to the magnetic graphene oxide powder obtained in step 1-1 and ultrasonically dispersed to obtain a uniform clear solution. Sodium hydroxide powder and chloroacetic acid powder were added in a mass ratio of 1:40 for the magnetic graphene oxide and sodium hydroxide powder and a mass ratio of 1:30 for the magnetic graphene oxide and chloroacetic acid powder. The mixture was ultrasonically reacted in an ice-water bath for 2 h and vacuum dried to obtain carboxylated magnetic graphene oxide.

[0101] Steps 1-3: Preparation of Tim4-rMGO magnetic beads

[0102] PBS buffer was added to the carboxylated magnetic graphene oxide powder obtained in steps 1-2 and ultrasonically dispersed to obtain a homogeneous, clear solution. EDC and NHS powders were then added at a mass ratio of 1:50 for carboxylated magnetic graphene oxide and 1:50 for NHS. After an ice bath reaction, the mixture was magnetically washed with pure water to activate the carboxyl groups on the surface of the magnetic graphene into a more reactive intermediate. The mixture was magnetically washed three times with PBS buffer. The Tim4 protein solution was then added at a mass ratio of 1:1500 for the activated carboxylated magnetic graphene oxide. After shaking at 4°C for 24 hours, the mixture was magnetically washed three times with PBS buffer to obtain Tim4-rMGO magnetic beads.

[0103] Example 5

[0104] The preparation method of Tim4-rMGO magnetic beads specifically comprises the following steps:

[0105] Step 1-1: Preparation of magnetic graphene oxide

[0106] Mix the monolayer graphene oxide with ethylene glycol solvent, add it into a Teflon high-temperature and high-pressure reactor, and ultrasonically disperse it for 5 minutes;

[0107] The three compounds were added to the monolayer graphene oxide dispersion in a mass ratio of 1:2 for graphene oxide and ferric chloride, 1:60 for graphene oxide and sodium acetate, and 1:5 for graphene oxide and polyethylene glycol 8000. After magnetic stirring, the reactor was transferred to a forced air drying oven at 200°C for reaction for 7 hours.

[0108] After the reaction is completed, the product is collected by centrifugation at a speed of 10,000 rpm, and magnetically washed three times with anhydrous ethanol and ultrapure water respectively, and then vacuum-dried at 60° C. to obtain magnetic graphene oxide.

[0109] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0110] 20 mL of pure water was added to the magnetic graphene oxide powder obtained in step 1-1 and ultrasonically dispersed to obtain a uniform clear solution. Sodium hydroxide powder and chloroacetic acid powder were added in a mass ratio of 1:50 for the magnetic graphene oxide and sodium hydroxide powder and a mass ratio of 1:23 for the magnetic graphene oxide and chloroacetic acid powder. The mixture was ultrasonically reacted in an ice-water bath for 2 h and vacuum dried to obtain carboxylated magnetic graphene oxide.

[0111] Steps 1-3: Preparation of Tim4-rMGO magnetic beads

[0112] PBS buffer was added to the carboxylated magnetic graphene oxide powder obtained in steps 1-2 and ultrasonically dispersed to obtain a homogeneous, clear solution. EDC and NHS powders were then added at a mass ratio of 1:100 for carboxylated magnetic graphene oxide and 1:100 for NHS. After an ice bath reaction, the mixture was magnetically rinsed with pure water to activate the carboxyl groups on the surface of the magnetic graphene into a more reactive intermediate. The mixture was magnetically rinsed three times with PBS buffer. The Tim4 protein solution was then added at a mass ratio of 1:1200 for the activated carboxylated magnetic graphene oxide. The mixture was shaken at 4°C for 24 hours and then magnetically rinsed three times with PBS buffer to obtain Tim4-rMGO magnetic beads.

[0113] Example 6

[0114] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0115] Step 2-1: Construction of Tim4-rMGO magnetic beads

[0116] In this embodiment, step 2-1 adopts Example 1 to obtain magnetic beads;

[0117] Step 2-2 Biological sample processing

[0118] The serum was centrifuged at 3000g and 10000g at 4°C for at least 30 minutes to separate larger impurities such as cell debris. The supernatant was taken to obtain the processed biological sample.

[0119] Steps 2-3: Exosome extraction

[0120] CaCl2 powder was added to the treated biological sample at a concentration of 1.5 mmol / L. After vortexing to dissolve, Tim4-rMGO magnetic beads were added, mixed thoroughly, and incubated at 4°C with shaking for 60 minutes to fully capture exosomes and obtain immunomagnetic bead-exosome complexes. The concentration of Tim4-rMGO magnetic beads in the treated biological sample was 3.2 mg / mL.

[0121] Steps 2-4: Exosome elution

[0122] The immunomagnetic beads-exosome complex in step 2-3 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. This was repeated twice, and EDTA solution was added at a concentration of 1 m mol / L. Ice bath sonication was performed for 10 minutes, and the supernatant obtained after magnetic adsorption was the exosome solution.

