Microporous membrane for exosome collection

Abstract

The invention belongs to the technical field of exosome collection, and particularly discloses a microporous membrane for exosome collection. The microporous membrane for exosome collection comprises a through hole layer, a membrane layer and a membrane layer, wherein through holes in the thickness direction are distributed in the through hole layer; the microporous layer is arranged on the through hole layer, and a microporous structure is distributed on the microporous layer; and the aperture of the through hole is less than 50nm. The microporous membrane disclosed by the invention is not easy to block when collecting the exosome, the maximum capacity of the membrane for collecting the exosome in unit area is improved, the collection efficiency is better, the loss amount of the exosome can be reduced, and the production efficiency is improved. The invention also provides a preparation method and application of the microporous membrane.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome extraction, and in particular to a microporous membrane for collecting exosomes. Background Art

[0002] Exosomes are small membrane vesicles containing complex RNA and proteins, with a particle size of about 30 to 200 nm. They are secreted by a variety of cells and body fluids and are used to express and regulate the biological activities of a variety of cells. Exosomes are widely distributed in body fluids such as peripheral blood, urine, saliva, cerebrospinal fluid, milk, ascites, and amniotic fluid. They participate in a variety of physiological processes in the body, such as cell communication and anti-tumor immunity, and are closely related to the occurrence and development of a variety of diseases. Exosomes have potential application value due to their special structure and function. Therefore, the separation and purification methods of exosomes have also become a key issue in scientific research.

[0003] At present, the commonly used methods for extracting exosomes in laboratories are mainly ultracentrifugation, magnetic bead extraction and membrane purification. Among them, membrane purification is widely used because of its simple operation and rapid acquisition. However, the membranes used for exosome extraction are mostly traditional virus filtration membranes, which are expensive and have limited filtration volume. For large-volume extraction of exosomes, a large number of membranes are required, which has poor economic benefits and production efficiency.

[0004] Therefore, it is urgent to develop new filtration membranes for collecting exosomes to improve the extraction capacity and collection efficiency of exosomes extracted by membrane encapsulation. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a new microporous membrane for collecting exosomes. The microporous membrane of the present invention is not easy to be blocked when collecting exosomes, which increases the maximum capacity of the membrane per unit area to collect exosomes. Compared with traditional products, it has better collection efficiency, and can greatly reduce the loss of exosomes during the collection process, thereby improving production efficiency.

[0006] The invention also provides a method for preparing the microporous membrane.

[0007] The present invention also provides an exosome collection device.

[0008] The present invention also proposes the application of the microporous membrane.

[0009] In a first aspect of the present invention, there is provided a microporous membrane for collecting exosomes, comprising:

[0010] A through-hole layer, wherein the through-hole layer is provided with through-holes along the thickness direction, and the aperture of the through-holes is less than 50 nm;

[0011] A microporous layer is disposed on the through-hole layer, and microporous structures are distributed on the microporous layer.

[0012] According to a specific embodiment of the present invention, the microporous membrane for collecting exosomes provided by the present invention has at least the following beneficial effects: the microporous membrane of the present invention is not easy to be clogged when collecting exosomes, which improves the maximum capacity of the membrane per unit area for collecting exosomes, and has a better collection efficiency than traditional products; and can greatly reduce the amount of loss in the process of collecting exosomes, thereby improving production efficiency; the surface of the through-hole layer of the microporous membrane of the present invention is relatively smooth, and the exosomes are not easy to adhere to the surface of the filter membrane during the collection process, and the smaller pore size can play a good role in blocking exosomes; in addition, the microporous layer of the microporous membrane of the present invention has the function of adsorbing foreign proteins in the solution, which helps to reduce the situation where the membrane is clogged by accumulated proteins, and can also provide the microporous membrane with stronger mechanical strength, thereby supporting sufficient liquid pressure during the filtration process, reducing the risk of clogging, and significantly improving the collection capacity per unit area.

[0013] According to some embodiments of the present invention, the through-hole layer includes a polyethylene terephthalate (PET) film or a polycarbonate (PC) film.

[0014] According to some embodiments of the present invention, the through-hole layer is a PET film.

[0015] According to some embodiments of the present invention, the through hole is a straight through hole.

[0016] According to some embodiments of the present invention, the porosity of the through-hole layer is 5% to 25%.

