Method for extracting bifunctional modified magnetic beads and exosome in cerebrospinal fluid

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a bifunctional modified magnetic bead and an extraction method of exosomes in cerebrospinal fluid. Aiming at the defects of an existing magnetic bead capture method in the aspect of removing impure protein, the invention firstly provides a bifunctional modified magnetic bead, a CD63 antibody and a sulphobetaine type zwitterionic polymer are modified on the surface of the bifunctional modified magnetic bead, specific capture of the exosome is realized on the basis of antigen-antibody specific binding of CD63 and the exosome, and meanwhile, the adsorption effect on the impure protein is reduced. The magnetic beads are applied to extraction of exosomes in cerebrospinal fluid, and the technical defects that the sample size of the cerebrospinal fluid is small, and the recovery rate of a traditional method is not high are effectively overcome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical separation technology, specifically relating to a bifunctional modified magnetic bead, a method for preparing the magnetic bead, and its application in exosome extraction. It also provides a method for extracting exosomes from cerebrospinal fluid. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Exosomes, extracellular vesicles enclosed in a lipid bilayer membrane with a diameter of approximately 30-150 nanometers, are widely distributed in bodily fluids such as blood, cerebrospinal fluid, and urine, and serve as important mediators of intercellular signaling. Cerebrospinal fluid exosomes, in particular, originate directly from central nervous system cells (such as neurons, glial cells, and choroid plexus cells) and carry abundant central nervous system-specific biological information, making them key research subjects for elucidating the pathogenesis of neurological diseases and developing diagnostic biomarkers and targeted therapeutic vectors. In neurological disease research, cerebrospinal fluid exosomes carry central nervous system-specific biomarkers, such as Aβ oligomers in Alzheimer's disease and EGFRvIII mutant protein in gliomas, making them key targets for liquid biopsies.

[0004] From a clinical application perspective, the research value of cerebrospinal fluid exosomes is reflected in multiple dimensions: In the field of neurodegenerative diseases, the Aβ oligomers and tau protein phosphorylated isoforms they carry are potential biomarkers for the early diagnosis of Alzheimer's disease; in the field of nervous system tumors, molecules such as EGFRvIII mutant protein and miR-21 contained in exosomes secreted by glioma cells can provide a basis for tumor grading, prognostic assessment, and targeted therapy; in autoimmune encephalopathy (such as multiple sclerosis), the autoantibodies and inflammatory factors carried by exosomes can reflect disease activity. Furthermore, as natural nanoscale carriers, the high affinity of exosome membrane structures for target cells makes exosomes a promising candidate for targeted drug delivery in neurological diseases. For example, by modifying the targeting peptides on the surface of exosomes, drugs can be precisely delivered to lesions behind the blood-brain barrier, reducing systemic toxicity.

[0005] Currently, exosome extraction techniques mainly include ultracentrifugation, polymer precipitation, size exclusion chromatography, and magnetic bead immunocapture. Ultracentrifugation, as the traditional gold standard, can separate exosomes, but its recovery efficiency is low for minute cerebrospinal fluid samples (0.5-2 mL), and the intense physical forces generated by prolonged high-speed centrifugation can easily damage the exosome membrane structure. Polymer precipitation is simple to operate, but the co-precipitation of impurities caused by chemical reagents results in extracts containing large amounts of lipoproteins and soluble proteins, severely interfering with downstream analysis. Size exclusion chromatography, while gentle in its separation process, is time-consuming (up to several hours) and expensive, making it difficult to meet the time-sensitive requirements of clinical diagnosis. Magnetic bead immunocapture, with its targeting and high efficiency, utilizes magnetic beads modified with antibodies such as anti-CD63 / CD81 to specifically bind to exosomes, completing the processing of minute cerebrospinal fluid samples within 1-2 hours, making it the mainstream method.

