A method and apparatus for extracting body fluid exosomes

Abstract

This invention relates to the field of biological sample processing technology, and provides a method and apparatus for extracting exosomes from body fluids. The apparatus includes: a liquid-filling tube, with a filter at the opening for filtering body fluid samples, and an exosome capturing agent inside the liquid-filling tube; a cap for sealing the opening of the liquid-filling tube; and a magnetic sleeve, with a magnet built into its sidewall or bottom for accommodating the liquid-filling tube and adsorbing the magnetic components in the exosome capturing agent. The exosome capturing agent is composed of a mixture of magnetic microspheres conjugated with various exosome-specific antibodies. This invention eliminates the need for expensive equipment such as ultracentrifuges, completes extraction within one hour, achieves a recovery rate of 85%, a lipoprotein removal rate of 92%, and effectively protects the integrity of exosomes. It is suitable for rapid detection and home self-testing of various body fluids such as blood and urine.

Description

Technical Field

[0001] This invention relates to the field of biological sample processing technology, specifically a method and apparatus for extracting exosomes from bodily fluids. Background Technology

[0002] Exosomes are vesicles (30-150 nm) with a lipid bilayer structure secreted by cells. They carry various biomolecules such as proteins and nucleic acids and play important roles in physiological and pathological processes such as intercellular communication, immune regulation, and tumor metastasis. In recent years, exosomes have shown great potential as biomarkers for early disease diagnosis, treatment monitoring, and drug delivery. However, efficient extraction of exosomes remains a key technological bottleneck restricting their research and clinical application.

[0003] Currently, commonly used methods for exosome extraction mainly include ultracentrifugation, polymer precipitation, immunoaffinity chromatography, size exclusion chromatography, and nanofiltration, but all of them have significant drawbacks: Time-consuming: Ultracentrifugation and polymer precipitation methods usually take 4-8 hours, while size exclusion chromatography can even take 1-2 days, which is difficult to meet the needs of rapid clinical diagnosis. High equipment dependence: Ultracentrifugation requires expensive ultracentrifuges, while size exclusion chromatography relies on chromatographs or purifiers. The equipment purchase and maintenance costs are high, and the equipment is large and complex to operate, which limits its promotion in primary healthcare institutions and home self-testing scenarios. Complex operation and low throughput: Existing methods are cumbersome and involve multiple transfers, centrifugation or chromatography, which are highly dependent on the experience of personnel; and the number of samples processed at one time is limited (e.g., ultracentrifugation usually processes 6-8 samples, while chromatography usually processes 1 sample), which is difficult to meet the needs of high-throughput clinical testing. High risk of exosome damage: The high shear force generated by ultracentrifugation, the mechanical compression of nanofiltration membranes, and the toxicity of chemical precipitation reagents can easily lead to exosome membrane rupture, leakage or aggregation of contents, affecting downstream analysis; Recovery rate and purity are unstable: the recovery rate varies greatly among different body fluid samples (such as blood and urine) (20% to 90%), and impurities such as proteins and lipids are not completely removed in high-impurity samples, affecting the comparability of data.

[0004] Therefore, in view of the above situation, there is an urgent need to provide a method and device for extracting exosomes from bodily fluids in order to overcome the shortcomings in current practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method and apparatus for extracting exosomes from bodily fluids, effectively solving the problems mentioned in the background art.

[0006] This invention is implemented as follows: a device for extracting body fluid exosomes, comprising: A liquid-filling tube, wherein a filter for filtering body fluid samples is provided at the tube opening, and an exosome trapping agent is placed inside the liquid-filling tube; A cap is used to seal the opening of the liquid-filling tube; A magnetic sleeve, wherein a magnet is built into the side wall or bottom of the magnetic sleeve for accommodating the liquid tube and adsorbing the magnetic components in the exosome trapping agent; The exosome capturing agent is composed of a mixture of magnetic microspheres conjugated with various exosome-specific antibodies.

[0007] As a further aspect of the present invention: the filter includes a composite filter membrane, which includes an upper filter membrane and a lower filter membrane.

[0008] As a further aspect of the present invention: the magnetic microspheres have a particle size of 500 nm to 3 μm and are made of carboxylated polystyrene-based superparamagnetic microspheres.

[0009] As a further aspect of the present invention: the exosome-specific antibodies include anti-CD9, anti-CD63, anti-CD81, anti-Alix, and anti-TSG101 antibodies.

