Preparation method of reusable [APMIm] Br-rMGO magnetic beads and application of reusable [APMIm] Br-rMGO magnetic beads in exosome extraction

Abstract

The invention discloses a preparation method of reusable [APMIm] Br-rMGO magnetic beads and application of the reusable [APMIm] Br-rMGO magnetic beads in exosome extraction.The preparation method comprises the steps that a pure water solution of ionic liquid [APMIm] Br is added into an activated carboxylated magnetic graphene oxide magnetic bead solution, after an oscillation reaction, pure water magnetic attraction washing is conducted, and the [APMIm] Br-rMGO magnetic beads are obtained. The [APMIm] Br-rMGO magnetic bead can be applied to extraction of the exosome. The amino functionalized ionic liquid combined with the magnetic beads is stable in property, is easy to store and stable in shape after being combined with the magnetic bead nano material, and can be repeatedly used for a long time. When the exosome is extracted by adopting the method disclosed by the invention, the exosome of 30-160nm can be obtained; the magnetic bead nanometer material is low in cost, rapid in preparation and high in magnetism, and can be completely separated from the solution within 20 s.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome extraction, and specifically relates to a method for preparing reusable [APMIm]Br-rMGO magnetic beads and an application thereof 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.

[0004] In addition, the exosome kits developed by patents CN 117095895 A and CN 118258991 A all have the problem of non-reusability, which undoubtedly greatly increases the cost of exosome separation and purification. Summary of the Invention

[0005] 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 [APMIm]Br-rMGO magnetic beads and their application in exosome extraction.

[0006] In order to achieve the above-mentioned purpose of the invention, the technical solution provided by the present invention is:

[0007] A method for preparing reusable [APMIm]Br-rMGO magnetic beads comprises the following steps:

[0008] The activated carboxylated magnetic graphene oxide magnetic bead solution was added to the pure aqueous solution of ionic liquid [APMIm]Br, and after shaking reaction, the solution was washed by magnetic absorption with pure water to obtain [APMIm]Br-rMGO magnetic beads.

[0009] Furthermore, the mass ratio of the activated carboxylated magnetic graphene oxide beads to [APMIm]Br is 1:50-90.

[0010] Furthermore, the activated carboxylated magnetic graphene oxide bead solution is prepared by the following process:

[0011] Pure water was added to the carboxylated magnetic graphene oxide powder and ultrasonic dispersion was performed to obtain a uniform clear solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide powders were added, and the mixture was reacted in an ice bath and then magnetically washed with pure water to obtain an activated carboxylated magnetic graphene oxide bead solution.

[0012] Furthermore, the mass ratio of the carboxylated magnetic graphene oxide powder to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:50-100.

[0013] Furthermore, the mass ratio of the carboxylated magnetic graphene oxide powder to the N-hydroxysuccinimide powder is 1:50-100.

[0014] Furthermore, pure water was added to the carboxylated magnetic graphene oxide powder and ultrasonic dispersion was performed to obtain a uniform clear solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added and reacted for at least 30 minutes. N-hydroxysuccinimide powder was then added and reacted in an ice bath for more than 2 hours. Finally, magnetic washing was performed with pure water to obtain an activated carboxylated magnetic graphene oxide bead solution.

[0015] A reusable [APMIm]Br-rMGO magnetic bead.

[0016] Application of reusable [APMIm]Br-rMGO magnetic beads in the extraction of exosomes.

[0017] An exosome extraction kit comprises reusable [APMIm]Br-rMGO magnetic beads and an elution solution.

[0018] A method for extracting exosomes, comprising the following steps:

[0019] Reusable [APMIm]Br-rMGO magnetic beads are added to biological fluids, 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. Pure water 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.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] [APMIm]Br has good solubility and stability as an ionic liquid, and the quaternary ammonium cation (N + ) and the protonated amino group (-NH3 + ) gives it a strong positive charge, which can adsorb negatively charged groups on the surface of exosomes through electrostatic attraction. At the same time, the methyl and propyl chains of the imidazole ring are hydrophobic and can interact with the exosome membrane through hydrophobic interaction to enhance adsorption stability. The synergistic effect of hydrophobic interaction and electrostatic interaction can improve the capture efficiency, especially reducing impurity interference in complex biological samples (such as serum). In addition, the carboxylic acid groups of sialic acid on the surface of exosomes, the hydroxyl groups in the sugar chain, etc. can form a hydrogen bond network with the amino group or imidazole ring of [APMIm]Br to strengthen the binding. Therefore, the amino-functionalized ionic liquid bound to magnetic beads in the present invention is stable in nature, easy to store and stable in shape after being combined with magnetic bead nanomaterials, and can be reused for a long time. At the same time, it avoids the need for expensive equipment in the extraction process and has good application prospects in the field of exosome extraction.