[0006] However, high concentrations of albumin (accounting for over 60% of total protein) and immunoglobulins in cerebrospinal fluid (CSF) can non-specifically adhere to the surface of magnetic beads through hydrophobic interactions or electrostatic attraction, forming a difficult-to-remove "protein contaminant." Existing magnetic bead kits using neutral buffers (such as PBS) cannot effectively dissociate these impurities. Non-specific binding between antibodies and contaminating proteins, along with the hydrophobic adsorption of lipids onto magnetic beads, results in insufficient elution efficiency. Research data indicates that impurity proteins account for as much as 25-40% of CSF exosomes extracted using traditional magnetic bead methods, severely impacting exosome purity and hindering their application in the precise diagnosis of neurological diseases. For example, protein coronas obscure the characteristic morphology of exosomes under electron microscopy; high-abundance contaminating proteins mask low-abundance disease-related biomarkers in proteomics; and non-exosome-derived nucleic acids lead to false positive results during RNA sequencing. Therefore, a method that can solve these problems and achieve high-purity, high-recovery extraction of CSF exosomes is urgently needed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to overcome the deficiencies of current magnetic bead capture methods in the extraction of cerebrospinal fluid exosomes, and provides an improved method for extracting cerebrospinal fluid exosomes using magnetic bead capture. This method effectively removes impurities and proteins, significantly improves exosome purity, and ensures a high exosome recovery rate. It is suitable for micro-volume cerebrospinal fluid samples and provides reliable technical support for the early diagnosis of neurological diseases.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a bifunctional modified magnetic bead, wherein the magnetic bead is used as a substrate, and the surface of the magnetic bead is coupled with a CD63 antibody and grafted with a sulfobetaine-type zwitterionic polymer.

[0009] In the bifunctional modified magnetic beads provided in the first aspect, CD63, as an exosome-specific membrane protein, can achieve targeted capture of exosomes through antigen-antibody reactions. A sulfobetaine-type zwitterionic polymer is covalently grafted onto the surface of the antibody-conjugated magnetic beads. This polymer possesses a unique "hydration layer barrier effect": the positively and negatively charged groups in its molecular structure can adsorb water molecules through electrostatic interactions, forming a dense hydration film that prevents albumin, immunoglobulins, and other impurities from adhering to the magnetic bead surface through hydrophobic or electrostatic interactions. Experimental data show that grafting sulfobetaine reduces the amount of impurities adsorbed on the magnetic bead surface by 60-70%.

[0010] Furthermore, the preferred size of the magnetic beads is 200-300 nm. This particle size can ensure a large specific surface area, improve antibody modification density, and enable rapid separation by a magnetic rack (magnetic adsorption is completed within 5 minutes), balancing capture efficiency and ease of operation. Furthermore, superparamagnetic nanoparticles are selected.

[0011] Furthermore, the aforementioned CD63 antibody is a magnetic bead with a capture molecule specifically binding to the human CD63 protein conjugated to its surface. The capture molecule is preferably an antibody, more preferably a monoclonal antibody. In one specific embodiment of the present invention, a commercially available monoclonal antibody (clone number MEM-259) recognizing the second extracellular circular epitope of CD63 is used. Those skilled in the art will understand that other antibodies, aptamers, or ligands specifically binding to CD63 can also be used in this invention.

[0012] Furthermore, the monomer structure of the sulfobetaine-type zwitterionic polymer is [-CH2-C(CH3)(COO-CH2-CH2-N]. + (CH3)2-CH2-CH2-SO3 - The monomer 2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate has the CAS number 3637-26-1, a degree of polymerization between DP=200-300, and a molecular weight of 5.6×10⁻⁶. 4 -8.4×10 4 g / mol.

[0013] Furthermore, in the above-mentioned bifunctional modified magnetic beads, the mass ratio of the CD63 antibody to the sulfobetaine-type zwitterionic polymer is 1:10-1:50, preferably 1:20-1:30.

[0014] In a second aspect, the present invention provides a method for preparing the bifunctional modified magnetic beads described in the first aspect, comprising the following steps: placing the magnetic beads in a buffer solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and shaking at room temperature to activate the surface; adding the activated magnetic beads to a CD63 antibody solution for coupling to obtain antibody-modified magnetic beads; and resuspending the antibody-modified magnetic beads in a buffer solution of a sulfobetaine-type zwitterionic polymer for grafting to obtain bifunctional modified magnetic beads.

