A separation device having a pre-test reagent lever release structure and an extraction method

Abstract

The application relates to a separation device with a pre-test reagent lever release structure and an extraction method, the separation device comprising a rotating center, a sample inlet unit, a separation unit and a collection unit arranged on a bottom plate; the separation unit comprises a carrier storage pool, a carrier combination pool and a carrier filter pool arranged in sequence from a proximal end to a distal end of the rotating center, the carrier storage pool is provided with a loading liquid box, and the release of the loading liquid is realized through lever action of a first supporting table and a first thimble table; the separation unit further comprises an eluent pool provided with an eluent box, and the release of the eluent is realized through lever action of a second supporting table and a second thimble table. The separation device further comprises a top plate attached to the bottom plate, and the top plate is provided with a jack for pressing down the loading liquid box and the eluent box. The separation device is used for efficient capture and release of target objects in sample liquid, and is especially suitable for extraction of extracellular vesicles.

Description

[0001] This application is a divisional application, based on the patent application No. 202111604447.6 entitled "Separation device and method for extracting extracellular vesicles" filed on December 24, 2021. Technical Field

[0002] This application relates to the technical field of biological sample separation, and more specifically, to a separation device with a pre-embedded reagent lever release structure and a method for extracting extracellular vesicles. Background Technology

[0003] Extracellular vesicles are small, membrane-bound vesicles released from cells into the extracellular matrix. They participate in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth. They are widely present in various body fluids and cell supernatants and stably carry some important signaling molecules.

[0004] Extracellular vesicles are mainly composed of microvesicles and exosomes. Microvesicles are small vesicles that detach from the cell membrane after cell activation, damage, or apoptosis, with a diameter of about 100nm-1000nm. Exosomes are multivesicles that fuse with the cell membrane and are released into the extracellular space in the form of secretion, with a diameter of about 40nm-100nm.

[0005] Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and breast milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0006] Methods for extracting extracellular vesicles mainly include differential centrifugation, density gradient centrifugation, polymer precipitation (PEG-based precipitation), ultrafiltration, magnetic bead immunoassay, and reagent kit methods. Differential centrifugation is the most commonly used method for purifying extracellular vesicles, employing alternating low-speed and high-speed centrifugation to separate vesicle particles of similar size. Density gradient centrifugation utilizes the density gradient created by ultracentrifugation to enrich extracellular vesicles. PEG-based precipitation utilizes the property of polyethylene glycol (PEG) to co-precipitate with hydrophobic proteins and lipids; initially used for collecting viruses from serum and other samples, it is now also used to precipitate extracellular vesicles, its principle possibly related to competitive binding of free water molecules. Ultrafiltration centrifugation uses ultrafiltration membranes with different molecular weight cutoffs for selective separation. Magnetic bead immunoassay utilizes the specific markers (such as CD63 and CD9 proteins) on the surface of extracellular vesicles; after incubation with magnetic beads coated with anti-marker antibodies, the extracellular vesicles are adsorbed and separated. However, extracting extracellular vesicles using the above methods usually requires multiple sample additions and changes, which is time-consuming and cumbersome. Summary of the Invention

[0007] Therefore, it is necessary to provide a separation device that is simple to operate and has a high efficiency in extracting extracellular vesicles.

[0008] In the first aspect, the separation device provided in this application adopts the following technical solution: A separation device includes a rotating center and at least one extraction mechanism, the extraction mechanism including a sample injection unit, a separation unit, and a collection unit; the sample injection unit is used for inputting and storing sample liquid; the separation unit is connected to the sample injection unit and located downstream of the sample injection unit, and is used to capture or release the extracted target substance in the sample liquid; the collection unit is connected to the separation unit and located downstream of the separation unit, and is used to collect the extracted target substance released by the separation unit; the separation unit includes a stirring ball.

[0009] In some embodiments, the separation unit includes a carrier storage tank, a carrier binding tank, a carrier filtration tank, and an eluent tank. The carrier storage tank is used to add a loading solution containing a carrier capable of binding with the target analyte. The carrier binding tank is connected to both the injection unit and the carrier storage tank. The eluent tank is used to add an eluent capable of separating the target analyte from the carrier. The carrier filtration tank is connected to both the carrier binding tank and the eluent tank, and the carrier filtration tank is equipped with a carrier filter membrane for retaining the carrier. The carrier storage tank, carrier binding tank, and carrier filtration tank are distributed sequentially from the proximal end to the distal end of the rotation center. In some implementations, the separation unit further includes a separation cell, the sample cell being connected to the carrier binding cell via the separation cell, and the sample cell, the separation cell, and the carrier binding cell being distributed sequentially from the proximal end to the distal end of the rotation center.

[0010] In some embodiments, the separation tank includes a first chamber, a connecting portion, and a second chamber, wherein the first chamber is connected to the second chamber via the connecting portion, and the inner diameter of the connecting portion is smaller than the inner diameters of the first chamber and the second chamber.

[0011] In some embodiments, from the proximal end to the distal end of the rotation center, the separation unit further includes a diluent pool and a dilution mixing pool for loading sample diluent, the diluent pool being connected to the dilution mixing pool, and the separation pool being connected to the carrier binding pool through the dilution mixing pool; In some embodiments, the separation unit further includes an impurity filtration tank, the dilution mixing tank being connected to the carrier binding tank via the impurity filtration tank, and the impurity filtration tank being provided with an impurity filter membrane for intercepting impurities; In some implementations, the separation unit further includes a buffer tank, and the eluent tank is connected to the carrier filter tank through the buffer tank. The eluent tank, the buffer tank, and the carrier filter tank are distributed sequentially from the proximal end to the distal end of the rotation center.

[0012] In some implementations, the injection unit includes a sample dispensing port and a sample reservoir, the sample dispensing port being in communication with the sample reservoir for introducing sample solution into the sample reservoir, the sample reservoir being used to store the sample solution.

[0013] In some embodiments, the collection unit includes a target material collection tank and a waste liquid tank respectively connected to the carrier filtration tank, the target material collection tank being used to collect the extracted target material from the carrier filtration tank, and the waste liquid tank being used to collect the waste liquid from the carrier filtration tank; In some implementations, the separation unit further includes a flow direction selection tank, and the target material collection tank and the waste liquid tank are respectively connected to the carrier filtration tank through the flow direction selection tank. The liquid flows from the carrier filtration tank to the flow direction selection tank, and then from the flow direction selection tank to the target material collection tank or the waste liquid tank.

[0014] In some embodiments, the separation tank and the dilution mixing tank are connected via a first channel, the diluent tank and the dilution mixing tank are connected via a second channel, the carrier storage tank and the carrier binding tank are connected via a third channel, the eluent tank and the buffer tank are connected via a fourth channel, the buffer tank and the carrier filtration tank are connected via a fifth channel, and the carrier binding tank and the carrier filtration tank are connected via a sixth channel; the first channel, the second channel, the third channel, the fourth channel, the fifth channel and the sixth channel all alternately bend towards the rotation center and the edge of the separation device, and the second channel, the third channel, the fourth channel and the fifth channel are provided with capillary valves in each section extending towards the rotation center.

[0015] In some embodiments, the carrier filter tank has a carrier filter inlet and a carrier filter outlet, and a membrane placement groove is formed in the bottom wall between the carrier filter inlet and the carrier filter outlet, and the carrier filter membrane is embedded in the membrane placement groove.

[0016] In a second aspect, the present invention also provides a method for extracting exosomes using the above-described separation apparatus, the extraction method comprising the following steps: The sample solution is added to the injection unit; the rotation speed of the separation device is controlled so that the sample solution passes through the separation unit and the exosomes in the sample solution are captured. The exosomes captured by the separation unit are released into the collection unit.

[0017] In some embodiments, the step of controlling the rotational speed of the separation device includes: (1) Control the separation device to centrifuge at a first speed so that the sample liquid flows from the sample cell into the separation cell; (2) Control the separation device to centrifuge at the second speed in the opposite direction to step (1). After stopping the rotation, start the centrifugation clockwise until the third speed is reached and then stop. Then start the centrifugation counterclockwise until the third speed is reached and then stop the centrifugation and let it stand. This allows the liquid in the separation tank and the liquid in the dilution tank to flow into the dilution mixing tank to mix and then flow into the impurity filtration tank. After passing through the impurity filter membrane, the liquid reaches the carrier binding tank, and the liquid in the carrier storage tank flows into the carrier binding tank to mix with the filtered liquid. (3) Control the separation device to centrifuge at the fourth speed in the same direction as in step (1), so that the liquid in the carrier binding tank flows into the carrier filtration tank and passes through the carrier filter membrane to the waste liquid tank, the carrier is intercepted, and at the same time the liquid in the eluent tank flows into the buffer tank. (4) Control the separation device to centrifuge at the fourth speed in the opposite direction to step (1), so that the liquid in the buffer pool flows into the carrier filter pool and passes through the carrier filter membrane to reach the target collection pool.

[0018] In some implementations, the first speed is higher than the second speed, the second speed is higher than the third speed, and the fourth speed is higher than the third speed.

[0019] In some implementations, the first speed is 4000~7000 rpm, the second speed is 3000~5000 rpm, the third speed is 1500~4000 rpm, and the fourth speed is 7000~9000 rpm; the centrifugation time in step (1) is 10s~3min, the centrifugation time in step (2) is 5s~2min, the centrifugation time in step (3) is 10s~1min, and the centrifugation time in step (4) is 10s~1min.

