Method for purifying and separating exosome based on magnetic particles

Abstract

The invention relates to a method for purifying and separating exosomes based on magnetic particles, and relates to the technical field of biologication.The method comprises the specific steps that magnetic nano-particles are prepared through a coprecipitation method, Al-MOF layers are synthesized on the surfaces of the magnetic nano-particles, and magnetic Al-MOF nano-particles are formed; then, fixing a nucleic acid aptamer aiming at the CD63 protein on the surface of the nano-particle by utilizing an electrostatic adsorption effect; mixing the modified nanoparticles with a biological sample, and capturing exosomes in a magnetic separation device; activating a magnetic field to aggregate the magnetic nanoparticles capturing the exosomes, and releasing the exosomes through flushing and alkaline solution treatment; and finally, purifying the exosome through ultrafiltration operation. By introducing the magnetic separation device, the problems that the magnetic nanoparticles are easy to agglomerate and poor in dispersity in a biological sample are solved, so that the magnetic nanoparticles are more fully contacted with the exosome, and the contact efficiency with the exosome is improved, thereby more effectively capturing and purifying the exosome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for purifying and separating exosomes based on magnetic particles. Background Art

[0002] Exosomes are small vesicles secreted by cells, which have important biological functions and clinical application potential. Exosomes are a type of extracellular vesicles (EVs), which are tiny vesicles wrapped by a lipid bilayer. The diameter is usually between 30 and 150 nanometers. Nowadays, they specifically refer to discoid vesicles with a diameter of 40-100nm. Exosomes can be secreted by all cell types, including immune cells, neurons, epithelial cells, endothelial cells, embryonic cells, cancer cells and mesenchymal stem cells, etc., and are widely distributed in various body fluids such as blood, urine, saliva, milk, and bile. Exosomes are wrapped by a phospholipid bilayer membrane, which contains a variety of biological macromolecules such as proteins, lipids, RNA (such as mRNA and miRNA), and DNA. Exosomes have become a hot spot in global life science / basic medical research as indicators of disease diagnosis and carriers of disease treatment.

[0003] For example, the Chinese patent application number CN202110719284.X discloses a method for purifying and separating exosomes based on magnetic MOF, which relates to the field of biotechnology. The method is a magnetic separation method capable of purifying the exosomes, wherein a magnetic Al-MOF nanoparticle with CD63 nucleic acid aptamer adsorbed on the surface specifically captures the exosomes in a trace biological sample, and after magnetic separation and multiple washings, an alkaline solution such as ammonia water or low-concentration sodium hydroxide is added to release the exosomes from the magnetic Al-MOF nanoparticles, and finally the high-purity exosomes are obtained by ultrafiltration washing. The invention is simple and fast, and can ensure that high-purity exosomes are separated from trace samples.

[0004] In the above scheme, the magnetism of the magnetic nanoparticles themselves makes them easy to aggregate, affecting their dispersibility in biological samples. At the same time, the mixing method during the capture of exosomes does not have sufficient contact with the biological samples and cannot fully contact the exosomes. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a method for purifying and separating exosomes based on magnetic particles. By introducing a magnetic separation device, the uniform distribution of magnetic nanoparticles in a biological sample solution is promoted, thereby more effectively capturing exosomes.