[0015] Furthermore, the surface of the aforementioned magnetic nanoparticles should be clean before they participate in the reaction, and they can be washed with PBS buffer to remove impurities.

[0016] Furthermore, in the EDC and NHS buffer solution, the concentration of EDC is 40-60 mM, the concentration of NHS is 80-120 mM, and the pH of the buffer solution is 5.5-6.5.

[0017] Furthermore, the time for the above-mentioned room temperature oscillation is 20~40 min, even more preferably 22~38 min, and more preferably 25~35 min.

[0018] Furthermore, the concentration of the CD63 antibody solution is 4~6 μg / 100 μL magnetic beads, and the beads are refrigerated overnight to obtain antibody-modified magnetic beads.

[0019] Furthermore, the buffer solution of the sulfobetaine-type zwitterionic polymer has a concentration of 1~4 mg / mL.

[0020] In a third aspect, the present invention provides the application of the bifunctionalized magnetic beads described in the first aspect in the field of exosome capture.

[0021] In a fourth aspect, the present invention provides a method for extracting exosomes from cerebrospinal fluid, characterized in that the exosomes are captured by targeting and binding with the bifunctionalized magnetic beads described in the first aspect, and then the exosomes are dissociated from the magnetic beads by a composite elution buffer; the composite elution buffer uses a weakly acidic buffer solution as a matrix and includes a detergent and a competing peptide.

[0022] The above extraction method uses bifunctional magnetic beads as specific capture elements for exosomes, achieving exosome separation based on magnetism, followed by separation of exosomes from the magnetic beads using a composite elution buffer. The composite elution buffer uses an acidic buffer as a matrix. Antibody-antigen binding depends on hydrogen bonding and hydrophobic interactions under neutral conditions; weakly acidic conditions can disrupt these interactions, reducing antibody-antigen binding affinity (KD value from 10). -9 M rises to 10 -7M), providing a basis for exosome dissociation; the composite eluent also contains a competitive elution component. This component is a commercially available antigenic peptide that specifically binds to the anti-CD63 antibody on the magnetic beads. At high working concentrations, this peptide directly and efficiently occupies the antibody binding sites on the magnetic beads through kinetic competition, thereby displacing the captured exosomes and significantly improving elution efficiency; in addition, the detergent can disrupt the hydrophobic interaction between the exosome membrane and the magnetic bead surface by intercalating into the lipid bilayer, reducing interference from non-specific adsorption.

[0023] The above extraction method specifically includes the following steps: (1) Centrifuge the cerebrospinal fluid sample at 2000-4000×g for 10-20 min at 2-8℃ and retain the supernatant; (2) Add the bifunctional modified magnetic beads described in the first aspect to the supernatant above, and incubate at 2~8℃ and 100~300rpm for 0.5~2h. After incubation, separate the magnetic beads from the liquid based on magnetism. The low temperature environment can reduce protein denaturation, and the shaking can promote the full contact between the magnetic beads and exosomes. The targeted capture of exosomes is achieved by the specific binding of anti-CD63 antibody to CD63 protein on the surface of exosomes. (3) Add the magnetic beads to the composite elution solution and shake and incubate for 10-20 minutes. Collect the supernatant to obtain the product.

[0024] In step (1) above, large particulate impurities such as cells and cell debris in the sample are first removed by centrifugation, and the supernatant is used for subsequent extraction. This step can avoid large particulate matter interfering with the binding of magnetic beads and exosomes, thereby improving capture specificity; furthermore, the centrifugation temperature is 2~6℃, the centrifugation speed is 2500~3500×g, and the centrifugation time is 12~17min.

[0025] In step (2) above, the dosage ratio of supernatant to magnetic beads is 0.4~0.6mL:50μL.

[0026] In step (3) above, the composite eluent uses 10-30mM Tris-HCl buffer as the matrix, and further, the pH is 5.5-6.0, and also includes a detergent and a competitive peptide; the detergent is 0.05-0.1% Triton X-100, preferably 0.08%; the competitive peptide is a small peptide designed for the CD63 antigen epitope, and its concentration is 80-120μM.