[0020] In use, the separation device of this invention involves adding sample solution to the injection unit, filling the carrier storage tank with loading solution, and filling the eluent tank with eluent. Then, by controlling the rotation speed of the separation device, the sample solution and loading solution are brought to the carrier binding tank for incubation, where the carrier in the loading solution binds to the target extractant, such as exosomes, in the sample solution. Next, centrifugation via clockwise or counterclockwise rotation releases the sample solution and loading solution from the carrier binding tank to the carrier filtration tank, where they pass through a carrier filter membrane, trapping the carrier and its bound target extractant. Then, centrifugation in the opposite direction releases the eluent into the carrier filtration tank, where it passes through a carrier filter membrane, separating the target extractant from the carrier under the action of the eluent. The eluent is then collected to obtain the target extractant. This invention, using the above separation device, requires only one sample addition and, by controlling the rotation of the separation device, achieves rapid exosome extraction, facilitating the subsequent use of the extracted exosomes. The extraction process using the above-mentioned separation device is simple to operate, fast and efficient, requires low reagent consumption, and yields a large amount of exosomes. It solves the problems of traditional extraction methods, such as complicated operation, need for multiple sample additions and changes, and long time consumption. It can save reagent consumption, shorten extraction time, and reduce extraction costs. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the base plate of the separation device according to the first embodiment of this application; Figure 2 This is a schematic diagram of the structure of the base plate of the separation device according to the second embodiment of this application; Figure 3 yes Figure 2 A schematic diagram of the structure of the base plate of the separation device in the illustrated embodiment; Figure 4 This is a schematic diagram of the carrier binding pool of one embodiment of this application; Figure 5 a is Figure 3 A schematic diagram of the loading liquid box of the separation device in the illustrated embodiment is shown. Figure 5 b is Figure 3 A schematic diagram of the eluent box of the separation device in the illustrated embodiment; Figure 6 yes Figure 1 A schematic diagram of the top plate of the separation device in the illustrated embodiment; Figure 7 yes Figure 3 A schematic diagram of the top plate of the separation device in the illustrated embodiment; Figure 8 yes Figure 3 A schematic diagram of the separation tank of the separation device in the illustrated embodiment; Figure 9This is a schematic diagram of the structure of the base plate of the separation device according to the third embodiment of this application; Figure 10 yes Figure 9 A schematic diagram of the structure of the base plate of the separation device in the illustrated embodiment; Figure 11 yes Figure 9 A schematic diagram of the centrifuge chamber with an insertion barrier in the embodiment shown; Figure 12 yes Figure 9 Exploded view of the barrier and top plate of the embodiment shown; Figure 13 yes Figure 9 A schematic diagram of the top plate of the separation device in the illustrated embodiment; Figure 14 This is a schematic cross-sectional view of the bottom plate of the separation device in one embodiment of this application at the impurity filtration tank. Figure 15 This is a schematic cross-sectional view of the bottom plate of the separation device in one embodiment of this application at the carrier filter tank. Figure 16 This is a schematic diagram of the structure of a film substrate according to one embodiment of this application; Figure 17 This is a schematic diagram of centrifugation for the extraction method of Example 1; Figure 18 This is a particle size distribution chart of the exosomes extracted in Example 1. Figure 19 In the image, a is a 500 nm field-of-view electron microscope image of the exosomes extracted in Example 1, and b is a 1 μm field-of-view electron microscope image of the exosomes extracted in Example 1. Figure 20 This is a particle size distribution chart of the exosomes extracted in Example 2. Figure 21 In the image, a is a 500 nm field-of-view electron microscope image of the exosomes extracted in the experiment of detection example 2, and b is a 1 μm field-of-view electron microscope image of the exosomes extracted in the experiment of detection example 2. Figure 22 This is a particle size distribution chart of the exosomes extracted in Example 3. Figure 23 a is a 500nm electron microscope image of the exosomes extracted in Example 3. Figure 23 b is a 1μm field electron microscope image of the exosomes extracted in Example 3. Attached Figure

[0022] 10. Rotation center; 21. Sample injection unit; 2101. Sample cell; 2102. Sample dispensing port; 2201. Carrier storage cell; 2202. Carrier binding cell; 2203. Carrier filtration cell; 22031. Carrier filtration inlet; 22032. Carrier filtration outlet; 22033. Carrier membrane placement tank; 22034. Carrier membrane; 2204. Eluent cell; 2205. Separation cell; 22051. First chamber; 22052. Second chamber; 22053. Connecting part; 2206. Impurity filtration cell; 22061 22061. Impurity Filter Inlet; 22062. Impurity Filter Outlet; 22063. Impurity Filter Membrane Placement Tank; 22064. Impurity Filter Membrane; 2207. Diluent Tank; 2208. Diluent Mixing Tank; 2209. Buffer Tank; 2210. Residual Sample Tank; 2211. Flow Direction Selection Tank; 22111. Waste Liquid Outflow Path; 22112. Target Material Outflow Path; 2212. Waste Liquid Discharge Path; 2213. Target Material Discharge Path; 2301. Target Material Collection Tank; 2302. Waste Liquid Tank; 30. Stirring Ball; 40. Loading Liquid Box; 50. 60. Loading fluid release port; 70. Loading fluid insertion port; 80. Eluent container; 90. Eluent release port; 100. Eluent insertion port; 110. First support platform; 120. First ejector pin platform; 130. Second ejector pin platform; 140. First convex wall; 150. First concave wall; 160. Second convex wall; 170. Second concave wall; 180. Barrier element; 190. Placement port; 200. Fixing plate; 210. Protrusion; 220. Groove; 230. Membrane substrate; 240. Weight reduction and balancing chamber; 250. Vent; 260. First channel; 270, Second channel; 280, Third channel; 290, Fourth channel; 300, Fifth channel; 310, Sixth channel; 320, First flow channel; 330, Second flow channel; 340, Third flow channel; 350, Fourth flow channel; 360, Fifth flow channel; 370, Sixth flow channel; 380, Seventh flow channel; 390, Eighth flow channel; 400, Mixture transfer channel; 410, Fixing tank; 420, Sampling port; 430, Centrifuge; 440, Target material filter membrane; 450, Spring; 460, Base plate; 470, Top plate Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Reference Figure 1 and Figure 2 A separation apparatus according to one embodiment of this application includes a rotation center 10 and at least one extraction mechanism. The extraction mechanism includes a sample injection unit 21, a separation unit, and a collection unit. The sample injection unit 21 is used for the input and storage of sample liquid. The separation unit is connected to the sample injection unit 21 and located downstream of the sample injection unit 21, and is used to capture or release the extraction target analyte from the sample liquid. The collection unit is connected to the separation unit and located downstream of the separation unit, and is used to collect the extraction target analyte released by the separation unit.

[0026] Reference Figure 1 In some embodiments, the separation unit includes a carrier storage tank 2201, a carrier binding tank 2202, and a carrier filtration tank 2203. The carrier storage tank 2201 is used to hold a loading liquid containing a carrier capable of binding with the target analyte. The carrier binding tank 2202 is connected to both the sample tank 2101 and the carrier storage tank 2201. The carrier filtration tank 2203 is connected to the carrier binding tank 2202 and is equipped with a carrier filter membrane 22034 for retaining the carrier. The carrier storage tank 2201, carrier binding tank 2202, and carrier filtration tank 2203 are distributed sequentially from the proximal end to the distal end of the rotation center 10.

[0027] Reference Figure 1 In use, the separation device involves adding sample solution to the injection unit 21 and loading solution to the carrier storage tank 2201. The sample solution and loading solution are then directed to the carrier binding tank 2202 for incubation by controlling the rotation speed of the separation device. The carrier in the loading solution binds to the target extractant, such as exosomes, in the sample solution. Centrifugation, either clockwise or counterclockwise, releases the sample solution and loading solution from the carrier binding tank 2202 to the carrier filtration tank 2203. The carrier and its bound target extractant are then retained by the carrier filter membrane 22034. Further centrifugation separates the carrier and its bound target extractant within the carrier filtration tank 2203. The supernatant is then collected to obtain the target extractant.

[0028] Reference Figure 2 and Figure 3Optionally, the separation unit includes a carrier storage tank 2201, a carrier binding tank 2202, a carrier filtration tank 2203, and an eluent tank 2204. The carrier storage tank 2201 is used to add a loading solution containing a carrier capable of binding with the target analyte. The carrier binding tank 2202 is connected to both the sample tank 2101 and the carrier storage tank 2201. The eluent tank 2204 is used to add an eluent capable of separating the target analyte from the carrier. The carrier filtration tank 2203 is connected to both the carrier binding tank 2202 and the eluent tank 2204, and the carrier filtration tank 2203 is equipped with a carrier filter membrane 22034 for retaining the carrier. The carrier storage tank 2201, the carrier binding tank 2202, and the carrier filtration tank 2203 are distributed sequentially from the proximal end to the distal end of the rotation center 10.

[0029] In some embodiments, the loading solution comprises 87–113 mM Tris-propane buffer and 130–165 mM sodium chloride, with a pH of 6.23–6.78.

