Preparation method of reusable amphiphilic amino acid modified magnetic beads and application of reusable amphiphilic amino acid modified magnetic beads in exosome extraction
By preparing reusable amphiphilic amino acid-modified magnetic beads and capturing exosomes using reverse electrostatic complexation, the problems of low purity, poor structural integrity, and high cost in existing technologies have been solved, achieving efficient and low-cost exosome separation.
Patent Information
- Application Number
- CN202511839240.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-03-20
AI Technical Summary
Existing exosome extraction technologies suffer from low purity, poor structural integrity, and insufficient biological activity. Furthermore, the extracted materials cannot be reused, resulting in high separation costs and low efficiency, making it difficult to meet the needs of large-scale research and industrialization.
Reusable amphiphilic amino acid-modified magnetic beads are used to capture exosomes through reverse electrostatic complexation and achieve efficient separation through a gentle elution method. The surface of the magnetic beads is modified with Glu or Asp to expose amino and carboxyl groups, which form reverse electrostatic complexes with phosphocholine molecules on the surface of exosomes, achieving efficient capture and gentle elution.
It achieves efficient capture and structural integrity of exosomes, reduces separation costs, simplifies equipment requirements, improves operational flexibility, and is suitable for exosome extraction in multiple scenarios.
Smart Images
Figure CN121695840A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of exosome extraction, and particularly relates to a preparation method of reusable amphiphilic amino acid modified magnetic beads and application thereof in exosome extraction. BACKGROUND
[0002] Exosomes are a kind of extracellular vesicles with a diameter of 30-150 nm (average diameter of about 100 nm), which encapsulate DNA, RNA, lipids, metabolites, cytoplasmic proteins, cell surface proteins and other key cellular components. It is this unique composition that makes exosomes have great potential application value in immune response regulation, viral pathogenic transmission, pregnancy maintenance, and diagnosis and treatment of cardiovascular diseases, central nervous system related diseases, cancer and other diseases.
[0003] Realizing rapid capture and high-purity separation of exosomes has become a core bottleneck problem for promoting the application research of exosomes. The current mainstream separation methods all have significant defects, and the specific limitations are as follows:
[0004] Ultracentrifugation (UC): As the long-term "gold standard" for exosome separation, it has very obvious defects; not only is the operation time-consuming and lengthy, the equipment requirements are extremely high and the equipment cost is expensive, but also it is easy to cause aggregation or rupture of exosome vesicles during separation, which directly affects the separation effect and the reliability of subsequent research.
[0005] Polymer precipitation method (such as PEG precipitation method): Although it has the advantage of high recovery rate (usually more than 80%), it has obvious shortcomings; while precipitating exosomes, it is easy to cause co-precipitation of impurities such as lipoprotein, which seriously reduces the purity of exosome separation.
[0006] Size exclusion chromatography (SEC): Although it can better preserve the biological activity of exosomes, its throughput is low, which makes it difficult to meet the actual needs of large-scale sample processing, limiting its application in batch research or industrialization scenarios.
[0007] Immunocapture method: Although it has the outstanding advantages of high specificity, convenient operation and not damaging the integrity of exosome morphology, it also has significant shortcomings; not only is the separation efficiency low, which is not conducive to the subsequent downstream experiment, but also there are problems of high antibody cost and difficulty in effectively eluting exosomes from the capture system, which further compresses its actual application space.
[0008] In addition, the existing technology still has significant common problems: on the one hand, the PEG cannot be separated after co-precipitation with the exosomes, resulting in low purity, poor structural integrity, and insufficient biological activity of the extracted exosomes, which is difficult to meet the demand for high-quality exosomes in downstream precise research or clinical application; on the other hand, the extraction materials in the PEG precipitation method and the immunomagnetic bead method cannot be reused, resulting in the consumption of new consumables for each separation, greatly increasing the separation cost. The two problems are superimposed on each other, not only directly increasing the single operation cost of exosome separation and purification, but also limiting the batch processing efficiency due to the characteristics of the non-reusable materials, which forms a double restriction on the scale-up of related research and industrialization. SUMMARY
[0009] In view of the problems of low purity, poor structural integrity, insufficient biological activity of the extracted exosomes, and non-reusable extraction materials in the exosome extraction and separation technology, the application provides a preparation method of reusable amphiphilic amino acid modified magnetic beads and application of the magnetic beads in exosome extraction, which belongs to a technical scheme with mild capture conditions, small influence on exosomes in the capture and detachment processes, stable capture performance, easy and complete elution.
