A surface-modified film and its application

Abstract

This application relates to the technical field of biomembranes, specifically disclosing a surface-modified membrane and its applications. The surface-modified membrane includes a basement membrane and a surfactant supported on the basement membrane, wherein the surfactant is selected from at least one of cationic and amphoteric surfactants. The applications of this surface-modified membrane include the separation and / or extraction of extracellular secretory membrane structures. The surface-modified membrane has the advantages of efficient and rapid extraction of extracellular secretory membrane structures, resulting in high concentrations and suitable particle sizes of the extracted structures.

Description

Technical Field

[0001] This application relates to the technical field of biomembranes, and more specifically, to a surface-modified membrane and its applications. Background Technology

[0002] Filter membranes are a commonly used material in the biological field, and different membranes are selected depending on different functional needs. The common application of membranes is the separation and extraction of functional substances, such as proteins, cells, and extracellular secretory membrane structures (vesicles, etc.). Extracellular secretory membrane structures, such as vesicles and exosomes, contain a large number of important informational proteins, including transmembrane proteins and buoyancy proteins; they also contain nucleic acids and proteins related to programmed cell death. Therefore, extracellular secretory membrane structures carry important information. Many extracellular secretory membrane structures participate in the transport of metabolic waste and also play important roles in pathophysiological activities.

[0003] Methods for obtaining extracellular secretory membrane structures include ultracentrifugation, filtration centrifugation, immunomagnetic bead extraction, PEG precipitation, and kits. Ultracentrifugation is the most commonly used method, but it is time-consuming, complex, yields a small amount of target material, and has a low separation rate. Filtration centrifugation uses a filter membrane to separate extracellular secretory membrane structures, but this method results in low purity of the target material. Immunomagnetic bead extraction yields twice the amount of target protein as ultracentrifugation and filtration, but it is not suitable for all extracellular secretory membrane structures, is inefficient, and expensive. PEG precipitation yields extracellular secretory membrane structures with low purity and recovery rate, containing many impurities, uneven particle size, and producing polymers that are difficult to remove. Currently, commercially available exosome extraction kits are mainly based on PEG precipitation for extracellular vesicle extraction; the most commonly used kits are ExoQuick, miRCURY, and TEIR. Kit methods are simple to operate, but the obtained extracellular vesicles contain many impurities. In conclusion, there is currently no single method or kit that can meet all requirements for isolating ideal extracellular vesicles from various samples. Furthermore, existing biomembranes are insufficient to meet the demands for efficient and low-cost separation and extraction. Summary of the Invention

[0004] To address the issues of high cost, low efficiency, and complexity in existing extraction processes for extracellular secretory membrane structures, this application provides a surface-modified membrane and its application, which offers the advantages of high efficiency and low cost in extracting extracellular secretory membrane structures.

[0005] In a first aspect, this application provides a surface-modified film, employing the following technical solution:

[0006] A surface-modified film comprising a base film and a surfactant loaded on the base film; the surfactant is selected from at least one of cationic surfactants and amphoteric surfactants.

[0007] Optionally, the cationic surfactant is selected from stearamide-propyl dimethylamine, potassium fatty acid soap, sodium cocoyl glutamate, behenamidopropyl dimethylamine, quaternary ammonium salt Gemini surfactant, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, QAS Cn One or more of the following: polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether.

[0008] Optionally, the amphoteric surfactant is a betaine-type amphoteric surfactant, which is selected from at least one of cocamidopropyl betaine, dodecyl betaine, caprydecanopropyl betaine, and lauramidopropyl hydroxysulfonate betaine.

[0009] Optionally, the surfactant is a cationic surfactant and an amphoteric surfactant, wherein the molar ratio of the cationic surfactant to the amphoteric surfactant is (0.01~1.64):3.

[0010] Optionally, the surface-modified film is further loaded with a protective agent, which is selected from at least one of alkaloids, amino acids and bioethanol.

[0011] Optionally, the molar ratio of the alkaloid to the surfactant is 0.8 to 2.3:1.

[0012] Optionally, the molar ratio of the amino acid to the surfactant is 0.05 to 1.2:1.

[0013] Optionally, the molar ratio of the bio-alcohol to the surfactant is 0.24 to 1.5:1.

[0014] Secondly, this application provides an application of a surface-modified film, employing the following technical solution:

[0015] An application of a surface-modified membrane, the application including the separation and / or extraction of extracellular secretory membrane structures.

[0016] Optionally, the extracellular secretory membrane structure includes extracellular vesicles.

[0017] Thirdly, this application provides a method for extracting extracellular secretory membrane structures, employing the following technical solution:

[0018] A method for extracting extracellular secretory membrane structures, the extraction method comprising the following steps:

[0019] S1. Pre-treat biological samples to remove large particulate matter;

[0020] S2. After pretreatment, the biological sample is mixed with the loading solution, then centrifuged and passed through the above-mentioned surface-modified membrane, so that the extracellular secretory membrane structure in the biological sample is captured on the surface-modified membrane. The surface-modified membrane is loaded with a surfactant, which is selected from at least one of quaternary ammonium salt cationic surfactants and amphoteric surfactants.

[0021] S3. Elute the surface-modified membrane with elution buffer to obtain the extracellular secretory membrane structure collection solution.

[0022] Optionally, the cationic surfactant is selected from stearamide-propyl dimethylamine, potassium fatty acid soap, sodium cocoyl glutamate, behenamidopropyl dimethylamine, quaternary ammonium salt Gemini surfactant, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, QAS Cn One or more of the following: polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether; wherein n is a natural number less than or equal to 18.

[0023] Optionally, the quaternary ammonium salt type Gemini surfactant is a symmetrical Gemini surfactant or an asymmetrical Gemini surfactant.

[0024] Optionally, the amphoteric surfactant is a betaine-type amphoteric surfactant, which is selected from at least one of cocamidopropyl betaine, dodecyl betaine, caprydecanopropyl betaine, and lauramidopropyl hydroxysulfonate betaine.

[0025] Optionally, the surfactant is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant.

[0026] Optionally, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.01~1.64):3.

[0027] Optionally, when the quaternary ammonium salt cationic surfactant is selected from one or more of dodecyltrimethylammonium bromide (DTAB), hexadecyltrimethylammonium bromide (CTAB), polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.01~0.36):3.

[0028] Optionally, when the quaternary ammonium salt cationic surfactant is a symmetrical Gemini surfactant, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.23~0.45):3.

[0029] Optionally, when the quaternary ammonium salt cationic surfactant is an asymmetric Gemini surfactant, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.56~1.26):3.

[0030] Optionally, when preparing the surface-modified film, a protective agent is also added together with the surfactant; the protective agent is selected from at least one of alkaloids, amino acids and bioethanol.

[0031] Optionally, the alkaloid is betaine.

[0032] Optionally, the molar ratio of the alkaloid to the surfactant is 0.8 to 2.3:1.

[0033] Optionally, the amino acid is proline.

[0034] Optionally, the molar ratio of the amino acid to the surfactant is 0.05 to 1.2:1.

[0035] Optionally, the bioethanol is mannitol.

[0036] Optionally, the molar ratio of the bio-alcohol to the surfactant is 0.24 to 1.5:1.

[0037] By loading protective agents onto surface-modified membranes, the likelihood of extracellular secretory membrane structure being damaged in salt environments can be reduced. Alkaloids, especially betaine, can resist salt stress, thus protecting the extracellular secretory membrane structure. Bioethanol, especially mannitol, also protects the extracellular secretory membrane structure from rupture to some extent. Amino acids, especially proline, protect the extracellular secretory membrane structure by scavenging free radicals.

[0038] When alkaloids, amino acids, and bioethanol are used in combination, their protective effect on the extracellular secretory membrane structure is even better.

[0039] Optionally, the loading fluid comprises components at the following concentrations:

[0040] 87~113mM Tris-propane buffer, 130~165mM NaCl, pH 6.23~6.78.

[0041] Optionally, the loading fluid further includes a protective agent; the protective agent is selected from at least one of alkaloids, amino acids, and bioethanol.

[0042] By adding a protective agent to the loading solution, the extracellular secretory membrane structure is protected from being damaged in a salt environment.

[0043] Optionally, if the loading solution contains the alkaloid, the molar ratio of the alkaloid to NaCl is 0.25~0.46:1;

[0044] If the loading solution contains the amino acid, the molar ratio of the amino acid to NaCl is 0.13~0.35:1;

[0045] If the loading solution contains the bio-alcohol, the molar ratio of the bio-alcohol to NaCl is 0.12~0.19:1.

[0046] Optionally, the eluent comprises components at the following concentrations:

[0047] 38~62mM Tris-propane buffer, 0.8~2.3M NaCl, pH 6.13~6.52.

[0048] Optionally, the loading fluid further includes a protective agent; the protective agent is selected from at least one of alkaloids, amino acids, and bioethanol.

[0049] Optionally, if the loading solution contains the alkaloid, the molar ratio of the alkaloid to NaCl is 0.02~0.16:1;

[0050] If the loading solution contains the amino acid, the molar ratio of the amino acid to NaCl is 0.005~0.1:1;

[0051] If the loading solution contains the bio-alcohol, the molar ratio of the bio-alcohol to NaCl is 0.005~0.1:1.

[0052] Fourthly, this application provides a kit for implementing the above extraction method, employing the following technical solution:

[0053] A kit for implementing the above extraction method, the kit comprising the loading solution, the eluent, and a filtration device, the filtration device containing the surface-modified membrane.

[0054] In summary, this application has the following beneficial effects:

[0055] 1. This application uses a surface-modified membrane loaded with a suitable surfactant, including at least one of cationic surfactants and amphoteric surfactants, to achieve efficient separation and extraction of extracellular secretory membrane structures in biological samples.

[0056] 2. This application adds a protective agent to the surface-modified membrane to further prevent the extracellular secretory membrane structure from being damaged, thereby ensuring the integrity of the obtained extracellular secretory membrane structure and further increasing the concentration of the obtained extracellular secretory membrane structure. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the overall structure of the separation and extraction device in one embodiment of this application;

[0058] Figure 2 This is a schematic diagram of the overall structure of the separation and extraction device in another embodiment of this application;

[0059] Figure 3 This is a schematic diagram of the overall structure of the separation and extraction device in another embodiment of this application;

[0060] Figure 4 This is a top view of the base plate in another embodiment of this application;

[0061] Figure 5 This is a schematic diagram illustrating the structure of the sample introduction unit in another embodiment of this application;

[0062] Figure 6 This is a schematic diagram illustrating the relevant structure of the sample quantification cell in another embodiment of this application;

[0063] Figure 7 This is a schematic diagram illustrating the structure of the sample pretreatment unit in another embodiment of this application;

[0064] Figure 8 This is a schematic diagram illustrating the structure of the impurity filtration tank in another embodiment of this application;

[0065] Figure 9 This is a schematic diagram illustrating the structure of the loading fluid containing assembly in another embodiment of this application;

[0066] Figure 10 This is a schematic diagram illustrating the internal structure of the liquid-filled container in another embodiment of this application;

[0067] Figure 11 This is a schematic diagram illustrating the structure of the eluent tank and the carrier filter tank in another embodiment of this application;

[0068] Figure 12 This is a schematic diagram illustrating the internal structure of the carrier filter tank in another embodiment of this application;

[0069] Figure 13 This is a schematic diagram illustrating the structure of the eluent container component in another embodiment of this application;

[0070] Figure 14 This is a schematic diagram illustrating the internal structure of the eluent container component in another embodiment of this application;

[0071] Figure 15 This is a schematic diagram illustrating the enriched unit structure in another embodiment of this application;

[0072] Figure 16 This is a schematic diagram illustrating one possible distribution of the weight-reducing balancing pool on the bottom plate in another embodiment of this application;

[0073] Figure 17 This is another structural schematic diagram of the incubation pool in this application;

[0074] Figure 18 This is a comparison chart of extraction times when different extraction methods are used to extract exosomes. Among them, 1: microfluidic centrifuge plate extraction method of Example 10; 2: size exclusion method of Comparative Example 1; 3: ultracentrifugation method of Comparative Example 2; 4: PEG precipitation method of Comparative Example 3; 5: density gradient centrifugation method of Comparative Example 4.

[0075] Figure 19 This is a comparison of Western Blot results when exosomes from different samples were extracted using the method of Example 10;

[0076] Figure 20 This is a comparison of Western Blot results of exosomes in cell supernatants obtained by different extraction methods. Among them, ①: microfluidic centrifuge plate extraction method of Example 10; ②: size exclusion method of Comparative Example 1; ③: ultracentrifugation method of Comparative Example 2; ④: PEG precipitation method of Comparative Example 3; ⑤: density gradient centrifugation method of Comparative Example 4.

[0077] Figure 21This is a transmission electron microscope image of exosomes extracted using the method described in Example 10;

[0078] Figure 22 This is a scanning electron microscope image of exosomes extracted using the method described in Example 10;

[0079] Figure 23 The image shows the NTA map of exosomes extracted using the method described in Example 10.

[0080] Reference numerals: 1. Rotation center; 2. Extraction mechanism; 21. Sample injection unit; 211. Sample cell; 2111. Sample inlet; 212. Sample quantification cell; 213. Excess sample collection cell; 214. First flow channel; 215. Second flow channel; 216. First impurity collection cell; 217. Third flow channel; 22. Capture and release unit; 221. Loading liquid cell; 2211. Loading liquid container assembly; 22111. Loading liquid container; 22112. Second lid; 22113. Second liquid release port; 22114. Second sealing membrane; 22115. 22116, Second notch; 2212, Second ejector pin; 22121, Tenth flow channel; 2213, Second support platform; 2214, Second protrusion; 222, Eluent tank; 2221, Eluent container assembly; 22211, Eluent container box; 22212, First box cover; 22213, First liquid release hole; 22214, First sealing membrane; 22215, First concave wall; 22216, First notch; 2222, First ejector pin; 22221, Ninth flow channel; 2223, First support platform; 2224, First protrusion; 223. Incubation tank; 2231. Elastic rod; 2232. Elastic ball; 224. Carrier filtration tank; 2241. First membrane placement chamber; 2242. Mixture receiving chamber; 2243. First filtration assembly; 22431. First membrane substrate; 22432. Surface-modified membrane; 225. Fourth flow channel; 226. Mixture transfer flow channel; 227. Fifth flow channel; 228. Eighth flow channel; 23. Enrichment unit; 231. Target material tank; 232. Waste liquid tank; 233. Sixth flow channel; 234. Seventh flow channel; 24. Weight reduction and balancing tank; 241. Air channel. 25. Sample pretreatment unit; 251. Impurity filtration chamber; 2511. Second membrane placement chamber; 2512. Quantitative liquid receiving chamber; 2513. Second filtration assembly; 25131. Second membrane substrate; 25132. Microfiltration membrane; 252. Eleventh flow channel; 253. Twelfth flow channel; 254. Second impurity collection chamber; 3. Base plate; 4. Cover plate; 41. First pin hole; 42. Second pin hole; 43. Vent; 5. Centrifugal adsorption column; 51. First outer tube; 511. First sealing cap; 52. First inner tube; 521. Discharge tube; 6. Syringe. Detailed Implementation

[0081] This application provides a surface-modified film, which includes a base film and a surfactant loaded on the base film; the surfactant is selected from at least one of cationic surfactants and amphoteric surfactants.

[0082] In some embodiments, the cationic surfactant is selected from stearamide propyl dimethylamine, potassium fatty acid soap, sodium cocoyl glutamate, behenamidopropyl dimethylamine, quaternary ammonium salt Gemini surfactant, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, QAS Cn One or more of the following: polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether.

[0083] In some embodiments, the amphoteric surfactant is a betaine-type amphoteric surfactant selected from at least one of cocamidopropyl betaine, dodecyl betaine, caprydecanopropyl betaine, and lauramidopropyl hydroxysulfonate betaine.