[0017] According to some embodiments of the present invention, the porosity of the through-hole layer is 10% to 20%.

[0018] According to some embodiments of the present invention, the porosity of the through-hole layer is 12% to 18%.

[0019] The present invention controls the porosity of the through-hole layer to ensure that the through-hole layer film has sufficiently high filtering performance while having sufficient strength performance, and can avoid the occurrence of a large number of repeated pores caused by excessively high porosity.

[0020] According to some embodiments of the present invention, the through-hole layer has a thickness of 5 to 15 μm.

[0021] According to some embodiments of the present invention, the through-hole layer has a thickness of 7 to 10 μm.

[0022] According to some embodiments of the present invention, the diameter of the through holes of the through hole layer is 15-30 nm.

[0023] According to some embodiments of the present invention, the diameter of the through holes of the through hole layer is 20-25 nm.

[0024] When the microporous membrane of the present invention is used to collect exosomes, the exosome stock solution passes through the stock solution side of the through-hole layer to the filtrate side of the microporous layer and flows out. The thin thickness design of the through-hole layer can block the passage of exosomes while allowing the filtrate to pass smoothly. Because it is a straight through hole with a relatively smooth surface, exosomes are not easy to clog it and adhere to the surface of the filter membrane, which can play a role in better collecting exosomes.

[0025] According to some embodiments of the present invention, the microporous layer comprises a nylon membrane or a nitrocellulose membrane.

[0026] According to some embodiments of the present invention, the microporous layer is a nylon film.

[0027] According to some embodiments of the present invention, the porosity of the microporous layer is 85% to 97%.

[0028] According to some embodiments of the present invention, the porosity of the microporous layer is 90% to 95%.

[0029] According to some embodiments of the present invention, the microporous layer has a thickness of 80 to 200 μm.

[0030] According to some embodiments of the present invention, the microporous layer has a thickness of 100 to 150 μm.

[0031] According to some embodiments of the present invention, the diameter of the micropores of the microporous layer is 80 to 250 nm.

[0032] According to some embodiments of the present invention, the pore diameter of the micropores of the microporous layer is 100-200 nm.

[0033] According to some embodiments of the present invention, a thickness ratio of the microporous layer to the through-hole layer is 6 to 20:1.

[0034] According to some embodiments of the present invention, a thickness ratio of the microporous layer to the through-hole layer is 8 to 15:1.

[0035] The microporous layer used in the present invention has a strong dirt-receiving capacity, can adsorb foreign proteins in the exosome sample liquid, and helps to reduce the risk of clogging of the PET straight pore layer. In addition, the present invention uses a microporous layer with higher porosity and thicker thickness to further enhance its ability to adsorb foreign proteins. The microporous layer can also provide a stronger mechanical strength for the microporous membrane, thereby supporting sufficient liquid pressure during the filtration process, reducing the risk of clogging, thereby extending the service life of the microporous membrane and improving its collection capacity.

[0036] The second aspect of the present invention provides a method for preparing the microporous membrane according to the first aspect of the present invention, comprising the following steps:

[0037] S1, coating a nylon resin solution or a nitrocellulose solution on the irradiated film to form a wet film layer to obtain a composite film;

[0038] S2, immersing the composite film obtained in step S1 into a coagulation bath to form a microporous structure in the wet film layer;

[0039] S3, immersing the composite film treated in step S2 into an alkaline solution to etch the irradiated film, thereby obtaining a microporous film.

[0040] According to the specific embodiment of the present invention, the preparation method of the microporous membrane provided by the present invention has a simple and efficient process and low cost, and can be applied in large-scale industrial production; the preparation method of the microporous membrane provided by the present invention can prepare the microporous membrane product described in the first aspect of the present invention, so the preparation method also includes the aforementioned advantages of the microporous membrane of the present invention.

[0041] According to some embodiments of the present invention, the concentration of the nylon resin solution or the nitrocellulose solution in step S1 is 30 wt % to 50 wt %.

[0042] According to some embodiments of the present invention, the solvent of the nylon resin solution or the nitrocellulose solution in step S1 includes formic acid.

[0043] According to some embodiments of the present invention, the nylon resin includes at least one of nylon 6, nylon 66, nylon 1010, nylon 610, nylon 612, nylon 11 or nylon 12.