[0010] To achieve the above-mentioned purposes, the technical solution provided by the application is as follows:
[0011] A preparation method of reusable amphiphilic amino acid modified magnetic beads, which is characterized by comprising the following steps:
[0012] An activated carboxylated magnetic graphene oxide magnetic bead solution is added to a pure water solution of ethylenediamine (EDA), and after oscillation reaction for 6-72 h, pure water is magnetically washed to obtain EDA-rMGO magnetic beads;
[0013] An activated carboxylated glutamic acid (Glu) solution or an activated carboxylated aspartic acid (Asp) solution is added to a pure water dispersion of the EDA-rMGO magnetic beads, and after oscillation reaction for 6-72 h, pure water is magnetically washed to obtain amphiphilic Glu-rMGO magnetic beads and Asp-rMGO magnetic beads;
[0014] During the entire preparation process, the pH is controlled at 4.8-7.4, which is conducive to the reaction, and a conventional adjustment method can be used.
[0015] Further, in step 1), the mass ratio of the activated carboxylated magnetic graphene oxide magnetic beads and ethylenediamine (EDA) is 1:10-150;
[0016] In step 2), the mass ratio of the EDA-rMGO magnetic beads and the activated carboxylated glutamic acid (Glu) solution or the activated carboxylated aspartic acid (Asp) solution is 1:30-300.
[0017] Further, in step 1), the activated carboxylated magnetic graphene oxide magnetic bead solution is prepared by the following method:
[0018] Pure water is added to the carboxylated magnetic graphene oxide powder and ultrasonic dispersion is performed to obtain a uniform and clear solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are sequentially added, and reaction is performed at room temperature for at least 30 min to obtain the activated carboxylated magnetic graphene oxide magnetic bead solution;
[0019] During the whole preparation process, the pH is controlled at 4.8-7.4.
[0020] Further, the mass ratio of the carboxylated magnetic graphene oxide powder to 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1:50-300;
[0021] The mass ratio of the carboxylated magnetic graphene oxide powder to N-hydroxysuccinimide powder is 1:50-300.
[0022] Further, in step 2), the activated carboxylated glutamic acid solution or activated carboxylated aspartic acid solution is prepared by the following method:
[0023] Glutamic acid or aspartic acid is dissolved in deionized water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are sequentially added, and reaction is performed at room temperature for at least 30 min to obtain the activated carboxylated glutamic acid solution or activated carboxylated aspartic acid solution;
[0024] During the whole preparation process, the pH is controlled at 4.8-7.4.
[0025] Further, the molar ratio of the glutamic acid or aspartic acid to 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1:0.8-3;
[0026] The molar ratio of the glutamic acid or aspartic acid to N-hydroxysuccinimide is 1:0.8-3.
[0027] Herein, the application further provides a reusable amphiphilic amino acid modified magnetic bead prepared by the above method and application of the magnetic bead in extraction of exosomes.
[0028] Based on the above application, the application further provides an exosome extraction kit containing the reusable amphiphilic amino acid modified magnetic bead and an exosome extraction method, which specifically comprises the following steps:
[0029] The reusable amphiphilic amino acid modified magnetic bead is added to the impurity-removed biological fluid, and after sufficient mixing and uniformity, shock incubation is performed to obtain a mixed solution;
[0030] The magnetic beads in the mixed solution are adsorbed by magnetic force, the supernatant is discarded, the magnetic beads are washed with PBS buffer to obtain the washed magnetic bead-exosome complex;
[0031] An aqueous solution with a pH of 6-7.4 is added to the magnetic bead-exosome complex, and the exosomes adsorbed on the magnetic beads are eluted by ultrasonic oscillation, and then collected by magnetic adsorption. The supernatant is the extracted exosomes.
[0032] The advantages of the present application are:
[0033] The exosome membrane structure is rich in phosphatidylcholine, and the phosphocholine groups on the molecular surface have specific charge characteristics. The present application prepares a new type of amphiphilic Glu-rMGO magnetic bead or amphiphilic Asp-rMGO magnetic bead by chemical modification method. A large number of amino and carboxyl groups are exposed on the surface of the magnetic bead after Glu or Asp modification, which can form reverse electrostatic complexation with the sulfonic acid groups and amino groups in the phosphocholine molecules on the surface of the exosome. This reverse electrostatic complexation not only enables efficient capture of exosomes, but also effectively reduces the adsorption of impurities.
[0034] Compared with the disadvantage of co-precipitation of lipoprotein by polymer precipitation method, the present application forms reverse electrostatic complexation between the amphiphilic groups on the surface of the magnetic bead and the phosphocholine groups on the surface of the exosome, which not only enables efficient capture of exosomes, but also separates the exosomes from the surface of the magnetic bead by mild elution. Finally, the magnetic bead is recovered by an external magnetic field. More importantly, based on the reversibility of reverse electrostatic complexation, the magnetic bead not only enables efficient capture and mild elution of exosomes, but also effectively ensures the structural integrity and biological activity of the exosomes, and can be reused, significantly reducing the economic cost.