[0084] In some embodiments, the cationic surfactant is a quaternary ammonium salt type cationic surfactant, selected from quaternary ammonium salt type Gemini surfactants, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, QAS Cn One or more of the following: polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether.

[0085] In some embodiments, the quaternary ammonium cationic surfactant is a quaternary ammonium Gemini surfactant.

[0086] In some embodiments, the quaternary ammonium salt Gemini surfactant is a symmetrical Gemini surfactant or an asymmetrical Gemini surfactant.

[0087] The quaternary ammonium salt-type Gemini surfactant is commercially available and may be a quaternary ammonium salt-type Gemini surfactant purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd., or a quaternary ammonium salt-type Gemini twin cationic surfactant purchased from Shanghai Shenrong Chemical Technology Co., Ltd.

[0088] In some embodiments, the quaternary ammonium salt-type Gemini surfactant is obtained by preparation.

[0089] In some embodiments, the synthesis steps of the asymmetric quaternary ammonium salt type Gemini surfactant include: reacting a short-chain alkyl tertiary amine with 1,3-dibromoisopropanol to obtain an intermediate product, and then reacting the intermediate product with a long-chain alkyl tertiary amine to obtain the quaternary ammonium salt type Gemini surfactant; wherein the long-chain alkyl tertiary amine has 12, 16, or 18 carbon atoms on its alkyl chain, and the short-chain alkyl tertiary amine has 6 or 8 carbon atoms on its alkyl chain.

[0090] In some embodiments, the long-chain alkyl tertiary amine is any one of dodecyl tertiary amine, octadecyl tertiary amine, and hexadecyl tertiary amine.

[0091] In some embodiments, the short-chain alkyl tertiary amine is selected from either octylamine or hexylamine.

[0092] In some embodiments, the molar ratio of 1,3-dibromoisopropanol to the short-chain alkyl tertiary amine is 3-5:1; further, the molar ratio of 1,3-dibromoisopropanol to the short-chain alkyl tertiary amine may be 3.5:1, 4:1, or 4.5:1. The molar ratio of the long-chain alkyl tertiary amine to the intermediate product is 0.8-1.2:1; further, the molar ratio of the long-chain alkyl tertiary amine to the intermediate product is 0.9:1, 1:1, or 1.1:1.

[0093] In some embodiments, the long-chain alkyl tertiary amine has 18 carbon atoms on its alkyl chain, and the short-chain alkyl tertiary amine has 6 carbon atoms on its alkyl chain.

[0094] In some embodiments, the symmetrical quaternary ammonium salt type Gemini surfactant is synthesized from long-chain alkyl tertiary amines and second-generation brominated alkanes, or from long-chain alkyl tertiary amines, epichlorohydrin and hydrochloric acid; the long-chain alkyl tertiary amine has 8, 12 or 16 carbon atoms on its alkyl chain, and the second-generation brominated alkanes have 2, 3 or 4 carbon atoms on their alkyl chains.

[0095] In some embodiments, the alkyl chain of the long-chain alkyl tertiary amine has 12 or 16 carbon atoms.

[0096] In some embodiments, the long-chain alkyl tertiary amine is any one of N,N-dimethyldodecylamine, N,N-dimethyl-1,3-propanediamine, and N,N-dimethylhexadecylamine; and the second-generation brominated alkane is 1,3-dibromopropane.

[0097] In some embodiments, the quaternary ammonium salt cationic surfactant is QAS. Cn n can be any one of 0, 6, 8, 12, 14, or 18.

[0098] In some embodiments, the amphoteric surfactant is a betaine-type amphoteric surfactant, which may be cocamidopropyl betaine, such as product CAB-35; it may be dodecyl betaine, such as product BS-12; or it may be caprydecanopropyl betaine. In some embodiments, the betaine-type amphoteric surfactant is a sulfonic acid-type betaine-type amphoteric surfactant, further comprising 3-(N,N-dimethyldodecylammonium)propane sulfonate (SPPT); or it may be lauramidopropyl hydroxysulfonate betaine, such as product LHSB-35.

[0099] In some embodiments, the base membrane is an RC membrane.

[0100] In some embodiments, the surface-modified film is prepared by a method comprising the following steps: dissolving at least one of the quaternary ammonium salt cationic surfactant and the amphoteric surfactant in a first organic solvent until completely dissolved; then placing it in a base membrane, reacting with shaking at room temperature, and drying; and then washing with a second organic solvent and drying to obtain the final product.

[0101] In some implementations, the room temperature oscillation time is 40-56 hours; in other implementations, the room temperature oscillation time can be 43 hours, 46 hours, 49 hours, 52 hours, or 54 hours.

[0102] In some embodiments, the first organic solvent is methanol.

[0103] In some embodiments, the second organic solvent is an ethanol solution, wherein the ethanol solution is a 5-95% ethanol solution. When using the second organic solvent for washing, a single wash with 95% ethanol can be performed; alternatively, a gradient wash with ethanol solutions of varying concentrations can be used.

[0104] In some embodiments, the surfactant loaded during the preparation of the surface-modified film is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant.

[0105] In some embodiments, the surfactant used to prepare the surface-modified film is a quaternary ammonium cationic surfactant and an amphoteric surfactant. Optionally, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.01~1.64):3.

[0106] In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant; the quaternary ammonium salt cationic surfactant is selected from dodecyltrimethylammonium bromide (DTAB), hexadecyltrimethylammonium bromide (CTAB), polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, polyalkyltrialkylammonium chloride, 3-chloro-2- When one or more of hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether are used, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.01~0.36):3; the molar ratio can be, for example, 0.05:3, 0.1:3, 0.15:3, 0.2:3, 0.25:3, or 0.3:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium cationic surfactant and an amphoteric surfactant; when the ammonium cationic surfactant is a symmetrical Gemini surfactant, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.23~0.45):3; the molar ratio can be, for example, 0.25:3, 0.31:3, 0.38:3, or 0.42:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium cationic surfactant and an amphoteric surfactant; when the ammonium cationic surfactant is an asymmetrical Gemini surfactant, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.56~1.26):3; the molar ratio can be, for example, 0.65:3, 0.72:3, 0.84:3, 0.92:3, 1.06:3, or 1.18:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant; the ammonium salt cationic surfactant is QAS. CnWhen n is 0 or 18, and the amphoteric surfactant 3-(N,N-dimethyldodecylammonium)propane sulfonate is used, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (1.05~1.64):3; the molar ratio can be, for example, 1.16:3, 1.28:3, 1.36:3 or 1.52:3.

[0107] In some embodiments, a protective agent is added together with the surfactant during the preparation of the surface-modified film; the protective agent serves to prevent exosomes from being destroyed by osmotic pressure in a high-salt environment. The protective agent includes at least one of alkaloids, amino acids, and bioethanol. In some embodiments, the protective agent includes alkaloids and amino acids; in other embodiments, the protective agent includes alkaloids, bioethanol, and amino acids.

[0108] In some embodiments, the alkaloid is selected from betaine. In some embodiments, the molar ratio of the alkaloid to the surfactant is 0.8 to 2.3:1; optionally, the molar ratio of the alkaloid to the surfactant is 1.3:1, 1.6:1, 1.8:1, or 2.2:1.

[0109] In some embodiments, the amino acid is selected from proline. In some embodiments, the molar ratio of the amino acid to the surfactant is 0.05 to 1.2:1; optionally, the molar ratio of the amino acid to the surfactant is 0.1:1, 0.3:1, 0.7:1, 0.9:1, or 1.1:1.

[0110] In some embodiments, the bioethanol is selected from mannitol. In some embodiments, the molar ratio of the bioethanol to the surfactant is 0.24 to 1.5:1; optionally, the molar ratio of the bioethanol to the surfactant is 0.3:1, 0.46:1, 0.69:1, 0.82:1, 0.96:1, 1.14:1, 1.23:1, or 1.41:1.

[0111] This application also provides an application of a surface-modified membrane, the application including the separation and / or extraction of extracellular secretory membrane structures.

[0112] In this application, "application for separating and / or extracting extracellular secretory membrane structures" means either the application for separating extracellular secretory membrane structures, or the application for extracting extracellular secretory membrane structures, or the application for separating and extracting extracellular secretory membrane structures.

[0113] In some implementations, the extracellular secretory membrane structure includes extracellular vesicles and exosomes.

[0114] This application also provides a method for extracting extracellular secretory membrane structures, the extraction method comprising the following steps:

[0115] S1. Preprocess the biological samples;

[0116] S2. After pretreatment, the biological sample is mixed with the loading solution, then centrifuged and passed through a surface-modified membrane, so that the extracellular secretory membrane structure in the biological sample is captured on the surface-modified membrane. The surface-modified membrane is loaded with a surfactant, which is selected from at least one of cationic surfactants and amphoteric surfactants.

[0117] S3. Elute the surface-modified membrane with elution buffer to obtain the extracellular secretory membrane structure collection solution.

[0118] The biological samples that can be processed by the method of this application include, but are not limited to, whole blood, plasma, serum, urine, saliva, pleural effusion, peritoneal fluid, cerebrospinal fluid, cell culture supernatant, tears, semen, amniotic fluid, gastric juice, saliva, nasal mucus, bronchoalveolar lavage fluid, synovial fluid, bile, uterine mucus, and feces.

[0119] Extracellular secretory membrane structures include exovesicles and exosomes. Exosomes are extracellular vesicles containing complex RNA and proteins, with a size of approximately 30–150 nm. They primarily originate from multivesicles formed by the invagination of lysosomal microparticles within the cell, and are released into the extracellular matrix after the outer membrane of the multivesicle fuses with the cell membrane. Exosomes are currently considered specifically secreted membrane vesicles, participating in intercellular communication and carrying abundant molecular information from the original cells, and are considered potential biomarkers for early tumor diagnosis. Therefore, developing rapid and convenient extraction methods for tumor exosomes is crucial.

[0120] Currently, exosome extraction methods include ultracentrifugation, filtration centrifugation, immunomagnetic bead extraction, PEG precipitation, and kits. Ultracentrifugation is the most commonly used method, but it is time-consuming, complex, yields a small number of exosomes, and has a low separation rate. Filtration centrifugation uses a filter membrane to separate exosomes, but this method results in exosomes with low purity. Immunomagnetic bead extraction yields exosomes with twice the protein content of ultracentrifugation and filtration, but it is not suitable for all exosome extractions, is inefficient, and expensive. PEG precipitation produces exosomes with low purity and recovery rate, containing many impurities, exhibiting uneven particle size, and generating polymers that are difficult to remove. Currently, commercially available exosome extraction kits are mainly based on PEG precipitation, with ExoQuick, miRCURY, and TEIR being the most commonly used kits. While kit methods are simple to operate, they yield exosomes with many impurities. In conclusion, there is currently no absolute method or kit that can meet all requirements for isolating ideal exosomes from various types of samples.

[0121] The pretreatment step described in step S1 is to remove large particulate matter such as cellular impurities, cell debris, dead bodies, and large-diameter vesicles from the biological sample. This can be achieved by filtering the biological sample through a microfiltration membrane. The microfiltration membrane that can be selected here is a 0.22 μm filter membrane to remove impurities with a particle size greater than about 200 nm. Specifically, it can be, but is not limited to, a polyethersulfone (PES) aqueous filter membrane purchased from Beijing Mairuida Technology Co., Ltd.

[0122] In some embodiments, step S1 further includes a centrifugation step after membrane treatment, wherein the centrifugation speed is 2500~3500 rpm and the centrifugation time is 150~250 s.

[0123] The pretreated biological sample is then mixed with a loading solution, passed through a surface-modified membrane, and finally eluted with an elution buffer. In this process, the loading solution, surface-modified membrane, and elution buffer are used in combination: the loading solution is used to surface-modify the extracellular secretory membrane structure in the biological sample, facilitating the binding of the extracellular secretory membrane structure to the surface-modified membrane, thereby improving the purity and recovery rate of exosomes. After passing through the surface-modified membrane, the extracellular secretory membrane structure in the biological sample, due to its negatively charged surface, binds well to the quaternary ammonium cationic surfactants and / or amphoteric surfactants on the surface-modified membrane, thus achieving the separation of the extracellular secretory membrane structure from other substances in the biological sample. The subsequent elution step involves an elution buffer, where the selected elution buffer has a stronger binding affinity to exosomes than the cationic surfactants and / or amphoteric surfactants on the surface-modified membrane, thereby eluting the extracellular secretory membrane structure from the surface-modified membrane to obtain an extracellular secretory membrane structure collection solution. The extracellular secretory membrane structure is then obtained by centrifugation.

[0124] In some alternative embodiments, the cationic surfactant is selected from stearamidopropyl dimethylamine, potassium fatty acid soap, sodium cocoyl glutamate, behenamidopropyl dimethylamine, quaternary ammonium salt Gemini surfactant, dodecyltrimethylammonium bromide (DTAB), hexadecyltrimethylammonium bromide (CTAB), polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, QAS Cn One or more of the following: polyalkyltrialkylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether; wherein n is a natural number less than or equal to 18.

[0125] In some embodiments, the quaternary ammonium cationic surfactant is a quaternary ammonium Gemini surfactant.

[0126] In some embodiments, the quaternary ammonium salt Gemini surfactant is a symmetrical Gemini surfactant or an asymmetrical Gemini surfactant.

[0127] The quaternary ammonium salt-type Gemini surfactant is commercially available and may be a quaternary ammonium salt-type Gemini surfactant purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd., or a quaternary ammonium salt-type Gemini twin cationic surfactant purchased from Shanghai Shenrong Chemical Technology Co., Ltd.

[0128] In some embodiments, the quaternary ammonium salt-type Gemini surfactant is obtained by preparation.

[0129] In some embodiments, the synthesis steps of the asymmetric quaternary ammonium salt type Gemini surfactant include: reacting a short-chain alkyl tertiary amine with 1,3-dibromoisopropanol to obtain an intermediate product, and then reacting the intermediate product with a long-chain alkyl tertiary amine to obtain the quaternary ammonium salt type Gemini surfactant; wherein the long-chain alkyl tertiary amine has 12, 16, or 18 carbon atoms on its alkyl chain, and the short-chain alkyl tertiary amine has 6 or 8 carbon atoms on its alkyl chain.

[0130] In some embodiments, the long-chain alkyl tertiary amine is any one of dodecyl tertiary amine, octadecyl tertiary amine, and hexadecyl tertiary amine.

[0131] In some embodiments, the short-chain alkyl tertiary amine is selected from either octylamine or hexylamine.

[0132] In some embodiments, the molar ratio of 1,3-dibromoisopropanol to the short-chain alkyl tertiary amine is 3-5:1; further, the molar ratio of 1,3-dibromoisopropanol to the short-chain alkyl tertiary amine may be 3.5:1, 4:1, or 4.5:1. The molar ratio of the long-chain alkyl tertiary amine to the intermediate product is 0.8-1.2:1; further, the molar ratio of the long-chain alkyl tertiary amine to the intermediate product is 0.9:1, 1:1, or 1.1:1.

[0133] In some embodiments, the long-chain alkyl tertiary amine has 18 carbon atoms on its alkyl chain, and the short-chain alkyl tertiary amine has 6 carbon atoms on its alkyl chain.

[0134] In some embodiments, the symmetrical quaternary ammonium salt type Gemini surfactant is synthesized from long-chain alkyl tertiary amines and second-generation brominated alkanes, or from long-chain alkyl tertiary amines, epichlorohydrin and hydrochloric acid; the long-chain alkyl tertiary amine has 8, 12 or 16 carbon atoms on its alkyl chain, and the second-generation brominated alkanes have 2, 3 or 4 carbon atoms on their alkyl chains.

[0135] In some embodiments, the alkyl chain of the long-chain alkyl tertiary amine has 12 or 16 carbon atoms.