[0030] In some embodiments, the eluent comprises 38–62 mM Tris-propane buffer and 0.8–2.3 M sodium chloride, with a pH of 6.13–6.52.

[0031] Reference Figure 2 and Figure 3 In use, the separation device involves adding sample solution to the injection unit 21, adding loading solution to the carrier storage tank 2201, and adding eluent to the eluent tank 2204. The sample solution and loading solution are then incubated in the carrier binding tank 2202 by controlling the rotation speed of the separation device. The carrier in the loading solution binds to the target extractant, such as exosomes, in the sample solution. Centrifugation, either clockwise or counterclockwise, releases the sample solution and loading solution from the carrier binding tank 2202 to the carrier filter tank 2203, where they pass through the carrier filter membrane 22034, where the carrier and its bound target extractant are retained. Centrifugation, in the opposite direction to the previous step, releases the eluent into the carrier filter tank 2203, where it passes through the carrier filter membrane 22034, separating the target extractant from the carrier under the action of the eluent. The eluent is then collected to obtain the target extractant. This invention uses a separation device that requires only one sample addition. By controlling the rotation of the separation device, exosomes can be extracted quickly and conveniently for later use.

[0032] It is understood that the middle part of the separation device is the rotating mounting part, and the rotation center 10 of the separation device is the rotation center during centrifugation. It is understood that the target substance can be captured by the target substance on the carrier, so that the target substance and the carrier are retained by the carrier filter membrane 22034, and the eluent can dissociate the target substance in the sample solution from the carrier, so that it is washed into the target substance collection pool 2301.

[0033] In some implementations, the separation device is generally circular, comprising two extraction mechanisms evenly distributed around the center, capable of simultaneously extracting multiple samples, suitable for various liquid samples such as whole blood, plasma, and saliva. Of course, in other implementations, the separation device can also be other shapes, such as rectangular, polygonal, etc. The number of extraction mechanisms on the separation device can also be one, two, four, five, seven, etc.

[0034] Reference Figure 4 In some embodiments, a stirring ball 30 is placed in the carrier binding pool 2202. When the sample solution and the loading solution enter the carrier binding pool 2202 for incubation, the stirring ball 30 moves under the rotation of the separation device to stir the sample solution and the loading solution, which helps to mix the sample solution and the loading solution more evenly.

[0035] In some implementations, the stirring ball 30 is coated with a hydrophobic coating.

[0036] In some implementation schemes, refer to Figure 2 and Figure 5 The carrier storage tank 2201 is equipped with a loading liquid box 40, which is used to hold the loading liquid. In a specific example, the loading liquid box 40 has a loading liquid release hole 50 for releasing the loading liquid in the loading liquid box 40. Furthermore, the loading liquid box 40 is provided with a sealing membrane at the loading liquid release hole 50 for sealing the liquid in the loading liquid box 40.

[0037] Furthermore, referring to Figure 7 The separation device has a loading fluid insertion hole 60 at the position corresponding to the loading fluid release hole 50. The loading fluid can be released by inserting a tool into the loading fluid insertion hole 60 and puncturing the sealing membrane.

[0038] In some implementation schemes, refer to Figure 2 and Figure 5 An eluent container 70 is placed inside the eluent tank 2204. The eluent container 70 is used to hold the eluent. In a specific example, the eluent container 70 has an eluent release hole 80 for releasing the eluent in the eluent container 70. Furthermore, the eluent container 70 also has a sealing membrane at the eluent release hole 80 for sealing the eluent in the eluent container 70.

[0039] Furthermore, referring to Figure 7 The separation device has an eluent insertion hole 90 at the position corresponding to the eluent release hole 80. The eluent can be released by inserting a tool into the eluent insertion hole 90 and puncturing the sealing membrane.

[0040] In some embodiments, the loading liquid box 40 is bonded to the carrier storage tank 2201 by double-sided adhesive bonding or hot pressing; the eluent box 70 is bonded to the eluent tank 2204 by double-sided adhesive bonding or hot pressing.

[0041] In some implementation schemes, refer to Figure 2 and Figure 5 The loading liquid release hole 50 is opened on the bottom wall of the loading liquid box 40 and is located away from the center of the loading liquid box 40. The carrier storage pool 2201 is provided with a first support platform 100 and a first ejector platform 110. The upper surfaces of the first support platform 100 and the first ejector platform 110 are flush. The loading liquid box 40 is placed on the first support platform 100 and the first ejector platform 110. The position of the first ejector platform 110 corresponds to the position of the loading liquid release hole 50.

[0042] When the loading fluid needs to be released, a tool is inserted into the loading fluid socket 60 and the loading fluid box 40 is squeezed downwards. The first support platform 100 acts as a fulcrum, creating a lever effect. The first ejector platform 110 breaks the sealing film on the loading fluid box 40, allowing the loading fluid in the loading fluid box 40 to be released from the loading fluid release hole 50.

[0043] In some implementation schemes, refer to Figure 2 and Figure 5 The eluent release port 80 is located on the bottom wall of the eluent container 70 and is located away from the center of the eluent container 70. The eluent pool 2204 is provided with a second support platform 120 and a second ejector platform 130. The upper surfaces of the second support platform 120 and the second ejector platform 130 are flush. The eluent container 70 is placed on the second support platform 120 and the second ejector platform 130. The position of the second ejector platform 130 corresponds to the position of the eluent release port 80.

[0044] When the eluent needs to be released, insert a tool into the eluent insertion hole 90 and press down on the eluent box 70. The second support platform 120 acts as a fulcrum, creating a lever effect. The second ejector platform 130 breaks the sealing film on the eluent box 70, allowing the eluent in the eluent box 70 to be released from the eluent release hole 80.

[0045] In some implementation schemes, refer to Figure 2 The inner peripheral wall of the carrier storage pool 2201 is provided with a first convex wall 140, and the outer peripheral wall of the loading liquid box 40 is provided with a first concave wall 150. The first convex wall 140 and the first concave wall 150 cooperate with each other so that the loading liquid box 40 does not undergo large rotational displacement on the separation device. In some implementation schemes, refer to Figure 2The inner peripheral wall of the eluent tank 2204 is provided with a second convex wall 160, and the outer peripheral wall of the eluent box 70 is provided with a second concave wall 170. The second convex wall 160 and the second concave wall 170 cooperate with each other so that the eluent box 70 does not undergo large rotational displacement on the separation device.

[0046] In some implementation schemes, refer to Figure 1 The sample injection unit 21 includes a sample inlet 2102 and a sample cell 2101. The sample inlet 2102 is connected to the sample cell 2101 for inputting sample solution into the sample cell 2101, and the sample cell 2101 is used to store sample solution.

[0047] In some implementation schemes, refer to Figure 1 The collection unit includes a target material collection tank 2301 and a waste liquid tank 2302, both connected to the carrier filter tank 2203. The target material collection tank 2301 is used to collect the extracted target material from the carrier filter tank 2203, and the waste liquid tank 2302 is used to collect the waste liquid from the carrier filter tank 2203. Further, the target material collection tank 2301 and the waste liquid tank 2302 are respectively located on both sides of the carrier filter tank 2203 along the rotation direction, i.e., one is located on the clockwise side of the outlet of the carrier filter tank 2203, and the other is located on the counterclockwise side of the outlet of the carrier filter tank 2203.

[0048] In some implementation schemes, refer to Figure 1 The separation unit also includes a separation cell 2205. The sample cell 2101 is connected to the carrier binding cell 2202 through the separation cell 2205. The sample cell 2101, separation cell 2205, and carrier binding cell 2202 are distributed sequentially from the proximal end to the distal end of the rotation center 10. In this way, when whole blood or other samples are used as the sample solution, the sample solution can be released into the separation cell 2205 first. By centrifugation, the whole blood is separated into plasma (on the side closer to the rotation center 10) in the separation cell 2205, while blood cells and other sediments are located on the side farther from the rotation center 10. This helps to prevent blood cells and other sediments from entering the carrier binding cell 2202, thereby improving extraction efficiency and sensitivity.

[0049] In some implementation schemes, refer to Figure 1 and Figure 2The separation tank 2205 includes a first chamber 22051, a connecting part 22053, and a second chamber 22052. The first chamber 22051 is connected to the second chamber 22052 through the connecting part 22053, and the inner diameter of the connecting part 22053 is smaller than the inner diameter of the first chamber 22051 and the second chamber 22052. The first chamber 22051, the connecting part 22053, and the second chamber 22052 are distributed sequentially from the proximal end to the distal end. Thus, the separation tank 2205 consists of two chambers, upper and lower, with a constricted connecting portion 22053 in the middle. This design facilitates quantitative control of the first chamber 22051 (the upper chamber closer to the rotation center 10), reducing plasma loss and effectively preventing blood cells in the second chamber 22052 (the second chamber further from the rotation center 10) from flowing downstream. In short, without this constricted design, the interface between the upper plasma (near the rotation center 10) and the lower blood cells (far from the rotation center 10) after centrifugation would be large. To quantitatively extract 50 μL of plasma, the outlet of the separation tank 220521 would need to be as close as possible to the rotation center 10 to avoid the plasma-blood cell interface, preventing the extraction of blood cells and resulting in a significant amount of residual plasma. Furthermore, the large and unstable interface could lead to an increased extraction volume. Additionally, the downstream chamber is connected to the connecting portion 22053 of the separation tank 2205 to better facilitate the entry of the separated plasma into subsequent extraction processes. Optionally, the sample cell 210110 has a capacity of 140 μL to 200 μL, and the first chamber 22051 has a capacity of 50 μL.