[0035] The present application not only solves the shortcoming of high cost of "antibody elution" in immunoaffinity capture method, but also avoids the dependence on expensive large equipment in ultracentrifugation method, and overcomes the problem of reduced exosome activity caused by PEG residue in PEG precipitation method, providing a new direction for reducing the cost of exosome separation and improving operational flexibility.
[0036] In the present application, the Glu and Asp combined with the magnetic bead have stable chemical properties, are easy to store and stable in shape after being combined with the magnetic bead nanomaterial, and can be reused for a long time. The exosomes extracted by the method of the present application can obtain exosomes with a diameter distribution of 30-150 nm. The magnetic bead of the present application not only has stable properties and is easy to store, but also has the characteristics of low cost, rapid preparation and strong magnetism by using magnetic bead nanomaterial, and can be completely separated from the solution within 20 seconds, can be reused for a long time. In addition, this method does not need to rely on expensive equipment, and has good application prospect in the field of exosome extraction in multiple scenes. BRIEF DESCRIPTION OF DRAWINGS
[0037] Figure 1 Determination of the particle size of Glu-rMGO magnetic beads by a laser particle size analyzer;
[0038] Figure 2 SEM observation of the morphology of Asp-rMGO magnetic beads;
[0039] Figure 3 SEM observation of the morphology of magnetic beads for extracting exosomes;
[0040] Figure 4 Determination of the particle size of magnetic beads for extracting exosomes by a laser particle size analyzer;
[0041] Figure 5 Determination of the electric potential of magnetic beads for extracting exosomes by a Zeta potential analyzer;
[0042] Figure 6 Western blot determination of the marker proteins of magnetic beads for extracting exosomes. DETAILED DESCRIPTION
[0043] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the disclosure of the present application can be more thoroughly and completely understood.
[0044] The experimental methods described in the following examples are all conventional methods unless otherwise specified. The reagents and biological materials described are all commercially available unless otherwise specified.
[0045] Example 1
[0046] A preparation method of a reusable amphiphilic Glu-rMGO magnetic bead, comprising the following steps:
[0047] Step 1: Preparation of magnetic graphene oxide
[0048] Single-layer graphene oxide (industrial) is dispersed in ethylene glycol by ultrasonic method to obtain a uniform graphene oxide solution. Iron trichloride powder, sodium acetate powder and polyethylene glycol 8000 powder are added, and the mixture is uniformly stirred by magnetic stirring. Then, a closed high-temperature and high-pressure reaction is carried out (in a Teflon high-temperature and high-pressure reactor). After the reaction is completed, the precipitate (i.e. the product) is collected by centrifugation, and then washed with anhydrous ethanol and pure water by magnetic attraction. Finally, the magnetic graphene oxide powder is obtained by vacuum drying, which is denoted as rMGO.
[0049] The mass ratio of the single-layer graphene oxide and the ferric trichloride is 1:5 (the mass ratio of the single-layer graphene oxide and the ferric trichloride can be in the range of 1:2-10); the mass ratio of the single-layer graphene oxide and the sodium acetate is 1:20 (the mass ratio of the single-layer graphene oxide and the sodium acetate can be in the range of 1:15-80); the mass ratio of the single-layer graphene oxide and the polyethylene glycol 8000 is 1:2.5 (the mass ratio of the single-layer graphene oxide and the polyethylene glycol 8000 can be in the range of 1:1-5); the reaction temperature is 220°C, and the reaction time is 8 hours (the reaction temperature can be in the range of 200-250°C, and the reaction time can be in the range of 4-8 hours); and the dispersibility and the magnetic effect of the magnetic graphene oxide prepared according to the mass ratio are better.
[0050] Step 2: Preparation of carboxylated magnetic graphene oxide
[0051] The magnetic graphene oxide powder obtained in step 1 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then sodium hydroxide powder and chloroacetic acid powder are added, and the mixture is reacted and vacuum dried to obtain carboxylated magnetic graphene oxide powder, denoted as rMGO-COOH.
[0052] The mass ratio of the magnetic graphene oxide and the sodium hydroxide powder is 1:50 (the mass ratio of the magnetic graphene oxide and the sodium hydroxide powder can be in the range of 1:10-50); and the mass ratio of the magnetic graphene oxide and the chloroacetic acid powder is 1:30 (the mass ratio of the magnetic graphene oxide and the chloroacetic acid powder can be in the range of 1:20-30), and the ratio is better for the carboxyl group of the magnetic beads.
[0053] Step 3: Activation of carboxylated magnetic graphene oxide
[0054] The carboxylated magnetic graphene oxide powder obtained in step 2 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then EDC (1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide, N-hydroxysuccinimide) are sequentially added, and the mixture is reacted at room temperature for at least 2 hours, and then washed with pure water by magnetic attraction, so that the carboxyl groups on the surface of the magnetic graphene oxide are activated into an intermediate form which is more prone to reaction, to obtain an activated carboxylated magnetic graphene oxide magnetic bead solution, denoted as activated rMGO-COOH solution.