[0010] The present invention also provides a method for extracting exosomes from bodily fluids, using the apparatus described above, the method comprising the following steps: S1. Sample pretreatment: Open the cap and add the body fluid sample into the liquid collection tube. After the sample is filtered by the filter, it enters the liquid collection tube and comes into contact with the lyophilized exosome capture agent. S2. Reconstitution and Capture: Reconstitute the lyophilized exosome capture agent into the sample, shake well until the lyophilized powder dissolves, and let stand at room temperature to allow the multi-antibody on the surface of the magnetic microspheres to specifically bind with the exosomes to form a complex. S3. Magnetic separation: Insert the liquid-filling tube into the magnetic sleeve and use the magnetic field to adsorb the complex to the tube wall or bottom, then discard the supernatant. S4. Dissociation and Collection: Add a dissociation agent to the liquid-filling tube to dissociate the exosomes from the magnetic microspheres. Use a magnetic sleeve to re-adsorb the magnetic microspheres and collect the enriched material containing exosomes.

[0011] As a further aspect of the present invention: in S4, the dissociating agent is composed of dissociating agent A and dissociating agent B.

[0012] As a further aspect of the present invention: the dissociation agent A is a glycine-HCl solution and the dissociation agent B is NaHCO3.

[0013] As a further aspect of the present invention: in S4, the amount of the dissociation agent added is less than the amount of body fluid sample added in S1.

[0014] As a further aspect of the present invention: the preparation of the exosome capturing agent includes: The magnetic microspheres were cleaned with a coupling solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added and activated at room temperature. Add exosome-specific antibodies and react at room temperature; Add a sealing agent and seal at room temperature; Different antibody-conjugated magnetic microspheres were mixed in proportion, resuspended in tris(hydroxymethyl)aminomethane hydrochloride buffer containing bovine serum albumin, and then freeze-dried.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Development of multi-antibody synergistic exosome capture: Multiple monoclonal antibodies are combined to capture exosomes, significantly improving exosome binding efficiency, reducing interference from non-target proteins, ensuring an exosome capture rate (>80%), and ensuring efficient extraction of exosomes from precious body fluid samples. It is suitable for clinical research with scarce or small sample sizes, rapid clinical detection of diseases, and home self-testing.

[0016] 2. Reduce extraction costs: Avoid relying on expensive ultracentrifugation and purification equipment. Use magnetic nanoparticles coupled with exosome-specific antibodies (such as anti-CD9 / CD63 / CD81) to achieve a complete "mixing-capture-washing" process through magnetic sleeve separation. No instruments are required, making it easier to promote.

[0017] 3. Equipment-independent: It does not rely on specific equipment such as ultracentrifuges, but uses more economical and efficient materials and methods, making it suitable for scientific research, clinical diagnosis and home self-testing scenarios.

[0018] 4. One-step simplified operation process: Eliminates cumbersome steps such as centrifugation and purification, shortens separation time (<1 hour), adopts an economical and universal method, helps consistency among different laboratories, medical institutions and ordinary people without skills, improves the comparability and reproducibility of data, and achieves "ready to use".

[0019] 5. Protect the integrity of exosomes: Avoid damage to the exosome membrane structure by high shear force or harsh elution conditions to ensure that the separated exosomes are morphologically intact and their biological activity is not affected.

[0020] 6. Enhanced adaptability to high-impurity samples: For complex clinical body fluid samples (blood, urine, cerebrospinal fluid, ascites, etc.), the selective adsorption strategy is optimized to effectively remove interfering components such as impurities and lipids, improve separation efficiency, and meet the needs of high-sensitivity downstream analysis (such as proteomics, RNA sequencing, and clinical testing). Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The flowchart of a method for extracting exosomes from body fluids provided by the present invention.

[0023] Figure 2 This is a schematic diagram of the structure of a body fluid exosome extraction device provided by the present invention.

[0024] Figure 3 This is a schematic diagram of the extraction process for extracting exosomes from body fluids using a body fluid exosome extraction device.

[0025] In the attached diagram: 1-pipe cap, 2-filter, 3-liquid filling tube, 4-magnetic sleeve. Detailed Implementation

[0026] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The present invention will be further explained below with reference to specific embodiments.