[0050] In some implementation schemes, refer to Figure 8 The bottom of the connecting part 22053 is stepped or sloping, and the depth of the connecting part 22053 on the side closer to the impurity filter tank 2206 is less than the depth on the side farther away from the impurity filter tank 2206, which helps the liquid to be transported to the impurity filter tank 2206.

[0051] Optionally, the bottom of the connecting part 22053 is flat.

[0052] In some implementation schemes, refer to Figure 1 and Figure 2 The separation unit also includes an impurity filtration tank 2206. A connecting part 22053 communicates with a carrier binding tank 2202 via the impurity filtration tank 2206. The impurity filtration tank 2206 is equipped with an impurity filter membrane 22064 for trapping impurities. Thus, the separated sample solution is released into the impurity filtration tank 2206 and filtered through the impurity filter membrane 22064 to remove impurities such as blood cells, facilitating better mixing and incubation with the loading solution.

[0053] In some implementations, the impurity filter membrane 22064 may be a 0.22 μm microfiltration membrane.

[0054] In some implementation schemes, refer to Figure 1 From the proximal end to the distal end of the rotation center 10, the separation unit also includes a diluent pool 2207 and a dilution mixing pool 2208 for loading sample diluent. The diluent pool 2207 is connected to the dilution mixing pool 2208, and the separation pool 2205 is connected to the carrier binding pool 2202 through the dilution mixing pool 2208. In this way, the diluent and sample solution can be released into the dilution mixing pool 2208 for mixing, and the sample solution is diluted by the diluent in the diluent pool 2207 to facilitate better mixing and incubation with the loading solution.

[0055] In some implementation schemes, refer to Figure 1 The dilution mixing tank 2208 is connected to the carrier binding tank 2202 via an impurity filtration tank 2206. The impurity filtration tank 2206 is equipped with an impurity filter membrane 22064 for trapping impurities. In this way, the diluted sample solution can be released into the impurity filtration tank 2206 and filtered through the impurity filter membrane 22064 to remove impurities such as blood cells, so as to better mix and incubate with the loading solution.

[0056] In some embodiments, the pore size of the carrier filter membrane 22034 is 0.22 μm, and the pore size of the impurity filter membrane 22064 is 4~6 μm. It can be understood that the carrier particle size is larger than the pore size of the carrier filter membrane 22034.

[0057] In some implementation schemes, refer to Figure 1 The separation unit also includes a buffer tank 2209. The eluent tank 2204 is connected to the carrier filter tank 2203 via the buffer tank 2209. The eluent tank 2204, the buffer tank 2209, and the carrier filter tank 2203 are distributed sequentially from the proximal end to the distal end of the rotation center 10. Thus, by setting up a buffer tank 2209 between the eluent tank 2204 and the carrier filter tank 2203, the fluid process control capability can be enhanced.

[0058] In some implementation schemes, refer to Figure 1 The separation unit also includes a residual sample reservoir 2210, which is connected to the end of the separation reservoir 2205 near the rotation center 10. Thus, when the sample solution is added to the sample reservoir 2101 and centrifuged, excess sample solution will enter the adjacent residual sample reservoir 2210, preventing excessive sample solution.

[0059] In some implementation schemes, refer to Figure 2The separation unit also includes a flow direction selection tank 2211, a target material collection tank 2301, and a waste liquid tank 2302, which are respectively connected to the outlet of the carrier filter tank 2203 through the flow direction selection tank 2211. The liquid flows from the carrier filter tank 2203 to the flow direction selection tank 2211, and then from the flow direction selection tank 2211 to the target material collection tank 2301 or the waste liquid tank 2302. In this way, after passing through the carrier filter tank 2203, the liquid first reaches the flow direction selection tank 2211, and then can flow to the target material collection tank 2301 or the waste liquid tank 2302 more effectively according to the rotation direction of the centrifuge.

[0060] In some implementation schemes, refer to Figure 2 The flow direction selection pool 2211 includes a waste liquid outflow path 22111 and a target material outflow path 22112. One end of the waste liquid outflow path 22111 is connected to the carrier filter pool 2203 and the other end is connected to the waste liquid pool 2302. One end of the target material outflow path 22112 is connected to the waste liquid outflow path 22111 and the other end is connected to the target material collection pool 2301.

[0061] In some implementation schemes, refer to Figure 2 The waste liquid outflow path 22111 is curved, which helps to increase the length of the flow path and reduce the mixing degree of the eluent entering the waste liquid pool 2302.

[0062] In some implementation schemes, refer to Figure 2 The acute angle between the end of the target material outflow path 22112 and the waste liquid outflow path 22111 is 30-90 degrees, and optionally 60 degrees.

[0063] Optionally, refer to Figure 9 The separation unit also includes a centrifuge tank 430, a waste liquid discharge path 2212, and a target substance discharge path 2213. The centrifuge tank 430 is connected to the mixed liquid transmission channel 4. The centrifuge tank 430 is connected to the waste liquid tank 2302 through the waste liquid discharge path 2212, and to the target substance collection tank 2301 through the target substance discharge path 2213. A target substance filter membrane 440 is provided between the centrifuge tank 430 and the target substance discharge path 2213. The target substance filter membrane 440 is used for the extraction of target substances.

[0064] In some embodiments, the target filter membrane 440 is loaded with a surfactant selected from at least one of quaternary ammonium cationic surfactants and amphoteric surfactants.

[0065] In some implementation schemes, refer to Figure 11 and 12 The separation device is provided with a barrier 180 at the position of the carrier filter membrane 22034. The barrier 180 is used to prevent the liquid in the centrifuge 430 from flowing through the carrier filter membrane 22034.

[0066] In some implementation schemes, refer to Figure 12 and Figure 13 The separation device has a placement port 190 at the position of the barrier 180 for the barrier 180 to move in and out, and the barrier 180 is placed on the carrier filter membrane 22034 through the placement port 190.

[0067] In some implementation schemes, refer to Figure 11 The barrier 180 blocks the carrier filter membrane 22034 and the mixed liquid transmission channel 400 at the same time, preventing the liquid in the centrifuge 430 from flowing into the mixed liquid transmission channel 400.

[0068] Furthermore, referring to Figure 12 A fixing plate 200 is fixed to the top of the barrier 180. Two or more protrusions 210 are fixed on the fixing plate 200. Two or more grooves 220 are opened on the separation device. The grooves 220 correspond one-to-one with each protrusion 210. After the barrier 180 is placed in the carrier filter tank 2203 through the placement port 190, the protrusions 210 on the fixing plate 200 are inserted into the corresponding grooves 220 to prevent the barrier 180 from being displaced when the separation device rotates.

[0069] In some implementation schemes, refer to Figure 14 The impurity filtration tank 2206 has an impurity filtration inlet 22061 and an impurity filtration outlet 22062. An impurity filter membrane placement groove 22063 is formed in the bottom wall between the impurity filtration inlet 22061 and the impurity filtration outlet 22062. The impurity filtration inlet 22061 is used to temporarily store liquid that has not yet flowed through the impurity filter membrane 22064. The impurity filter membrane 22064 is embedded in the impurity filter membrane placement groove 22063, thereby facilitating the effective stabilization of the impurity filter membrane 22064.

[0070] In some implementation schemes, refer to Figure 15 The carrier filter tank 2203 has a carrier filter inlet 22031 and a carrier filter outlet 22032. A carrier filter membrane placement groove 22033 is formed in the bottom wall between the carrier filter inlet 22031 and the carrier filter outlet 22032. The carrier filter inlet 22031 is used to temporarily store liquid that has not yet flowed through the carrier filter membrane 22034. The carrier filter membrane 22034 is embedded in the carrier filter membrane placement groove 22033, which helps to effectively stabilize the carrier filter membrane 22034.

[0071] In some implementation schemes, refer to Figure 14The depth of the impurity filter membrane placement groove 22063 is greater than the depth of the impurity filter inlet 22061 and the impurity filter outlet 22062. If the depth of the impurity filter membrane placement groove 22063 is less than the depth of the impurity filter inlet 22061 and the impurity filter outlet 22062, then the impurity filter membrane 22064 cannot be properly fixed vertically, and the gap between the bottom of the impurity filter membrane placement groove 22063 and the bottom of the impurity filter membrane 22064 cannot guarantee that the fluid flows through the middle of the impurity filter membrane 22064.

[0072] In some implementation schemes, refer to Figure 15 The depth of the carrier filter membrane placement groove 22033 is greater than the depth of the carrier filter inlet 22031 and the carrier filter outlet 22032. If the depth of the carrier filter membrane placement groove 22033 is less than the depth of the carrier filter inlet 22031 and the carrier filter outlet 22032, then the carrier filter membrane 22034 cannot be properly fixed vertically, and the gap between the carrier filter membrane placement groove 22033 and the bottom of the carrier filter membrane 22034 cannot guarantee that the fluid flows through the middle of the carrier filter membrane 22034.