[0027] Compared with the prior art, the beneficial effects of the present invention are: The most critical challenge in cerebrospinal fluid (CSF) exosome extraction lies in the efficient extraction and high-purity separation of minute samples. Clinically obtained CSF samples are typically only 1-2 mL, and for some special patients (such as children or critically ill patients), the sample volume may even be less than 0.5 mL, while the exosome concentration per milliliter of CSF is only 10. 8 -10 9 The samples contain numerous proteins (total protein concentration approximately 0.15-0.45 g / L), lipids, and cell debris. Existing extraction technologies struggle to balance "recovery rate" and "purity": traditional methods may result in the loss of low-abundance biomarkers due to excessively low recovery rates, or the introduction of large amounts of contaminating proteins due to insufficient purity, interfering with downstream detection and severely limiting the application of exosomes in clinical diagnosis.

[0028] This invention addresses this technical challenge by innovatively combining magnetic bead surface functionalization with a composite elution system to construct an exosome extraction protocol suitable for trace amounts of cerebrospinal fluid samples. This method not only efficiently captures exosomes (recovery rate ≥85%) but also significantly reduces contamination by other proteins (protein removal rate ≥90%), providing high-quality samples for downstream proteomics analysis (e.g., mass spectrometry), nucleic acid analysis (e.g., qPCR, high-throughput sequencing), and morphological observation (e.g., transmission electron microscopy). The technological achievements can be directly applied to the development of clinical diagnostic kits for neurological diseases, basic exosome research in research institutions, and the development of exosome drug carriers in biopharmaceutical companies. Furthermore, the technical approach of this invention can be extended to the extraction of exosomes from other body fluids (e.g., blood, urine), and is particularly suitable for complex biological matrices with limited sample volume and high levels of contaminating proteins, providing a new solution for separation technology in the field of extracellular vesicles. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a flowchart of the method for extracting cerebrospinal fluid exosomes in this invention; Figure 2 The graph shows the recovery rate of exosomes and the effect of removing contaminating proteins in Example 2 and Comparative Examples 1-2. Figure 2 In the middle, A is the histogram of exosome recovery rate. Figure 2 B is a histogram showing the removal efficiency of extraneous proteins from exosomes; Figure 3 These are morphological comparison images of exosomes extracted in Example 2 and Comparative Examples 1-2; Figure 3 Image A is a transmission electron microscope image of exosomes extracted using the method in Comparative Example 1. Figure 3 Image B is a transmission electron microscope image of exosomes extracted using the method in Example 2; Figure 4 The results of Western blot verification of exosomes extracted in Example 2 and Comparative Examples 1-3; Figure 5 Detection of particle size distribution and concentration of exosomes in raw cerebrospinal fluid and exosomes extracted in Example 2; Figure 5 A in the diagram shows the particle size distribution of exosomes in the raw cerebrospinal fluid. Figure 5 Figure B shows the particle size distribution of exosomes extracted in Example 2. Detailed Implementation

[0031] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] In the context of this specification, the word "including" is considered to mean "particularly including". It should not be interpreted as "consisting of only".

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0035] Example 1 In this embodiment, a bifunctional modified magnetic bead is first provided, which uses superparamagnetic nanoparticles as a substrate for surface modification. The modification method includes the following steps: 1. Activation: Take 100 μL of magnetic beads and wash them three times with 0.01 M PBS buffer (pH 7.4). After each wash, place the beads on a magnetic rack and let them stand for 2 minutes. Discard the supernatant to remove impurities. Resuspend the washed magnetic beads in 1 mL of activation buffer (0.1 M MES, pH 6.0) containing 50 mM MEDC and 100 mM NHS. Activate the beads by shaking at 200 rpm for 30 minutes at room temperature (25 °C) to activate the carboxyl groups on the surface of the magnetic beads.