[0028] Please see Figure 2 and Figure 3 An embodiment of the present invention provides a body fluid exosome extraction device, comprising: The liquid-filling tube 3 is equipped with a filter 2 for filtering body fluid samples at the tube opening, and the liquid-filling tube 3 contains an exosome trapping agent. Cap 1 is used to seal the opening of the liquid-filling tube 3; Magnetic sleeve 4, with a magnet built into its side wall or bottom, is used to contain the liquid tube 3 and adsorb the magnetic components in the exosome trapping agent. The exosome capturing agent is composed of a mixture of magnetic microspheres conjugated with various exosome-specific antibodies.

[0029] In this embodiment, the shape and size of the cap 1 are not limited, as long as its shape and size match those of the filter 2, and it is used to prevent external contamination. The filter 2 includes a composite filter membrane, which comprises an upper filter membrane and a lower filter membrane. The upper filter membrane has a pore size of 1.2 μm, and the lower filter membrane has a pore size of 0.45 μm. Based on physical sieving, only substances smaller than this pore size (including exosomes, proteins, nucleic acids, etc.) are allowed to pass through, while larger particles are blocked on the filter membrane. This removes cells and impurities from the body fluid sample. As a key pretreatment step, it prevents large particles from being non-specifically adsorbed onto the immunomagnetic beads, which can significantly improve the purity and efficiency of exosome extraction. The filling tube 3 is used to hold the immunomagnetic bead antibody complex solution for lyophilization. It is also used to pour body fluids into it later. It is the core reaction tube in the entire extraction process. The tube wall may or may not be marked with graduations, and the shape is not limited. The size and volume of the filling tube 3 are determined according to the application (e.g., 5 mL). The filling tube 3 is filled with the immunomagnetic bead antibody complex solution (including but not limited to CD9, CD63, CD81, Alix, TSG101 antibody-conjugated magnetic beads), and lyophilized according to the lyophilization process for exosome capture. The magnetic sleeve 4 is a convenient magnetic separation device with a strong magnet built into its side wall or bottom. When the sample-containing tube 3 is inserted into the sleeve, the magnetic field can quickly attract immunomagnetic beads (such as magnetic beads coupled with exosome antibodies to capture exosomes in the sample) to the tube wall or bottom near the magnetic field, while unbound impurities (such as free proteins, nucleic acids, etc.) remain in the supernatant. By directly pouring out the supernatant, the magnetic bead-exosome complex will be firmly fixed to the tube wall or bottom. Subsequently, the sample-containing tube 3 is removed from the magnetic sleeve 4, and the magnetic beads are removed from the magnetic field, allowing for exosome collection or washing steps. This device uses chemical covalent coupling to immobilize exosome monoclonal antibodies onto the surface of magnetic microspheres (e.g., magnetic beads with a particle size of 500nm-3μm, carboxylated polystyrene-based superparamagnetic microspheres), and uses a magnetic field to separate exosomes from the sample.

[0030] Please see Figures 1-3 This invention also provides a method for extracting exosomes from bodily fluids, using the aforementioned apparatus, and the method includes the following steps: S1. Sample pretreatment: Open the cap 1 and add the body fluid sample into the liquid filling tube 3. After the sample is filtered by the filter 2, it enters the liquid filling tube 3 and comes into contact with the lyophilized exosome capturing agent. The filter 2 removes interfering substances. S2. Reconstitution and Capture: Reconstitute the lyophilized exosome capture agent into the sample, gently shake for 20-40 seconds until the lyophilized powder is dissolved, and let stand at room temperature for 20-40 minutes to allow the multi-antibody on the surface of the magnetic microspheres to specifically bind with the exosomes to form a complex. S3, Magnetic separation: Insert the liquid-filling tube 3 into the magnetic sleeve 4, and use the magnetic field to adsorb the complex to the tube wall or bottom, and discard the supernatant; S4. Dissociation and Collection: Add a dissociation agent to the liquid tube 3 to dissociate the exosomes from the magnetic microspheres. Use the magnetic sleeve 4 to re-adsorb the magnetic microspheres and collect the enriched exosomes. The dissociation agent consists of dissociation agent A and dissociation agent B. The amount of dissociation agent added is less than the amount added to the body fluid sample to achieve the purpose of exosome concentration.