[0136] In some embodiments, the long-chain alkyl tertiary amine is any one of N,N-dimethyldodecylamine, N,N-dimethyl-1,3-propanediamine, and N,N-dimethylhexadecylamine; and the second-generation brominated alkane is 1,3-dibromopropane.

[0137] In some embodiments, the quaternary ammonium salt cationic surfactant is QAS. Cn n can be any one of 0, 6, 8, 12, 14, or 18.

[0138] In some implementations, n is either 0 or 18.

[0139] In some embodiments, the amphoteric surfactant is a betaine-type amphoteric surfactant, which may be cocamidopropyl betaine, such as product CAB-35; it may be dodecyl betaine, such as product BS-12; or it may be caprydecanopropyl betaine. In some embodiments, the betaine-type amphoteric surfactant is a sulfonic acid-type betaine-type amphoteric surfactant, further comprising 3-(N,N-dimethyldodecylammonium)propane sulfonate (SPPT); or it may be lauramidopropyl hydroxysulfonate betaine, such as product LHSB-35.

[0140] In some embodiments, the surface-modified film is prepared by a method comprising the following steps: dissolving at least one of the quaternary ammonium salt cationic surfactant and the amphoteric surfactant in a first organic solvent until completely dissolved; then placing it in a base membrane, reacting with shaking at room temperature, and drying; and then washing with a second organic solvent and drying to obtain the final product.

[0141] In some embodiments, the base membrane is an RC membrane.

[0142] In some implementations, the room temperature oscillation time is 40-56 hours; in other implementations, the room temperature oscillation time can be 43 hours, 46 hours, 49 hours, 52 hours, or 54 hours.

[0143] In some embodiments, the first organic solvent is methanol.

[0144] In some embodiments, the second organic solvent is an ethanol solution, wherein the ethanol solution is a 5-95% ethanol solution. When using the second organic solvent for washing, a single wash with 95% ethanol can be performed; alternatively, a gradient wash with ethanol solutions of varying concentrations can be used.

[0145] In some embodiments, the surfactant loaded during the preparation of the surface-modified film is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant.

[0146] In some embodiments, the surfactant used to prepare the surface-modified film is a quaternary ammonium cationic surfactant and an amphoteric surfactant. Optionally, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.01~1.64):3.

[0147] In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant; the quaternary ammonium salt cationic surfactant is selected from dodecyltrimethylammonium bromide (DTAB), hexadecyltrimethylammonium bromide (CTAB), polyethylene oxide trimethylammonium chloride, N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-tetradecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, polyalkyltrialkylammonium chloride, 3-chloro-2- When one or more of hydroxypropyltrimethylammonium oxide, N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium bromide, N-hexadecyl-N,N-di(2-hydroxyethyl)-N-methylammonium bromide, and 2,4,4-trichloro-2-dihydroxydiphenyl ether are used, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (0.01~0.36):3; the molar ratio can be, for example, 0.05:3, 0.1:3, 0.15:3, 0.2:3, 0.25:3, or 0.3:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium cationic surfactant and an amphoteric surfactant; when the ammonium cationic surfactant is a symmetrical Gemini surfactant, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.23~0.45):3; the molar ratio can be, for example, 0.25:3, 0.31:3, 0.38:3, or 0.42:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium cationic surfactant and an amphoteric surfactant; when the ammonium cationic surfactant is an asymmetrical Gemini surfactant, the molar ratio of the quaternary ammonium cationic surfactant to the amphoteric surfactant is (0.56~1.26):3; the molar ratio can be, for example, 0.65:3, 0.72:3, 0.84:3, 0.92:3, 1.06:3, or 1.18:3. In some embodiments, when preparing the surface-modified film, the surfactant is a quaternary ammonium salt cationic surfactant or an amphoteric surfactant; the ammonium salt cationic surfactant is QAS. Cn When n is 0 or 18, and the amphoteric surfactant 3-(N,N-dimethyldodecylammonium)propane sulfonate is used, the molar ratio of the quaternary ammonium salt cationic surfactant to the amphoteric surfactant is (1.05~1.64):3; the molar ratio can be, for example, 1.16:3, 1.28:3, 1.36:3 or 1.52:3.

[0148] In some embodiments, a protective agent is added together with the surfactant during the preparation of the surface-modified film; the protective agent serves to prevent the extracellular secretory membrane structure from being damaged by osmotic pressure in a high-salt environment. The protective agent includes at least one of alkaloids, amino acids, and bioethanol. In some embodiments, the protective agent includes alkaloids and amino acids; in other embodiments, the protective agent includes alkaloids, bioethanol, and amino acids.

[0149] In some embodiments, the alkaloid is selected from betaine. In some embodiments, the molar ratio of the alkaloid to the surfactant is 0.8 to 2.3:1; optionally, the molar ratio of the alkaloid to the surfactant is 1.3:1, 1.6:1, 1.8:1, or 2.2:1.

[0150] In some embodiments, the amino acid is selected from proline. In some embodiments, the molar ratio of the amino acid to the surfactant is 0.05 to 1.2:1; optionally, the molar ratio of the amino acid to the surfactant is 0.1:1, 0.3:1, 0.7:1, 0.9:1, or 1.1:1.

[0151] In some embodiments, the bioethanol is selected from mannitol. In some embodiments, the molar ratio of the bioethanol to the surfactant is 0.24 to 1.5:1; optionally, the molar ratio of the bioethanol to the surfactant is 0.3:1, 0.46:1, 0.69:1, 0.82:1, 0.96:1, 1.14:1, 1.23:1, or 1.41:1.

[0152] In some embodiments, the loading solution comprises 130–170 mM NaCl; optionally, the NaCl concentration is 140 mM, 152 mM, 160 mM, or 168 mM. In some embodiments, the loading solution further comprises 91–103 mM Tris-propane buffer at a pH of 6.45–6.7; optionally, the Tris-propane concentration is 94 mM, 98 mM, or 102 mM.

[0153] In some embodiments, the loading fluid further includes the protective agent, which includes at least one selected from alkaloids, amino acids, and bioethanol. In some embodiments, the protective agent includes alkaloids and amino acids; in other embodiments, the protective agent includes alkaloids, bioethanol, and amino acids.

[0154] In some embodiments, the alkaloid is selected from betaine. In some embodiments, the molar ratio of the alkaloid to NaCl is 0.25 to 0.46:1; optionally, the molar ratio of the alkaloid to NaCl is 0.28:1, 0.32:1, 0.37:1, 0.4:1, or 0.43:1.

[0155] In some embodiments, the amino acid is selected from proline. In some embodiments, the molar ratio of the amino acid to NaCl is 0.13 to 0.35:1; optionally, the molar ratio of the amino acid to NaCl is 0.16:1, 0.19:1, 0.22:1, 0.26:1, 0.29:1, 0.32:1, or 0.34:1.

[0156] In some embodiments, the bioethanol is selected from mannitol. In some embodiments, the molar ratio of the bioethanol to NaCl is 0.12 to 0.19:1; optionally, the molar ratio of the bioethanol to NaCl is 0.14:1, 0.16:1, or 0.18:1.

[0157] In some embodiments, the volume ratio of the biological sample to the loading fluid is 0.4 to 1.6:1. Optionally, the volume ratio of the biological sample to the loading fluid is 0.53:1, 0.68:1, 0.76:1, 0.94:1, 1.21:1, or 1.47:1.

[0158] In some embodiments, the eluent comprises 1–2.5 M NaCl; optionally, the concentration of the NaCl is 1.2 M, 1.45 M, 1.53 M, 1.62 M, 1.83 M, 1.96 M, or 2.37 M. In some embodiments, the eluent further comprises 42–58 mM Tris-propane buffer with a pH of 6.2–6.41; optionally, the concentration of Tris-propane is 45 mM, 48 mM, 53 mM, or 56 mM.

[0159] In some embodiments, the eluent further includes the protective agent, which includes at least one selected from alkaloids, amino acids, and bioethanol. In some embodiments, the protective agent includes alkaloids and amino acids; in other embodiments, the protective agent includes alkaloids, bioethanol, and amino acids.

[0160] In some embodiments, the alkaloid is selected from betaine. In some embodiments, the molar ratio of the alkaloid to NaCl is 0.02 to 0.16:1; optionally, the molar ratio of the alkaloid to NaCl is 0.034:1, 0.058:1, 0.077:1, 0.092:1, 0.126:1, or 0.149:1.

[0161] In some embodiments, the amino acid is selected from proline. In some embodiments, the molar ratio of the amino acid to NaCl is 0.005 to 0.1:1; optionally, the molar ratio of the amino acid to NaCl is 0.018:1, 0.035:1, 0.063:1, 0.086:1, or 0.097:1.

[0162] In some embodiments, the bioethanol is selected from mannitol. In some embodiments, the molar ratio of the bioethanol to NaCl is 0.005 to 0.1:1; optionally, the molar ratio of the bioethanol to NaCl is 0.018:1, 0.035:1, 0.063:1, 0.086:1, or 0.097:1.

[0163] In some implementations, the centrifugation speed during step S2 is 3500~4500 rpm and the centrifugation time is 30~70 s.

[0164] In some implementations, the extraction method uses a separation extraction device to extract the extracellular secretory membrane structure.

[0165] In some embodiments, the separation and extraction device includes a rotating center and at least one extraction mechanism, the extraction mechanism including a sample introduction unit, a capture-release unit, and an enrichment unit; the sample introduction unit is used for inputting and storing biological samples; the capture-release unit is provided with the surface-modified membrane, the capture-release unit is connected to the sample introduction unit and located downstream of the sample introduction unit, and is used to capture or release target extracts from the biological samples; the enrichment unit is connected to the capture-release unit and located downstream of the capture-release unit, and is used to enrich the target extracts released by the capture-release unit.

[0166] When using the separation and extraction device to separate and extract extracellular secretory membrane structures in biological samples, the biological sample is first added to the sample injection unit of the separation and extraction device, and then flows into the capture and release unit. The extracellular secretory membrane structures in the biological sample are captured onto the surface-modified membrane set in the capture and release unit. The captured extracellular secretory membrane structures are then released from the surface-modified membrane and finally enriched in the enrichment unit, thus obtaining a collection solution containing extracellular secretory membrane structures with high purity and concentration.

[0167] In some embodiments, the central part of the separation device is the rotating mounting part, and the rotation center of the separation and extraction device is the rotation center during centrifugation. In some embodiments, the extraction mechanism can be one or more, such as two, three, four, five, or six.

[0168] In some implementations, the sample introduction unit includes a sample cell and a sample introduction port formed on the sample cell, the sample introduction port being in communication with an internal cavity of the sample cell, the sample cell being used to store biological samples.

[0169] In some embodiments, the sample introduction unit further includes a sample quantification cell and an excess sample containment cell. A first flow channel connecting the sample cell and the sample quantification cell is provided, and a second flow channel connecting the sample quantification cell and the excess sample containment cell is also provided. When using the separation and extraction device to separate and extract extracellular secretory membrane structures within biological samples, the biological sample is first added to the sample introduction cell. Subsequently, after the biological sample flows into the sample quantification cell, an excess is typically added, causing the excess biological sample to flow into the excess sample containment cell. The amount of biological sample processed each time is the maximum volume of the sample quantification cell.

[0170] In some implementations, the first flow channel is a capillary flow channel; setting the first flow channel as a capillary flow channel allows biological samples in the sample cell to enter the sample quantification cell more smoothly and quickly under the siphon effect.

[0171] In some embodiments, the sample introduction unit further includes a first impurity collection cell, and a third flow channel connecting the first impurity collection cell and the sample quantification cell is provided. In some embodiments, the first impurity collection cell is located on the side of the sample quantification cell away from the center of rotation. In this embodiment, it facilitates the removal of high molecular weight impurities from the sample by centrifugation, so that the impurities are collected in the first impurity collection cell.

[0172] In some embodiments, the third flow channel is a constricted channel; the side connecting the sample quantification cell and the first impurity collection cell has a gradually rising structure, which can be a step-like gradual rise or a slope-like gradual rise. This structural design facilitates the smoother entry of impurities in the sample quantification cell into the first impurity collection cell under centrifugal force, while the purified biological sample enters the capture and release unit.

[0173] In some implementations, the outlet of the sample quantification cell is connected to the inlet of the capture and release unit.

[0174] In some embodiments, the separation and extraction device further includes several weight-reducing and balancing tanks, and an air passage connecting the excess sample holding tank and one weight-reducing and balancing tank is provided. Air holes are provided on the base plate at positions corresponding to the weight-reducing and balancing tanks to allow gas generated during centrifugation to be discharged at any time, thus connecting the separation and extraction device with the external atmospheric pressure.

[0175] In some embodiments, at least one of the weight-reducing balancing cells connected to the excess sample containment cell is provided with pores.

[0176] In some embodiments, the capture and release unit includes a loading liquid tank, an elution liquid tank, an incubation tank, and a carrier filter tank; the incubation tank is connected to the outlet ends of the sample injection unit and the loading liquid tank, respectively, and the carrier filter tank is connected to the outlet ends of the incubation tank and the elution liquid tank, respectively, and the carrier filter tank is provided with the surface modification membrane; the loading liquid tank, the incubation tank, and the carrier filter tank are distributed sequentially from the proximal end to the distal end of the rotation center.

[0177] In some implementations, the incubation pool and the sample quantification pool are connected at their outlets.

[0178] The loading fluid tank can be used to add the loading fluid, and the eluent tank can be used to add the eluent. In some embodiments, one or more eluent tanks are provided; wherein the types of eluents added to the multiple eluent tanks can be the same or different.

[0179] In some embodiments, a fourth flow channel is provided between the loading liquid tank and the incubation tank, enabling communication between them. In some embodiments, the incubation tank is located on the side of the loading liquid tank away from the center of rotation; or the incubation tank and the loading liquid tank are located on the same radial line.

[0180] In some embodiments, the capture and release unit further includes a mixed liquid transport channel connecting the incubation tank and the carrier filtration tank. In some embodiments, the mixed liquid transport channel is an arc-shaped channel to facilitate the transport of the liquid contained therein under centrifugal force.

[0181] In some embodiments, a fifth channel is provided between the mixed liquid transport channel and the incubation tank. In some embodiments, the inner diameter of the fifth channel is smaller than the inner diameter of the mixed liquid transport channel. In some embodiments, the fifth channel is a capillary channel, allowing the liquid in the incubation tank to flow smoothly to the mixed liquid transport channel through the siphon effect of the capillary channel. In one example, the mixed liquid transport channel is an arc-shaped channel, a structure that allows the liquid to enter the next processing unit under centrifugal force.

[0182] In some embodiments, the carrier filtration chamber includes a first membrane placement chamber and a mixture receiving chamber, the mixture receiving chamber being disposed on a side near the rotation center relative to the first membrane placement chamber, the first membrane placement chamber being provided with a first filtration assembly, the first filtration assembly including the surface-modified membrane.

[0183] In some embodiments, the opening depth of the first membrane placement cavity is greater than the opening depth of the mixture receiving cavity; this facilitates the entry of liquid in the mixture receiving cavity into the first membrane placement cavity under the action of gravity.

[0184] In some implementations, the first filter assembly is inserted into the first membrane placement cavity.

[0185] In some embodiments, the first filtration assembly includes a first membrane substrate and the surface-modified membrane. The first membrane substrate has a window in the middle for liquid to pass through, and the surface-modified membrane is disposed on the side of the first membrane substrate near the rotation center. The first membrane substrate supports the surface-modified membrane, such that the surface-modified membrane is inserted into a first membrane placement cavity.

[0186] After the loading solution and the sample from the sample quantification cell both enter the incubation cell, they are mixed in the incubation cell and then enter the mixed solution containment chamber of the carrier filtration cell. Then, they pass through the surface modification membrane set in the first membrane placement chamber, so that substances in the biological sample, including extracellular secretory membrane structures, are trapped on the surface modification membrane. The remaining substances flow out from the hollow part of the membrane substrate after passing through the surface modification membrane.