[0036] 2. Antibody Coupling: After activation, place the magnetic beads on a magnetic rack and let them stand for 2 minutes. Discard the supernatant and wash twice with 0.01M PBS. Then add 100μL of PBS solution containing 5μg of anti-CD63 antibody (antibody diluted with PBS to 50μg / mL), and incubate overnight (12 hours) at 4°C with shaking at 150rpm to allow the antibody to couple to the surface of the magnetic beads via amide bonds.

[0037] 3. Blocking unreacted sites: After incubation, wash the magnetic beads three times with 0.01M PBS to remove unbound free antibodies. Add 1 mL of PBS solution containing 1% bovine serum albumin (BSA), and incubate at room temperature with shaking for 30 minutes to block unreacted activation sites on the surface of the magnetic beads, thus avoiding non-specific binding in subsequent experiments.

[0038] 4. Zwitterionic polymer grafting: Discard the blocking solution, wash the magnetic beads twice with 0.01M PBS, resuspend them in 1mL of reaction buffer (0.1M Tris-HCl, pH 7.4) containing 2mg / mL sulfobetaine, and react at room temperature with shaking at 200rpm for 2 hours to allow sulfobetaine to be covalently grafted onto the surface of the magnetic beads (the grafting rate can reach over 85% as determined by fluorescent labeling).

[0039] 5. Storage: After the reaction is complete, wash the magnetic beads three times with 0.01M PBS, and finally resuspend them in 100μL of PBS containing 0.02% sodium azide. Store at 4℃ for up to 30 days.

[0040] Example 2 This embodiment provides a method for separating exosomes from cerebrospinal fluid, including targeted binding of bifunctional modified magnetic beads prepared in Example 1 to exosomes in cerebrospinal fluid. The specific steps of this separation method are as follows: 1. Cerebrospinal Fluid Sample Pretreatment: Remove the cerebrospinal fluid sample frozen at -80℃ and thaw it slowly in an ice box (avoid rapid thawing at room temperature to prevent exosome membrane rupture). Immediately after thawing, centrifuge at 3000×g for 15 minutes at 4℃ (centrifugation radius 8cm). Transfer the supernatant to a new 1.5mL centrifuge tube to remove cells, cell debris, and large particulate impurities. If the sample is not to be processed immediately, it can be aliquoted and stored again at -80℃, avoiding repeated freeze-thaw cycles more than twice.

[0041] 2. Magnetic bead-targeted binding of exosomes: Take 0.5 mL of pretreated cerebrospinal fluid supernatant and add 50 μL of the bifunctional modified magnetic beads prepared in Example 1 (the concentration of magnetic beads was adjusted to 1 × 10⁻⁶ by counting). 9After gently mixing by pipetting, place the mixture in a 4°C constant temperature shaker and incubate at 180 rpm for 1 hour to allow the anti-CD63 antibody on the surface of the magnetic beads to specifically bind to the CD63 protein on the surface of the exosomes.

[0042] After incubation, place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed. Carefully discard the supernatant (avoid aspirating the magnetic beads). Wash the magnetic beads three times with 0.01M PBS buffer (pH 7.4), adding 1 mL of PBS each time. Gently pipette to resuspend the magnetic beads, then place them on a magnetic rack and let them stand for 3 minutes. Discard the supernatant to remove unbound proteins and other impurities.

[0043] 3. Separation of exosomes using a composite elution system Preparation of the compound elution buffer: Prepare 20mM Tris-HCl buffer (pH 5.8) in advance, add Triton X-100 to the final concentration of 0.08%, add CD63 specific competitive peptide to the final concentration of 100μM, mix thoroughly, filter through a 0.22μm filter membrane for sterilization, and store at 4℃ for 7 days.

[0044] Elution procedure: Add 100 μL of composite elution buffer to the washed magnetic beads, gently blow to completely resuspend the magnetic beads, and incubate at room temperature (25°C) at 200 rpm for 15 minutes (during the shaking process, gently invert the centrifuge tube once every 5 minutes to ensure that the elution buffer is in full contact with the magnetic beads).

[0045] Collecting exosomes: After incubation, place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed. Then, carefully aspirate the supernatant (about 90-95 μL) into a new centrifuge tube. This is the extracted exosome solution, which can be used immediately for downstream experiments or stored at -80°C (for short-term storage, it is recommended to use it within 1 week; for long-term storage, it needs to be aliquoted to avoid repeated freeze-thaw cycles).