[0031] In practical applications, first unscrew the cap 1 and collect a body fluid sample (1 mL or 5 mL, depending on the specific situation); add the collected sample to the liquid-filling tube 3, and let the sample pass through the filter 2 to remove interfering substances (e.g., blood cells in blood samples, impurities in urine samples, etc.); after the sample flows through the filter membrane, it flows into the liquid-filling tube 3, reconstitute the lyophilized reagent, and gently shake for a certain period of time (e.g., 20-40 seconds) until the lyophilized powder dissolves, and let the immunomagnetic beads capture exosomes and stand at room temperature (e.g., 20-40 minutes); put the magnetic sleeve 4 on the liquid-filling tube 3, the magnetic beads will precipitate, discard the supernatant, and obtain the exosome-capturing magnetic beads. After removing the magnetic sleeve 4, the precipitate at the bottom of the tube is the extracted exosome magnetic beads; add dissociation agents, consisting of dissociation agent A (e.g., 0.1M glycine-HCl solution, pH=3.0) and dissociation agent B (e.g., 1.0 M NaHCO3, pH=8.3), and gently shake the liquid-filling tube 3 to separate the magnetic beads and exosomes; (the amount of dissociation agent added is less than the amount of sample added to achieve the purpose of exosome concentration); put the magnetic sleeve 4 on the liquid-filling tube 3 to aggregate the magnetic beads, and collect the enriched product containing exosomes.

[0032] In a more specific example, the steps for preparing the exosome trapping agent are as follows: 1. Preparation of immunomagnetic bead antibody complexes Take an appropriate amount of carboxyl magnetic beads and wash them repeatedly with a coupling solution (e.g., 10-100mM 2-morpholine ethanesulfonic acid buffer, pH 5.0-6.0); Two crosslinking agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, are added to chemically activate the carboxyl groups on the surface of the magnetic beads, transforming them into active esters that can react efficiently with the primary amino groups of antibody molecules. This activation process lasts for tens of minutes (e.g., 10-30 minutes) at room temperature. After activation, the target antibody (including but not limited to CD63 (lysosome-associated membrane protein 3), CD81 (target antigen-1 of anti-proliferation antibody), Alix (apoptosis-inducing factor 6 interacting protein), TSG101 (tumor susceptibility gene 101)) is directly added to the system; During the several-hour room temperature reaction, the antibody covalently links to the activated magnetic bead surface via stable amide bonds. After the reaction, the unbound antibody is removed, and a blocking agent (e.g., 2%-10% BSA solution) is added for incubation (e.g., incubation conditions: room temperature 18-25℃, rotation speed 50-200 rpm, time 30 min-6 h). Finally, after thorough cleaning to remove excess blocking agent, the coated magnetic beads are resuspended in a storage solution (e.g., 20-100mM tris(hydroxymethyl)aminomethane hydrochloride buffer containing 0.1%-1% bovine serum albumin, pH 7.0-8.0) and can then be stored at 2-8°C.

[0033] 2. Preparation of immunomagnetic bead antibody complex solution Immunomagnetic bead antibody complexes of CD9 (leukocyte differentiation antigen 9), CD63, CD81, Alix, and TSG101 are mixed in different ratios (e.g., 5:5:3:1:1-1:1:1:1:1) to obtain exosome capture agents, which are then dispensed according to the required amount.

[0034] The effects of the method of the present invention compared with those of existing methods are as follows: Table 1 below shows that Comparative Example 1 uses conventional immunoaffinity assay to separate exosomes from urine, Comparative Example 2 uses ultracentrifugation to separate exosomes from urine, and Example 1 uses the method of the present invention to extract exosomes from urine. The number of exosomes in the samples and products of Example 1 and Comparative Examples 1-2 was detected using NTA (nanoparticle tracking analysis); the lipoprotein concentration in the samples and products of Example 1 and Comparative Examples 1-2 was detected using lipoprotein electrophoresis; and the total protein concentration in the samples and products of Example 1 and Comparative Examples 1-2 was detected using the BCA total protein assay. The results of the comparison are shown in Table 1. Table 1. Results of the comparison

[0035] The results are analyzed as follows: 1. The exosome recovery rate of Example 1 was 85%, which is greater than that of Comparative Examples 1-2 (65% and 70%), indicating a higher exosome yield. 2. The residual contaminating proteins in Example 1 were comparable to those in Comparative Examples 1-2, and the purity of exosomes was comparable; 3. The processing time of Example 1 is much shorter than that of Comparative Examples 1-2, thus shortening the processing time; 4. The lipoprotein removal rate of Example 1 is comparable to that of Comparative Examples 1-2.