[0187] In some embodiments, the side of the carrier filter tank away from the rotation center is a plane perpendicular to the bottom surface of the tank. In some embodiments, the side of the carrier filter tank away from the rotation center gradually rises from the bottom to the surface of the tank; this can be a stepped rise from the bottom to the surface, or a sloping rise from the bottom to the surface. This gradual rise facilitates the transport of liquid from one treatment tank to the next.

[0188] In some embodiments, the enrichment unit includes a target material pool and a waste liquid pool, both connected to the carrier filtration pool. The target material pool is used to collect target extracts from the carrier filtration pool, and the waste liquid pool is used to collect waste liquid from the carrier filtration pool.

[0189] In some embodiments, a sixth flow channel is provided between the carrier filter tank and the waste liquid tank, so that the waste liquid flowing out of the carrier filter tank flows into the waste liquid tank through the sixth flow channel. In some embodiments, the sixth flow channel is a straight flow channel; in some embodiments, the sixth flow channel is a curved flow channel, and the curve helps to increase the flow path.

[0190] In some embodiments, a seventh flow channel is provided between the carrier filter tank and the target material tank, allowing the target material flowing out of the carrier filter tank to flow into the target material tank through the seventh flow channel. In some embodiments, the inlet end of the seventh flow channel is connected to the sixth flow channel. In some embodiments, the connection end of the seventh flow channel and the sixth flow channel forms an acute angle or a right angle. In some embodiments, the connection end of the seventh flow channel and the sixth flow channel forms an angle of 30-90°; for example, 37°, 42°, 48°, 56°, 64°, 72°, 79°, or 84°. The acute angle or right angle between the seventh and sixth flow channels facilitates the transport of the target material to the target material tank; furthermore, when the acute angle or right angle between the seventh and sixth flow channels and the sixth flow channel is a curved flow channel, the degree to which the eluent containing the target material enters the waste liquid tank can be reduced, thus reducing the loss of the target material.

[0191] In some embodiments, the eluent tank is located on the side of the carrier filter tank closer to the center of rotation. In some embodiments, an eighth flow channel is provided between the eluent tank and the carrier filter tank, connecting the two.

[0192] In some implementations, the target material pool and the waste liquid pool are arranged on the same side or opposite side with respect to the sixth flow channel.

[0193] In some implementations, the target material pool and at least one weight-reducing and balancing pool are connected by an air passage; an air hole is provided at the position corresponding to the at least one weight-reducing and balancing pool connected to the target material pool on the bottom plate so that the gas generated during centrifugation can be discharged at any time, so that the separation and extraction device is connected to the external atmospheric pressure.

[0194] In some implementations, the eluent pool is a circular pool or a polygonal pool, such as a square pool, a pentagonal pool, or a hexagonal pool.

[0195] In some embodiments, the eluent tank further includes an eluent container assembly disposed within the eluent tank. The eluent container assembly includes an eluent container box and a first lid. The first lid and the eluent container box are shaped to match each other, and when the eluent container box and the first lid are placed in the eluent tank, the upper surface of the first lid is substantially flush with the upper surface of the eluent tank. A first ejector pin is disposed on the bottom surface of the eluent tank near the eighth flow channel. A first liquid release hole is opened on the bottom surface of the eluent container box. A first sealing membrane is also disposed on the bottom surface of the eluent container box to seal the ejector pin hole.

[0196] Using the above method, the eluent can be pre-placed in the eluent container. During extraction, the first sealing membrane ruptures under the action of the first ejector pin, releasing the eluent from the container. The eluent flows out from the eighth flow channel. This method avoids the step of adding additional eluent, allowing the separation and extraction of the target analyte to be achieved in a single step by simply adding the biological sample.

[0197] In some embodiments, a first support platform is provided on the bottom surface of the eluent tank; the first support platform is used to support the eluent container. When the first ejector pin squeezes the eluent container through the first liquid release hole, the first support platform acts as a fulcrum for the eluent container, creating a lever effect, which facilitates the first ejector pin to break through the first sealing membrane on the eluent container, thereby causing the first sealing membrane to rupture and releasing the eluent.

[0198] In some embodiments, the first sealing membrane is disposed on the inner or outer side of the bottom surface of the eluent container.

[0199] In some embodiments, a ninth flow channel is formed on the upper surface of the first ejector pin, the ninth flow channel pointing towards the eighth flow channel. This arrangement facilitates the flow of eluent from the eluent container to the eighth flow channel.

[0200] In some embodiments, a first protrusion is formed on the sidewall of the eluent tank, and a first concave wall is formed on the outer side of the eluent container, with the first protrusion and the first concave wall engaging. This arrangement facilitates centrifugation, ensuring that the first needle is positioned above the first liquid release hole, allowing the first needle to puncture the first sealing membrane in time, thus enabling the eluent to flow out from the first needle hole. Furthermore, a first notch is formed at a corresponding position on the first lid to engage with the first protrusion. In some embodiments, the first protrusion can be an arc-shaped protrusion, a polygonal protrusion, or any shape that allows the eluent container to be relatively fixed in position.

[0201] In some implementations, the eluent tank and its structure are similar to those of the loading tank.

[0202] In some implementations, the loading liquid pool is a circular pool or a polygonal pool, such as a square pool, a pentagonal pool, or a hexagonal pool.

[0203] In some embodiments, the loading liquid pool further includes a loading liquid holding assembly disposed within the loading liquid pool. The loading liquid holding assembly includes a loading liquid holding box and a second box cover. The second box cover and the loading liquid holding box are matched in shape, and when the eluent holding box and the second box cover are placed in the loading liquid pool, the upper surface of the second box cover is substantially flush with the upper surface of the loading liquid pool. A second ejector pin is disposed on the bottom surface of the loading liquid pool near the fourth flow channel. A second liquid release hole is opened on the bottom surface of the loading liquid holding box. A second sealing membrane is also disposed on the bottom surface of the loading liquid holding box to seal the second liquid release hole.

[0204] Using the above method, the loading fluid can be pre-placed in the loading fluid container. During extraction, the second sealing membrane ruptures under the action of the second ejector pin, thereby releasing the loading fluid from the container. The loading fluid flows out from the fourth flow channel. This method avoids the step of adding additional loading fluid, allowing the separation and extraction of the target analyte to be achieved in a single step by simply adding a biological sample.

[0205] In some embodiments, a second support platform is provided on the bottom surface of the loading liquid pool; the second support platform is used to support the loading liquid container. When the second ejector pin squeezes the loading liquid container through the second liquid release hole, the second support platform acts as a fulcrum for the loading liquid container, generating a lever effect, which facilitates the second ejector pin to break through the second sealing membrane on the loading liquid container, thereby causing the second sealing membrane to rupture and releasing the loading liquid.

[0206] In some embodiments, the second sealing membrane is disposed on the inner or outer side of the bottom surface of the loading liquid container.

[0207] In some embodiments, a tenth flow channel is formed on the upper surface of the second ejector pin, the tenth flow channel pointing towards the fourth flow channel. This arrangement facilitates the flow of loading fluid from the loading fluid container to the fourth flow channel.

[0208] In some embodiments, a second protrusion is formed on the side wall of the loading liquid pool, and a second concave wall is formed on the outer side of the loading liquid container, with the second protrusion and the second concave wall cooperating. This arrangement facilitates centrifugation, ensuring that the second pin is positioned above the second liquid release hole, allowing the second pin to puncture the second sealing membrane in time and thus enabling the loading liquid to flow out from the second liquid release hole. Furthermore, a second recess is formed at a corresponding position on the second container cover, cooperating with the second protrusion.

[0209] In some implementations, the first seal is made of aluminum-plastic film; the second seal is made of aluminum-plastic film.

[0210] In some embodiments, the separation and extraction apparatus further includes a sample pretreatment unit, which includes an impurity filtration pool connected to the incubation pool, and the impurity filtration pool is provided with a microfiltration membrane for retaining impurities.

[0211] In some implementations, the impurity filtration tank and the carrier filtration tank have similar structural configurations.

[0212] In some embodiments, the impurity filtration tank includes a second membrane placement chamber and a metering liquid receiving chamber, the metering liquid receiving chamber being disposed on a side near the rotation center relative to the second membrane placement chamber, the second membrane placement chamber being provided with a second filtration assembly, the second filtration assembly including the microfiltration membrane.

[0213] In some embodiments, the opening depth of the second membrane placement cavity is greater than the opening depth of the metering liquid receiving cavity; this facilitates the entry of liquid in the metering liquid receiving cavity into the second membrane placement cavity under the action of gravity.

[0214] In some embodiments, the second filter assembly is inserted into the second membrane placement cavity.

[0215] In some embodiments, the second filtration assembly includes a second membrane substrate and the microfiltration membrane, wherein the second membrane substrate is hollow in the middle, and the microfiltration membrane is disposed on the side of the second membrane substrate near the rotation center. The second membrane substrate supports the microfiltration membrane, such that the microfiltration membrane is inserted into the second membrane placement cavity.

[0216] In some embodiments, an eleventh flow channel is provided between the impurity filtration cell and the sample quantitative cell, connecting the two. In some embodiments, the inlet end of the eleventh flow channel is connected to the third flow channel. In some embodiments, the eleventh flow channel is a capillary channel, which siphons the solution in the sample quantitative cell into the impurity filtration cell through the siphon effect of the eleventh flow channel.

[0217] In some implementations, a twelfth flow channel is provided between the impurity filtration tank and the incubation tank to connect the two.

[0218] After the sample from the sample quantification cell enters the impurity filtration cell, it first passes through the quantitative liquid containment chamber, and then through the microfiltration membrane set in the second membrane placement chamber. This allows high molecular weight and / or large particle size substances in the biological sample to be trapped on the microfiltration membrane, while the remaining substances flow out from the hollow part of the second membrane substrate after passing through the microfiltration membrane.

[0219] In some embodiments, the side of the impurity filtration tank away from the rotation center is a plane perpendicular to the bottom of the tank. In some embodiments, the side of the impurity filtration tank away from the rotation center gradually rises from the bottom to the surface of the tank; this can be a stepped rise from the bottom to the surface, or a sloping rise from the bottom to the surface. This gradual rise facilitates the transport of liquid from one treatment tank to the next.

[0220] In some embodiments, the sample pretreatment unit further includes a second impurity collection tank, and a thirteenth flow channel connecting the impurity filtration tank and the second impurity collection tank is provided.

[0221] In some embodiments, the separation and extraction device includes a matching base plate and a cover plate, wherein the processing pool, flow channel, air channel, cavity, and air hole are formed on the base plate.

[0222] The "matching settings" refer to the shape being compatible, and the location, shape, and size of the mounting holes and air vents being compatible.

[0223] In some embodiments, a rotation center, a pin hole, and an air hole are provided at corresponding positions on the cover plate. For example, a first pin hole is provided on the cover plate at a position corresponding to the first pin; a second pin hole is provided on the cover plate at a position corresponding to the second pin.

[0224] In some embodiments, the material used to prepare the main body of the separation device can be a polymer such as polymethyl methacrylate (PMMA), polycarbonate (PC), or polyvinyl chloride (PVC); optionally, polycarbonate (PC) can be used. In some embodiments, the materials used to prepare the base plate and cover plate can be polymers such as polymethyl methacrylate (PMMA), polycarbonate (PC), or polyvinyl chloride (PVC); optionally, polycarbonate (PC) can be used.

[0225] In some embodiments, the base plate and cover plate can be bonded by double-sided adhesive or by thermoforming. In some embodiments, the eluent container and the first lid can be bonded by double-sided adhesive or by thermoforming. In some embodiments, the loading liquid container and the second lid can be bonded by double-sided adhesive or by thermoforming. Besides using the same polymer material as the base plate, the eluent container and the loading liquid container can also be a single-sided adhesive film with a hydrophobic adhesive layer. The flow channels can be modified with a modifier to achieve hydrophilicity, allowing the liquid to flow out smoothly. In some embodiments, the thickness of the base plate is 3-8 mm, for example, 4 mm, 4.5 mm, 5 mm, and 5.5 mm.

[0226] In some embodiments, when extracting the target extract from a organism using the above-described separation and extraction device, a loading liquid container is installed in the loading liquid tank of the separation and extraction device, and the loading liquid container is filled with loading liquid. A surface-modified membrane is disposed in the first membrane placement chamber of the carrier filtration tank; a microfiltration membrane is disposed in the second membrane placement chamber of the impurity filtration tank. An eluent container is provided in the eluent tank, and the eluent container contains an eluent tank. The method for extracting the target extract from a organism using the above-described separation and extraction device includes the following steps:

[0227] I. The biological sample is added into the sample pool, causing the separation and extraction device to rotate at high speed at the first rotation speed, so that the sample quantitative pool is filled with the biological sample, and at the same time, some impurities enter the first impurity collection pool.

[0228] II. After standing for a period of time, the biological sample gradually enters the impurity filtration tank due to the siphon effect of the eleventh flow channel; at the same time, the second pin punctures the second sealing membrane, allowing the loading liquid to flow out from the loading liquid tank.

[0229] III. The separation and extraction device is made to rotate at high speed at the second rotation speed, so that the biological sample entering the impurity filtration pool passes through the microfiltration membrane and enters the incubation pool; at the same time, the loading liquid flowing out from the loading liquid pool also enters the incubation pool; the biological sample and the loading liquid are mixed in the incubation pool.

[0230] IV. Control the separation and extraction device to rotate at high-low-high speeds in a cycle to promote accelerated mixing of the two solutions in the incubation tank;

[0231] V. Stop and let stand for a period of time to allow the fifth flow channel to be filled with the mixture;

[0232] VI. The separation and extraction device rotates at high speed, so that the mixture enters the carrier filtration tank and passes through the surface-modified membrane, thereby trapping the extracellular secretory membrane structure in the mixture on the surface-modified membrane, while the waste liquid enters the waste liquid tank.

[0233] VII. Stop and let stand for a period of time; during this period, the first pin will puncture the first sealing membrane, allowing the eluent to flow out from the eluent pool;

[0234] VIII. The separation and extraction device is rotated at high speed, so that the eluent passes through the surface-modified membrane and the extracellular secretory membrane structure is eluted from the surface-modified membrane and then enters the target cell.

[0235] In some implementations, the first rotational speed is 2500~5500 rpm; for example, it can be 2750 rpm, 3000 rpm, 3250 rpm, 3500 rpm, 3750 rpm, 4000 rpm, 4250 rpm, 4500 rpm, 4750 rpm, 5000 rpm, or 5250 rpm. The duration of the first rotational speed is 120~480 s; for example, it can be 155 s, 185 s, 235 s, 275 s, 335 s, 395 s, 415 s, or 475 s.

[0236] In some implementations, the settling time in step II is 5 to 50 seconds; for example, it can be 9 seconds, 17 seconds, 23 seconds, 29 seconds, 32 seconds, 38 seconds, 43 seconds, or 47 seconds.

[0237] In some embodiments, the second rotational speed is 2000~4500 rpm; for example, it can be 2250 rpm, 2500 rpm, 2750 rpm, 3000 rpm, 3250 rpm, 3500 rpm, 3750 rpm, 4000 rpm, or 4250 rpm. The duration of the second rotational speed is 15~120 s; for example, it can be 25 s, 45 s, 75 s, 95 s, or 115 s.