[0073] In some implementation schemes, refer to Figure 14 The bottom of the impurity filter outlet 22062 is stepped or sloping, and the side away from the impurity filter inlet 22061 is higher than the side near the impurity filter inlet 22061, which helps the liquid to be transported to the carrier binding tank 2202.

[0074] In some implementation schemes, refer to Figure 15 The bottom of the carrier filter outlet 22032 is stepped or sloping, and the side away from the carrier filter inlet 22031 is higher than the side near the carrier filter inlet 22031, which helps the liquid to be transferred to the flow selection pool 2211.

[0075] Optionally, refer to Figure 16 The carrier filter membrane 22034 is attached to the membrane substrate 230 and, together with the membrane substrate 230, is embedded in the carrier filter membrane placement groove 22033. Optionally, the impurity filter membrane 22064 is also attached to the membrane substrate 230 and, together with the membrane substrate 230, is embedded in the impurity filter membrane placement groove 22063. This overcomes the problem of thin filter membranes that are easily deformed. The operation is simple and effectively and stably places the filter membrane in the flow path.

[0076] Furthermore, the depths (in the thickness direction of the separation device) of the carrier filter inlet 22031, the carrier filter outlet 22032, the impurity filter inlet 22061, and the impurity filter outlet 22062 are 0.1mm-7.8mm, the depths of the carrier filter membrane placement groove 22033 and the impurity filter membrane placement groove 22063 are 0.2mm-7.8mm, and the widths of the carrier filter membrane placement groove 22033 and the impurity filter membrane 22064 are 0.2mm-5mm.

[0077] In some implementation schemes, refer to Figure 2 and Figure 9 The separation device also includes several weight-reducing and balancing chambers 240 to reduce the overall weight of the separation device. The distribution of these chambers ensures that the center of gravity of the separation device is as close to the center as possible, allowing for stable operation and minimizing vibration during high-speed rotation. In some embodiments, the weight-reducing and balancing chambers are independent of each other or connected via air passages. In further embodiments, at least one weight-reducing and balancing chamber 240 has an air vent 250 to ensure that the entire flow path is connected to atmospheric pressure.

[0078] In some implementation schemes, refer to Figure 1 The separation tank 2205 and the dilution mixing tank 2208 are connected via a first channel 260; the diluent tank 2207 and the dilution mixing tank 2208 are connected via a second channel 270; the carrier storage tank 2201 and the carrier binding tank 2202 are connected via a third channel 280; the elution tank 2204 and the buffer tank 2209 are connected via a fourth channel 290; the buffer tank 2209 and the carrier filtration tank 2203 are connected via a fifth channel 300; and the carrier binding tank 2202 and the carrier filtration tank 2203 are connected via a sixth channel 310. Thus, during high-speed centrifugation, the centrifugal force is large, exceeding the capillary force, and the channels cannot be fully filled with sample liquid, preventing the siphon effect from being triggered. When the centrifugal force is reduced to a suitable range, the capillary force exceeds the centrifugal force, triggering the siphon effect. Therefore, the flow of liquid can be controlled through centrifugation.

[0079] In some implementation schemes, refer to Figure 1 The first channel 260, the second channel 270, the third channel 280, the fourth channel 290, the fifth channel 300, and the sixth channel 310 all alternately bend towards the rotation center 10 and the edge of the separating device, thereby forming one or more inverted U-shaped structures. Each of the second channel 270, the third channel 280, the fourth channel 290, and the fifth channel 300 has a capillary valve in each section extending towards the rotation center 10. It can be understood that the bends of each channel towards the edge of the separating device are closer to the rotation center 10 than their upstream chambers.

[0080] Furthermore, the depth of each channel (in the thickness direction of the separation device) can be 0.2 mm, and the shape of the capillary valve can be circular, square or various polygons, with a diameter of 1.5 mm and a depth (in the thickness direction of the separation device) of 0.5 mm.

[0081] In such Figure 1 In the illustrated implementation scheme, the first channel 260, the second channel 270, the third channel 280, the fifth channel 300, and the sixth channel 310 each bend towards the rotation center 10 once, forming one inverted U-shaped structure. The fourth channel 290 bends towards the rotation center 10 twice, forming two inverted U-shaped structures. The fourth channel 290 employs a two-stage bend, which, in conjunction with a buffer pool 2209, enables time-domain process control of the fluid, i.e., delays the time it takes for the fluid to flow into the carrier filter pool 2203.

[0082] Furthermore, the bend in the first channel 260 is located on the clockwise side of the separation tank 2205, and the bend in the second channel 270 is located on the clockwise side of the diluent tank 2207. This allows for better synchronization between the diluent tank 2207 and the separation tank 2205; that is, when plasma is extracted from the separation tank 2205 by counter-clockwise rotation, the solution in the diluent tank 2207 also flows out synchronously.

[0083] Optionally, refer to Figure 2 and Figure 3 The sample cell 2101 is connected to the separation cell 2205 through the first flow channel 320. The separation cell 2205 is connected to the waste liquid chamber through the second flow channel 330. The separation cell 2205 is connected to the impurity filter cell 2206 through the third flow channel 340. The carrier binding cell 2202 is connected to the fourth flow channel 350. The end of the fourth flow channel 350 away from the carrier binding cell 2202 is connected to the mixed liquid transfer flow channel 400. The end of the mixed liquid transfer flow channel 400 away from the fourth flow channel 350 is connected to the carrier filter cell 2203. The carrier storage cell 2201 is connected to the carrier binding cell 2202 through the fifth flow channel 360. The eluent cell 2204 is connected to the carrier filter cell 2203 through the sixth flow channel 370.

[0084] In some implementation schemes, refer to Figure 2 and Figure 3 The upper surface of the first ejector plate 110 has a seventh flow channel 380, and the upper surface of the second ejector plate 130 has an eighth flow channel 390. After the sealing aluminum-plastic film is punctured, its edges may tightly adhere to the periphery of the first ejector plate 110 or the second ejector plate 130, hindering the flow of liquid. The seventh flow channel 380 and the eighth flow channel 390 serve to guide the flow and help prevent the above situation from occurring.

[0085] In some implementation schemes, refer to Figure 2 and Figure 3 The first flow channel 320, the third flow channel 340, the fourth flow channel 350, and the target material outflow path 22112 all bend towards the rotation center 10, thereby forming one or more inverted U-shaped structures. Each section of the first flow channel 320, the third flow channel 340, and the fourth flow channel 350 extending towards the rotation center 10 is equipped with a capillary valve. The inverted U-shaped structure serves to control the fluid and delay its movement.

[0086] In some embodiments, the first flow channel 320, the third flow channel 340, and the fourth flow channel 350 are hydrophilically modified with a modifier. Optionally, the modifier is a hydrogel solution or polyethylene oxide. After hydrophilic modification, the liquid can flow better in the capillary path, forming a capillary-siphon valve.

[0087] In some implementations, the depth of each flow channel (in the thickness direction of the separation device) is 0.1-0.5 mm, preferably 0.2 mm; the shape of the capillary valve is circular, square or various polygons, with a diameter of 1.0-2.0 mm, preferably 1.5 mm, and a depth (in the thickness direction of the separation device) of 0.3-0.8 mm, preferably 0.5 mm.

[0088] In such Figure 2 In the illustrated embodiment, reference is made to Figure 2 and Figure 3 The first flow channel 320, the third flow channel 340, the fourth flow channel 350 and the target material outflow channel all bend toward the rotation center 10 once, thus forming an inverted U-shaped structure.

[0089] In some implementation schemes, refer to Figure 4 The carrier bonding tank 2202 has a connecting hole at the position where it connects with the fourth flow channel 350. A spring 450 is provided at the position of the connecting hole in the carrier bonding tank 2202. One end of the spring 450 is fixedly connected to the carrier bonding tank 2202 in the length direction. The length direction of the spring 450 is set perpendicular to the diameter direction of the connecting hole. The inner radius of the spring 450 is smaller than the radius of the stirring ball 30. When the stirring ball 30 moves to the position of the spring 450, the stirring ball 30 squeezes the spring 450, and the spring 450 undergoes elastic deformation. Under the action of the elastic force, the stirring ball 30 moves away from the spring 450. The setting of the spring 450 can prevent the stirring ball 30 from entering the fourth flow channel 350 through the connecting hole and can also prevent the stirring ball 30 from blocking the connecting hole and thus hindering the flow of liquid.

[0090] In some embodiments, the separation device has a central rotating hole for mounting on the centrifugal shaft of the centrifuge, thereby facilitating centrifugation. In some embodiments, the carrier storage tank 2201, the eluent tank 2204, and the diluent tank 2207 are each provided with corresponding sample addition holes 2102 to facilitate the addition of the corresponding solutions.

[0091] In some implementations, the separation device is processed by methods such as CNC machining, laser engraving, soft lithography, 3D printing, and injection molding, but is not limited to these.

[0092] In some implementation schemes, refer to Figure 9 and Figure 10 The separation device includes a bottom plate 460 and a top plate 470. The bottom plate 460 has corresponding sample pool 2101, carrier storage pool 2201, carrier binding pool 2202, carrier filtration pool 2203, eluent pool 2204, target analyte collection pool 2301, waste liquid pool 2302, separation pool 2205, dilution pool 2207, dilution mixing pool 2208, impurity filtration pool 2206, residual sample pool 2210, buffer pool 2209, centrifuge pool 430, and weight reduction and balancing chamber 240, etc. The depth (thickness direction of the separation device) is 4-8mm, preferably 2mm.