[0044] According to some embodiments of the present invention, the irradiated film in step S1 is a product obtained by using a heavy ion accelerator to accelerate ions to penetrate a PET film or a PC film.

[0045] According to some embodiments of the present invention, the ions accelerated by the heavy ion accelerator are krypton ions or bromide ions.

[0046] According to some embodiments of the present invention, the heavy ion accelerator is set to accelerate ions to penetrate the film with an energy of 5 to 100 GeV and a time of 10 to 200 s.

[0047] According to some embodiments of the present invention, the energy set by the heavy ion accelerator when accelerating ions to penetrate the film is 10 to 30 GeV, and the time is 20 to 50 s.

[0048] According to some embodiments of the present invention, the irradiated film has a thickness of 5 to 15 μm.

[0049] According to some embodiments of the present invention, the irradiated film has a thickness of 7 to 10 μm.

[0050] According to some embodiments of the present invention, the coagulation bath in step S2 comprises at least one of a formic acid solution, a methanol solution or a citric acid solution.

[0051] According to some embodiments of the present invention, the immersion of the film in the coagulation bath described in step S2 is divided into two steps: first, the film is immersed in a coagulation bath 1 containing 10wt% to 25wt% formic acid and 8wt% to 20wt% methanol for 3 to 6 minutes, and then the film is immersed in a coagulation bath 2 containing 4wt% to 10wt% citric acid and 5wt% to 12wt% methanol for 0.5 to 3 minutes.

[0052] According to some embodiments of the present invention, the immersion of the film in a coagulation bath described in step S2 is divided into two steps: first, the film is immersed in a coagulation bath 1 containing 10wt% to 20wt% formic acid and 10wt% to 20wt% methanol for 3 to 6 minutes, and then the film is immersed in a coagulation bath 2 containing 5wt% to 10wt% citric acid and 5wt% to 10wt% methanol for 1 to 2 minutes.

[0053] According to some embodiments of the present invention, step S2 further comprises the steps of cleaning and drying the film after the wet film layer is formed into a microporous structure.

[0054] According to some embodiments of the present invention, the alkaline solution in step S3 includes at least one of a sodium hydroxide solution or a potassium hydroxide solution.

[0055] According to some embodiments of the present invention, the alkaline solution in step S3 is a mixed solution of sodium hydroxide and potassium hydroxide.

[0056] According to some embodiments of the present invention, the concentration of the alkaline solution is 1 to 10 mol / L.

[0057] According to some embodiments of the present invention, the temperature of the alkaline solution is 50-80°C.

[0058] According to some embodiments of the present invention, step S3 further includes cleaning and drying the film after the etching step.

[0059] According to some embodiments of the present invention, the cleaning in step S2 and step S3 is performed by using deionized water.

[0060] According to some embodiments of the present invention, the drying temperature in step S2 and step S3 is 100-150°C.

[0061] According to a third aspect of the present invention, there is provided an exosome collection device, comprising a filter, wherein the filter is provided with the microporous membrane for collecting exosomes according to the first aspect of the present invention.

[0062] The fourth aspect of the present invention provides the use of the microporous membrane described in the first aspect of the present invention in collecting exosomes, microorganisms or biomacromolecules.

[0063] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0065] Figure 1 This is a schematic cross-sectional structure diagram of a microporous membrane for collecting exosomes according to Example 1 of the present invention;

[0066] Figure 2 This is a schematic diagram of the microporous membrane for collecting exosomes of the present invention;

[0067] Figure 3 It is a schematic diagram of the structure of the exosome collection device in the exosome collection test of the present invention;

[0068] Reference numerals: 1-PET straight-pore layer; 2-nylon microporous layer. DETAILED DESCRIPTION

[0069] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0070] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0071] Example 1

[0072] This embodiment provides a microporous membrane for collecting exosomes, such as Figure 1 As shown, the microporous membrane for collecting exosomes in this embodiment has a PET straight pore layer 1 and a nylon microporous layer 2, the PET straight pore layer is provided with straight through holes arranged along the thickness direction, and the nylon microporous layer coated on the PET straight pore layer is provided with a microporous structure.