[0006] The present invention provides a method for purifying and isolating exosomes based on magnetic particles, which specifically includes the following steps: Step 1, preparing Fe3O4 magnetic nanoparticles by coprecipitation method; Step 2, synthesizing a layer of Al-MOF on the surface of the magnetic nanoparticles to prepare magnetic Al-MOF nanoparticles, and preparing them into an aqueous dispersion; Step 3: Adding nucleic acid aptamers targeting CD63 protein to the aqueous dispersion of magnetic Al-MOF nanoparticles to 3+ The CD63 nucleic acid aptamer is fixed on the magnetic Al-MOF nanoparticles by electrostatic adsorption with the phosphate group in the CD63 nucleic acid aptamer skeleton, thereby forming magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer; Step 4, mixing the magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer with a trace amount of biological sample; placing the mixed solution in a magnetic separation device, and the exosomes are captured on the surface of the magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer; Step 5, activating magnetic field 1 and magnetic field 2, so that the magnetic nanoparticles containing captured exosomes are aggregated and adsorbed on both ends of the device under the action of the magnetic field; Step 6: Use filter paper or a pipette to remove the unbound solution at both ends of the device; then, rinse the device with pure water or PBS solution, keeping the magnetic field on during the rinsing process to prevent the magnetic nanoparticles from falling off; repeat the rinsing several times until there are no obvious impurities in the rinsing solution; Step 7, introducing ammonia water with a pH value of 9 or sodium hydroxide alkaline solution with a pH value of 11 into both ends of the magnetic separation device, so that the alkaline solution fully contacts and rinses the magnetic nanoparticles in the device, and after standing for 5-10 minutes, using a pipette or a pipette to collect the rinsing liquid, which contains exosomes released from the magnetic nanoparticles; Step 8: Transfer the washing liquid collected in step 7 to a 100K ultrafiltration tube for ultrafiltration, and repeat the ultrafiltration washing several times until there are no obvious impurities in the ultrafiltrate to obtain a purified exosome suspension. Furthermore, in step 4, the magnetic separation device is a structure with a convex middle and narrowed two ends, the convex middle part is the diffusion area, and the narrowed parts at both ends are the collection areas; magnetic fields are set at both ends of the device, namely magnetic field one and magnetic field two, and the magnetic field strengths of magnetic field one and magnetic field two are both set within the range of 0.5-1T; the device can be flipped upside down and rotated laterally.

[0007] Furthermore, the method for capturing exosomes by a magnetic separation device is specifically as follows: activating magnetic field one and magnetic field two to cause the magnetic nanoparticles to gather at two ends of the device; Turn off the magnetic field to allow the magnetic particles originally adsorbed on the upper magnetic field to fall off naturally. At this time, rotate the separation device horizontally at a speed of 5-20 rpm for 10-30 seconds; The separation device is flipped 180 degrees up and down, and the magnetic particles originally adsorbed on the magnetic fields at both ends fall off naturally through the alternating adsorption of magnetic field one and magnetic field two, and enter the biological sample solution again. After flipping, the separation device is rotated horizontally at a speed of 5-20 rpm; The magnetic field is controlled continuously as well as the upside-down flipping and lateral rotation are performed until the magnetic nanoparticles are evenly distributed in the biological sample solution, thus completing the capture of exosomes.

[0008] Furthermore, the material of the diffusion zone in the magnetic field separation device is a flexible material that can shrink or expand.

[0009] Furthermore, the flexible material is any one of silicone, rubber or polyurethane.

[0010] Furthermore, a ring-shaped magnetic field is also arranged in the diffusion zone of the magnetic field separation device, which is the magnetic field three, and the magnetic field strength of the magnetic field three is 0.1-1T.

[0011] Furthermore, γ-Fe2O3 magnetic nanoparticles are also used, and the ratio of Fe3O4 magnetic nanoparticles to γ-Fe2O3 magnetic nanoparticles is (1-5):1.

[0012] Furthermore, two smaller middle protrusions are provided outside the narrowed portion of the magnetic separation device, which serve as pre-dispersion areas.

[0013] Furthermore, two annular magnetic fields are set in the pre-dispersion area, namely magnetic field four and magnetic field five.

[0014] Furthermore, the magnetic field strengths of the magnetic field four and the magnetic field five are both set within the range of 0.1-3T.