[0093] In some implementation schemes, refer to Figure 6 A fixing groove 410 corresponding to the membrane placement groove is provided on the top plate 470, so that the filter membrane can be better sealed and fixed when the top plate 470 and the bottom plate 460 are attached.

[0094] Optionally, refer to Figure 7 The top plate 470 is provided with corresponding sample loading holes 2102, loading liquid insertion holes 60, elution liquid insertion holes 90 and air holes 250.

[0095] In some implementations, the thickness of the base plate 460 is 3-8 mm, preferably 6 mm.

[0096] In some implementation schemes, the main material of the separation device can be selected from polymer materials such as polymethyl methacrylate (PMMA), polycarbonate (PC), and polyvinyl chloride (PVC), with polycarbonate (PC) being preferred.

[0097] In some implementations, the top plate 470 may be made of the same polymer material as the bottom plate 460, or it may be a single-sided adhesive film with a hydrophobic adhesive layer.

[0098] In some implementations, the top plate 470 and the bottom plate 460 are bonded by double-sided adhesive bonding or thermoforming bonding.

[0099] An embodiment of the present invention provides a method for extracting extracellular vesicles, using the above-described separation apparatus, comprising the following steps: Add the sample solution to the injection unit 21; control the rotation speed of the separation device so that the sample solution passes through the separation unit and the extracellular vesicles in the sample solution are captured. Extracellular vesicles captured by the separation unit are released into the collection unit.

[0100] In a specific example, the steps for controlling the rotational speed of the separation device include: (1) Control the separation device to centrifuge at the first speed so that the sample liquid flows from the sample cell 2101 into the separation cell 2205; (2) Control the separation device to centrifuge at the second speed in the opposite direction to step (1). After stopping the rotation, start the centrifugation clockwise until the third speed is reached and then stop. Then start the centrifugation counterclockwise until the third speed is reached and then stop the centrifugation and let it stand. This allows the liquid in the separation tank 2205 and the liquid in the dilution tank 2207 to flow into the dilution mixing tank 2208 to mix and then flow into the impurity filtration tank 2206. After passing through the impurity filter membrane 22064, the liquid reaches the carrier binding tank 2202. The liquid in the carrier storage tank 2201 flows into the carrier binding tank 2202 and mixes with the filtered liquid. (3) Control the separation device to centrifuge at the fourth speed in the same direction as in step (1), so that the liquid in the carrier binding pool 2202 flows into the carrier filter pool 2203 and passes through the carrier filter membrane 22034 to reach the waste liquid pool 2302, where the carrier is intercepted, and at the same time the liquid in the eluent pool 2204 flows into the buffer pool 2209. (4) Control the separation device to centrifuge at the fourth speed in the opposite direction to step (1), so that the liquid in the buffer pool 2209 flows into the carrier filter pool 2203 and passes through the carrier filter membrane 22034 to reach the target material collection pool 2301.

[0101] In a specific example, the first speed is higher than the second speed, the second speed is higher than the third speed, and the fourth speed is higher than the third speed. Further, the first speed is 4000~7000 rpm, the second speed is 3000~5000 rpm, the third speed is 1500~4000 rpm, and the fourth speed is 7000~9000 rpm. Further, the centrifugation time in step (1) is 10 s~3 min, the centrifugation time in step (2) is 5 s~2 min, the centrifugation time in step (3) is 10 s~1 min, and the centrifugation time in step (4) is 10 s~1 min.

[0102] In a specific example, the steps for controlling the rotational speed of the separation device include: S1. Control the separation device to rotate at a speed of V1 for a rotation time of T1, so that the sample liquid flows from the sample cell 2101 into the separation cell 2205; S2. Stop rotating and let stand for T2. Use a tool to squeeze the loading liquid box 40 to release the loading liquid inside the loading liquid box 40. S3. Control the separation device to rotate at a speed of V2 for a rotation time of T3, so that the sample solution flows from the separation cell 2205 through the impurity filter membrane 22064 to the carrier binding cell 2202, and the loading liquid flows to the carrier binding cell 2202 to mix with the sample solution. S4. Control the separation device to rotate in a cycle of speed V3-speed V4. The rotation time of V3 is T4, the rotation time of V4 is T5, and V3 is higher than V4. The rotation directions of V3 and V4 are the same or opposite. The number of times V3 and V4 alternate operation is 3-15 times. S5. Stop rotating and let stand still for T6. S6. Control the separation device to rotate at a speed of V5 for a rotation time of T7. The liquid in the carrier binding tank 2202 flows into the carrier filtration tank 2203 and passes through the carrier filter membrane 22034 to reach the waste liquid tank 2302, where the carrier is retained. S7. Stop rotating and let stand for T8. Use a tool to squeeze the eluent container 70 to release the eluent inside the eluent container 70. S8. Control the separation device to rotate at a speed of V6 for a rotation time of T9. The liquid in the carrier filter tank 2203 passes through the carrier filter membrane 22034 and reaches the target material collection tank 2301.

[0103] In a specific example In step S1, V1 is 2500-5500 rpm, and T1 is 120-480 s; In step S2, T2 is 5-50s; In step S3, V2 is 2000-4500 rpm, and T3 is 15s-120s; In step S4, V3 is 2500-4000 rpm, T4 is 3-20s, V5 is 300-2000 rpm, and T5 is 3-20s. In step S5, T6 is 5-50 seconds; In step S6, V5 is 2000-5000 rpm, and T7 is 20-120 seconds; In step S7, T8 is 5-40 seconds; In step S8, V6 is 2500-6500 rpm, and T9 is 30-120 seconds.

[0104] In a specific example, the steps for controlling the rotational speed of the separation device include: S1. Control the separation device to rotate at a speed of A1 for a rotation time of B1, so that the sample liquid flows from the sample cell 2101 into the separation cell 2205; S2. Stop rotating and let stand for B2 hours. Use a tool to squeeze the loading liquid box 40 to release the loading liquid inside the loading liquid box 40. S3. Control the separation device to rotate at a speed of A2 for a rotation time of B3, so that the sample solution flows from the separation cell 2205 through the impurity filter membrane 22064 to the carrier binding cell 2202, and the loading liquid flows to the carrier binding cell 2202 to mix with the sample solution. S4. Control the separation device to rotate in a cycle of speed A3-speed A4. The rotation time of A3 is B4, the rotation time of A4 is B5, and A3 is higher than A4. The rotation directions of A3 and A4 are the same or opposite. The number of times A3 and A4 alternate operation is 3-15 times. S5. Stop rotating and let stand still for B6 hours. S6. Control the separation device to rotate at a speed of A5 for a rotation time of B7. The liquid in the carrier binding tank 2202 flows into the carrier filtration tank 2203 and passes through the carrier filter membrane 22034 to reach the waste liquid tank 2302, where the carrier is retained. S7. Stop rotating and let stand for B8. During the standing period, insert the first barrier 180 and the second barrier 180 into the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of A6 for a rotation time of B9. The liquid in the carrier filter tank 2203 passes through the target filter membrane 440 and reaches the target collection tank 2301.

[0105] In a specific example In step S1, A1 is 2500-5500 rpm, and B1 is 120-480 s; In step S2, B2 is 5-50 seconds; In step S3, A2 is 2000-4500 rpm, and B3 is 15s-120s; In step S4, A3 is 2500-4000 rpm, B4 is 3-20s, A5 is 300-2000 rpm, and B5 is 3-20s. In step S5, B6 is 5-50 seconds; In step S6, A5 is 2000-5000 rpm, and B7 is 20-120 seconds; In step S7, B8 is 5-40 seconds; In step S8, A6 is 2500-6500 rpm, and B9 is 30-120 seconds. Example Example

[0106] Specifically, one embodiment of a method for extracting exosomes from whole blood employs the following... Figure 1 and Figure 6 The separation device shown is implemented using the following method: The first step involves preloading whole blood into the sample pool, preloading PBS solution into the diluent pool 2207 (due to the presence of the capillary valve on the second channel 270, the PBS solution only fills the flow path between the capillary valve and the diluent pool 2207 under the action of capillary force), preloading loading solution into the carrier storage pool 2201 (due to the presence of the capillary valve on the third channel 280, the loading solution only fills the flow path between the capillary valve and the carrier storage pool 2201 under the action of capillary force), and preloading 100 μL of elution solution into the elution pool 2204 (due to the presence of the capillary valve on the fourth channel 290, the elution solution only fills the flow path between the downstream first capillary valve and the elution pool 2204 under the action of capillary force).

[0107] The second step involves controlling the rotation of the separation device, which includes the following steps: like Figure 17 As shown in section 0~t1, the separation device rotates clockwise at 5000 rpm for 1 minute and then stops. Whole blood solution flows from sample cell 2101 into separation cell 2205, and excess whole blood flows into residual sample cell 2210. Plasma is separated in separation cell 2205. The plasma is located in the first chamber 22051 of separation cell 2205, which is closer to the rotation center 10, while the second chamber 22052, which is farther from the rotation center 10, is filled with blood cells, white blood cells, etc. At the same time, during high-speed rotation, a capillary valve on the second channel 270, the third channel 280, and the fourth channel 290 breaks down. When the separation device stops rotating, the flow path between the two capillary valves in the second channel 270, the third channel 280, and the fourth channel 290 is filled under the action of capillary force.