[0022] The method of the present invention is used to extract exosomes, and exosomes of 30-160 nm can be obtained. In addition, the magnetic bead nanomaterials used in the present invention are low in cost, fast to prepare, and have strong magnetism, and can be completely separated from the solution within 20 seconds.

[0023] In the present invention, [APMIm]Br-rMGO magnetic beads utilize physical adsorption to bind to exosomes based on electrostatic adsorption and size confinement effects. During elution, only the pH value of the elution solution needs to be changed, which can achieve rapid release and capture of exosomes without damaging the activity of the exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The scanning electron microscope and EDS images of the [APMIm]Br-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 EDS analysis of the C element distribution on the material surface, (c) is the EDS analysis of the O element distribution on the material surface, (d) is the EDS analysis of the distribution of all elements on the material surface, (e) is the EDS analysis of the Fe element distribution on the material surface, and (f) is the EDS analysis of the N element distribution on the material surface;

[0025] Figure 2 This is a transmission electron micrograph of the exosomes obtained in Example 2 of the present invention;

[0026] Figure 3 This is the NTA nanoparticle size analysis of the cell exosomes obtained in Example 2 of the present invention;

[0027] Figure 4 The effect of [APMIm]Br-rMGO magnetic beads obtained in Example 1 of the present invention on the concentration of isolated exosome proteins was repeated. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 1. A method for preparing reusable [APMIm]Br-rMGO magnetic beads, comprising the following steps:

[0031] Step 1-1, preparation of magnetic graphene oxide: single-layer 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 evenly, and then a closed high-temperature and high-pressure reaction is carried out. 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, recorded as rMGO.

[0032] Step 1-2, preparing carboxylated magnetic graphene oxide: adding pure water to the magnetic graphene oxide powder obtained in step 1-1 and performing ultrasonic dispersion to obtain a uniform clear solution, then adding sodium hydroxide powder and chloroacetic acid powder, mixing and reacting, and vacuum drying to obtain carboxylated magnetic graphene oxide powder, recorded as rMGO-COOH.

[0033] Step 1-3, activation of carboxylated magnetic graphene oxide: pure water is added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonically dispersed to obtain a uniform clear solution, and then EDC (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) powders are added. After the reaction in an ice bath, the carboxyl groups on the surface of the magnetic graphene are activated to an intermediate form that is more easily reactive, thereby obtaining an activated carboxylated magnetic graphene oxide bead solution, which is recorded as an activated rMGO-COOH solution.

[0034] Step 1-4, preparation of [APMIm]Br-rMGO magnetic beads: Weigh semi-solid [APMIm]Br in pure water, shake to dissolve, and then add to the activated rMGO-COOH solution obtained in Step 1-3. Shake at room temperature, then rinse with pure water by magnetic absorption to obtain [APMIm]Br-rMGO magnetic beads. Long-term storage of the obtained magnetic beads should be in pure water at 4°C.

[0035] [APMIm]Br-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.

[0036] Furthermore, in step 1-1, the mass ratio of the monolayer graphene oxide to ferric chloride is 1:2-10;

[0037] The mass ratio of the monolayer graphene oxide to sodium acetate is 1:20-80;

[0038] The mass ratio of the monolayer graphene oxide to polyethylene glycol 8000 is 1:1-5;

[0039] The reaction temperature is 200-250° C. and the reaction time is 5-8 hours.

[0040] The magnetic graphene oxide prepared within the above mass ratio range has better dispersibility and magnetic effect.

[0041] 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.

[0042] 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.

[0043] Furthermore, in steps 1-4, the mass ratio of the activated carboxylated magnetic graphene oxide beads to [APMIm]Br is 1:50-90;

[0044] The monolayer graphene oxide, ethylene glycol, sodium acetate powder, ferric chloride powder, PEG 8000 powder, EDC powder, NHS powder, and [APMIm]Br used in the above preparation process are all reagents that can be prepared by yourself or purchased.