[0238] In some embodiments, the high speed in step IV is 2500~4000 rpm; for example, it can be 2750 rpm, 3000 rpm, 3250 rpm, 3500 rpm, or 3750 rpm; the duration of the high speed is 3~20 s; for example, it can be 5 s, 8 s, 12 s, 16 s, or 19 s. In some embodiments, the low speed in step IV is 300~2000 rpm; for example, it can be 450 rpm, 800 rpm, 1250 rpm, 1500 rpm, 1750 rpm, or 1900 rpm; the duration of the low speed is 3~20 s; for example, it can be 5 s, 8 s, 12 s, 16 s, or 19 s. In some embodiments, the rotation direction of the high speed and low speed can be the same or opposite. In some embodiments, the number of high-low-high speed alternations can be 3~15 times; for example, 5 times, 8 times, 11 times, or 13 times.

[0239] In some implementations, the settling time in step V can be 5 to 50 seconds; for example, 15 seconds, 22 seconds, 28 seconds, 34 seconds, 41 seconds, or 47 seconds.

[0240] In some implementations, the high speed in step VI is 2000~5000 rpm; for example, it can be 2250 rpm, 2500 rpm, 2750 rpm, 3000 rpm, 3250 rpm, 3500 rpm, 3750 rpm, 4000 rpm, 4250 rpm, 4500 rpm or 4750 rpm; the duration of the high speed is 20~120s; for example, it can be 35s, 65s, 85s, 105s or 115s.

[0241] In some implementations, the settling time in step VII can be 5 to 40 seconds; for example, 10 seconds, 17 seconds, 22 seconds, 29 seconds, or 37 seconds.

[0242] In some implementations, the high speed in step VIII is 2500~6500 rpm; for example, it can be 2750 rpm, 3350 rpm, 3950 rpm, 4550 rpm, 4900 rpm, 5450 rpm or 5900 rpm; the duration of the high speed is 30~120s; for example, it can be 35s, 65s, 85s, 105s or 115s.

[0243] In some embodiments, this application also provides a kit for the isolation and extraction of extracellular secretory membrane structures. The kit includes the isolation and extraction device, which contains the surface-modified membrane, a loading solution pre-embedded therein, and an elution solution. In some embodiments, the isolation and extraction device further includes the microfiltration membrane.

[0244] In some implementations, the separation and extraction device is a centrifugal adsorption column. The separation and extraction device includes a first outer tube with one end open and a first inner tube with both ends open and sleeved inside the first outer tube. The other end of the first outer tube is closed. There is a certain accommodating space between the end of the first inner tube near the closed end of the first outer tube and the closed end of the first outer tube. A surface modification film is provided at the end of the first inner tube near the closed end of the first outer tube.

[0245] In some implementations, the open end of the first outer tube is provided with a first sealing cap.

[0246] In some implementations, a liquid outlet pipe is provided at one end of the first inner tube near the closed end of the first outer tube, and the inner diameter of the liquid outlet pipe is smaller than the inner diameter of the first inner tube.

[0247] In some embodiments, there are at least two centrifugal adsorption columns, at least one of which has a surface-modified membrane disposed in the first inner tube, and at least one of which has a microfiltration membrane disposed in the first inner tube.

[0248] When using the above-mentioned separation and extraction device to separate and extract extracellular secretory membrane structures, the following steps are included:

[0249] S1. Add the biological sample into the first inner tube of the centrifugal adsorption column equipped with a microfiltration membrane, then centrifuge and collect the filtrate for later use.

[0250] S2. After mixing the filtrate and the loading liquid, place them in the first inner tube of a centrifugal adsorption column equipped with a surface-modified membrane, and then let them stand until all the filtrate has passed through the surface-modified membrane. Discard the filtrate.

[0251] S3. Add the eluent to the first inner tube equipped with the surface-modified membrane. After all the eluent has passed through the surface-modified membrane, centrifuge and discard the supernatant to obtain the target sample.

[0252] In some implementations, the centrifugation speed in step S1 is 2000~5000 rpm; for example, it can be 2250 rpm, 2500 rpm, 2750 rpm, 3000 rpm, 3250 rpm, 3500 rpm, 3750 rpm, 4000 rpm, 4250 rpm, 4500 rpm or 4750 rpm; the centrifugation time is 20~120 s; for example, it can be 35 s, 65 s, 85 s, 105 s or 115 s.

[0253] In some implementations, the centrifugation speed in step S3 is 2500~6500 rpm; for example, it can be 2750 rpm, 3350 rpm, 3950 rpm, 4550 rpm, 4900 rpm, 5450 rpm or 5900 rpm; the duration of the high speed is 30~120s; for example, it can be 35s, 65s, 85s, 105s or 115s.

[0254] The separation and extraction kit includes at least one of the above-described centrifugal adsorption columns, with a surface-modified membrane disposed within the column; the kit also includes a loading solution and an elution solution. Further, the separation and extraction kit includes at least two of the above-described centrifugal adsorption columns, with a surface-modified membrane disposed within at least one centrifugal adsorption column and a microfiltration membrane disposed within at least one centrifugal adsorption column.

[0255] In some embodiments, the separation and extraction device includes at least one syringe, and the needle insertion end of at least one of the syringes is provided with a surface-modified film.

[0256] In some embodiments, the separation and extraction device includes at least two syringes, at least one of which has a surface-modified membrane at the needle insertion end and at least one of which has a microfiltration membrane at the needle insertion end.

[0257] When using the above-mentioned separation and extraction device to separate and extract extracellular secretory membrane structures, the following steps are included:

[0258] S1. Add the biological sample into a syringe equipped with a microfiltration membrane and collect the filtrate for later use.

[0259] S2. After mixing the filtrate and loading solution, place them in a syringe equipped with a surface-modified membrane, and then let them stand until the mixture has completely passed through the surface-modified membrane. Discard the filtrate.

[0260] S3. Add the eluent to the syringe equipped with the surface modification membrane. After all the eluent has passed through the surface modification membrane, centrifuge the eluent, discard the supernatant, and obtain the target sample.

[0261] In some implementations, step S1 can be performed by allowing the biological sample to pass through the microfiltration membrane by letting it stand or by pressing down the piston rod.

[0262] In some implementations, the centrifugation speed in step S3 is 2500~6500 rpm; for example, it can be 2750 rpm, 3350 rpm, 3950 rpm, 4550 rpm, 4900 rpm, 5450 rpm or 5900 rpm; the duration of the high speed is 30~120s; for example, it can be 35s, 65s, 85s, 105s or 115s.

[0263] The separation and extraction kit includes at least one syringe as described above, with a surface-modified membrane disposed within the syringe; the kit also includes a loading solution and an elution solution. Further, the separation and extraction kit includes at least two syringes, with a surface-modified membrane disposed within at least one syringe and a microfiltration membrane disposed within at least one syringe.

[0264] In some implementations, the syringe can also be replaced with a solid-phase extraction column.

[0265] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0266] Preparation of surface-modified films

[0267] Example 1: Preparation of surface-modified films

[0268] Dissolve 8 mM dodecyltrimethylammonium bromide (DTAB) in 10 mL of methanol, mix well at room temperature and ensure DTAB is fully dissolved; place in an RC membrane (RC membrane purchased from Sartorius / Whatman), react with shaking at room temperature for 40 h, and dry in a vacuum drying oven at 60 °C; then wash three times with 95% ethanol, dry in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which is stored at room temperature for later use.

[0269] Example 2 of surface modification film preparation

[0270] Dissolve 8.6 mM hexadecyltrimethylammonium bromide (CTAB) in 10 mL of methanol, mix well at room temperature and ensure CTAB is fully dissolved; place in an RC membrane, shake at room temperature for 48 h, and dry in a vacuum drying oven at 60 °C; then wash three times with 95% ethanol, dry in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which is stored at room temperature for later use.

[0271] Example 3: Preparation of surface-modified film

[0272] Dissolve 5.6 mM of quaternary ammonium salt-type Gemini surfactant (gemini cationic surfactant purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.) in 10 mL of water at 45℃, mix well and ensure the quaternary ammonium salt-type Gemini surfactant is fully dissolved; place in an RC membrane, shake at room temperature for 54 h, dry in a vacuum drying oven at 60℃ to obtain a quaternized filter membrane, and store at room temperature for later use.

[0273] Example 4: Preparation of surface-modified film

[0274] The preparation of asymmetric quaternary ammonium salt-type Gemini surfactants includes the following steps:

[0275] N,N-dimethylhexylamine and 1,3-dibromoisopropanol were mixed in a 1:4 molar ratio, and anhydrous ethanol was added. The mixture was stirred at 70°C for 12 h and then cooled. After removing the ethanol by rotary evaporation, the mixture was extracted with diethyl ether, allowed to stand for 10 min, and the lower viscous liquid was collected. After filtration, the filtrate was evaporated by rotary evaporation to remove the diethyl ether, yielding the intermediate product 2-hydroxy-3-bromohexane ammonium bromide. 2-hydroxy-3-bromohexane ammonium bromide was mixed with N,N-dimethyloctadecylamine in a 1:1 molar ratio and reacted at 80°C for 24 h. After cooling, the reactants were evaporated by rotary evaporation to remove the ethanol. The mixture was then recrystallized from the filtrate using a 1:10 volume ratio of ethanol / ethyl acetate, and the filtrate was recrystallized three times using a 1:8 volume ratio of ethanol / anhydrous diethyl ether to obtain the final product.

[0276] The preparation of the surface-modified film includes the following steps:

[0277] The asymmetric quaternary ammonium salt-type Gemini surfactant prepared above at 3.8 mM was dissolved in 10 mL of ethanol, mixed well, and the quaternary ammonium salt-type Gemini surfactant was fully dissolved; it was placed in an RC membrane, and the reaction was carried out with shaking at room temperature for 54 h. It was then dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0278] Example 5: Preparation of surface-modified film

[0279] The preparation of a symmetrical quaternary ammonium salt-type Gemini surfactant includes the following steps: 0.1 mol of alkyl dimethyl tertiary amine, 0.05 mol of dihydrocarbon, and 50 mL of ethanol are added to a three-necked flask, and the mixture is heated and stirred under reflux for 48 h. After the reaction solution cools slightly, it is rotary evaporated to a slightly viscous substance. The product is recrystallized with a mixture of ethyl acetate. To ensure sufficient purity, recrystallization is performed three times using a 1:10 volume ratio ethanol / ethyl acetate mixture. The resulting solid product is transferred to a vacuum drying oven and dried under vacuum at 50 °C for 12 h to obtain the symmetrical quaternary ammonium salt-type Gemini surfactant.

[0280] The preparation of the surface-modified film includes the following steps:

[0281] The symmetrical quaternary ammonium salt-type Gemini surfactant prepared above at 4.7 mM was dissolved in 10 mL of ethanol, mixed well and the quaternary ammonium salt-type Gemini surfactant was fully dissolved; placed in an RC membrane, and reacted with shaking at room temperature for 50 h, and dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0282] Example 6: Preparation of surface-modified film

[0283] 5.3mM QAS C0 (Purchased from Macklin) Dissolve in 10 mL of water, mix well and stir for 2 hours, then adjust QAS. C0 Dissolve completely; place in RC membrane, shake at room temperature for 24 h, dry in a vacuum drying oven at 60℃, wash three times with 95% ethanol, dry in a vacuum drying oven at 60℃ to obtain quaternized filter membrane, store at room temperature for later use.

[0284] Example 7: Preparation of surface-modified films

[0285] 5.8mM QAS C18 (Purchased from Macklin) Dissolve in 10 mL of water, mix well and stir for 2 hours, then adjust QAS. C18 Dissolve completely; place in RC membrane, shake at room temperature for 24 h, dry in a vacuum drying oven at 60℃, wash three times with 95% ethanol, dry in a vacuum drying oven at 60℃ to obtain quaternized filter membrane, store at room temperature for later use.

[0286] Example 8 of surface modification film preparation

[0287] 6.4 mM cocamidopropyl betaine (product model CAB-35, C) 19 H 38 Dissolve N2O3 in 10 mL of water, mix and stir to fully dissolve cocamidopropyl betaine; place in an RC membrane, shake at room temperature for 42 h, dry in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, and store at room temperature for later use.

[0288] Example 9 of surface modification film preparation

[0289] Dissolve 3.7 mM capryloyl propyl betaine (product model ODAB-35, available from Wuhan Kangqiong Biomedical Technology Co., Ltd.) in 10 mL of ethanol, mix and stir until the cocamidopropyl betaine is fully dissolved; place in an RC membrane, shake at room temperature for 52 h, dry in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, and store at room temperature for later use.

[0290] Example 10 of surface modification film preparation

[0291] 4.6 mM 3-(N,N-dimethyldodecylammonium)propane sulfonate (SPPT, CAS 14933-08-5) was dissolved in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The solution was then placed in an RC membrane and reacted with shaking at room temperature for 24 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0292] Example 11 of surface modification film preparation

[0293] 0.24 mM DTAB and 6 mM cocamidopropyl betaine (DTAB to cocamidopropyl betaine molar ratio of 0.12:3) were dissolved in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The solution was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0294] Example 12 of surface modification film preparation

[0295] 1.8mM QAS C0 and 4.5mM SPPT (QAS) C0 The solution (with a molar ratio of 1.2:3) to SPPT was dissolved in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The solution was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0296] Example 13 of surface modification film preparation

[0297] 1.8mM QAS C18 and 4.5mM SPPT (QAS) C18 The solution (with a molar ratio of 1.2:3) to SPPT was dissolved in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The solution was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0298] Example 14: Preparation of surface-modified films

[0299] 1.8mM QAS C0 4.5mM SPPT (QAS) C0 The molar ratio of SPPT to betaine (1.2:3) and 7.6 mM betaine (molar ratio of betaine to surfactant 1.2:1) were dissolved in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0300] Example 15: Preparation of Surface Modified Film

[0301] 1.8mM QAS C0 4.5mM SPPT (QAS) C0 The proline (in a molar ratio of 1.2:3 to SPPT) and 2.5 mM proline (in a molar ratio of 0.4:1 to surfactant) were placed in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0302] Example 16: Preparation of Surface Modified Film

[0303] 1.8mM QAS C0 4.5mM SPPT (QAS) C0 The 3.8 mM mannitol (with a molar ratio of 1.2:3 to SPPT) and surfactant (with a molar ratio of 0.6:1 to 3.8 mM) were placed in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0304] Example 17: Preparation of Surface Modified Film

[0305] 1.8mM QAS C0 4.5mM SPPT (QAS) C0 The mixture of SPPT (in a molar ratio of 1.2:3), 7.6 mM betaine, and 2.5 mM proline was placed in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0306] Example 18: Preparation of surface-modified films

[0307] 1.8mM QAS C0 4.5mM SPPT (QAS) C0 The mixture of SPPT (in a molar ratio of 1.2:3), 7.6 mM betaine, 2.5 mM proline, and 3.8 mM mannitol was placed in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0308] Example 19: Preparation of Surface Modified Film

[0309] 1.8mM QAS C18 4.5mM SPPT (QAS) C18 The mixture of SPPT (in a molar ratio of 1.2:3), 7.6 mM betaine, 2.5 mM proline, and 3.8 mM mannitol was placed in 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The mixture was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0310] Example 20 of surface modification film preparation

[0311] 6.4 mM cocamidopropyl betaine (product model CAB-35, C) 19 H 38 N2O3) and 7.7 mM betaine, 2.6 mM proline and 3.8 mM mannitol were added to 10 mL of water, mixed and stirred until cocamidopropyl betaine was fully dissolved; placed in an RC membrane, and reacted at room temperature with shaking for 42 h, and dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0312] Example 21 of surface modification film preparation

[0313] 4.6 mM SPPT, 5.5 mM betaine, 1.8 mM proline, and 2.8 mM mannitol were added to 10 mL of methanol and mixed at room temperature for 2 h to ensure complete dissolution. The solution was then placed in an RC membrane and reacted with shaking at room temperature for 48 h. The membrane was then dried in a vacuum drying oven at 60 °C. After washing three times with 95% ethanol, the membrane was dried in a vacuum drying oven at 60 °C to obtain a quaternized filter membrane, which was stored at room temperature for later use.