[0015] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: First, by introducing a magnetic separation device, the uniform distribution of magnetic nanoparticles in the biological sample solution is promoted, so as to more effectively capture exosomes. The magnetic separation device is a structure with a convex middle and narrowed at both ends. The convex middle part is the diffusion area, and the narrowed parts at both ends are the collection area. Magnetic field one and magnetic field two are set at both ends of the device; the magnetic separation device uses the adsorption effect of the magnetic field on the magnetic nanoparticles, so that the magnetic Al-MOF nanoparticles modified with CD63 nucleic acid aptamers gather at the two ends of the device under the action of the magnetic field; when the magnetic field is turned off or changes direction, the magnetic nanoparticles will lose the restraint of the magnetic field, so that they can fall off from the device and be redispersed in the biological sample solution; Second, flexible materials such as silicone, rubber and polyurethane have good elasticity and plasticity, and can be deformed under external forces, including contraction and expansion; these materials can maintain structural integrity during deformation and are not easy to break or produce permanent deformation; this property enables the diffusion zone to adjust its spatial configuration according to actual needs, thereby optimizing fluid dynamic conditions; magnetic separation technology relies on the specific binding of magnetic nanoparticles to exosomes in biological samples. When flexible materials are used as diffusion zones, they can change fluid dynamic conditions by adjusting their shape and size according to the different contents or types of biological samples and magnetic nanoparticles, thereby affecting the dispersion state of magnetic nanoparticles; the contraction or expansion of the diffusion zone can change the flow rate and direction of the fluid, as well as the contact area between the magnetic nanoparticles and the biological sample solution, thereby optimizing the binding efficiency of the magnetic nanoparticles with the target molecules; Third, by introducing a circular magnetic field and adjusting the ratio of Fe3O4 and γ-Fe2O3 magnetic nanoparticles, the capture efficiency and separation purity of exosomes were further improved; the magnetic field strength directly affects the magnetic force on the magnetic nanoparticles, which in turn affects their capture efficiency. According to the different ratios of Fe3O4 and γ-Fe2O3 magnetic nanoparticles, the strength of the magnetic field three is adjusted. By adjusting the strength of the magnetic field three, the distribution and capture efficiency of the magnetic nanoparticles in the biological sample solution can be optimized; when the ratio of γ-Fe2O3 is high, the magnetic field strength can be appropriately increased to enhance its attraction to the magnetic nanoparticles; when the ratio of Fe3O4 is high, the magnetic field strength can be appropriately reduced to avoid excessive attraction leading to particle aggregation and blockage; Fourth, by adding a pre-dispersion zone and adjusting the magnetic field configuration, the capture process of exosomes is further optimized. The magnetic nanoparticles Fe3O4 and γ-Fe2O3 will be affected by the magnetic force in the magnetic field, and the movement trajectory and distribution state will be controlled by the magnetic field strength and gradient; when the biological sample solution flows through channels of different shapes and structures, the flow rate, flow direction and pressure distribution will change; by further optimizing the design of the channel structure, the flow characteristics of the biological sample solution can be adjusted, thereby improving the mixing and separation effect of the magnetic particles and the biological sample; Two middle raised devices, namely the pre-dispersion zone, are added to the outside of the narrowed part of the magnetic separation device. By setting an annular magnetic field, magnetic field four and magnetic field five, the magnetic particles can be pre-dispersed before entering the main separation area, which can avoid the aggregation of magnetic nanoparticles in narrow channels and improve the dispersion uniformity in the biological sample solution; the introduction of magnetic field four and magnetic field five increases the magnetic field control force on the magnetic particles, and by adjusting the intensity and gradient of these two magnetic fields, the movement trajectory and distribution state of the magnetic particles in the pre-dispersion zone and the main separation zone can be more accurately controlled; the optimized magnetic separation device can realize the combination and separation of magnetic particles and exosomes more quickly, thereby shortening the separation time and improving work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the structure of a magnetic separation device according to Example 1 of the present invention; Figure 2 This is a schematic structural diagram of a magnetic separation device according to Embodiment 2 of the present invention; Figure 3 This is a schematic structural diagram of a magnetic separation device according to Embodiment 3 of the present invention; Figure 4 This is a schematic diagram of the structure of the magnetic separation device of Example 4 of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0018] Example 1 A method for purifying and separating exosomes based on magnetic particles, specifically comprising the following steps: Step 1, preparing Fe3O4 magnetic nanoparticles by coprecipitation method; The preparation method of magnetic nanoparticles is as follows: at room temperature of 30°C, 5 mL of ultrapure water and 5 mL of 0.3 mmol / mL FeCl3 aqueous solution are placed in a three-necked bottle, vacuumized for 30 minutes to remove the air in the bottle, and continuously filled with nitrogen for 5 minutes. While maintaining the nitrogen flow and continuous stirring, the stirring speed is 1000 rpm, a pH meter is inserted from the rubber sealing cover of the right side of the three-necked bottle, 2.5 mmol of disodium terephthalate is added, stirred and dissolved at a stirring speed of 1000 rpm for 10 minutes, 1 mmol of FeCl2·4H2O particles is added, stirred and dissolved at a stirring speed of 1000 rpm for 15 minutes, sodium hydroxide solution is added, and the reaction is terminated after continuous stirring at 1000 rpm for 20 minutes, a permanent magnet is used to attract the magnetic nanoparticles outside the three-necked bottle, and after removing the clarified liquid, the ultrapure water is used to repeatedly wash 5 times to obtain Fe3O4 magnetic nanoparticles.