[0055] The mass ratio of the carboxylated magnetic graphene oxide and the EDC is 1:56; and the mass ratio of the carboxylated magnetic graphene oxide and the NHS is 1:60.
[0056] Step 4: Preparation of EDA-rMGO magnetic beads
[0057] EDA was weighed in pure water, shaken to dissolve, then the activated rMGO-COOH solution obtained in step 3 was added, and the reaction was carried out at room temperature for 72 h, and then the EDA-rMGO magnetic beads were obtained by washing with pure water. The mass ratio of activated carboxylated magnetic graphene oxide magnetic beads to EDA was 1:150. The long-term storage condition of the obtained EDA-rMGO magnetic beads was a pure water system, stored at 4°C.
[0058] Step 5: Preparation of Glu-rMGO magnetic beads
[0059] EDA-rMGO magnetic beads were weighed and dispersed in pure water, then the activated carboxylated Glu solution was added, and the reaction was carried out at room temperature for 72 h, and then the amphiphilic Glu-rMGO magnetic beads were obtained by washing with pure water. The mass ratio of EDA-rMGO magnetic beads to activated carboxylated Glu solution was 1:50.
[0060] The preparation method of the activated carboxylated Glu solution was as follows: glutamic acid was dissolved in deionized water, EDC and NHS were added in turn, and the reaction was carried out at room temperature for at least 30 min, and then the activated carboxylated Glu solution was obtained by washing with pure water. The molar ratio of glutamic acid to EDC and NHS was 1:0.8;
[0061] During the entire preparation process, the pH was controlled at 4.8-7.4. The particle size of the prepared amphiphilic Glu-rMGO magnetic beads was as shown in Figure 1 The magnetic bead particle size was uniformly distributed at about 1000 nm, and the long-term storage condition was a pure water system, stored at 4°C. The magnetic beads not only maintained good water dispersibility in water, PBS buffer and cell culture solution, etc., but also ensured good magnetism, which could be quickly (<20 s) separated from the solution by magnetic attraction. The exosomes in the plasma were extracted by using the magnetic beads, the exosome structure was complete, the biological activity was sufficient, and after 5 repeated times, the separation purity remained above 80%.
[0062] Example 2
[0063] A preparation method of a reusable amphiphilic Asp-rMGO magnetic bead, comprising the following steps:
[0064] Step 1: Preparation of magnetic graphene oxide
[0065] The single-layer graphene oxide is mixed with ethylene glycol solvent, added into a Teflon high-temperature and high-pressure reaction kettle, and ultrasonically dispersed for 5 min; according to the mass ratio of single-layer graphene oxide to ferric chloride of 1:3, the mass ratio of single-layer graphene oxide to sodium acetate of 1:30, and the mass ratio of single-layer graphene oxide to polyethylene glycol 8000 of 1:2, the above three compounds are added into the single-layer graphene oxide dispersion liquid, and then uniformly stirred by magnetic force; then the reaction kettle is transferred into a blast drying oven and reacted at 200 DEG C for 6 h; after the reaction is completed, the product is collected by centrifugation at a speed of 10000 rpm, and then washed with anhydrous ethanol and ultrapure water by magnetic force for three times respectively, and then vacuum dried at 60 DEG C to obtain magnetic graphene oxide.
[0066] Step 2: Preparation of carboxylated magnetic graphene oxide
[0067] After the magnetic graphene oxide powder obtained in step 1 is added with 20 mL of pure water and ultrasonically dispersed to obtain a uniform and clear solution, sodium hydroxide powder and chloroacetic acid powder are added according to the mass ratio of magnetic graphene oxide to sodium hydroxide powder of 1:20 and the mass ratio of magnetic graphene oxide to chloroacetic acid powder of 1:35, and then ultrasonically reacted in an ice water bath for 2 h, and then vacuum dried to obtain carboxylated magnetic graphene oxide.
[0068] Step 3: Activation of carboxylated magnetic graphene oxide
[0069] After the carboxylated magnetic graphene oxide powder obtained in step 2 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, EDC and NHS are sequentially added according to the mass ratio of carboxylated magnetic graphene oxide to EDC of 1:40 and the mass ratio of carboxylated magnetic graphene oxide to NHS of 1:40, and then reacted at room temperature for at least 2 hours, and then washed with pure water by magnetic force, so that the carboxyl groups on the surface of the magnetic graphene are activated into an intermediate form which is more prone to reaction, to obtain an activated carboxylated magnetic graphene oxide magnetic bead solution, which is denoted as activated rMGO-COOH solution.