[0045] 2. A method for extracting exosomes using the [APMIm]Br-rMGO magnetic beads comprises the following steps:

[0046] Step 2-1, biological sample processing: The obtained biological fluid (such as serum, cell culture supernatant, other body fluids such as saliva, urine, etc.) is centrifuged at 3000g and 10000g at 4°C to separate larger impurities such as cell debris, and the supernatant is obtained to obtain the processed biological sample.

[0047] Step 2-2, exosome extraction: [APMIm]Br-rMGO magnetic beads were added to the treated biological sample obtained in step 2-1, mixed thoroughly, and incubated with shaking to fully capture exosomes to obtain a mixed solution.

[0048] Step 2-3, washing: magnetically adsorb the magnetic beads in the mixed solution in step 2-2, discard the supernatant, and wash the magnetic beads with PBS buffer to obtain washed magnetic bead-exosome complexes;

[0049] Step 2-4, exosome elution: pure water is added to the magnetic bead-exosome complex obtained in step 2-3, and the exosomes adsorbed on the magnetic beads are eluted by ultrasonic vibration. The magnetic beads are then adsorbed by magnetic force, and the supernatant is collected. The supernatant is the extracted and separated exosome product.

[0050] Furthermore, in step 2-2, the incubation conditions are 4° C. and the incubation time is 10-30 min; the activity of the exosomes can be guaranteed to the greatest extent; in step 2-2, the mass of [APMIm]Br-rMGO magnetic beads in each 1 mL of treated biological sample is 1-2 mg.

[0051] In step 2-4, the exosome elution solution is ultrapure water with a pH of 7-8, the elution time is 10-30 min, and the optimal elution condition is an ice bath.

[0052] Based on the principle of this magnetic bead design, after the exosomes captured in the "magnetic bead-exosome" complex are eluted with an eluent, the magnetic beads and the exosome solution can be separated by magnetic attraction. The separated magnetic beads can be reused at least three times.

[0053] 3. An exosome extraction kit, comprising [APMIm]Br-rMGO magnetic beads and PBS buffer.

[0054] The principle of the present invention is: based on the good solubility and stability of [APMIm]Br as an ionic liquid, and the quaternary ammonium cation (N + ) and the protonated amino group (-NH3 + ) imparts a strong positive charge, allowing it to adsorb negatively charged groups on the exosome surface through electrostatic attraction. Simultaneously, the methyl and propyl chains of the imidazole ring are hydrophobic, interacting with the exosome membrane through hydrophobic interactions, enhancing adsorption stability. The synergistic hydrophobic and electrostatic interactions improve capture efficiency, particularly in complex biological samples such as serum, reducing impurity interference. Furthermore, the carboxylic acid groups of sialic acid on the exosome surface and the hydroxyl groups of the sugar chains can form hydrogen bond networks with the amino groups or imidazole rings of [APMIm]Br, strengthening the binding.

[0055] The following are specific examples.

[0056] Example 1

[0057] The preparation method of [APMIm]Br-rMGO magnetic beads specifically comprises the following steps:

[0058] Step 1-1: Preparation of magnetic graphene oxide

[0059] 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;

[0060] 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.

[0061] 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.

[0062] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0063] 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.

[0064] Steps 1-3: Preparation of [APMIm]Br-rMGO magnetic beads

[0065] Pure water was added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonically dispersed to obtain a homogeneous, clear solution. EDC and NHS powders were then added in a ratio of 1:50 (mass ratio) of carboxylated magnetic graphene oxide to EDC and 1:50 (mass ratio) of carboxylated magnetic graphene oxide to NHS, respectively. After the reaction was allowed to proceed in an ice bath, the surface carboxyl groups of the magnetic graphene were activated to form a more reactive intermediate. Furthermore, a corresponding amount of semi-solid [APMIm]Br was weighed in a ratio of 1:62.5 (mass ratio) of carboxylated magnetic graphene oxide to [APMIm]Br. The solution was then dissolved by shaking and added to the activated carboxylated magnetic graphene oxide solution. The reaction was allowed to proceed by shaking at room temperature for 24 hours, followed by magnetic washing with pure water to obtain [APMIm]Br-rMGO magnetic beads.