[0019] Step 2, synthesizing a layer of Al-MOF on the surface of the magnetic nanoparticles to prepare magnetic Al-MOF nanoparticles, and preparing them into an aqueous dispersion; Step 3: Adding nucleic acid aptamers targeting CD63 protein to the aqueous dispersion of magnetic Al-MOF nanoparticles to 3+The CD63 nucleic acid aptamer is fixed on the magnetic Al-MOF nanoparticles by electrostatic adsorption with the phosphate group in the CD63 nucleic acid aptamer skeleton, thereby forming magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer; Step 4, mixing the magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer with a trace amount of biological sample; Figure 1 As shown, the mixed solution is placed in a magnetic separation device, and the exosomes are captured on the surface of magnetic Al-MOF nanoparticles modified with CD63 nucleic acid aptamers; The magnetic separation device is a structure with a convex middle and narrowed ends. The convex middle part is a diffusion area, and the narrowed parts at both ends are collection areas. Magnetic fields are set at both ends of the device, namely magnetic field one and magnetic field two. The magnetic field strengths of magnetic field one and magnetic field two are both set within the range of 0.5-1T. The device can be flipped upside down and rotated horizontally. The method for capturing exosomes by a magnetic separation device is specifically as follows: activating magnetic field one and magnetic field two to cause the magnetic nanoparticles to gather at two ends of the device; Turn off the magnetic field to allow the magnetic particles originally adsorbed on the upper magnetic field to fall off naturally. At this time, rotate the separation device horizontally at a speed of 5-20 rpm for 10-30 seconds; The separation device is flipped 180 degrees up and down, and the magnetic particles originally adsorbed on the magnetic fields at both ends fall off naturally through the alternating adsorption of magnetic field one and magnetic field two, and enter the biological sample solution again. After flipping, the separation device is rotated horizontally at a speed of 5-20 rpm; The magnetic field is controlled continuously as well as the upside-down flipping and lateral rotation are performed until the magnetic nanoparticles are evenly distributed in the biological sample solution, thus completing the capture of exosomes.

[0020] Step 5, activating magnetic field 1 and magnetic field 2, so that the magnetic nanoparticles containing captured exosomes are aggregated and adsorbed on both ends of the device under the action of the magnetic field; Step 6: Use filter paper or a pipette to remove the unbound solution at both ends of the device; then, rinse the device with pure water or PBS solution, keeping the magnetic field on during the rinsing process to prevent the magnetic nanoparticles from falling off; repeat the rinsing several times until there are no obvious impurities in the rinsing solution; Step 7, introducing ammonia water with a pH value of 9 or sodium hydroxide alkaline solution with a pH value of 11 into both ends of the magnetic separation device, so that the alkaline solution fully contacts and rinses the magnetic nanoparticles in the device, and after standing for 5-10 minutes, using a pipette or a pipette to collect the rinsing liquid, which contains exosomes released from the magnetic nanoparticles; Step 8: Transfer the washing liquid collected in step 7 to a 100K ultrafiltration tube for ultrafiltration, and repeat the ultrafiltration washing several times until there are no obvious impurities in the ultrafiltrate to obtain a purified exosome suspension. The comparative example was prepared by a method for purifying and separating exosomes based on a magnetic MOF according to a Chinese patent application number CN202110719284.X; the difference between the comparative example and this embodiment is that no magnetic separation device is used; Performance testing; Exosome concentration analysis; The obtained exosome suspension was added to the exclusion column. When the liquid was completely drained, 2.5 ml PBS was added and the effluent was collected. The effluent was transferred to an ultrafiltration tube and centrifuged at 3000-5000 g for 1-5 min at 4°C. 200-500 μL of the retentate was retained and the concentration of the exosomes in the retentate was detected using a nanoparticle tracking analyzer (ZetaView PMX110). After testing, the exosome concentration of the comparative ratio was 5.0×10 6 / μL; the detection result of the exosome concentration in this example is 1.5×10 7 Pieces / μL.