[0070] Step 4: Preparation of EDA-rMGO magnetic beads
[0071] According to the mass ratio of carboxylated magnetic graphene oxide to EDA of 1:62.5, the corresponding mass of EDA is weighed in pure water, dissolved by shaking, and then added into the activated rMGO-COOH solution obtained in step 3, and then shaken and reacted at room temperature for 24 h, and then washed with pure water by magnetic force, to obtain EDA-rMGO magnetic beads.
[0072] Step 5: Preparation of Asp-rMGO magnetic beads
[0073] The EDA-rMGO magnetic beads are weighed and dispersed in pure water. An activated carboxyl Asp solution is added to the pure water dispersion of the EDA-rMGO magnetic beads at a mass ratio of 1:200 of the EDA-rMGO magnetic beads to the activated carboxyl Asp solution. After 24 hours of reaction at room temperature, the pure water is magnetically washed, and the amphiphilic Asp-rMGO magnetic beads are obtained.
[0074] The activated carboxyl Asp solution is prepared by dissolving aspartic acid in deionized water, sequentially adding EDC and NHS, and magnetically washing with pure water after at least 30 minutes of reaction at room temperature. The molar ratio of aspartic acid to EDC and NHS is 1:3.
[0075] During the entire preparation process, the pH is controlled at 4.8-7.4. The morphology of the prepared amphiphilic Asp-rMGO magnetic beads is shown in Figure 2 The particle size distribution is 500-1000 nm. The long-term storage condition is a pure water system at 4°C. The magnetic beads can not only maintain good water dispersibility in water, PBS buffer, and cell culture solution, but also ensure good magnetism, allowing rapid (<20s) separation from the solution by magnetic attraction. The exosomes extracted from saliva using the magnetic beads have complete structure and sufficient biological activity. After three repetitions, the separation purity remains above 85%.
[0076] Example 3
[0077] A method for preparing reusable amphiphilic Glu-rMGO magnetic beads includes the following steps:
[0078] Step 1: Preparation of magnetic graphene oxide
[0079] Single-layer graphene oxide is mixed with ethylene glycol solvent and added to a Teflon high-temperature high-pressure reaction kettle. Ultrasonic dispersion is performed for 5 minutes. The three compounds are added to the single-layer graphene oxide dispersion liquid at a mass ratio of 1:2 of single-layer graphene oxide to ferric chloride, 1:80 of single-layer graphene oxide to sodium acetate, and 1:5 of single-layer graphene oxide to polyethylene glycol 8000. After magnetic stirring, the reaction kettle is transferred to a blast drying oven for reaction at 230°C for 4 hours. After the reaction is completed, the product is collected by centrifugation at 10000 rpm, and is sequentially washed with anhydrous ethanol and ultrapure water by magnetic attraction for three times each. Vacuum drying at 60°C obtains the magnetic graphene oxide.
[0080] Step 2: Preparation of carboxylated magnetic graphene oxide
[0081] The carboxylated magnetic graphene oxide powder obtained in step 2 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then EDC and NHS are sequentially added in a mass ratio of 1:80 and 1:100 of the carboxylated magnetic graphene oxide and EDC and the carboxylated magnetic graphene oxide and NHS, respectively. After reacting at room temperature for at least 2 hours, the surface carboxyl groups of the magnetic graphene are activated into an intermediate form which is more prone to reaction, to obtain an activated carboxylated magnetic graphene oxide magnetic bead solution, denoted as activated rMGO-COOH solution.
[0082] Step 3: Activating the carboxylated magnetic graphene oxide
[0083] The carboxylated magnetic graphene oxide powder obtained in step 2 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then EDC and NHS are sequentially added in a mass ratio of 1:80 and 1:100 of the carboxylated magnetic graphene oxide and EDC and the carboxylated magnetic graphene oxide and NHS, respectively. After reacting at room temperature for at least 2 hours, the surface carboxyl groups of the magnetic graphene are activated into an intermediate form which is more prone to reaction, to obtain an activated carboxylated magnetic graphene oxide magnetic bead solution, denoted as activated rMGO-COOH solution.
[0084] Step 4: Preparing EDA-rMGO magnetic beads
[0085] The EDA-rMGO magnetic beads are dispersed in pure water, and an activated carboxyl Glu solution is added to the EDA-rMGO magnetic bead dispersion in pure water in a mass ratio of 1:70 of the EDA-rMGO magnetic beads and the activated carboxyl Glu solution. After shaking and reacting at room temperature for 24 hours, the EDA-rMGO magnetic beads are washed with pure water by magnetic attraction, to obtain amphiphilic Glu-rMGO magnetic beads. The magnetic beads are used to extract exosomes from urine, and the exosomes have complete structure and sufficient biological activity. After 4 repeated extractions, the separation purity is still maintained at more than 83%. In the entire preparation process, the pH is controlled at 4.8-7.4.