[0046] Comparative Example 1 In this embodiment, a method for separating exosomes using magnetic beads is provided, the steps of which are as follows: 1. Sample pretreatment: Centrifuge cerebrospinal fluid at 4℃ and 2000×g for 10 min, discard the precipitate (cell debris), and keep the supernatant for later use (no dilution required to avoid sample loss).

[0047] 2. Preparation of magnetic beads: Take CD63 antibody-conjugated magnetic beads and vortex to mix well; wash twice with PBS at a ratio of 30-50 μL of magnetic beads per 1 mL of cerebrospinal fluid, and resuspend to prepare the working solution of magnetic beads.

[0048] 3. Targeted binding: Mix the magnetic bead working solution with the cerebrospinal fluid supernatant thoroughly, and incubate at 4°C and 150 rpm for 60 min (vortex gently for 10 s every 15 min).

[0049] 4. Washing and purification: Adsorb magnetically for 2 min, discard the supernatant; wash 3 times with 0.5 mL PBST (containing 0.05% Tween-20), and finally resuspend the magnetic bead-exosome complex in 10-30 μL PBS for later use.

[0050] Comparative Example 2 In this embodiment, a method for ultracentrifugation separation of exosomes is provided, the specific steps of which are as follows: 1. Sample pretreatment: Take cerebrospinal fluid samples, centrifuge at 2000×g for 15 min at 4℃, and discard the precipitate (a small amount of cell debris); filter the supernatant through a 0.22μm filter membrane to remove impurities >220nm (to avoid clogging the ultracentrifuge tube).

[0051] 2. High-speed centrifugation to remove impurities: After filtration, transfer the supernatant to a high-speed centrifuge tube, centrifuge at 4℃ and 10000×g for 30 min, discard the precipitate (apoptotic bodies, large vesicles), and retain the supernatant.

[0052] 3. Ultracentrifugation enrichment: Transfer the supernatant to a pre-cooled ultracentrifuge tube, add exosome-free PBS to the rated volume of the centrifuge tube (e.g., 5 mL), with a balance weight difference ≤ 0.1 g; centrifuge at 4℃, 100000×g for 70 min, discard the supernatant, and the milky white precipitate at the bottom of the tube is the crude exosome extract.

[0053] 4. Washing and purification: Add 0.5-1 mL of pre-cooled PBS to the precipitate and gently resuspend by pipetting; centrifuge again at 4℃ and 100,000×g for 70 min, and discard the supernatant.

[0054] 5. Resuspension and preservation: Resuspend the precipitate in 10-50 μL of pre-cooled PBS (adapt the volume to subsequent detection), aliquot and freeze at -80℃ (avoid repeated freeze-thaw cycles).

[0055] Performance verification The exosomes prepared in Example 2 and Comparative Examples 1-2 were tested, and the results are as follows: 1. Exosome purity detection SDS-PAGE electrophoresis analysis: Take 20 μL of the extracted exosome solution, add 5 μL of 5×SDS loading buffer, boil at 100℃ for 5 minutes, cool, and then load onto a 12% separating gel for electrophoresis (stacking gel voltage 80V, separating gel voltage 120V, total time approximately 90 minutes). After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 2 hours, and destain with destaining solution (methanol:acetic acid:water = 4:1:5) until the background is clear. Analyze the electrophoretic pattern using ImageJ software and calculate the proportion of gray values ​​of contaminating protein bands (mainly albumin 66kDa and immunoglobulin heavy chain 50kDa) to the total protein gray values.

[0056] The results showed that the proportion of miscellaneous proteins in the exosomes extracted in Example 2 was 8.2±1.5%, while that in Comparative Example 1 (conventional magnetic bead method) was 34.6±3.8%, and that in Comparative Example 2 (ultracentrifugation method) was 22.5±4.1%.