[0021] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: By introducing a magnetic separation device, the uniform distribution of magnetic nanoparticles in the biological sample solution is promoted, so as to more effectively capture exosomes. The magnetic separation device is a structure with a convex middle and narrowed at both ends. The convex middle part is the diffusion area, and the narrowed parts at both ends are the collection area. Magnetic field one and magnetic field two are set at both ends of the device. The magnetic separation device uses the adsorption effect of the magnetic field on magnetic nanoparticles, so that the magnetic Al-MOF nanoparticles modified with CD63 nucleic acid aptamers gather at the two ends of the device under the action of the magnetic field; when the magnetic field is turned off or changes direction, the magnetic nanoparticles will lose the restraint of the magnetic field, so they can fall off the device and redisperse into the biological sample solution; By flipping the device upside down and rotating it horizontally, as well as the alternating adsorption of the magnetic field, the magnetic nanoparticles and the biological sample solution can be effectively separated and mixed. At the same time, this physical separation method can avoid the use of chemical reagents and reduce the contamination and damage to the biological sample. The magnetic separation device uses the magnetic field to rapidly aggregate magnetic nanoparticles in the biological sample solution, thereby increasing the chance of contact with exosomes and the capture efficiency; the flipping and rotation of the device further promotes the uniform mixing of magnetic nanoparticles and exosomes, ensuring the comprehensiveness and uniformity of the capture; Through simple magnetic field switching and device movement, magnetic nanoparticles can be quickly separated from biological sample solutions without the need for complex centrifugation or filtration operations; this greatly shortens the separation time and improves work efficiency; the magnetic separation process is a gentle physical separation method that has little effect on the activity of biological samples and can maintain the integrity and biological activity of exosomes.

[0022] Example 2 The above-mentioned Example 1 introduces a magnetic separation device to promote the uniform distribution of magnetic nanoparticles in the biological sample solution, thereby more efficiently and fully capturing exosomes. In order to further improve the effective capture of exosomes, further improvements are made on the basis of Example 1.

[0023] like Figure 2 As shown, the material of the diffusion zone in the magnetic field separation device is a flexible material that can shrink or expand; The flexible material is any one of silicone, rubber or polyurethane.

[0024] Experiments were conducted on this solution based on Example 1. The difference from Example 1 is that silica gel is used in the diffusion zone of the magnetic field separation device; After testing, the exosome concentration of this example was 1.7×10 7 Pieces / μL.

[0025] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Flexible materials such as silicone, rubber and polyurethane have good elasticity and plasticity, and can deform under external forces, including contraction and expansion; these materials can maintain structural integrity during deformation and are not prone to rupture or permanent deformation; this property enables the diffusion zone to adjust its spatial configuration according to actual needs, thereby optimizing fluid dynamics conditions; Magnetic separation technology relies on the specific binding of magnetic nanoparticles to exosomes in biological samples. When the flexible material is used as the diffusion area, the fluid dynamic conditions can be changed by adjusting its shape and size according to the different contents or types of biological samples and magnetic nanoparticles, thereby affecting the dispersion state of magnetic nanoparticles. The contraction or expansion of the diffusion area can change the flow rate and direction of the fluid, as well as the contact area between the magnetic nanoparticles and the biological sample solution, thereby optimizing the binding efficiency of the magnetic nanoparticles and the target molecules. The contraction or expansion of the flexible material can more accurately control the dispersion state of the magnetic nanoparticles in the biological sample solution, so that they can cover the surface of the target molecule more evenly. By adjusting the contraction or expansion of the diffusion zone, the contact area and contact time between the magnetic nanoparticles and the biological sample can be precisely controlled, thereby improving the efficiency of the magnetic nanoparticles in capturing exosomes. For biological samples and magnetic nanoparticles of different contents or types, the viscosity of the sample, the concentration of the magnetic nanoparticles and the number of exosomes are all different. The flexible diffusion zone can be adaptively adjusted according to the characteristics of the sample, and its shape and size can be flexibly adjusted to ensure that the magnetic nanoparticles reach the best dispersion state in the sample solution to meet different separation requirements, thereby enhancing the adaptability and flexibility of the device. The deformation of flexible materials can change the flow pattern of the fluid in the diffusion area, promoting more effective binding of magnetic nanoparticles to exosomes; by precisely controlling the dispersion state of magnetic nanoparticles, flexible materials can reduce the occurrence of nonspecific binding, thereby improving separation purity and the accuracy of subsequent analysis.