[0314] Example 1

[0315] A centrifugal adsorption column 5, such as Figure 1As shown, it includes a first outer tube 51 with one end open and a first inner tube 52 with both ends open and sleeved inside the first outer tube 51. The other end of the first outer tube 51 is closed. There is a certain space between the end of the first inner tube 52 near the closed end of the first outer tube 51 and the closed end of the first outer tube 51. A surface modification film 22432 is provided on the end of the first inner tube 52 near the closed end of the first outer tube 51.

[0316] The outer diameter of the first inner tube 52 is the same as the inner diameter of the first outer tube 51 (the attached diagram is for illustrative purposes only), meaning the first inner tube 52 is snapped into the first outer tube 51. A first sealing cap 511 is provided at the open end of the first outer tube 51 to prevent liquid splashing during centrifugation. The structure of the end of the first inner tube 52 near the sealed end of the first outer tube 51 is similar to the design of the needle insertion end of a syringe. An outlet tube 521 is provided at this end of the first inner tube 52 near the sealed end of the first outer tube 51. The inner diameter of the outlet tube 521 is smaller than the inner diameter of the first inner tube 52, reducing the possibility of liquid splashing onto the side wall of the first outer tube 51 when it exits the first inner tube 52, thus preventing poor extraction results. In addition, there are two centrifugal adsorption columns 5, namely the first centrifugal adsorption column and the second centrifugal adsorption column. The difference between the first centrifugal adsorption column and the second centrifugal adsorption column is that: one end of the liquid outlet tube 521 of the first inner tube 52 of the first centrifugal adsorption column is clamped with a microfiltration membrane 25132 (not shown in the figure), and one end of the liquid outlet tube 521 of the first inner tube 52 of the second centrifugal adsorption column is clamped with a surface modification membrane 22432.

[0317] The volume of the first and second centrifugal adsorption columns is 3 mL.

[0318] A method for extracting exosomes, implemented using a centrifugal adsorption column, includes the following steps:

[0319] S1. Take 500 μL of whole blood sample, add it to the first centrifugal adsorption column, and then centrifuge at 2500 rpm for 60 s. Collect the filtrate for later use to obtain plasma.

[0320] S2. The plasma and loading solution are mixed at a 1:1 volume ratio (200 μL each) and placed in a second centrifugal adsorption column. The second centrifugal adsorption column is equipped with a surface-modified membrane 22432. In this embodiment, the surface-modified membrane 22432 used is prepared by surface-modified membrane preparation example 1 and is loaded with DTAB. Then, the mixture is allowed to stand until all the mixture has passed through the surface-modified membrane 22432. The first inner tube 52 in the second centrifugal adsorption column 5 is then removed, the filtrate is discarded, and the first inner tube 52 is then installed into the first outer tube 51.

[0321] S3. Then, the eluent is added to the first inner tube 52 of the second centrifugal adsorption column 5, and the surface-modified membrane 22432 loaded with exosomes is eluted with the eluent. The high concentration of salt solution in the eluent causes the exosomes to detach from the surface-modified membrane 22432, and the exosome collection solution is obtained.

[0322] The loading solution mainly consists of 100 mM Tris-propane, 150 mM NaCl, and pH 6.5. The eluent mainly consists of 50 mM Tris-propane, 1 M NaCl, and pH 6.36.

[0323] Example 2

[0324] The difference between this embodiment and Embodiment 1 is that the surface-modified film used in this embodiment is prepared by Surface Modified Film Preparation Example 2, and CTAB is loaded on it.

[0325] The loading solution consists mainly of 100 mM Tris-propane, 150 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2 M NaCl, and pH 6.36.

[0326] Everything else is the same as in Example 1.

[0327] Example 3

[0328] A method for extracting exosomes, using syringes, specifically two syringes 6: a first syringe and a second syringe. For example... Figure 2 As shown, a surface-modified membrane 22432 is fixedly disposed at one end of the first syringe near the needle insertion end; a microfiltration membrane 25132 (not shown in the figure) is fixedly disposed at one end of the second syringe near the needle insertion end. The volume of the first syringe and the second syringe is 3 mL.

[0329] The extraction method includes the following steps:

[0330] S1. Take 500 μL of whole blood sample and add it to the second syringe. Press the piston rod of the second syringe to make the whole blood sample pass through the microfiltration membrane. Collect the filtrate for later use to obtain plasma.

[0331] S2. The plasma and loading solution are mixed at a volume ratio of 1:1 (200 μL each) and placed in a first syringe. The first syringe is equipped with a surface-modified membrane. In this embodiment, the surface-modified membrane used is prepared by Surface Modification Membrane Preparation Example 3 and is loaded with a quaternary ammonium salt-type Gemini surfactant. The mixture is then allowed to stand until all the mixture has passed through the surface-modified membrane, and the filtrate is discarded.

[0332] S3. Then, add 300 μL of eluent to the first syringe, press the piston rod of the first syringe, and elute the surface-modified membrane loaded with exosomes with the eluent. The high concentration of salt solution in the eluent causes the exosomes to detach from the surface-modified membrane, and the exosome collection solution is obtained.

[0333] The loading solution mainly consists of 100 mM Tris-propane, 168 mM NaCl, and pH 6.5. The eluent mainly consists of 50 mM Tris-propane, 2.45 M NaCl, and pH 6.36.

[0334] Example 4

[0335] The difference between this embodiment and Example 1 is that the surface-modified film used in this embodiment was prepared by Example 4 of surface-modified film preparation, and it is loaded with an asymmetric quaternary ammonium salt type Gemini surfactant.

[0336] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0337] Everything else is the same as in Example 1.

[0338] Example 5

[0339] The difference between this embodiment and Example 1 is that the surface-modified film used in this embodiment is prepared by Example 5 of surface-modified film preparation, and is loaded with a symmetrical quaternary ammonium salt type Gemini surfactant.

[0340] The loading solution consists mainly of 100 mM Tris-propane, 145 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.1 M NaCl, and pH 6.36.

[0341] Everything else is the same as in Example 1.

[0342] Example 6

[0343] A method for extracting exosomes, comprising using a separation and extraction device, such as a microfluidic centrifuge disc, to complete the extraction. Figure 3 As shown, the microfluidic centrifuge disc includes a base plate 3 and a cover plate 4.

[0344] like Figure 4 As shown, a rotation center 1 and an extraction mechanism 2 are provided on the base plate 3. The extraction mechanism 2 includes a sample injection unit 21, a capture and release unit 22, and an enrichment unit 23. A surface modification film 22432 (see reference) is disposed inside the capture and release unit 22. Figure 12 The capture and release unit 22 is connected to the sample injection unit 21 and is located downstream of the sample injection unit 21. It is used to capture or release target extracts from biological samples. The enrichment unit 23 is connected to the capture and release unit 22 and is located downstream of the capture and release unit 22. It is used to enrich exosomes released from the capture and release unit 22.

[0345] The center of the base plate 3 is the rotating mounting part, and the rotation center 1 is the rotation center during centrifugal operation.

[0346] like Figure 3 and Figure 5 As shown, the sample injection unit 21 includes a sample cell 211, and a sample injection hole 2111 is provided at a corresponding position on the cover plate 4. Whole blood samples are injected through the sample injection hole 2111. Figure 5 and Figure 6 As shown, the sample introduction unit 21 also includes a sample quantification cell 212 and a excess sample holding cell 213. A first flow channel 214, which is a capillary channel, is provided between the sample cell 211 and the sample quantification cell 212 to allow the biological sample in the sample cell 211 to enter the sample quantification cell 212 more smoothly and quickly under the siphon effect. A second flow channel 215 is provided between the sample quantification cell 212 and the excess sample holding cell 213 to connect the two. In addition, the excess sample holding cell 213 is connected to one of the weight reduction and balancing cells 24, and the two are connected through an air channel 241, such as... Figure 3 As shown, the cover plate 4 is provided with air holes 43 at the corresponding positions to facilitate timely venting during centrifugation. During sample injection, the biological sample is first added to the injection cell, and then the biological sample flows into the sample quantification cell 212, so that the excess biological sample flows into the excess sample holding cell 213. The amount of biological sample processed each time is the maximum holding volume of the sample quantification cell 212.

[0347] The sample injection unit 21 also includes a first impurity collection cell 216, which is located on the side of the sample quantitative cell 212 away from the rotation center 1. A third flow channel 217 is provided between the first impurity collection cell 216 and the sample quantitative cell 212 to connect the two. The third flow channel 217 is a constricted channel, and the side connecting the sample quantitative cell 212 and the first impurity collection cell 216 is stepped, so that impurities in the sample quantitative cell 212 can enter the first impurity collection cell 216 more smoothly under the action of centrifugal force.

[0348] like Figure 7 As shown, the capture and release unit 22 includes a loading liquid tank 221 and an eluent tank 222 (see reference). Figure 11 ), incubation tank 223 and carrier filtration tank 224 (reference) Figure 11The incubation pool 223 is connected to the outlet of the sample injection unit 21 and the loading liquid pool 221, respectively. The carrier filter pool 224 is connected to the outlet of the incubation pool 223 and the elution liquid pool 222, respectively. The carrier filter pool 224 is provided with a surface modification membrane 22432. The surface modification membrane 22432 selected in this embodiment is prepared by surface modification membrane preparation example 6 and is loaded with QASCO. In addition, the loading liquid pool 221, the incubation pool 223 and the carrier filter pool 224 are distributed sequentially from the proximal end to the distal end of the rotation center 1.

[0349] like Figure 7 As shown, a sample pretreatment unit 25 is also provided on the base plate 3 between the sample injection unit 21 and the capture unit. The sample pretreatment unit 25 includes an impurity filtration cell 251 and a second impurity collection cell 254. The upstream of the impurity filtration cell 251 is connected to the downstream of the sample quantification cell 212, and the impurity filtration cell 251 is also connected to the second impurity collection cell 254. An eleventh flow channel 252 is provided between the impurity filtration cell 251 and the sample quantification cell 212 to connect the two, and the liquid inlet end of the eleventh flow channel 252 is connected to the third flow channel 217. The eleventh flow channel 252 is a capillary channel, so that the solution in the sample quantification cell 212 is smoothly siphoned into the impurity filtration cell 251 under the siphon effect.

[0350] like Figure 8 As shown, the impurity filtration tank 251 has a second membrane placement chamber 2511 and a metering liquid receiving chamber 2512. The metering liquid receiving chamber 2512 is located on the side closer to the rotation center 1 relative to the second membrane placement chamber 2511, and a second filter assembly 2513 is inserted into the second membrane placement chamber 2511. Furthermore, the depth of the second membrane placement chamber 2511 is greater than the depth of the metering liquid receiving chamber 2512; this facilitates the entry of liquid from the metering liquid receiving chamber 2512 into the second membrane placement chamber 2511 under gravity. Additionally, the side of the impurity filtration tank 251 away from the rotation center 1 is stepped from the bottom to the surface, which facilitates the transport of liquid from the previous treatment tank to the next treatment tank.

[0351] The second filtration assembly 2513 includes a second membrane substrate 25131 for supporting the microfiltration membrane 25132 and the microfiltration membrane 25132. The second membrane substrate 25131 has a window in the middle to allow liquid to pass through. The microfiltration membrane 25132 is bonded to the side of the second membrane substrate 25131 near the rotation center 1.

[0352] A twelfth flow channel 253 is provided between the impurity filtration pool 251 and the incubation pool 223, connecting the two. When the sample from the sample quantification pool 212 enters the impurity filtration pool 251, it first passes through the quantitative liquid receiving chamber 2512, and then through the microfiltration membrane 25132 set in the second membrane placement chamber 2511. This allows high molecular weight and / or large particle size substances in the biological sample to be trapped on the microfiltration membrane 25132, while the remaining substances flow out from the hollow part of the second membrane substrate 25131 after passing through the microfiltration membrane 25132 and enter the incubation pool 223.

[0353] like Figure 7 As shown, loading liquid also enters the incubation tank 223 simultaneously, and the loading liquid tank 221 is used for loading liquid. The loading liquid tank 221 is a circular tank located on the side of the incubation tank 223 near the rotation center 1, and a fourth flow channel 225 is provided between it and the incubation tank 223 to connect them. The loading liquid tank 221 includes a loading liquid holding assembly 2211 disposed within the loading liquid tank 221, such as... Figure 9 As shown, the loading fluid container assembly 2211 includes a loading fluid container 22111 and a second lid 22112. The second lid 22112 matches the shape of the loading fluid container, and when the loading fluid container 22111 and the second lid 22112 are placed in the loading fluid pool 221, the upper surface of the second lid 22112 is substantially flush with the upper surface of the loading fluid pool 221. (Reference) Figure 7 and Figure 9 A second ejector pin 2212 is provided on the bottom surface of the loading liquid pool 221 near the fourth flow channel 225; a tenth flow channel 22121 is formed on the upper surface of the second ejector pin 2212, such as... Figure 10 As shown, the tenth flow channel 22121, pointing towards the fourth flow channel 225, has a second liquid release hole 22113 on its bottom surface of the liquid-filled container 22111. A second sealing film 22114, made of aluminum-plastic film, is also provided on the outer side of the bottom surface of the liquid-filled container 22111 to seal the second liquid release hole 22113. This second seal is easily punctured. Furthermore, a second support platform 2213 is provided on the bottom surface of the liquid-filled pool 221; the second support platform 2213 is used to support the liquid-filled container 22111.

[0354] During separation and extraction, the second ejector pin 2212 squeezes the loading liquid container 22111 through the second liquid release hole 22113. The second support platform 2213 acts as a fulcrum for the loading liquid container 22111, and through leverage, the second ejector pin 2212 ruptures the second sealing membrane 22114 on the loading liquid container 22111, thereby releasing the loading liquid. This method avoids the step of adding additional loading liquid, allowing the separation and extraction of the target analyte to be achieved in a single step by simply adding the biological sample.

[0355] To prevent the loading liquid container assembly 2211 from shifting position arbitrarily within the loading liquid pool 221 during centrifugation, a second protrusion 2214 is provided on the side wall of the loading liquid pool 221; correspondingly, a second recess 22116 that mates with the second protrusion 2214 is provided at a corresponding position on the second cover 22112; and a second concave wall 22115 is provided at a corresponding position on the outer side of the loading liquid container 22111. After the loading liquid container assembly 2211 is placed in the loading liquid pool 221, the second protrusion 2214, the second recess 22116, and the second concave wall 22115 engage. This arrangement ensures that during centrifugation, the second ejector pin 2212 is positioned above the second liquid release hole 22113, and that the second ejector pin 2212 can promptly puncture the second sealing membrane 22114, allowing the loading liquid to flow out from the second liquid release hole 22113 and enter the carrier filter pool 224.

[0356] like Figure 7 As shown, a fifth flow channel 227 and a mixed liquid transport flow channel 226 are sequentially connected between the incubation tank 223 and the carrier filter tank 224. The fifth flow channel 227 is a capillary channel, which allows the liquid in the incubation tank 223 to flow smoothly to the mixed liquid transport flow channel 226 through the siphon effect of the capillary channel; the mixed liquid transport flow channel 226 is an arc-shaped flow channel, which facilitates the transport of the liquid contained therein to the next stage under the action of centrifugal force.

[0357] like Figure 11 As shown, the carrier filter tank 224 includes a first membrane placement chamber 2241 and a mixed liquid receiving chamber 2242. The mixed liquid receiving chamber 2242 is located on the side near the rotation center 1 relative to the first membrane placement chamber 2241. The opening depth of the first membrane placement chamber 2241 is greater than the opening depth of the mixed liquid receiving chamber 2242, so that the liquid in the mixed liquid receiving chamber 2242 can enter the first membrane placement chamber 2241 under the action of gravity.