[0086] Step 5: Preparing Glu-rMGO magnetic beads
[0087] The EDA-rMGO magnetic beads are dispersed in pure water, and an activated carboxyl Glu solution is added to the EDA-rMGO magnetic bead dispersion in pure water in a mass ratio of 1:70 of the EDA-rMGO magnetic beads and the activated carboxyl Glu solution. After shaking and reacting at room temperature for 24 hours, the EDA-rMGO magnetic beads are washed with pure water by magnetic attraction, to obtain amphiphilic Glu-rMGO magnetic beads. The magnetic beads are used to extract exosomes from urine, and the exosomes have complete structure and sufficient biological activity. After 4 repeated extractions, the separation purity is still maintained at more than 83%. In the entire preparation process, the pH is controlled at 4.8-7.4.
[0088] Example 4
[0089] A preparation method of a reusable amphiphilic Asp-rMGO magnetic bead, specifically comprising the following steps:
[0090] Step 1: Preparing magnetic graphene oxide
[0091] The single-layer graphene oxide is mixed with ethylene glycol solvent, added into a Teflon high-temperature and high-pressure reaction kettle, and ultrasonically dispersed for 5 min; the single-layer graphene oxide, the ferric chloride and the sodium acetate are added into the single-layer graphene oxide dispersion liquid in a mass ratio of 1:10, 1:15 and 1:3 respectively, and after magnetic stirring, the reaction kettle is transferred into a blast drying oven for reaction at 250℃ for 5 h; after the reaction is completed, the product is collected by centrifugation at a speed of 10000 rpm, and washed with anhydrous ethanol and ultrapure water for three times respectively by magnetic attraction, and vacuum dried at 60℃ to obtain the magnetic graphene oxide.
[0092] Step 2: Preparation of carboxylated magnetic graphene oxide
[0093] The magnetic graphene oxide powder obtained in step 1 is added with 20 mL of pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then the sodium hydroxide powder and the chloroacetic acid powder are added in a mass ratio of 1:50 and 1:23 respectively, and ultrasonically reacted in an ice water bath for 2 h, and vacuum dried to obtain the carboxylated magnetic graphene oxide.
[0094] Step 3: Activation of carboxylated magnetic graphene oxide
[0095] The carboxylated magnetic graphene oxide powder obtained in step 2 is added with pure water and ultrasonically dispersed to obtain a uniform and clear solution, and then the EDC and the NHS are added in a mass ratio of 1:50 and 1:50 respectively, and reacted at room temperature for at least 2 hours, and then washed with pure water by magnetic attraction, so that the carboxyl groups on the surface of the magnetic graphene are activated into an intermediate form which is more prone to reaction, to obtain an activated carboxylated magnetic graphene oxide magnetic bead solution, denoted as activated rMGO-COOH solution.
[0096] Step 4: Preparation of EDA-rMGO magnetic beads
[0097] The EDA is weighed in pure water in a mass ratio of 1:80 with respect to the carboxylated magnetic graphene oxide, and after being dissolved by shaking, it is added into the activated rMGO-COOH solution obtained in step 3, and after shaking reaction at room temperature for 24 h, it is washed with pure water by magnetic attraction, to obtain the EDA-rMGO magnetic beads.
[0098] Step 5: Preparation of Asp-rMGO magnetic beads
[0099] EDA-rMGO magnetic beads were weighed and dispersed in pure water. Activated carboxyl Asp solution was added to the pure water dispersion of EDA-rMGO magnetic beads at a mass ratio of 1:50 of carboxylated magnetic graphene oxide and activated carboxyl Asp solution. After 24 hours of reaction at room temperature, the mixture was washed with pure water by magnetic attraction. The amphiphilic Asp-rMGO magnetic beads were obtained. The magnetic beads were used to extract exosomes from bone marrow mesenchymal stem cell culture solution. The exosomes were complete in structure and sufficient in biological activity. After 5 repetitions, the separation purity remained above 80%. The pH was controlled at 4.8-7.4 throughout the entire preparation process.
[0100] Example 5
[0101] A method for extracting and separating plasma exosomes using amphiphilic Glu-rMGO magnetic beads, comprising the following steps:
[0102] Step 1: Biological sample processing
[0103] The plasma was diluted by mixing with PBS at a ratio of 1:1. The diluted plasma was centrifuged at 3000 g and 17000 g at 4°C for 15 min and 30 min, respectively, to separate larger impurities such as cell debris. The supernatant was obtained as the processed biological sample.
[0104] Step 2: Exosome extraction
[0105] The biological sample obtained in step 1 was mixed with the amphiphilic Glu-rMGO magnetic bead solution. The mass of the amphiphilic Glu-rMGO magnetic beads in 1 mL of the biological sample was 0.6 mg. The mixture was placed on a shaking incubator and incubated at 4°C for 5 min to obtain an immunomagnetic bead-exosome complex.