[0026] Example 3 The above-mentioned Example 2 sets the material of the diffusion zone in the magnetic field separation device as a flexible material, thereby more efficiently and fully capturing exosomes, thereby enhancing the adaptability and flexibility of the device. In order to further improve the effective capture of exosomes, further improvements are made on the basis of Example 2.

[0027] like Figure 3 As shown, a circular magnetic field is also set in the diffusion zone of the magnetic field separation device, which is magnetic field three, and the magnetic field strength of magnetic field three is 0.1-1T; the magnetic nanoparticles also include γ-Fe2O3 magnetic nanoparticles, and the ratio of Fe3O4 magnetic nanoparticles to γ-Fe2O3 magnetic nanoparticles is (1-5):1.

[0028] The present invention is conducted on the basis of Example 2. The difference from Example 3 is that a circular magnetic field is set in the diffusion zone of the magnetic field separation device, which is the magnetic field three, and the magnetic field strength of the magnetic field three is 0.1-1T; the magnetic nanoparticles also include γ-Fe2O3 magnetic nanoparticles, and the ratio of Fe3O4 magnetic nanoparticles to γ-Fe2O3 magnetic nanoparticles is 3:1; After testing, the exosome concentration of this example was 2.2×10 7 Pieces / μL.

[0029] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: By introducing a circular magnetic field and adjusting the ratio of Fe3O4 and γ-Fe2O3 magnetic nanoparticles, the capture efficiency and separation purity of exosomes were further improved; the magnetic field strength directly affects the magnetic force on the magnetic nanoparticles, which in turn affects their capture efficiency. According to the different ratios of Fe3O4 and γ-Fe2O3 magnetic nanoparticles, the strength of the magnetic field three is adjusted. By adjusting the strength of the magnetic field three, the distribution and capture efficiency of the magnetic nanoparticles in the biological sample solution can be optimized; when the ratio of γ-Fe2O3 is high, the magnetic field strength can be appropriately increased to enhance its attraction to the magnetic nanoparticles; when the ratio of Fe3O4 is high, the magnetic field strength can be appropriately reduced to avoid excessive attraction leading to particle aggregation and blockage; The magnetic field gradient determines the difference in force on the magnetic nanoparticles in the magnetic field, thus affecting their separation effect. By adjusting the magnetic field gradient of the annular magnetic field, when the ratio of Fe3O4 and γ-Fe2O3 magnetic nanoparticles changes, it is ensured that the magnetic field gradient can fully distinguish and guide particles of different magnetic properties to move in different directions. It can more accurately guide particles of different magnetic properties to move in different directions, thereby improving the separation effect and further improving the distribution and capture efficiency of magnetic nanoparticles in biological sample solutions. Fe3O4 and γ-Fe2O3 magnetic nanoparticles have different magnetic properties. The ratio of the two magnetic nanoparticles can be flexibly adjusted according to the specific biological samples and exosome characteristics. When higher capture efficiency is required, the proportion of Fe3O4 can be increased; and when better separation effect is required, the proportion of γ-Fe2O3 can be increased to meet different separation requirements.