[0066] See also Figure 1 In (a)-(f), it can be seen that this material exhibits the lamellar structure unique to graphene, and there are also microspheres attached to its surface. After EDS analysis, these are Fe3O4 attached to the surface. In addition, its nitrogen element distribution is mainly concentrated on the surface of the material, proving the successful grafting of [APMIm]Br protein.

[0067] Example 2

[0068] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0069] Step 2-1: Construction of [APMIm]Br-rMGO magnetic beads

[0070] In this example, step 2-1 adopts the operation of Example 1 to obtain [APMIm]Br-rMGO magnetic beads;

[0071] Step 2-2 Biological sample processing

[0072] The 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. The supernatant was taken to obtain the processed biological sample.

[0073] Steps 2-3: Exosome extraction

[0074] After the biological sample obtained in step 2-2 is evenly mixed with [APMIm]Br-rMGO magnetic beads, the mass of [APMIm]Br-rMGO magnetic beads per 1 mL of biological sample is 1 mg, and incubated on a shaking shaker at 4°C for 10 min to obtain the immunomagnetic bead-exosome complex;

[0075] Steps 2-4: Exosome elution

[0076] The mixed solution in step 2-2 was subjected to magnetic adsorption for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. After repeating twice, ultrapure water with pH = 7.5 was added, and ice bath sonication was performed for 10 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0077] Identification of exosomes:

[0078] (1) Exosomes obtained by electron microscopy:

[0079] 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.

[0080] (2) Exosome particle size determination:

[0081] Based on Example 2, the obtained exosome solution was subjected to NTA particle size detection, and the particle size thereof was found to be consistent with the exosome particle size of 50-150 nm, see Figure 3 , it can be seen that the particle size is about 130.4nm, the data is credible and consistent with the size of exosome particles.

[0082] (3) Determination of the concentration of extracted exosome protein by BCA kit:

[0083] Take an appropriate amount of the exosome solution prepared in Example 2 and add it to RIPA lysis buffer at a ratio of 1:1, and ultrasonically disrupt for 30 seconds; centrifuge at 12000g and 4°C for 10 minutes, and take the supernatant as the sample; add the sample to a 96-well plate, 20 μL per well, and repeat 3 wells for each sample; add 200 μL of working solution, and let it stand at 37°C for 20 minutes; measure the OD value at 562nm with a microplate reader, and calculate the protein concentration according to the standard curve, see Figure 4 It can be seen that the extraction efficiency of the magnetic beads after one recovery is 73% of the previous one, and after two recovery it is 64%.

[0084] 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.

[0085] Example 3

[0086] The preparation method of [APMIm]Br-rMGO magnetic beads specifically comprises the following steps:

[0087] Step 1-1: Preparation of magnetic graphene oxide

[0088] 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;

[0089] The three compounds were added to the monolayer graphene oxide dispersion at a mass ratio of 1:2 for graphene oxide and ferric chloride, 1:80 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 8 hours.

[0090] 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.

[0091] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0092] 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.

[0093] Steps 1-3: Preparation of [APMIm]Br-rMGO magnetic beads

[0094] Pure water was added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonic dispersion was performed to obtain a uniform clear solution. EDC and NHS powders were added in a mass ratio of 1:70 for carboxylated magnetic graphene oxide and EDC and a mass ratio of 1:100 for carboxylated magnetic graphene oxide and NHS, and the mixture was reacted in an ice bath and then magnetically washed with pure water. The carboxyl groups on the surface of the magnetic graphene were activated to an intermediate form that was more reactive, thereby obtaining an activated carboxylated magnetic graphene oxide bead solution, which was recorded as an activated rMGO-COOH solution.

[0095] Step 1-4: Weigh the corresponding mass of semi-solid [APMIm]Br in pure water at a mass ratio of 1:50 between carboxylated magnetic graphene oxide and [APMIm]Br, shake to dissolve, and then add it to the activated rMGO-COOH solution. After shaking at room temperature for 24 hours, wash it with pure water magnetically to obtain [APMIm]Br-rMGO magnetic beads.

[0096] Example 4

[0097] The preparation method of [APMIm]Br-rMGO magnetic beads specifically comprises the following steps:

[0098] Step 1-1: Preparation of magnetic graphene oxide

[0099] 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;

[0100] The three compounds were added to the monolayer graphene oxide dispersion at a mass ratio of 1:10 for graphene oxide and ferric chloride, 1:20 for graphene oxide and sodium acetate, and 1:3 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.