[0123] Example 7

[0124] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0125] Step 2-1: Construction of Tim4-rMGO magnetic beads

[0126] In this embodiment, step 2-1 adopts Example 1 to obtain magnetic beads;

[0127] Step 2-2 Biological sample processing

[0128] The saliva was centrifuged at 3000g and 10000g at 4°C for at least 30 minutes to separate larger impurities such as cell debris. The supernatant was taken to obtain the processed biological sample.

[0129] Steps 2-3: Exosome extraction

[0130] CaCl2 powder was added to the treated biological sample at a concentration of 2 mmol / L. After vortexing to dissolve, Tim4-rMGO magnetic beads were added, mixed thoroughly, and incubated at 4°C with shaking for 20 minutes to fully capture exosomes and obtain immunomagnetic bead-exosome complexes. The concentration of Tim4-rMGO magnetic beads in the treated biological sample was 3.5 mg / mL.

[0131] Steps 2-4: Exosome elution

[0132] The immunomagnetic beads-exosome complex in step 2-3 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. This was repeated twice, and EDTA solution was added at a concentration of 1 mmol / L. Ice bath ultrasonication was performed for 20 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0133] Example 8

[0134] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0135] Step 2-1: Construction of Tim4-rMGO magnetic beads

[0136] In this embodiment, step 2-1 adopts Example 1 to obtain magnetic beads;

[0137] Step 2-2 Biological sample processing

[0138] The urine was centrifuged at 3000g and 10000g at 4°C for at least 30 minutes to separate larger impurities such as cell debris. The supernatant was taken to obtain the processed biological sample.

[0139] Steps 2-3: Exosome extraction

[0140] CaCl2 powder was added to the treated biological sample at a concentration of 1 mmol / L. After vortexing to dissolve, Tim4-rMGO magnetic beads were added, mixed thoroughly, and incubated at 4°C with shaking for 40 minutes to fully capture exosomes and obtain immunomagnetic bead-exosome complexes. The concentration of Tim4-rMGO magnetic beads in the treated biological sample was 3 mg / mL.

[0141] Steps 2-4: Exosome elution

[0142] The immunomagnetic beads-exosome complex in step 2-3 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. This was repeated twice, and EDTA solution was added at a concentration of 1 mmol / L. Ice bath ultrasonication was performed for 15 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0143] The above description is merely a description of the preferred embodiment of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiment, and variations in the specific structure are permitted. Any variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing reusable Tim4-rMGO magnetic beads, characterized in that: The following steps are involved: Sterile enzyme-free water was added to the Tim4 protein powder, and after dissolving, the powder was added to the activated carboxylated magnetic graphene oxide solution. After shaking reaction, the powder was magnetically washed with pure water to obtain Tim4-rMGO magnetic beads.

2. The method for preparing reusable Tim4-rMGO magnetic beads according to claim 1, wherein The mass ratio of Tim4 protein to activated carboxylated magnetic graphene oxide is 1:1000-1500.

3. The method for preparing reusable Tim4-rMGO magnetic beads according to claim 1, wherein: Activated carboxylated magnetic graphene oxide is prepared by the following process: PBS buffer was added to the carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a clear solution, EDC and NHS powder were added, and the mixture was reacted in an ice bath and then magnetically washed to obtain an activated carboxylated magnetic graphene oxide solution.

4. The method for preparing reusable Tim4-rMGO magnetic beads according to claim 3, wherein: The amount ratio of carboxylated magnetic graphene oxide to EDC is 1:50-100.

5. The method for preparing reusable Tim4-rMGO magnetic beads according to claim 3, characterized in that: The mass ratio of carboxylated magnetic graphene oxide to NHS is 1:50-100.

6. The method for preparing reusable Tim4-rMGO magnetic beads according to claim 3, characterized in that: PBS buffer was added to the carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a clear solution. EDC was added and reacted for at least 30 minutes. NHS was then added and reacted for at least 2 hours. Finally, the solution was reacted in an ice bath and then washed by magnetic adsorption to obtain an activated carboxylated magnetic graphene oxide solution.

7. Reusable Tim4-rMGO magnetic beads prepared according to the method according to any one of claims 1 to 6.

8. Use of reusable Tim4-rMGO magnetic beads prepared according to the method according to any one of claims 1 to 6 in extracting exosomes.

9. An exosome extraction kit, characterized in that: The method comprises reusable Tim4-rMGO magnetic beads prepared by the method according to any one of claims 1 to 6 and an eluent.

10. A method for extracting exosomes, characterized in that: The following steps are involved: CaCl2 powder and the reusable Tim4-rMGO magnetic beads prepared by the method according to any one of claims 1 to 6 are added to the biological fluid, mixed evenly, and incubated by shaking to obtain a mixed solution, the magnetic beads in the mixed solution are magnetically adsorbed, and the supernatant is discarded to obtain a washed magnetic bead-exosome complex; an eluent is added to the magnetic bead-exosome complex, the exosomes adsorbed on the magnetic beads are eluted by ultrasonic vibration, and the supernatant is collected by magnetic adsorption, and the supernatant is the extracted exosomes.

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