[0030] Example 4 The above-mentioned Example 3 improves the capture efficiency and separation purity of exosomes by introducing a circular magnetic field and adjusting the ratio of Fe3O4 and γ-Fe2O3 magnetic nanoparticles, and also enhances the adaptability and flexibility of the device. In order to further improve the effective capture of exosomes, further improvements are made on the basis of Example 3.

[0031] like Figure 4 As shown, two smaller middle protrusion devices are added outside the narrowed part of the magnetic separation device, which is the pre-dispersion area; two annular magnetic fields are set in the pre-dispersion area, namely magnetic field four and magnetic field five; the magnetic field strength of magnetic field four and magnetic field five is 0.1-3T.

[0032] Experiments were conducted on the basis of Example 3 for this solution. The difference from Example 3 is that two smaller middle protrusions are added outside the narrowing part of the magnetic separation device, which are pre-dispersion areas; and an annular magnetic field is set in the pre-dispersion area, which are magnetic field four and magnetic field five; the magnetic field strengths of magnetic field four and magnetic field five are 0.1-3T respectively; After testing, the exosome concentration of this example was 2.7×10 7Pieces / μL.

[0033] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: By adding a pre-dispersion zone and adjusting the magnetic field configuration, the capture process of exosomes was further optimized. The magnetic nanoparticles Fe3O4 and γ-Fe2O3 will be affected by the magnetic force in the magnetic field, and the movement trajectory and distribution state will be controlled by the magnetic field strength and gradient. When the biological sample solution flows through channels of different shapes and structures, the flow rate, flow direction and pressure distribution will change. By further optimizing the design of the channel structure, the flow characteristics of the biological sample solution can be adjusted, thereby improving the mixing and separation effect of the magnetic particles and the biological sample. Two intermediate protrusions, namely the pre-dispersion zone, are added outside the narrowed portion of the magnetic separation device. By setting an annular magnetic field, magnetic field four and magnetic field five, the magnetic particles can be pre-dispersed before entering the main separation zone, which can prevent the magnetic nanoparticles from aggregating in the narrow channel and improve the dispersion uniformity in the biological sample solution. The introduction of magnetic field four and magnetic field five increases the magnetic field control force on the magnetic particles. By adjusting the strength and gradient of the two magnetic fields, the movement trajectory and distribution state of the magnetic particles in the pre-dispersion zone and the main separation zone can be more accurately controlled. The setting of the pre-dispersion zone enables the magnetic particles to be better dispersed before entering the main separation area, thereby increasing their contact opportunities with exosomes and improving the capture efficiency; by enhancing the magnetic field control, the magnetic particles that capture exosomes can be more effectively separated from the unbound magnetic particles and the biological sample solution, thereby improving the separation purity; the introduction of the pre-dispersion zone and magnetic fields four and five enables the magnetic separation device to process more complex and diverse biological samples, improving the adaptability of the magnetic separation device; by adjusting the magnetic field strength and gradient, as well as the structure of the pre-dispersion zone, it can adapt to the characteristics and separation requirements of different samples; the optimized magnetic separation device can achieve the combination and separation of magnetic particles and exosomes more quickly, thereby shortening the separation time and improving work efficiency.