[0108] like Figure 17 As shown in segment t2~t4, the centrifuge is started, and the separation device is controlled to rotate counterclockwise at 4000 rpm for 1 minute before stopping. It then starts rotating clockwise, reaching 3000 rpm before decelerating and stopping. It then accelerates counterclockwise to 3000 rpm before decelerating and stopping. During high-speed rotation, the plasma in separation tank 2205 flows into dilution mixing tank 2208 under siphon force, mixing with the PBS diluent solution flowing out of diluent tank 2207 in dilution mixing tank 2208 and flowing into impurity filtration tank 2206. After passing through the membrane, it enters carrier binding tank 2202 and remains there. The loading solution in carrier storage tank 2201 also flows into carrier binding tank 2202 and mixes with the diluted plasma solution. During the mixing process, the plasma and loading solution complete incubation.

[0109] like Figure 17As shown in t5~t6, after mixing is complete, the separation device is stopped and allowed to stand for a while. At this time, the sixth channel 310 is filled with the mixed liquid under the action of capillary force, and the fourth channel 290 is completely filled with the eluent. The separation device is then started and rotated clockwise at 8000 rpm for 30 seconds and then stopped. The mixed liquid in the carrier binding tank 2202 flows through the carrier filter membrane 22034 and is selected to enter the waste liquid tank 2302 in the flow direction selection tank 2211. At the same time, under high-speed rotation, the eluent in the eluent tank 2204 flows into the buffer tank 2209 and breaks through the capillary valve on the fifth channel 300. After a period of time, the eluent in the buffer tank 2209 fills the fifth channel 300.

[0110] like Figure 17 As shown in t7~t8, the separation device is started and rotated counterclockwise at 8000 rpm for 30 seconds and then stopped. The eluent in the buffer tank 2209 flows through the carrier filter membrane 22034 and is selected in the flow direction selection tank 2211 to enter the target material collection tank 2301. Example

[0111] Specifically, one embodiment of a method for extracting microvesicles from whole blood employs, as follows: Figure 1 and Figure 6 The separation device shown is implemented using the following method: The first step involves preloading whole blood into the sample pool, preloading PBS solution into the diluent pool 2207 (due to the presence of the capillary valve on the second channel 270, the PBS solution only fills the flow path between the capillary valve and the diluent pool 2207 under the action of capillary force), preloading loading solution into the carrier storage pool 2201 (due to the presence of the capillary valve on the third channel 280, the loading solution only fills the flow path between the capillary valve and the carrier storage pool 2201 under the action of capillary force), and preloading 100 μL of elution solution into the elution pool 2204 (due to the presence of the capillary valve on the fourth channel 290, the elution solution only fills the flow path between the downstream first capillary valve and the elution pool 2204 under the action of capillary force).

[0112] The second step involves controlling the rotation of the separation device, which includes the following steps: like Figure 17As shown in section 0~t1, the separation device rotates clockwise at 5000 rpm for 1 minute and then stops. Whole blood solution flows from sample cell 2101 into separation cell 2205, and excess whole blood flows into residual sample cell 2210. Plasma is separated in separation cell 2205. The plasma is located in the first chamber 22051 of separation cell 2205, which is closer to the rotation center 10, while the second chamber 22052, which is farther from the rotation center 10, is filled with blood cells, white blood cells, etc. At the same time, during high-speed rotation, one capillary valve on the second channel 270, the third channel 280, and the fourth channel 290 breaks down. When the separation device stops rotating, under the action of capillary force, the flow path between the two capillary valves in the second channel 270, the third channel 280, and the fourth channel 290 is filled.

[0113] like Figure 17 As shown in segment t2~t4, the centrifuge is started, and the separation device is controlled to rotate counterclockwise at 4000 rpm for 1 minute before stopping. It then starts rotating clockwise, reaching 3000 rpm before decelerating and stopping. It then accelerates counterclockwise to 3000 rpm before decelerating and stopping. During high-speed rotation, the plasma in separation tank 2205 flows into dilution mixing tank 2208 under siphon force, mixing with the PBS diluent solution flowing out of diluent tank 2207 in dilution mixing tank 2208 and flowing into impurity filtration tank 2206. After passing through the membrane, it enters carrier binding tank 2202 and remains there. The loading solution in carrier storage tank 2201 also flows into carrier binding tank 2202 and mixes with the diluted plasma solution. During the mixing process, the plasma and loading solution complete incubation.

[0114] like Figure 17 As shown in t5~t6, after mixing is complete, the separation device is stopped and allowed to stand for a while. At this time, the sixth channel 310 is filled with the mixed liquid under the action of capillary force, and the fourth channel 290 is completely filled with the eluent. The separation device is then started and rotated clockwise at 8000 rpm for 30 seconds and then stopped. The mixed liquid in the carrier binding tank 2202 flows through the carrier filter membrane 22034 and is selected to enter the waste liquid tank 2302 in the flow direction selection tank 2211. At the same time, under high-speed rotation, the eluent in the eluent tank 2204 flows into the buffer tank 2209 and breaks through the capillary valve on the fifth channel 300. After a period of time, the eluent in the buffer tank 2209 fills the fifth channel 300.

[0115] like Figure 17 As shown in t7~t8, the separation device is started and rotated counterclockwise at 8000 rpm for 30 seconds and then stopped. The eluent in the buffer tank 2209 flows through the carrier filter membrane 22034 and is selected in the flow direction selection tank 2211 to enter the target material collection tank 2301. Example

[0116] Specifically, one embodiment of a method for extracting exosomes from whole blood employs the following... Figure 2 and Figure 7 The separation device shown is implemented using the following method: The first step involves preloading 500 μL of whole blood into the sample pool, preloading 120 μL of loading solution into the loading solution cartridge 40, and placing the loading solution cartridge 40 into the carrier storage pool 2201. The second step involves preloading 250 μL of elution solution into the elution solution cartridge 70, and placing the elution solution cartridge 70 into the elution pool 2204.

[0117] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 4500 rpm for 150 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 30 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 110 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 2500 rpm for 20 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at 3000 rpm for 5 seconds, then rotate clockwise at 500 rpm for 5 seconds, and repeat this cycle 10 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 30 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 60 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 30 seconds. During the standing period, use the ejector pin to press down on the eluent box 70 through the eluent insertion hole 90. The second support platform 120 acts as a fulcrum and creates a lever effect. The second ejector pin platform 130 then breaks the sealing aluminum-plastic film on the eluent box 70, causing the eluent box 70 to rupture. The eluent in the eluent box 70 is released from the eluent release hole 80 and flows through the sixth flow channel 370 to the carrier filter tank 2203 to elute the solution in the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 5000 rpm for 50 seconds. The liquid after being eluted by the eluent flows through the carrier filter membrane 22034. A small portion fills the gap of the waste liquid outflow path 22111 and is then transferred to the target material collection tank 2301 by the target material outflow path 22112. The solution in the target material collection tank 2301 is the eluted exosome. Example

[0118] Specifically, one embodiment of a method for extracting exosomes from whole blood employs the following... Figure 2 and Figure 7 The separation device shown is implemented using the following method: The first step involves preloading 350 μL of whole blood into the sample pool, preloading 130 μL of loading solution into the loading solution cartridge 40, and placing the loading solution cartridge 40 into the carrier storage pool 2201. The second step involves preloading 200 μL of elution solution into the elution solution cartridge 70, and placing the elution solution cartridge 70 into the elution pool 2204.

[0119] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 3500 rpm for 180 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 40 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 110 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 3500 rpm for 30 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at a speed of 2500 rpm for 8 seconds, then rotate clockwise at a speed of 300 rpm for 8 seconds. Repeat this cycle 5 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 40 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 40 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 40 seconds. During the standing period, use the ejector pin to press down on the eluent box 70 through the eluent insertion hole 90. The second support platform 120 acts as a fulcrum and creates a lever effect. The second ejector pin platform 130 then breaks the sealing aluminum-plastic film on the eluent box 70, causing the eluent box 70 to rupture. The eluent in the eluent box 70 is released from the eluent release hole 80 and flows through the sixth flow channel 370 to the carrier filter tank 2203 to elute the solution in the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 3500 rpm for 50 seconds. The liquid after being eluted by the eluent flows through the carrier filter membrane 22034. A small portion fills the gap of the waste liquid outflow path 22111 and is then transferred to the target material collection tank 2301 by the target material outflow path 22112. The solution in the target material collection tank 2301 is the eluted exosome. Example

[0120] Specifically, one embodiment of a method for extracting exosomes from whole blood employs the following... Figure 2 and Figure 7 The separation device shown is implemented using the following method: The first step involves preloading 400 μL of whole blood into the sample pool, preloading 120 μL of loading solution into the loading solution cartridge 40, and placing the loading solution cartridge 40 into the carrier storage pool 2201. The second step involves preloading 300 μL of elution solution into the elution cartridge 70, and placing the elution cartridge 70 into the elution pool 2204.