[0101] 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.

[0102] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0103] 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.

[0104] Steps 1-3: Preparation of [APMIm]Br-rMGO magnetic beads

[0105] Pure water was added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonic dispersion was performed to obtain a uniform clear solution. EDC and NHS powders were added in a mass ratio of 1:50 for carboxylated magnetic graphene oxide and EDC, and a mass ratio of 1:50 for carboxylated magnetic graphene oxide and NHS. After the reaction in an ice bath, the mixture was magnetically washed with pure water. The carboxyl groups on the surface of the magnetic graphene were activated to an intermediate form that was more reactive, thereby obtaining an activated carboxylated magnetic graphene oxide bead solution, which was recorded as an activated rMGO-COOH solution.

[0106] Step 1-4: Weigh the corresponding mass of semi-solid [APMIm]Br in pure water at a mass ratio of 1:80 between carboxylated magnetic graphene oxide and [APMIm]Br, shake to dissolve, and then add it to the activated rMGO-COOH solution. After shaking at room temperature for 24 hours, wash it with pure water magnetically to obtain [APMIm]Br-rMGO magnetic beads.

[0107] Example 5

[0108] The preparation method of [APMIm]Br-rMGO magnetic beads specifically comprises the following steps:

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

[0110] 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;

[0111] The three compounds were added to the monolayer graphene oxide dispersion at a mass ratio of 1:6 for graphene oxide and ferric chloride, 1:50 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 6 hours.

[0112] 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.

[0113] Step 1-2: Preparation of carboxylated magnetic graphene oxide

[0114] 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.

[0115] Steps 1-3: Preparation of [APMIm]Br-rMGO magnetic beads

[0116] Pure water was added to the carboxylated magnetic graphene oxide powder obtained in step 1-2 and ultrasonic dispersion was performed to obtain a uniform clear solution. EDC and NHS powders were added in a mass ratio of 1:100 for carboxylated magnetic graphene oxide and EDC and a mass ratio of 1:80 for carboxylated magnetic graphene oxide and NHS, and the mixture was reacted in an ice bath and then magnetically washed with pure water. The carboxyl groups on the surface of the magnetic graphene were activated to an intermediate form that was more reactive, thereby obtaining an activated carboxylated magnetic graphene oxide bead solution, which was recorded as an activated rMGO-COOH solution.

[0117] Step 1-4: Weigh the corresponding mass of semi-solid [APMIm]Br in pure water at a mass ratio of 1:90 between carboxylated magnetic graphene oxide and [APMIm]Br, shake to dissolve, and then add it to the activated rMGO-COOH solution. After shaking at room temperature for 24 hours, wash it with pure water magnetically to obtain [APMIm]Br-rMGO magnetic beads.

[0118] Example 6

[0119] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0120] Step 2-1: Construction of [APMIm]Br-rMGO magnetic beads

[0121] In this example, step 2-1 adopts the operation of Example 1 to obtain [APMIm]Br-rMGO magnetic beads;

[0122] Step 2-2 Biological sample processing

[0123] The serum was centrifuged at 3000g and 10000g at 4°C for 30 minutes to separate larger impurities such as cell debris, and the supernatant was taken to obtain the processed biological sample.

[0124] Steps 2-3: Exosome extraction

[0125] After the biological sample obtained in step 2-2 was evenly mixed with the [APMIm]Br-rMGO magnetic bead solution, the mass of [APMIm]Br-rMGO magnetic beads per 1 mL of biological sample was 1 mg, and incubated on a shaking shaker at 4°C for 15 min to obtain the immunomagnetic bead-exosome complex;

[0126] Steps 2-4: Exosome elution

[0127] The mixed solution in step 2-2 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. After repeating twice, ultrapure water with pH = 8 was added, and ice bath sonication was performed for 25 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0128] Example 7

[0129] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0130] Step 2-1: Construction of [APMIm]Br-rMGO magnetic beads

[0131] In this example, step 2-1 adopts the operation of Example 1 to obtain [APMIm]Br-rMGO magnetic beads;

[0132] Step 2-2 Biological sample processing

[0133] The saliva was centrifuged at 3000g and 10000g at 4°C for 40 minutes to separate larger impurities such as cell debris. The supernatant was taken to obtain the processed biological sample.