[0106] Step 3: Resuspension of exosomes
[0107] The magnetic bead-exosome complex obtained in step 2 was magnetically separated and precipitated for 15 min. After the supernatant was removed, a water solution with pH=7.4 was added to resuspend the precipitate, obtaining a magnetic bead-exosome mixture resuspended in ultrapure water.
[0108] Step 4: Elution and collection of exosomes
[0109] The magnetic bead-exosome mixture obtained in step 3 was subjected to ice bath ultrasonic shaking for 5 min to separate the magnetic beads and exosomes. The magnetic beads were precipitated by magnetic attraction, and the supernatant was collected, which was the exosome solution. The morphology of the obtained plasma exosomes is shown in Figure 3 The particle size distribution was 50-150 nm.
[0110] Example 6
[0111] A method for extracting and separating saliva exosomes by using amphiphilic Asp-rMGO magnetic beads, comprising the following steps:
[0112] Step 1: biological sample processing
[0113] The saliva is centrifuged at 3000 g and 17000 g at 4°C, respectively, for 40 min, to separate the cell fragments and other large impurities, and the supernatant is obtained as the processed biological sample.
[0114] Step 2: exosome extraction
[0115] The biological sample obtained in step 1 is mixed with the amphiphilic Asp-rMGO magnetic beads, and the mass of the amphiphilic Asp-rMGO magnetic beads in 1 mL of the biological sample is 0.5 mg, which is placed on a shaking incubator and incubated at 4°C for 20 min to obtain an immunomagnetic bead-exosome complex.
[0116] Step 3: Resuspension of exosomes
[0117] The magnetic bead-exosome complex obtained in step 2 is magnetically separated and precipitated for 1 min, and after the supernatant is removed, a water solution with pH=7.4 is added to resuspend the precipitate, to obtain a magnetic bead-exosome mixture resuspended in ultrapure water.
[0118] Step 4: elution and collection of exosomes
[0119] The magnetic bead-exosome mixture obtained in step 3 is subjected to ice bath ultrasonic oscillation for 5 min to separate the magnetic beads and exosomes, the magnetic beads are precipitated by magnetic adsorption, and the supernatant is collected, which is the exosome solution. The particle size of the obtained saliva exosomes is shown in Figure 4 , and the average particle size distribution is 110 nm.
[0120] Example 7
[0121] A method for extracting and separating urine exosomes by using amphiphilic Glu-rMGO magnetic beads, comprising the following steps:
[0122] Step 1: biological sample processing
[0123] The urine is mixed and diluted with PBS at a ratio of 1:1, and the diluted urine is centrifuged at 3000 g and 17000 g at 4°C, respectively, for 15 min and 30 min, to separate the cell fragments and other large impurities, and the supernatant is obtained as the processed biological sample.
[0124] Step 2: exosome extraction
[0125] The biological sample obtained in step 1 is mixed with the amphiphilic Glu-rMGO magnetic bead solution, and the mass of the amphiphilic Glu-rMGO magnetic beads in 1 mL of the biological sample is 0.6 mg. The mixture is placed on a shaking table and incubated at 4°C for 5 min to obtain an immunomagnetic bead-exosome complex.
[0126] Step 3: Resuspension of exosomes
[0127] The magnetic bead-exosome complex obtained in step 2 is magnetically separated and precipitated for 15 min. After the supernatant is removed, the precipitate is resuspended in a water solution with pH = 7.4 to obtain a magnetic bead-exosome mixture resuspended in ultrapure water.
[0128] Step 4: Elution and collection of exosomes
[0129] The magnetic bead-exosome mixture obtained in step 3 is subjected to ice bath ultrasonic vibration for 5 min to separate the magnetic beads and exosomes. The magnetic beads are precipitated by magnetic adsorption, and the supernatant is collected, which is the exosome solution. The potential of the obtained urine exosomes is shown in Figure 5 , which is -20 mV, i.e. the potential of the exosomes.
[0130] Example 8
[0131] A method for extracting and separating bone marrow mesenchymal stem cell exosomes using amphiphilic Asp-rMGO magnetic beads, comprising the following steps:
[0132] Step 1: Treatment of biological sample
[0133] The animal bone marrow mesenchymal stem cell culture supernatant is first centrifuged at 3000 g and then at 17000 g at 4°C for 15 min and 30 min, respectively, to separate the cell debris and other large impurities. The supernatant is obtained to obtain the treated biological sample.
[0134] Step 2: Extraction of exosomes
[0135] The biological sample obtained in step 1 is mixed with the amphiphilic Asp-rMGO magnetic bead solution, and the mass of the amphiphilic Asp-rMGO magnetic beads in 1 mL of the biological sample is 5 mg. The mixture is placed on a shaking table and incubated at 4°C for 10 min to obtain a magnetic bead-exosome complex.