[0057] 2. Exosome recovery rate detection NTA assay: 10 μL of raw cerebrospinal fluid sample (supernatant after centrifugation at 3000×g) and 10 μL of extracted exosome solution were taken and diluted 100-fold with 0.01M PBS. The samples were analyzed using a NanoSight NS300 nanoparticle tracking analyzer (detection temperature 25℃, 3 tests per sample, 60 seconds per test, camera level 13). Particle concentration (particles / mL) and particle size distribution (particles in the 30-150 nm range were defined as exosomes) were analyzed using NTA software. Figure 2 As shown, the exosome concentration in the original cerebrospinal fluid sample was (2.1±0.3)×10⁻⁶. 9 The concentration of the extracted exosome solution was (9.2±0.8)×10⁻⁶ cells / mL. 8 Cells / mL, recovery rate calculated: (9.2 × 10⁻⁶) 8 ×0.1L) / (2.1×10 9 (×0.5L)×100%=87.6%, which is significantly higher than the 58.2±6.5% of the traditional magnetic bead method and the 27.5±5.2% of the ultracentrifugation method.

[0058] Recovery of exosome markers: The content of CD63 protein in exosomes was detected by ELISA kit. The concentration of CD63 in the original cerebrospinal fluid sample was (156±18) pg / mL, and the concentration of CD63 in the extracted exosome solution was (68±7) pg / 100μL. The recovery rate was calculated as (68pg) / (156pg / mL×0.5mL)×100%=87.1%, which was consistent with the NTA results, verifying the reliability of the method.

[0059] 3. Verification of exosome morphology and integrity Transmission electron microscopy (TEM) observation: 10 μL of the extracted exosome solution was added to a copper mesh (200 mesh, carbon support membrane), allowed to stand at room temperature for 5 minutes, and excess liquid was absorbed with filter paper. 2% phosphotungstic acid solution (pH 7.0) was added for negative staining for 5 minutes, the stain was absorbed with filter paper, and the solution was air-dried at room temperature. Observation and photography were performed using a JEOL JEM-1400 TEM (accelerating voltage 80 kV). The results showed that the exosomes exhibited typical cup-shaped or spherical structures with diameters ranging from 50 to 120 nm. The membrane structure was intact and clear, with no obvious protein corona covering the surface. In contrast, exosomes extracted using the traditional magnetic bead method had a large number of irregular electron-dense materials (impure proteins) on their surface, resulting in a blurred morphology.

[0060] Membrane integrity assay: The Calcein-AM / PI double staining method was used. Extracted exosomes were incubated with Calcein-AM (final concentration 5 μM) at 37°C for 30 minutes. After washing away the free dye, PI (final concentration 1 μM) was added, and the samples were analyzed by flow cytometry. Figure 3 The results showed that 92.5% of the exosomes were Calcein-AM positive / PI negative (membrane intact), which was significantly higher than the 78.3% of the traditional magnetic bead method (due to interference from other proteins causing partial membrane structure damage).

[0061] 4. Downstream analysis and compatibility verification Western blot: 20 μg of total exosomal protein was subjected to SDS-PAGE, transferred to a PVDF membrane, and incubated with anti-CD63 (1:1000), immunoglobulin heavy chain (1:5000), and anti-albumin (1:5000) antibodies, respectively. ECL chemiluminescence was then used for color development. Figure 4 As shown, the CD63 band is clear (molecular weight 55kDa), while the albumin (66kDa) and immunoglobulin heavy chain (50kDa) bands are significantly weakened; whereas the albumin and immunoglobulin heavy chain bands of the traditional magnetic bead method are 5-8 times brighter than those of this method, and the CD63 band is diffused due to interference from other proteins.