[0358] like Figure 12As shown, a first filter assembly 2243 is inserted into the first membrane placement cavity 2241. The first filter assembly 2243 includes a first membrane substrate 22431 for supporting the surface-modified membrane 22432 and the surface-modified membrane 22432. The first membrane substrate 22431 is hollow in the middle, and the surface-modified membrane 22432 is disposed on the side of the first membrane substrate 22431 near the rotation center 1. When the loading solution and the sample from the sample quantitative cell 212 both enter the incubation cell 223, they are mixed in the incubation cell 223 and then enter the mixture receiving cavity 2242 of the carrier filter cell 224. After passing through the surface-modified membrane 22432 disposed in the first membrane placement cavity 2241, the extracellular secretory membrane structure of the biological sample is trapped on the surface-modified membrane 22432, and the remaining substances flow out from the hollow part of the first membrane substrate 22431 after passing through the surface-modified membrane 22432.

[0359] The side of the carrier filter tank 224 away from the rotation center 1 is stepped, which is conducive to the transport of liquid from the upper treatment tank to the lower treatment tank.

[0360] The downstream of the carrier filter tank 224 is connected to the enrichment unit 23; the upstream of the carrier filter tank 224 is connected to the eluent tank 222.

[0361] The eluent tank 222 is located on the side of the carrier filter tank 224 near the rotation center 1, and the eluent tank 222 is a square tank. For example... Figure 11 As shown, an eighth flow channel 228 is provided between the eluent tank 222 and the carrier filter tank 224 to connect the two.

[0362] like Figure 13 As shown, the eluent tank 222 includes an eluent container 2221 disposed within the eluent tank 222. The eluent container 2221 includes an eluent container 22211 and a first lid 22212. The first lid 22212 and the eluent container 22211 are matched in shape. When the eluent container 22211 and the first lid 22212 are placed in the eluent tank 222, the upper surface of the first lid 22212 is substantially flush with the upper surface of the eluent tank 222.

[0363] like Figure 11 As shown, a first ejector pin 2222 is provided on the bottom surface of the eluent tank 222 near the eighth flow channel 228. A ninth flow channel 22221 is formed on the upper surface of the first ejector pin 2222, and the ninth flow channel 22221 points towards the eighth flow channel 228. A first support platform 2223 is integrally formed on the bottom surface of the eluent tank 222 away from the hole of the first ejector pin 2222; as shown Figure 14As shown, the bottom surface of the eluent container 22211 has a first liquid release hole 22213. A first sealing membrane 22214, which seals the hole of the first ejector pin 2222, is also adhered to the bottom surface of the eluent container 22211. The material of the first sealing membrane 22214 is the same as that of the second sealing membrane 22114. During extraction, when the first ejector pin 2222 squeezes the eluent container 22211 through the first liquid release hole 22213, the first support platform 2223 acts as a fulcrum for the eluent container 22211, creating a lever effect. This facilitates the first ejector pin 2222 breaking the first sealing membrane 22214 on the eluent container 22211, thereby rupturing the first sealing membrane 22214 and releasing the eluent. This method achieves one-time separation and extraction of the target analyte within the biological sample.

[0364] Furthermore, a first protrusion 2224 is provided on the side wall of the eluent tank 222, a first concave wall 22215 is provided on the outer side surface of the eluent container 22211, and a first recess 22216 that mates with the first protrusion 2224 is provided at a corresponding position on the first lid 22212. This arrangement allows the eluent container assembly 2221 to be stably fixed inside the eluent tank 222.

[0365] like Figure 15 As shown, the enrichment unit 23 includes a target material pool 231 and a waste liquid pool 232, which are respectively connected to the carrier filtration pool 224. The target material pool 231 is used to collect the target extract from the carrier filtration pool 224, and the waste liquid pool 232 is used to collect the waste liquid from the carrier filtration pool 224.

[0366] A sixth flow channel 233 is provided between the carrier filter tank 224 and the waste liquid tank 232, allowing the waste liquid flowing out of the carrier filter tank 224 to flow into the waste liquid tank 232 through the sixth flow channel 233. The sixth flow channel 233 is a curved flow channel, and the curve helps to increase the flow path. When the mixture passing through the carrier filter tank 224 passes through the surface modification membrane 22432, the extracellular secretory membrane structure in the biological sample is trapped on the surface modification membrane 22432, and the waste liquid enters the waste liquid tank 232 through the sixth flow channel 233.

[0367] A seventh flow channel 234 is provided between the carrier filter tank 224 and the target material tank 231, allowing the target material flowing out of the carrier filter tank 224 to flow into the target material tank 231 through the seventh flow channel 234. The inlet end of the seventh flow channel 234 is connected to the sixth flow channel 233. Furthermore, the connection between the seventh flow channel 234 and the sixth flow channel 233 forms a 60° acute angle, which facilitates the transport of the target material to the target material tank 231. In addition, when the seventh flow channel 234 and the sixth flow channel 233 are arranged at an acute or right angle, and the sixth flow channel 233 is a curved flow channel, the degree to which the eluent containing the target material enters the waste liquid tank 232 can be reduced, minimizing the loss of the target material.

[0368] Furthermore, the target pool 231 and a weight reduction and balancing pool 24 are connected by an air passage, and air holes 43 are provided at corresponding positions on the cover plate 4.

[0369] like Figure 3 As shown, a rotation center 1, a ejector pin hole, and an air hole 43 are provided at corresponding positions on the cover plate 4. Specifically, a first ejector pin hole 41 is provided on the cover plate 4 at the position corresponding to the first ejector pin 2222; a second ejector pin hole 42 is provided on the cover plate 4 at the position corresponding to the second ejector pin 2212. Furthermore, as... Figure 16 As shown, a plurality of weight-reducing and balancing pools 24 are provided on the base plate 3. The plurality of weight-reducing and balancing pools 24 are provided circumferentially on the base plate 3 with the rotation center 1 as the center.

[0370] The main material for preparing the separation device is polycarbonate (PC) polymer material, that is, the material for preparing the bottom plate 3 and the cover plate 4 is polycarbonate (PC).

[0371] The base plate 3 and the cover plate 4 are thermo-bonded. The eluent container 22211 and the first cover 22212 are thermo-bonded. The loading fluid container 22111 and the second cover 22112 are thermo-bonded. The eluent container and the loading fluid container 22111 are made of a hydrophobic material. The thickness of the base plate 3 is 6 mm.

[0372] A method for isolating and extracting extracellular secretory membrane structures includes the following steps:

[0373] I. Add 500 μL of whole blood sample into sample cell 211, and make the separation and extraction device rotate at high speed of 4500 rpm for 150 s, so that the sample quantitative cell 212 (the volume of sample quantitative cell 212 is 120 μL) is filled with biological sample. At the same time, some impurities enter the first impurity collection cell 216 to obtain plasma.

[0374] II. After standing for 30 seconds, the plasma in the sample quantitative cell 212 gradually enters the impurity filter cell 251 due to the siphon effect of the eleventh flow channel 252; at the same time, the second pin 2212 punctures the second sealing membrane 22114, allowing the loading liquid (the pre-embedded amount of loading liquid is 120μL) to flow out from the loading liquid cell 221.

[0375] III. The separation and extraction device is rotated at a high speed of 2500 rpm for 20 seconds, so that the plasma entering the impurity filtration pool 251 passes through the microfiltration membrane 25132, filtering out large particulate impurities, and the rest enters the incubation pool 223; at the same time, the loading liquid flowing out of the loading liquid pool 221 also enters the incubation pool 223; the biological sample and the loading liquid are mixed in the incubation pool 223.

[0376] IV. Control the separation and extraction device to rotate at a high-low-high speed cycle to promote the accelerated mixing of the two solutions in the incubation tank 223; after rotating at a high speed of 3000 rpm for 5 seconds, rotate at a high speed of 500 rpm for 5 seconds, and then rotate at a high speed of 3000 rpm for 5 seconds, repeating the high-low-high cycle 10 times.

[0377] V. Stop and let stand for 30 seconds to fill the fifth flow channel 227 with the mixture;

[0378] VI. The separation and extraction device is rotated at a high speed of 3000 rpm for 60 seconds, so that the mixture enters the carrier filter tank 224 and passes through the surface modification membrane 22432, thereby trapping the exosomes in the mixture on the surface modification membrane 22432, while the waste liquid enters the waste liquid tank 232.

[0379] VII. Stop standing for 30 seconds; during this period, the first ejector pin 2222 punctures the first sealing membrane 22214, allowing the eluent (pre-embedded amount of eluent is 250μL) to flow out from the eluent pool 222;

[0380] VIII. The separation and extraction device is rotated at a high speed of 5000 rpm for 50 seconds, so that the eluent passes through the surface-modified membrane 22432 and the extracellular secretory membrane structure is eluted from the surface-modified membrane 22432 and then enters the target material pool 231.

[0381] An isolation and extraction kit includes the above-described isolation and extraction device, which contains a surface-modified membrane and a microfiltration membrane, and is loaded with 120 μL of loading solution and 250 μL of elution solution.

[0382] Example 7

[0383] The difference between this embodiment and Embodiment 6 is that the surface-modified film used in this embodiment is prepared by Example 7, and QAS is loaded on it. C18 ;

[0384] A method for isolating and extracting extracellular secretory membrane structures includes the following steps:

[0385] I. Add 350 μL of whole blood sample into sample cell 211, and make the separation and extraction device rotate at a high speed of 3500 rpm for 180 s, so that sample quantitative cell 212 (the volume of sample quantitative cell 212 is 120 μL) is filled with plasma sample, and at the same time, some impurities enter the first impurity collection cell 216.

[0386] II. After standing for 40 seconds, the plasma sample gradually enters the impurity filtration pool 251 due to the siphon effect of the eleventh flow channel 252. At the same time, the second pin 2212 punctures the second sealing membrane 22114, allowing the loading liquid (the pre-embedded amount of loading liquid is 120μL) to flow out from the loading liquid pool 221.

[0387] III. The separation and extraction device is rotated at a high speed of 3500 rpm for 30 seconds, so that the plasma sample entering the impurity filtration pool 251 passes through the microfiltration membrane 25132, and some large-particle-size and high-molecular-weight impurities are intercepted, while the rest enters the incubation pool 223; at the same time, the loading liquid flowing out of the loading liquid pool 221 also enters the incubation pool 223; the biological sample and the loading liquid are mixed in the incubation pool 223.

[0388] IV. Control the separation and extraction device to rotate at a high-low-high speed cycle to promote the accelerated mixing of the two solutions in the incubation tank 223; after rotating at a high speed of 2500 rpm for 8 seconds, rotate at a high speed of 300 rpm for 8 seconds, and then rotate at a high speed of 2500 rpm for 8 seconds, repeating the high-low-high cycle 5 times.

[0389] V. Stop and let stand for 40 seconds to fill the fifth flow channel 227 with the mixture;

[0390] VI. The separation and extraction device is rotated at a high speed of 2500 rpm for 40 seconds, so that the mixture enters the carrier filter tank 224 and passes through the surface modification membrane 22432, thereby retaining the exosomes in the mixture on the surface modification membrane 22432, while the waste liquid enters the waste liquid tank 232.

[0391] VII. Stop standing for 40 seconds; during this period, the first ejector pin 2222 punctures the first sealing membrane 22214, allowing the eluent (pre-embedded amount of eluent is 200μL) to flow out from the eluent pool 222;

[0392] VIII. The separation and extraction device is rotated at a high speed of 3500 rpm for 50 seconds, so that the eluent passes through the surface modification membrane 22432 and the exosomes are eluted from the surface modification membrane 22432 and then enter the target cell 231.

[0393] The rest is the same as in Example 6.

[0394] Example 8

[0395] A method for extracting exosomes is disclosed, employing two syringes: a first syringe and a second syringe. A surface-modified filter membrane is fixed to the end of the first syringe near the needle insertion point; a microfiltration membrane is fixed to the end of the second syringe near the needle insertion point. The volume of both syringes is 3 mL.

[0396] The extraction method includes the following steps:

[0397] S1. Take 500 μL of whole blood sample and add it to the second syringe. Press the piston rod of the second syringe to make the whole blood sample pass through the microfiltration membrane. Collect the filtrate for later use to obtain plasma.

[0398] S2. The plasma and loading solution are mixed at a volume ratio of 1:1 (200 μL each) and placed in a first syringe. The first syringe is equipped with a surface-modified membrane. In this embodiment, the surface-modified membrane used is prepared by Surface Modification Membrane Preparation Example 8 and is loaded with cocamidopropyl betaine. The mixture is then allowed to stand until all the mixture has passed through the surface-modified membrane, and the filtrate is discarded.

[0399] S3. Then, add 300 μL of eluent to the first syringe, press the piston rod of the first syringe, and elute the surface-modified membrane loaded with exosomes with the eluent. The high concentration of salt solution in the eluent causes the exosomes to detach from the surface-modified membrane, and the exosome collection solution is obtained.

[0400] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0401] Example 9

[0402] The difference between this embodiment and Example 8 is that the surface-modified film used in this embodiment is prepared by Example 9 of surface-modified film preparation, and is loaded with octadecanopropyl betaine.

[0403] The rest is the same as in Example 8.

[0404] Example 10

[0405] The difference between this embodiment and embodiment 6 is that the surface-modified film used in this embodiment is prepared by surface-modified film preparation example 10, and SPPT is loaded on it;

[0406] A method for separating and extracting exosomes includes the following steps:

[0407] I. Add 400 μL of whole blood sample into sample cell 211, and make the separation and extraction device rotate at a high speed of 3000 rpm for 180 s, so that sample quantitative cell 212 (the volume of sample quantitative cell 212 is 120 μL) is filled with plasma sample, and at the same time, some impurities enter the first impurity collection cell 216.

[0408] II. After standing for 60 seconds, the plasma sample gradually enters the impurity filtration pool 251 due to the siphon effect of the eleventh flow channel 252. At the same time, the second pin 2212 punctures the second sealing membrane 22114, allowing the loading liquid (the pre-embedded amount of loading liquid is 120μL) to flow out from the loading liquid pool 221.

[0409] III. The separation and extraction device is rotated at a high speed of 3000 rpm for 20 seconds, so that the plasma sample entering the impurity filtration pool 251 passes through the microfiltration membrane 25132. The microfiltration membrane 25132 retains some large-particle-size and high-molecular-weight impurities, and the rest enters the incubation pool 223. At the same time, the loading liquid flowing out of the loading liquid pool 221 also enters the incubation pool 223. The biological sample and the loading liquid are mixed in the incubation pool 223.

[0410] IV. Control the separation and extraction device to rotate at a high-low-high speed cycle to promote the accelerated mixing of the two solutions in the incubation tank 223; after rotating at a high speed of 3000 rpm for 10 seconds, rotate at a high speed of 800 rpm for 10 seconds, and then rotate at a high speed of 3000 rpm for 10 seconds, repeating the high-low-high cycle 15 times.

[0411] V. Stop and let stand for 60 seconds to fill the fifth flow channel 227 with the mixture;

[0412] VI. The separation and extraction device is rotated at a high speed of 3000 rpm for 40 seconds, so that the mixture enters the carrier filter tank 224 and passes through the surface modification membrane 22432, thereby trapping the exosomes in the mixture on the surface modification membrane 22432, while the waste liquid enters the waste liquid tank 232.

[0413] VII. Stop standing for 60 seconds; during this period, the first ejector pin 2222 punctures the first sealing membrane 22214, allowing the eluent (pre-embedded amount of eluent is 300μL) to flow out from the eluent pool 222;

[0414] VIII. The separation and extraction device is rotated at a high speed of 4000 rpm for 50 seconds, so that the eluent passes through the surface modification membrane 22432 and the exosomes are eluted from the surface modification membrane 22432 and then enter the target cell 231.

[0415] The rest is the same as in Example 6.

[0416] Example 11

[0417] The difference between this embodiment and Example 1 is that the surface-modified film used in this embodiment was prepared by Example 11 of surface-modified film preparation, and it is loaded with DTAB and cocamidopropyl betaine.