[0034] It should be noted that in the above embodiments, the description of each embodiment has its own emphasis, and for parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0035] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0036] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for purifying and separating exosomes based on magnetic particles, characterized in that: The specific steps include: Step 1, preparing Fe3O4 magnetic nanoparticles by coprecipitation method; Step 2, synthesizing a layer of Al-MOF on the surface of the magnetic nanoparticles to prepare magnetic Al-MOF nanoparticles, and preparing them into an aqueous dispersion; Step 3: Adding nucleic acid aptamers targeting CD63 protein to the aqueous dispersion of magnetic Al-MOF nanoparticles to 3+ The CD63 nucleic acid aptamer is fixed on the magnetic Al-MOF nanoparticles by electrostatic adsorption with the phosphate group in the CD63 nucleic acid aptamer skeleton, thereby forming magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer; Step 4, mixing the magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer with a trace amount of biological sample; placing the mixed solution in a magnetic separation device, and the exosomes are captured on the surface of the magnetic Al-MOF nanoparticles modified with the CD63 nucleic acid aptamer; Step 5, activating magnetic field 1 and magnetic field 2, so that the magnetic nanoparticles containing captured exosomes are aggregated and adsorbed on both ends of the device under the action of the magnetic field; Step 6: Use filter paper or a pipette to remove the unbound solution at both ends of the device; then, rinse the device with pure water or PBS solution, keeping the magnetic field on during the rinsing process to prevent the magnetic nanoparticles from falling off; repeat the rinsing several times until there are no obvious impurities in the rinsing solution; Step 7, introducing ammonia water with a pH value of 9 or sodium hydroxide alkaline solution with a pH value of 11 into both ends of the magnetic separation device, so that the alkaline solution fully contacts and rinses the magnetic nanoparticles in the device, and after standing for 5-10 minutes, using a pipette or a pipette to collect the rinsing liquid, which contains exosomes released from the magnetic nanoparticles; Step 8: Transfer the washing liquid collected in step 7 to a 100K ultrafiltration tube for ultrafiltration, and repeat the ultrafiltration washing several times until there are no obvious impurities in the ultrafiltrate to obtain a purified exosome suspension.

2. The method for purifying and isolating exosomes based on magnetic particles according to claim 1, characterized in that: In step 4, the magnetic separation device is a structure with a convex middle and narrowed two ends, the convex middle part is the diffusion area, and the narrowed parts at both ends are the collection areas; magnetic fields are set at both ends of the device, namely magnetic field one and magnetic field two, and the magnetic field strengths of magnetic field one and magnetic field two are both set within the range of 0.5-1T; the device can be flipped upside down and rotated laterally.

3. The method for purifying and isolating exosomes based on magnetic particles according to claim 2, characterized in that: The method for capturing exosomes by a magnetic separation device is specifically as follows: activating magnetic field one and magnetic field two to cause the magnetic nanoparticles to gather at two ends of the device; Turn off the magnetic field to allow the magnetic particles originally adsorbed on the upper magnetic field to fall off naturally. At this time, the separation device is rotated horizontally at a speed of 5-20 rpm for 10-30 seconds; The separation device is flipped 180 degrees up and down, and the magnetic particles originally adsorbed on the magnetic fields at both ends fall off naturally through the alternating adsorption of magnetic field one and magnetic field two, and enter the biological sample solution again. After flipping, the separation device is rotated horizontally at a speed of 5-20 rpm; The magnetic field is continuously controlled as well as flipped upside down and rotated laterally until the magnetic nanoparticles are evenly distributed in the biological sample solution, thus completing the capture of exosomes.

4. The method for purifying and isolating exosomes based on magnetic particles according to claim 1, characterized in that: The material of the diffusion zone in the magnetic field separation device is a flexible material and can shrink or expand.

5. The method for purifying and isolating exosomes based on magnetic particles according to claim 4, characterized in that: The flexible material is any one of silicone, rubber or polyurethane.

6. The method for purifying and isolating exosomes based on magnetic particles according to claim 4, characterized in that: A circular magnetic field is also arranged in the diffusion zone of the magnetic field separation device, which is the magnetic field three. The magnetic field strength of the magnetic field three is 0.1-1T.

7. The method for purifying and isolating exosomes based on magnetic particles according to claim 6, characterized in that: The magnetic nanoparticles also include γ-Fe2O3 magnetic nanoparticles, and the ratio of Fe3O4 magnetic nanoparticles to γ-Fe2O3 magnetic nanoparticles is (1-5):

1.

8. The method for purifying and isolating exosomes based on magnetic particles according to claim 6, characterized in that: Two smaller middle protrusions are also provided outside the narrowed portion of the magnetic separation device, which serve as pre-dispersion areas.

9. The method for purifying and isolating exosomes based on magnetic particles according to claim 8, characterized in that: Two annular magnetic fields are set in the pre-dispersion area, namely magnetic field four and magnetic field five.

10. The method for purifying and isolating exosomes based on magnetic particles according to claim 8, characterized in that: The magnetic field strengths of magnetic field four and magnetic field five are both set within the range of 0.1-3T.

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