[0073] In this embodiment, the porosity of the PET straight hole layer 1 is 10% to 20%, the thickness is 7 to 10 μm, and the pore diameter of the straight through hole is 20 to 25 nm.

[0074] In this embodiment, the porosity of the nylon microporous layer 2 is 85% to 97%, the thickness is 100 to 150 μm, and the pore size is 100 to 200 nm.

[0075] In this embodiment, the PET straight hole layer 1 is formed by a PET film which is penetrated by krypton or bromide ions through a heavy ion accelerator and then etched by an alkaline solution to obtain straight through holes.

[0076] In this embodiment, the raw material of the nylon microporous layer 2 is nylon 6 or nylon 66.

[0077] The schematic diagram of the exosome collection using the microporous membrane in this embodiment is as follows Figure 2 shown.

[0078] Example 2

[0079] The present embodiment provides a microporous membrane for collecting exosomes. The microporous membrane for collecting exosomes in the present embodiment comprises a PET straight pore layer 1 and a nylon microporous layer 2. The PET straight pore layer is provided with straight through holes arranged along the thickness direction, and the nylon microporous layer coated on the PET straight pore layer is provided with a microporous structure.

[0080] In this embodiment, the porosity of the PET straight hole layer 1 is 15%, the thickness is 10 μm, and the pore diameter of the straight through hole is 25 nm.

[0081] In this embodiment, the nylon microporous layer 2 has a porosity of 95%, a thickness of 100 μm, and a pore size of 150 nm.

[0082] In this embodiment, the microporous membrane for collecting exosomes is prepared by the following steps:

[0083] 1) using formic acid to dissolve nylon 6 resin to obtain a mixed solution with a concentration of 43 wt%;

[0084] 2) Preparation of PET irradiated film: using a heavy ion accelerator to accelerate krypton ions to penetrate a 10 μm thick polyethylene terephthalate (PET) film, setting the energy of the heavy ion accelerator to 17 GeV and the penetration time to 30 s, to obtain a PET irradiated film;

[0085] 3) coating the mixed solution of step 1) on the PET irradiated film of step 2), controlling the wet film thickness formed by the mixed solution to be 220 μm, to obtain a thin film;

[0086] 4) using deionized water to prepare a coagulation bath 1 containing 15 wt % formic acid and 15 wt % methanol, immersing the film coated in step 3) in the coagulation bath 1 for 5 minutes to perform preliminary pore formation;

[0087] 5) using deionized water to prepare a coagulation bath 2 containing 5 wt% citric acid and 10 wt% methanol, immersing the film initially porous in step 4 in the coagulation bath 2 for 1 min, so that the nylon layer forms a microporous layer with an equivalent pore size of about 150 nm;

[0088] 6) After washing the film with deionized water, place it in an oven and dry it at 130°C for 5 minutes;

[0089] 7) The film was placed in a mixed solution containing 1 mol / L sodium hydroxide and 0.2 mol / L potassium hydroxide, and etched at 65°C for 6 min to form through holes with a pore size of 25 nm in the PET layer;

[0090] 8) After washing the film with deionized water, place it in an oven and dry it at 130° C. for 5 min.

[0091] The above steps finally prepared a microporous membrane composed of a PET straight pore layer (porosity 15%, thickness 10 μm, pore size 25 nm) and a nylon microporous layer (porosity 95%, thickness 100 μm, pore size 150 nm).

[0092] Example 3

[0093] The present embodiment provides a microporous membrane for collecting exosomes. The microporous membrane for collecting exosomes in the present embodiment comprises a PET straight pore layer 1 and a nylon microporous layer 2. The PET straight pore layer is provided with straight through holes arranged along the thickness direction, and the nylon microporous layer coated on the PET straight pore layer is provided with a microporous structure.

[0094] In this embodiment, the porosity of the PET straight hole layer 1 is 10%, the thickness is 7 μm, and the pore diameter of the straight through hole is 20 nm.

[0095] In this embodiment, the nylon microporous layer 2 has a porosity of 95%, a thickness of 140 μm, and a pore size of 200 nm.