[0418] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0419] Everything else is the same as in Example 1.

[0420] Example 12

[0421] The difference between this embodiment and Embodiment 6 is that the surface-modified film used in this embodiment is prepared by Example 12 of surface-modified film preparation, and QAS is loaded on it. C0 And SPPT.

[0422] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0423] Furthermore, the incubation tank structure of the microfluidic centrifuge tray differs. Specifically:

[0424] like Figure 17 As shown, the incubation tank 223 also includes several elastic rods 2231. One end of each elastic rod 2231 is fixed to the side wall of the incubation tank 223, or it can be integrally formed. The other end of each elastic rod 2231 has an integrally formed elastic ball 2232. Because it consists of elastic rods 2231 and elastic balls 2232, the forces acting on them are inconsistent during liquid flow and centrifugation. This causes the elastic balls 2232 and elastic rods 2231 to drive the liquid flow, thus accelerating the mixing and ensuring uniform mixing of the liquid in the incubation tank 223. Furthermore, elastic rods 2231 and / or elastic balls 2232 are not placed near the fourth flow channel 225, the fifth flow channel 227, and the twelfth flow channel 253 to prevent the elastic balls 2232 and / or elastic rods 2231 from clogging the flow channels.

[0425] The rest is the same as in Example 6.

[0426] Example 13

[0427] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 13, and QAS is loaded on it. C18 And SPPT.

[0428] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0429] The rest is the same as in Example 12.

[0430] Example 14

[0431] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 14 of surface-modified film preparation, and QAS is loaded on it. C18 SPPT and betaine.

[0432] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0433] The rest is the same as in Example 12.

[0434] Example 15

[0435] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 15 of surface-modified film preparation, and QAS is loaded on it. C0 SPPT and proline.

[0436] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0437] The rest is the same as in Example 12.

[0438] Example 16

[0439] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 16 of surface-modified film preparation, and QAS is loaded on it. C0 SPPT and mannitol.

[0440] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0441] The rest is the same as in Example 12.

[0442] Example 17

[0443] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 17, and QAS is loaded on it. C0 SPPT, betaine, and proline.

[0444] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0445] The rest is the same as in Example 12.

[0446] Example 18

[0447] The difference between this embodiment and Embodiment 12 is that the surface-modified film used in this embodiment is prepared by Example 18, and QAS is loaded on it. C0 SPPT, betaine, mannitol and proline.

[0448] The loading solution consists mainly of 100 mM Tris-propane, 170 mM NaCl, and pH 6.5. The eluent consists mainly of 50 mM Tris-propane, 2.5 M NaCl, and pH 6.36.

[0449] The rest is the same as in Example 12.

[0450] Example 19

[0451] The difference between this embodiment and Embodiment 18 is that the surface-modified film used in this embodiment is prepared by Example 19, and QAS is loaded on it. C18 SPPT, betaine, mannitol and proline.

[0452] The rest is the same as in Example 18.

[0453] Example 20

[0454] The difference between this embodiment and Example 18 is that the surface-modified film used in this embodiment is prepared by Surface Modified Film Preparation Example 20, and is loaded with cocamidopropyl betaine, betaine, mannitol and proline.

[0455] The rest is the same as in Example 18.

[0456] Example 21

[0457] The difference between this embodiment and Example 18 is that the surface-modified film used in this embodiment is prepared by Example 21 of surface-modified film preparation, and is loaded with SPPT, betaine, mannitol and proline.

[0458] The rest is the same as in Example 18.

[0459] Example 22

[0460] The difference between this embodiment and Embodiment 10 is that in step S1, the same volume of the processed whole blood sample is replaced with cell supernatant.

[0461] In this approach, the cells that can be selected include, but are not limited to, any one of A549 cells, H1299 cells, H23 cells, and Beas-2b cells. In this example, A549 cells, which are commercially available, were selected.

[0462] Step S1 is as follows:

[0463] Obtaining cell supernatant: The obtained A549 cells were cultured using existing methods to obtain cell culture medium, and centrifuged at 3000g for 25min to obtain initial cell supernatant; then 40mL of initial cell supernatant was taken and pre-concentrated to the same volume as the whole blood sample to obtain cell supernatant;

[0464] Place the cell supernatant, loading solution, and elution solution in the corresponding positions on the microfluidic centrifuge tray.

[0465] Everything else is the same as in Example 10.

[0466] Example 23

[0467] The difference between this embodiment and Embodiment 10 is that step S1 replaces the processed whole blood sample with pleural and peritoneal fluid of equal volume. Step S1 specifically involves:

[0468] Acquisition of pleural and peritoneal fluid: Samples were obtained from patients at Maoming People's Hospital; 20 mL of initial pleural and peritoneal fluid was taken from the patient and then pre-concentrated to the same volume as the whole blood sample to obtain the pleural and peritoneal fluid.

[0469] Place the pleural and peritoneal fluid, loading fluid, and eluent in the appropriate positions on the microfluidic centrifuge disc.

[0470] Everything else is the same as in Example 10.

[0471] Example 24

[0472] The difference between this embodiment and Embodiment 10 is that step S1 replaces the processed whole blood sample with an equal volume of urine. Step S1 specifically involves:

[0473] Urine collection: Take 20 mL of initial urine, then pre-concentrate it to the same volume as the whole blood sample to obtain urine.

[0474] Place the urine, loading fluid, and eluent in the appropriate positions on the microfluidic centrifuge disc.

[0475] Everything else is the same as in Example 10.

[0476] Example 25

[0477] The difference between this embodiment and implementation 10 is that step S1 replaces the processed whole blood sample with an equal volume of plasma. Step S1 specifically involves:

[0478] Plasma acquisition: Whole blood was centrifuged at 3000 rpm for 200 s to obtain plasma.

[0479] Place the plasma, loading solution, and eluent in their respective positions on the microfluidic centrifuge tray.

[0480] Everything else is the same as in Example 10.

[0481] Example 26

[0482] The difference between this embodiment and Embodiment 10 is that step S1 replaces the processed whole blood sample with an equal volume of saliva. Step S1 specifically involves:

[0483] Saliva collection: Take 20 mL of initial saliva, then pre-concentrate it to the same volume as the whole blood sample to obtain saliva.

[0484] Place the saliva, loading solution, and elution solution in the appropriate positions on the microfluidic centrifuge disc.

[0485] Everything else is the same as in Example 10.

[0486] Comparative Example 1

[0487] Exosome extraction using the size exclusion method includes the following steps:

[0488] Size-based resistance method:

[0489] I. Separation column and sample preparation

[0490] Separation column preparation: (1) Take a size exclusion separation column (purchased from Shanghai Huaying Biotechnology, product model qEVoriginal / 35nm) and equilibrate the column at room temperature;

[0491] (2) Add PBS buffer to the separation column in step (1) and perform column equilibration;

[0492] Sample preparation: (1) Take 1 mL of whole blood and centrifuge at 1500 g for 10 min, and collect the supernatant;

[0493] (2) Centrifuge the supernatant from step (1) at 2000g for 10 min and collect the supernatant;

[0494] (3) Pass the supernatant from step (2) through a 0.22 μm filter membrane to obtain the filtrate;

[0495] II. Add all the filtrate obtained in step (3) into the separation column and start collecting the effluent;

[0496] III. Add 2.5 mL of buffer solution to the separation column and continue to collect the eluent.

[0497] In this process, the first 3 mL of the effluent collected in steps II and III is waste liquid, and the remaining 2.5 mL is exosome collection liquid.

[0498] Comparative Example 2

[0499] Ultracentrifugation extraction of exosomes includes the following steps:

[0500] I. Processing of whole blood samples

[0501] (1) Centrifuge 1 mL of whole blood sample at 220g and 4℃ for 10 min and collect the supernatant;

[0502] (2) Centrifuge the supernatant obtained in step (1) at 2000g and 4℃ for 20min and collect the supernatant;

[0503] (3) Centrifuge the supernatant obtained in step (2) at 15000g and 4℃ for 30min and collect the supernatant;

[0504] (4) Filter the supernatant obtained in step (3) with a 0.22μm needle filter to remove particles with a diameter of 220nm or more and obtain the filtrate.

[0505] II. Transfer all the supernatant obtained in step (4) of step I to an ultracentrifuge tube, centrifuge at 120000g and 4℃ for 70min, and take the precipitate.

[0506] III. Add an appropriate amount of PBS (about 100 μL) to the precipitate obtained in step II, resuspend the exosome precipitate, and repeat step II.

[0507] IV. Add an appropriate amount of PBS (100 μL) to the precipitate obtained in step III, and resuspend the exosome precipitate to obtain the final product.

[0508] Comparative Example 3

[0509] PEG precipitation method for exosome extraction: Purchase the Gisele Biotech exosome extraction kit and extract exosomes according to the kit's operating procedures.

[0510] Comparative Example 4

[0511] Exosomes were extracted using density gradient centrifugation, including the following steps:

[0512] I. Whole blood sample pretreatment:

[0513] (1) Take 1 mL of whole blood sample and centrifuge at 2000g for 10 min, then take the supernatant;

[0514] (2) Filter the supernatant obtained in step (1) with a 0.22 μm filter to obtain the filtrate.

[0515] II. Add 2 mL of 60 wt% iodixanol to the bottom of the centrifuge tube, add the filtrate obtained in step (2), and then centrifuge at 10000 g and 4 °C for 3 h. Discard the supernatant and keep 3 mL at the bottom of the centrifuge tube.

[0516] III. Add 2 mL of 20 wt%, 10 wt%, and 5 wt% iodixanol to the filtrate from step II in sequence, centrifuge at 10000 g and 4 °C for 18 h, and then collect the 7th mL, which is the exosome collection solution.

[0517] Performance testing

[0518] (a) Extraction time distribution of each step in each extraction method

[0519] The time distribution of each stage during exosome extraction using the methods described in Example 10 and Comparative Examples 1-4 is recorded in the following figures. Figure 18 .from Figure 18 The data results show that the extraction time of exosomes using the method of this application is significantly shorter, only 12 minutes, which is much shorter than the extraction time of 2.5 to 22 hours for the other four extraction methods.

[0520] (ii) Western Blot analysis of exosomes

[0521] Western blotting was performed using an SDS-PAGE gel preparation kit (commonly available). Protein lysates were elute by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked at room temperature for 1 h, and then incubated with primary antibody overnight at 4 °C.

[0522] The following antibodies were used for Western blot analysis: anti-CD63 (Abcam), anti-CD81 (Abcam), anti-CD9 (Abcam), anti-Alix (Abcam), anti-TSG101 (Abcam), and anti-GAPDH (Abcam). All primary antibodies were used at a dilution of 1:1000.

[0523] The polyvinylidene fluoride membrane was washed three times with TBST (1×TBS and 0.5wt% Tween-20, pH 7.4), and then immersed in HRP-conjugated anti-rabbit IgG as a secondary antibody (1:3000) at room temperature for 60 minutes. Immunoassay was then performed using enhanced chemiluminescence (Pall).

[0524] The exosome collection fluids from Examples 10 and 22-26 were tested, and the results are shown in the table below. Figure 19 The exosome collection fluids from Comparative Examples 1-4 were analyzed, and the results are shown in the table below. Figure 20 . Figure 19 The results show that the extraction method of this application can be used to extract exosomes from different biological samples. Figure 20 The results show that when exosomes are extracted from biological samples using the method of this application, the protein concentrations of Alix, GAPDH, CD81, CD9, and CD63 are high, and the concentrations of each protein are significantly higher than those obtained by size exclusion method, ultracentrifugation method, and PEG precipitation method.

[0525] (III) Transmission electron microscopy of exosomes

[0526] The transmission electron microscopy (TEM) method for exosome detection was as follows: 10 μL of exosome suspension was dropped onto a clean sealing film. A carbon-coated copper mesh was then placed on top of the exosome suspension and suspended for 90 seconds. The mesh was then removed, and excess liquid was absorbed from the side with filter paper before detection. TEM detection was performed on the exosomes obtained in Example 10, and the results are shown below. Figure 21 .

[0527] (iv) Scanning electron microscopy examination

[0528] The exosomes obtained in Example 10 were subjected to scanning electron microscopy by Funa Scientific Instruments (Shanghai) Co., Ltd. The results are shown below. Figure 22 The results showed that the exosomes prepared in this embodiment were particles with a particle size distribution of about 100 nm.

[0529] (v) Determination of exosome particle size range

[0530] The exosomes obtained in Example 10 were subjected to exosome size range determination by Southern Medical University. The results are shown in [Figure 1]. Figure 23 The results showed that the exosomes prepared in this embodiment had a particle size distribution within the exosome particle size distribution range, approximately 100 nm, and the exosome concentration was 10. 6 / mL.

[0531] The particle size range and concentration of the exosomes prepared in Examples 1-9 and 11-21 were determined, and the results are shown in Table 1.

[0532] Table 1. Exosome particle size range and concentration obtained from different implementation schemes.

[0533]

[0534] The data in Table 1 show that, by comparing Examples 14-16 with Example 12, the use of the protective agent effectively protects exosomes, resulting in a higher concentration of extracted exosomes. This conclusion can also be drawn by comparing Examples 13 and 19, Examples 8 and 20, and Examples 10 and 21.

[0535] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Use of a surface-modified membrane for isolating and / or extracting an extracellular secretory membrane structure, characterized in that The surface modification film includes a base film and a surfactant loaded on the base film, the surfactant being a quaternary ammonium salt type cationic surfactant and an amphoteric surfactant; the ammonium salt type cationic surfactant being QAS Cn , n being 0 or 18, and the amphoteric surfactant being 3-(N,N-dimethyl dodecyl ammonium) propane sulfonate (SPPT).

2. Use according to claim 1, characterized in that, The molar ratio of the cationic surfactant and the amphoteric surfactant is (1.05-1.64):

3.

3. Use according to claim 1, characterized in that, The surface modification membrane further carries a protective agent selected from at least one of alkaloids, amino acids and bio-alcohols.

4. Use according to claim 3, characterized in that, The molar ratio of the alkaloids and the surfactant is 0.8-2.3:1, the molar ratio of the amino acids and the surfactant is 0.05-1.2:1, and the molar ratio of the bio-alcohols and the surfactant is 0.24-1.5:

1.

5. Use according to claim 4, characterized in that, The extracellular secretion membrane structure includes extracellular vesicles and exosomes.

6. Use according to claim 5, characterized in that, The application comprises the following steps, S1, pretreating a biological sample; S2, mixing the pretreated biological sample with a loading solution, then centrifuging and passing through a surface modification membrane, so that the extracellular secretion membrane structure in the biological sample is captured on the surface modification membrane, the surface modification membrane carries a surfactant selected from at least one of cationic surfactants and amphoteric surfactants; S3, eluting the surface modification membrane with an elution solution to obtain an extracellular secretion membrane structure collection solution.

7. Use according to claim 6, characterized in that, The biological sample includes, but is not limited to, whole blood, plasma, serum, urine, pleural effusion, cerebrospinal fluid, cell culture supernatant, tears, semen, amniotic fluid, gastric juice, saliva, nasal discharge, bronchoalveolar lavage fluid, synovial fluid, bile, uterine mucus and feces.

8. Use according to claim 6, characterized in that, The loading solution includes 130-170mM NaCl.

9. Use of the surface-modified membrane according to any one of claims 1 to 4 for the preparation of a kit for isolating and / or extracting an extracellular secretory membrane structure, characterized in that, The kit includes a loading solution, an elution solution and a filtration device containing the surface modification membrane of any one of claims 1-4.

10. Use according to claim 9, characterized in that, The loading solution includes 87-113mM Tris-propane buffer, 130-165mM NaCl, pH 6.23-6.78; and the elution solution includes 38-62mM Tris-propane buffer, 0.8-2.3M NaCl, pH 6.13-6.52.

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