[0062] 5. Exosome particle size distribution The particle size distribution of exosomes extracted from Comparative Example 1 and Example 2 was measured, and the results are as follows: Figure 5 As shown, Figure 5 Figure A shows the distribution pattern of exosomes in the raw cerebrospinal fluid. It can be seen that the particle size distribution of exosomes ranges from 80 to 990 particles / mL. Within this range, the particle size distribution of exosomes is relatively dispersed, with several sharp peaks appearing. Figure 5 B represents exosomes extracted using the method described in Example 2, with a particle size distribution compared to... Figure 5 The exosomes extracted using the method in Example 2 are more uniform in size. This uniformity in particle size reduces the interference of exosome heterogeneity, improving the reproducibility of downstream experimental data and the accuracy of functional mechanism studies.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-functional modified magnetic bead, characterized in that, Using magnetic beads as a substrate, CD63 antibodies are coupled to the surface of the magnetic beads and grafted with sulfobetaine-type zwitterionic polymers.

2. The bifunctional modified magnetic bead as described in claim 1, characterized in that, The magnetic beads have a size of 200-300 nm; the magnetic beads are superparamagnetic nanoparticles.

3. The method for preparing the bifunctional modified magnetic beads according to claim 1 or 2, characterized in that, The process includes the following steps: placing magnetic beads in a buffer solution of EDC and NHS and shaking at room temperature to activate the surface; adding the activated magnetic beads to a CD63 antibody solution for coupling to obtain antibody-modified magnetic beads; and resuspending the antibody-modified magnetic beads in a buffer solution of a sulfobetaine-type zwitterionic polymer for grafting to obtain bifunctional modified magnetic beads.

4. The method for preparing bifunctional modified magnetic beads as described in claim 3, characterized in that, In the EDC and NHS buffer solution, the concentration of EDC is 40-60 mM, the concentration of NHS is 80-120 mM, and the pH of the buffer solution is 5.5-6.

5.

5. The method for preparing bifunctional modified magnetic beads as described in claim 3, characterized in that, The room temperature oscillation time is 20-40 min, more specifically, 22-38 min, and even more preferably, 25-35 min; Alternatively, the concentration of the CD63 antibody solution is 4~6 μg / 100 μL magnetic beads, and the magnetic beads are refrigerated overnight to obtain antibody-modified magnetic beads; Alternatively, the buffer solution of the sulfobetaine-type zwitterionic polymer has a concentration of 1~4 mg / mL.

6. The application of the bifunctionalized magnetic beads as described in claim 1 or 2 in the field of exosome capture.

7. A method for extracting exosomes from cerebrospinal fluid, characterized in that, The magnetic beads modified with bifunctionality as described in claim 1 or 2 are used to target and bind to exosomes in cerebrospinal fluid to achieve the capture of exosomes, and then the exosomes are dissociated from the magnetic beads by a composite eluent; the composite eluent uses a weakly acidic buffer as a matrix and includes detergent and competitive peptides.

8. The method for extracting exosomes from cerebrospinal fluid as described in claim 7, characterized in that, The extraction method specifically includes the following steps: (1) Centrifuge the cerebrospinal fluid sample at 2000-4000×g for 10-20 min at 2-8℃ and retain the supernatant; (2) Add the bifunctional modified magnetic beads as described in claim 1 or 2 to the supernatant above, and incubate at 2~8℃ and 100~300rpm for 0.5~2h. After incubation, separate the magnetic beads from the liquid based on magnetism. The low temperature environment can reduce protein denaturation, and the shaking can promote full contact between the magnetic beads and exosomes. The targeted capture of exosomes is achieved by the specific binding of anti-CD63 antibody to CD63 protein on the surface of exosomes. (3) Add the magnetic beads to the composite elution solution and shake and incubate for 10-20 minutes. Collect the supernatant to obtain the product.

9. The method for extracting exosomes from cerebrospinal fluid as described in claim 8, characterized in that, In step (1), the centrifugation temperature is 2~6℃, the centrifugation speed is 2500~3500×g, and the centrifugation time is 12~17min; In step (2), the dosage ratio of the supernatant to the magnetic beads is 0.4~0.6mL:50μL.

10. The method for extracting exosomes from cerebrospinal fluid as described in claim 8, characterized in that, In step (3), the composite eluent uses 10-30mM Tris-HCl buffer as the matrix, and further, the pH is 5.5-6.0, and also includes a detergent and a competitive peptide; the detergent is 0.05-0.1% Triton X-100, preferably 0.08%; the concentration of the competitive peptide is 80-120μM.

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