[0036] In summary, this invention employs a multi-antibody strategy, specifically an immunomagnetic microsphere freeze-drying technology based on a multi-antibody combined capture strategy. By coupling various exosome-specific monoclonal antibodies (such as CD9, CD63, and CD81) to the surface of magnetic nanoparticles and mixing them in optimized proportions, the exosome binding efficiency is significantly improved. This exosome capture agent utilizes the principle of specific immune binding and magnetic separation between antibodies and exosomes to achieve rapid exosome extraction. The freeze-drying process ensures the long-term stability of the exosome extraction device, facilitating storage and transportation.

[0037] By adding a dissociation agent to release exosomes, the exosomes are separated from the magnetic beads under the action of the magnetic sleeve 4, enabling rapid, one-step extraction of exosomes.

[0038] It offers high protection of exosome integrity and bioactivity, and operates under mild conditions, avoiding irreversible aggregation of PEG precipitates or damage to the exosome membrane caused by ultracentrifugation shear forces.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A device for extracting exosomes from bodily fluids, characterized in that, include: The liquid filling tube (3) is provided with a filter (2) for filtering body fluid samples at the tube opening, and the liquid filling tube (3) contains an exosome capture agent. A cap (1) is used to seal the opening of the liquid-filling tube (3); A magnetic sleeve (4) has a magnet built into its sidewall or bottom to accommodate the liquid tube (3) and adsorb the magnetic components in the exosome trapping agent; The exosome capturing agent is composed of a mixture of magnetic microspheres conjugated with various exosome-specific antibodies.

2. The exosome extraction device according to claim 1, characterized in that, The filter (2) includes a composite filter membrane, which includes an upper filter membrane and a lower filter membrane.

3. The exosome extraction device according to claim 1, characterized in that, The magnetic microspheres have a particle size of 500 nm to 3 μm and are made of carboxylated polystyrene-based superparamagnetic microspheres.

4. The exosome extraction device according to claim 1, characterized in that, The exosome-specific antibodies include anti-CD9, anti-CD63, anti-CD81, anti-Alix, and anti-TSG101 antibodies.

5. A method for extracting exosomes from bodily fluids, using the apparatus as described in any one of claims 1 to 4, characterized in that, The method includes the following steps: S1. Sample pretreatment: Open the cap (1), add the body fluid sample into the liquid filling tube (3), and after the sample is filtered by the filter (2), it enters the liquid filling tube (3) and comes into contact with the lyophilized exosome capture agent. S2. Reconstitution and Capture: Reconstitute the lyophilized exosome capture agent into the sample, shake well until the lyophilized powder dissolves, and let stand at room temperature to allow the multi-antibody on the surface of the magnetic microspheres to specifically bind with the exosomes to form a complex. S3, Magnetic separation: Insert the liquid-filling tube (3) into the magnetic sleeve (4) and use the magnetic field to adsorb the complex to the tube wall or bottom, and discard the supernatant; S4. Dissociation and collection: Add a dissociation agent to the liquid tube (3) to dissociate the exosomes from the magnetic microspheres. Use the magnetic sleeve (4) to adsorb the magnetic microspheres again and collect the enrichment containing the exosomes.

6. The method for extracting exosomes from bodily fluids according to claim 5, characterized in that, In S4, the dissociating agent consists of dissociating agent A and dissociating agent B.

7. The method for extracting exosomes from bodily fluids according to claim 6, characterized in that, The dissociation agent A is a glycine-HCl solution, and the dissociation agent B is NaHCO3.

8. The method for extracting exosomes from bodily fluids according to claim 5, characterized in that, In S4, the amount of dissociating agent added is less than the amount of body fluid sample added in S1.

9. The method for extracting exosomes from bodily fluids according to claim 5, characterized in that, The preparation of the exosome capturing agent includes: The magnetic microspheres were cleaned with a coupling solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added and activated at room temperature. Add exosome-specific antibodies and react at room temperature; Add a sealing agent and seal at room temperature; Different antibody-conjugated magnetic microspheres were mixed in proportion, resuspended in tris(hydroxymethyl)aminomethane hydrochloride buffer containing bovine serum albumin, and then freeze-dried.

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