[0134] Steps 2-3: Exosome extraction

[0135] After the biological sample obtained in step 2-2 was evenly mixed with [APMIm]Br-rMGO magnetic beads, the mass of [APMIm]Br-rMGO magnetic beads per 1 mL of biological sample was 2 mg, and incubated on a shaking shaker at 4°C for 20 min to obtain the immunomagnetic bead-exosome complex;

[0136] Steps 2-4: Exosome elution

[0137] The mixed solution in step 2-2 was subjected to magnetic adsorption for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. After repeating twice, ultrapure water with pH = 8 was added, and ice bath ultrasonication was performed for 20 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0138] Example 8

[0139] A method for extracting and separating extracellular vesicles, comprising the following steps:

[0140] Step 2-1: Construction of [APMIm]Br-rMGO magnetic beads

[0141] In this example, step 2-1 adopts the operation of Example 1 to obtain [APMIm]Br-rMGO magnetic beads;

[0142] Step 2-2 Biological sample processing

[0143] The saliva was centrifuged at 3000g and 10000g at 4°C for 35 minutes to separate larger impurities such as cell debris, and the supernatant was taken to obtain the processed biological sample.

[0144] Steps 2-3: Exosome extraction

[0145] After the biological sample obtained in step 2-2 was evenly mixed with [APMIm]Br-rMGO magnetic beads, the mass of [APMIm]Br-rMGO magnetic beads per 1 mL of biological sample was 1.5 mg, and incubated on a shaking shaker at 4°C for 30 min to obtain the immunomagnetic bead-exosome complex;

[0146] Steps 2-4: Exosome elution

[0147] The mixed solution in step 2-2 was magnetically adsorbed for 20 seconds, the supernatant was discarded, sterile PBS buffer was added, and the supernatant was discarded after magnetic adsorption. After repeating twice, ultrapure water with pH = 7 was added, and ice bath ultrasonication was performed for 30 minutes. The supernatant obtained after magnetic adsorption was the exosome solution.

[0148] 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.

[0149] 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 [APMIm]Br-rMGO magnetic beads, characterized in that: The following steps are involved: The activated carboxylated magnetic graphene oxide magnetic bead solution was added to the pure aqueous solution of ionic liquid [APMIm]Br, and after shaking reaction, the solution was washed by magnetic absorption with pure water to obtain [APMIm]Br-rMGO magnetic beads.

2. The method for preparing reusable [APMIm]Br-rMGO magnetic beads according to claim 1, wherein The mass ratio of activated carboxylated magnetic graphene oxide beads to [APMIm]Br is 1:50-90.

3. The method for preparing reusable [APMIm]Br-rMGO magnetic beads according to claim 1, wherein The activated carboxylated magnetic graphene oxide bead solution was prepared by the following process: Pure water was added to the carboxylated magnetic graphene oxide powder and ultrasonic dispersion was performed to obtain a uniform clear solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide powders were added, and the mixture was reacted in an ice bath and then magnetically washed with pure water to obtain an activated carboxylated magnetic graphene oxide bead solution.

4. The method for preparing reusable [APMIm]Br-rMGO magnetic beads according to claim 3, wherein The mass ratio of the carboxylated magnetic graphene oxide powder to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:50-100.

5. The method for preparing reusable [APMIm]Br-rMGO magnetic beads according to claim 3, wherein The mass ratio of the carboxylated magnetic graphene oxide powder to the N-hydroxysuccinimide powder is 1:50-100.

6. The method for preparing reusable [APMIm]Br-rMGO magnetic beads according to claim 3, wherein Pure water was added to the carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a uniform clear solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added and reacted for at least 30 minutes. N-hydroxysuccinimide powder was then added and reacted in an ice bath for more than 2 hours. Finally, the solution was magnetically washed with pure water to obtain an activated carboxylated magnetic graphene oxide bead solution.

7. Reusable [APMIm]Br-rMGO magnetic beads prepared according to the method according to any one of claims 1 to 6.

8. Use of reusable [APMIm]Br-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 invention comprises reusable [APMIm]Br-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: The reusable [APMIm]Br-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; pure water is added to the magnetic bead-exosome complex, the exosomes adsorbed on the magnetic beads are eluted by ultrasonic vibration, and then the supernatant is collected by magnetic adsorption, and the supernatant is the extracted exosomes.

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