[0096] In this embodiment, the microporous membrane for collecting exosomes is prepared by the following steps:

[0097] 1) using formic acid to dissolve nylon 66 resin to obtain a mixed solution with a concentration of 39 wt%;

[0098] 2) Preparation of PET irradiated film: using a heavy ion accelerator to accelerate bromide ions to penetrate a 7 μm thick PET film, setting the energy of the heavy ion accelerator to 15 GeV and the penetration time to 25 s, to obtain a PET irradiated film;

[0099] 3) coating the mixed solution of step 1) on the PET irradiated film of step 2), controlling the wet film thickness formed by the mixed solution to be 300 μm, to obtain a thin film;

[0100] 4) using deionized water to prepare a coagulation bath 1 containing 20 wt % formic acid and 10 wt % methanol, immersing the film coated in step 3) in the coagulation bath 1 for 5 minutes to perform preliminary pore formation;

[0101] 5) using deionized water to prepare a coagulation bath 2 containing 5 wt% citric acid and 10 wt% methanol, immersing the film initially porous in step 4 in the coagulation bath 2 for 2 minutes, so that the nylon layer forms a microporous layer with an equivalent pore size of about 200 nm;

[0102] 6) After washing the film with deionized water, place it in an oven and dry it at 130°C for 5 minutes;

[0103] 7) The film was placed in a mixed solution containing 1.5 mol / L sodium hydroxide and 0.1 mol / L potassium hydroxide, and etched at 65°C for 6 min, so that straight through holes with a pore size of 20 nm appeared in the PET layer;

[0104] 8) After washing the film with deionized water, place it in an oven and dry it at 130°C for 5 minutes;

[0105] The above steps finally prepared a microporous membrane composed of a PET straight pore layer (porosity 10%, thickness 7 μm, pore size 20 nm) and a nylon microporous layer (porosity 95%, thickness 140 μm, pore size 200 nm).

[0106] Comparative Example 1

[0107] This comparative example provides a microporous membrane for collecting exosomes, and the preparation steps are as follows:

[0108] 1) Preparation of PET irradiated film: using a heavy ion accelerator to accelerate krypton ions to penetrate a 10 μm thick PET film, setting the energy of the heavy ion accelerator to 17 GeV and the penetration time to 30 s, to obtain a PET irradiated film;

[0109] 2) placing the film in a mixed solution containing 1 mol / L sodium hydroxide and 0.2 mol / L potassium hydroxide, and etching at 65°C for 6 min, so that straight through holes with a pore size of 25 nm appear in the PET layer;

[0110] 3) After washing the film with deionized water, place it in an oven and dry it at 130° C. for 5 min.

[0111] The above steps finally prepare a PET microporous membrane without a nylon layer.

[0112] Exosome collection test:

[0113] 1. Take 50cm respectively 2 The microporous membrane prepared in Example 2 and Comparative Example 1 is loaded Figure 3 The exosome collection test was performed in the exosome collection device shown in the figure. The exosome content of 3L was about 3.76×10 10 Since Comparative Example 1 is a single-layer PET film, in order to avoid the film from breaking during collection, the liquid pressure is set to 0.3 bar, while Example 1 uses a conventional 3 bar liquid pressure for filtration collection.

[0114] In the collection test of the microporous membrane of Example 2, the exosome stock solution passes through the microporous membrane from the PET straight pore layer side (stock solution side) of the microporous membrane, and the filtrate flows out from the nylon microporous layer side (filtrate side), as shown in the schematic diagram Figure 2 shown.

[0115] Results: The microporous membrane of Comparative Example 1 became clogged after the filtered filtrate reached 37 mL, while the microporous membrane of Example 2 became clogged after the filtered filtrate reached 2.1 L.

[0116] The test results show that the design of the microporous membrane of the present invention can significantly increase the filtration volume of exosomes per unit area, thereby greatly improving the collection capacity and production efficiency of exosomes. It is analyzed that the single-layer PET straight pore layer cannot provide greater liquid pressure for the collection of exosomes, and the turbulence in the cavity after the single-layer membrane filtration is serious, and protein aggregation is prone to occur, making the filter membrane more prone to clogging. Although increasing the thickness of the PET straight pore layer can improve the strength of the film, it will bring about a significant increase in the difficulty and cost of preparation. The longer the straight-through hole length, the greater the film resistance will be, which may be more likely to cause clogging. Therefore, the use of this solution to combine a thinner PET straight pore layer and a thicker nylon microporous layer to obtain a composite microporous membrane can achieve the best technical effect.