[0136] Step 3: Resuspension of exosomes
[0137] The magnetic bead-exosome complex obtained in step 2 is magnetically separated and precipitated for 20 min. After the supernatant is removed, the precipitate is resuspended in a water solution with pH = 7.4 to obtain a magnetic bead-exosome mixture resuspended in ultrapure water.
[0138] Step 4: Elution and collection of exosomes
[0139] The magnetic bead-exosome mixture obtained in step 3 is subjected to ice bath ultrasonic oscillation for 1 min to separate the magnetic beads and the exosomes, the magnetic beads are precipitated by magnetic adsorption, and the supernatant is collected, which is the exosome solution. The marker proteins of the obtained bone marrow mesenchymal stem cell exosomes are shown in FIG. 4, and it can be seen from the marker proteins that the extracted substance is exosomes. Figure 6
[0140] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application.
Claims
1. A method for preparing reusable amphiphilic amino acid-modified magnetic beads, characterized in that, Includes the following steps: 1) Add an activated carboxylated magnetic graphene oxide bead solution to a pure aqueous solution of ethylenediamine, shake the reaction, and then wash with pure water using magnetic adsorption to obtain EDA-rMGO magnetic beads; 2) Add a glutamic acid solution or an aspartic acid solution with activated carboxyl groups to the pure aqueous dispersion of EDA-rMGO magnetic beads, shake the reaction, and then wash with pure water by magnetic adsorption to obtain amphiphilic Glu-rMGO magnetic beads or amphiphilic Asp-rMGO magnetic beads. Throughout the entire preparation process, the pH was controlled between 4.8 and 7.
4.
2. The method for preparing reusable amphiphilic amino acid-modified magnetic beads according to claim 1, characterized in that: In step 1), the mass ratio of activated carboxylated magnetic graphene oxide beads to ethylenediamine is 1:10-150; In step 2), the mass ratio of EDA-rMGO magnetic beads to glutamic acid solution or aspartic acid solution with activated carboxyl groups is 1:30-300.
3. The method for preparing reusable amphiphilic amino acid-modified magnetic beads according to claim 1 or 2, characterized in that: In step 1), the activated carboxylated magnetic graphene oxide bead solution is prepared by the following method: Pure water was added to carboxylated magnetic graphene oxide powder and ultrasonically dispersed to obtain a uniform and clear solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially and reacted at room temperature for at least 30 min to obtain an activated carboxylated magnetic graphene oxide bead solution. Throughout the entire preparation process, the pH was controlled between 4.8 and 7.
4.
4. The method for preparing reusable amphiphilic amino acid-modified magnetic beads according to claim 3, characterized in that: The mass ratio of the carboxylated magnetic graphene oxide powder to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:50-300; The mass ratio of the carboxylated magnetic graphene oxide powder to N-hydroxysuccinimide is 1:50-300.
5. The method for preparing reusable amphiphilic amino acid-modified magnetic beads according to claim 1 or 2, characterized in that: In step 2), the glutamic acid solution with activated carboxyl groups or the aspartic acid solution with activated carboxyl groups is prepared by the following method: Dissolve glutamic acid or aspartic acid in deionized water, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in sequence, and react at room temperature for at least 30 min to obtain a glutamic acid solution with activated carboxyl groups or an aspartic acid solution with activated carboxyl groups. Throughout the entire preparation process, the pH was controlled between 4.8 and 7.
4.
6. The method for preparing reusable amphiphilic amino acid-modified magnetic beads according to claim 5, characterized in that: The molar ratio of the glutamic acid or aspartic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:0.8-3; The molar ratio of glutamic acid or aspartic acid to N-hydroxysuccinimide is 1:0.8-3.
7. A reusable amphiphilic amino acid-modified magnetic bead, characterized in that: It is prepared by any one of the preparation methods described in claims 1-6.
8. The application of reusable amphiphilic amino acid-modified magnetic beads prepared by any of the preparation methods described in claims 1-6 in the extraction of exosomes.
9. An exosome extraction kit, characterized in that: Includes reusable amphiphilic amino acid-modified magnetic beads prepared by any of the preparation methods described in claims 1-6, and an eluent.
10. A method for extracting exosomes, characterized in that, Includes the following steps: The reusable amphiphilic amino acid-modified magnetic beads prepared by any of the preparation methods described in claims 1-6 are added to a biological fluid after impurities have been removed, and after thorough mixing and incubation by shaking, a mixed solution is obtained. Magnetic beads were magnetically adsorbed into the mixture. The supernatant was discarded, and the magnetic beads were washed with PBS buffer to obtain the washed magnetic bead-exosome complex. An aqueous solution with a pH of 6-7.4 was added to the magnetic bead-exosome complex. The exosomes adsorbed on the magnetic beads were eluted by ultrasonic vibration. The supernatant was then collected by magnetic adsorption. The supernatant is the extracted exosome.