[0117] 2. Take 50cm respectively 2 The microporous membrane prepared in Example 2 and the conventional virus filtration membrane (polyethersulfone membrane with a thickness of 130 μm and an equivalent pore size of 30 nm) were loaded into Figure 3 The exosome collection test was performed in the exosome collection device shown in the figure, and 3 bar liquid pressure was used for filtration collection. The exosome content of 1L was about 3.76×10 10 The exosomes were collected from the cell culture fluid with a concentration of 100 mL / ml, and the volume of the culture fluid was concentrated to 100 mL. The exosome content in the collected fluid was measured using a nanoparticle size detector.

[0118] Results: The concentration of exosomes collected by the microporous membrane of Example 2 was 2.39×1011 / ml, the collection efficiency was 63.5%, and the concentration of exosomes collected by traditional filtration membrane was 1.33×10 11 / ml, and the collection efficiency was 35.5%. In addition, the traditional virus filter membrane had been clogged at the end of the collection, indicating that 1L of cell culture medium was already 50cm 2 The collection limit of the traditional virus filtration membrane was exceeded, and further collection tests were performed on the microporous membrane of Example 2, and it was found that its filtration capacity was greater than 2L.

[0119] The test results show that the microporous membrane of the present invention has significantly higher collection efficiency and collection capacity than traditional virus filtration membranes. Analysis shows that the microscopic surface of traditional virus filtration membranes is not smooth, and some large particles are easily hung on the membrane during tangential flow filtration, resulting in pore blockage. The microporous membrane of this scheme can overcome this technical problem well. In addition, exosomes are also more likely to attach to traditional virus filtration membranes, which will also lead to a decrease in the collection efficiency of exosomes.

[0120] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A microporous membrane for collecting exosomes, characterized in that: include: A through-hole layer, wherein the through-hole layer is provided with through-holes along the thickness direction, and the aperture of the through-holes is less than 50 nm; A microporous layer is disposed on the through-hole layer, and microporous structures are distributed on the microporous layer.

2. The microporous membrane according to claim 1, characterized in that The through-hole layer comprises a PET film or a PC film; And / or, the microporous layer comprises a nylon membrane or a nitrocellulose membrane.

3. The microporous membrane according to claim 1, characterized in that The porosity of the through-hole layer is 5% to 25%; And / or, the thickness of the through-hole layer is 5 to 15 μm; And / or, the diameter of the through holes of the through hole layer is 15-30 nm.

4. The microporous membrane according to claim 1, characterized in that The porosity of the microporous layer is 85% to 97%; and / or, the thickness of the microporous layer is 80 to 200 μm; And / or, the pore diameter of the micropores of the microporous layer is 80 to 250 nm.

5. A method for preparing a microporous membrane according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, coating a nylon resin solution or a nitrocellulose solution on the irradiated film to form a wet film layer to obtain a composite film; S2, immersing the composite film obtained in step S1 into a coagulation bath to form a microporous structure in the wet film layer; S3, immersing the composite film treated in step S2 into an alkaline solution to etch the irradiated film, thereby obtaining a microporous film.

6. The preparation method according to claim 5, characterized in that: The irradiated film in step S1 is a product obtained by using a heavy ion accelerator to accelerate ions to penetrate a PET film or a PC film; Preferably, the ions accelerated by the heavy ion accelerator are krypton ions or bromide ions; Preferably, the heavy ion accelerator is set to accelerate ions to penetrate the film with an energy of 5 to 100 GeV and a time of 20 to 40 seconds.

7. The preparation method according to claim 5, characterized in that: The coagulation bath described in step S2 includes at least one of a formic acid solution, a methanol solution or a citric acid solution.

8. The preparation method according to claim 5, characterized in that: The alkaline solution described in step S3 includes at least one of a sodium hydroxide solution or a potassium hydroxide solution; Preferably, the concentration of the alkaline solution is 1 to 10 mol / L; Preferably, the temperature of the alkaline solution is 50-80°C.

9. An exosome collection device, comprising a filter, characterized in that: The filter is provided with the microporous membrane for collecting exosomes according to any one of claims 1 to 4.

10. Use of the microporous membrane according to any one of claims 1 to 4 in collecting exosomes, microorganisms or biomacromolecules.

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