[0121] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 3000 rpm for 180 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 60 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 110 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 3000 rpm for 20 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at 3000 rpm for 10 seconds, then rotate clockwise at 800 rpm for 10 seconds, and repeat this cycle 15 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 60 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 40 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 20 seconds. During the standing period, use the ejector pin to press down on the eluent box 70 through the eluent insertion hole 90. The second support platform 120 acts as a fulcrum and creates a lever effect. The second ejector pin platform 130 then breaks the sealing aluminum-plastic film on the eluent box 70, causing the eluent box 70 to rupture. The eluent in the eluent box 70 is released from the eluent release hole 80 and flows through the sixth flow channel 370 to the carrier filter tank 2203 to elute the solution in the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 4000 rpm for 50 seconds. The liquid after being eluted by the eluent flows through the carrier filter membrane 22034. A small portion fills the gap of the waste liquid outflow path 22111 and is then transferred to the target material collection tank 2301 by the target material outflow path 22112. The solution in the target material collection tank 2301 is the eluted exosome. Example

[0122] Specifically, one embodiment of a method for extracting microvesicles from whole blood employs, as follows: Figure 2 and Figure 7 The separation device shown is implemented using the following method: The first step involves preloading 500 μL of whole blood into the sample pool, preloading 120 μL of loading solution into the loading solution cartridge 40, and placing the loading solution cartridge 40 into the carrier storage pool 2201. The second step involves preloading 250 μL of elution solution into the elution solution cartridge 70, and placing the elution solution cartridge 70 into the elution pool 2204.

[0123] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 4500 rpm for 150 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 30 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 110 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 2500 rpm for 20 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at 3000 rpm for 5 seconds, then rotate clockwise at 500 rpm for 5 seconds, and repeat this cycle 10 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 30 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 60 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 30 seconds. During the standing period, use the ejector pin to press down on the eluent box 70 through the eluent insertion hole 90. The second support platform 120 acts as a fulcrum and creates a lever effect. The second ejector pin platform 130 then breaks the sealing aluminum-plastic film on the eluent box 70, causing the eluent box 70 to rupture. The eluent in the eluent box 70 is released from the eluent release hole 80 and flows through the sixth flow channel 370 to the carrier filter tank 2203 to elute the solution in the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 5000 rpm for 50 seconds. The liquid after being eluted by the eluent flows through the carrier filter membrane 22034. A small portion fills the gap of the waste liquid outflow path 22111 and is then transferred to the target material collection tank 2301 by the target material outflow path 22112. The solution in the target material collection tank 2301 is the eluted microvesicle. Example

[0124] Specifically, one embodiment of a method for extracting exosomes from whole blood employs the following... Figure 9 The separation device shown is implemented using the following method: The first step involves preloading 500 μL of whole blood into the sample pool, preloading 120 μL of loading solution into the loading solution cartridge 40, and placing the loading solution cartridge 4000 into the carrier storage pool 2201.

[0125] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 4500 rpm for 150 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 30 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 11000 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 2500 rpm for 20 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at 3000 rpm for 5 seconds, then rotate clockwise at 500 rpm for 5 seconds, and repeat this cycle 10 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 30 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 60 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 30 seconds. During the standing period, insert the first barrier 180 and the second barrier 180 into the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 5000 rpm for 50 seconds. After centrifugation, part of the liquid flows through the target material filter membrane 440 and is transferred to the target material collection tank 2301 through the target material outflow path 22112. The solution in the target material collection tank 2301 is the exosome. Example

[0126] Specifically, one embodiment of a method for extracting microvesicles from whole blood employs, as follows: Figure 9 The separation device shown is implemented using the following method: The first step involves preloading 500 μL of whole blood into the sample pool, preloading 120 μL of loading solution into the loading solution cartridge 40, and then placing the loading solution cartridge 40 into the carrier storage pool 2201.

[0127] The second step involves controlling the rotation of the separation device, which includes the following steps: S1. Control the separation device to rotate clockwise at a speed of 4500 rpm for 150 seconds, so that the sample liquid flows from the sample pool into the separation pool 2205, and the excess sample liquid flows into the residual sample pool 2210. Plasma is separated in the separation pool 2205. The plasma is located in the first chamber 22051 of the separation pool 2205 near the rotation center 10, and the second chamber 22052 away from the rotation center 10 is filled with blood cells, white blood cells, etc. S2. Stop rotating and let stand for 30 seconds. Due to capillary effect, the third flow channel 340 is filled with plasma. During the standing period, use the ejector pin to squeeze the loading liquid box 40 downward through the loading liquid insertion hole 60. The first support platform 100 acts as a fulcrum and a lever action is generated. The first ejector pin platform 110 then breaks the sealing aluminum-plastic film on the loading liquid box 40, causing the loading liquid box 40 to rupture. The loading liquid in the loading liquid box 40 is released from the loading liquid release hole 50. S3. Control the separation device to rotate clockwise at a speed of 2500 rpm for 20 seconds. At this time, the filled third channel 340 will have a siphon effect. The plasma in the first chamber 22051 will flow out through the third channel 340, pass through the impurity filter membrane 22064 and flow to the carrier binding pool 2202. The loading liquid will flow through the fifth channel 360 to the carrier binding pool 2202 and mix with the plasma. S4. Control the separation device to rotate in a high-low speed cycle. First, rotate clockwise at 3000 rpm for 5 seconds, then rotate clockwise at 500 rpm for 5 seconds, and repeat this cycle 10 times to promote the mixing of plasma and loading solution in carrier binding pool 2202. S5. Stop rotating and let stand for 30 seconds. The mixed liquid in the carrier binding tank 2202 flows out and fills the fourth flow channel 350. S6. Control the separation device to rotate at a speed of 3000 rpm for 60 seconds. At this time, the fourth flow channel 350, which has been filled with the mixed liquid in step S5, will have a siphon effect. After the mixed liquid in the carrier binding tank 2202 reaches the carrier filter membrane 22034, it will reach the waste liquid tank 2302 through the waste liquid outflow path 22111. S7. Stop rotating and let stand for 30 seconds. During the standing period, insert the first barrier 180 and the second barrier 180 into the carrier filter tank 2203. S8. Control the separation device to rotate at a speed of 5000 rpm for 50 seconds. After centrifugation, part of the liquid flows through the target material filter membrane 440 and is transferred to the target material collection tank 2301 through the target material outflow path 22112. The solution in the target material collection tank 2301 is the microvesicle.

[0128] Exosomes were extracted from plasma using an ultrafiltration method, and the steps are as follows: Take 200 μL of plasma, dilute it with 10 mL of PBS, centrifuge it at 220 g and 4 °C for 10 min, and collect the supernatant; The supernatant collected in step 1 was centrifuged at 2000g and 4℃ for 20 min, and the supernatant was collected. The supernatant collected in step 2 was centrifuged at 15000g and 4℃ for 30 min (T3=30 min) and the supernatant was collected. The supernatant collected in step 3 was filtered through a 0.22 μm syringe filter to remove particles larger than 220 nm in diameter; All supernatant was transferred to an ultracentrifuge tube and centrifuged at 120,000 g and 4°C for 70 min. Discard the supernatant, add 100 μL PBS to resuspend the exosome precipitate, and repeat step 5); Discard the supernatant, add 100 μL PBS to resuspend the exosome precipitate, and obtain the exosome solution.

[0129] Detection Example 1 Example 1 was tested, and after elution with 100 μL of elution buffer, approximately 95 μL of sample collection solution was finally obtained in target collection cell 2301. The sample solution was then subjected to NTA (Nanoparticle Tracking Analysis) to track exosomes, such as... Figure 18 As shown, the exosome concentration was 3E+6 cells / mL at a particle size of approximately 110 nm. The sample solution was examined using a electron microscope (SEM). Figure 19 In the figure, Figure a shows an electron microscope image of exosomes seen at a 500 nm field of view, and Figure b shows an electron microscope image of exosomes seen at a 1 μm field of view.

[0130] Detection Example 2 Example 3 was tested, and after elution with 200 μL of elution buffer, approximately 190 μL of sample collection solution was finally obtained in target collection cell 2301. The sample solution was then subjected to NTA (Nanoparticle Tracking Analysis) to track exosomes, such as... Figure 20 As shown, at a particle size of approximately 100.9 nm, the exosome concentration was approximately 3.7E+6 cells / mL. The sample solution was examined using a electron microscope (SEM), as shown... Figure 21 In the figure, Figure a shows an electron microscope image of exosomes seen at a 500 nm field of view, and Figure b shows an electron microscope image of exosomes seen at a 1 μm field of view.

[0131] Detection Example 3 The control group was tested, and NTA (Nanoparticle Tracking Analysis) was performed on the sample solution to track exosomes, such as... Figure 22As shown, at a particle size of approximately 133.9 nm, the exosome concentration was approximately 2.3E+6 cells / mL. The sample solution was examined using a electron microscope (SEM), as shown... Figure 23 In the figure, Figure a shows an electron microscope image of exosomes seen at a 500 nm field of view, and Figure b shows an electron microscope image of exosomes seen at a 1 μm field of view.

[0132] Compared with the comparative examples, the extraction process using the separation device provided in this application (Examples 1-7) is simple to operate, fast and efficient, requires low reagent consumption, and yields a large amount of exosomes. It solves the shortcomings of traditional extraction methods, such as complicated operation, need for multiple sample additions and changes, and long time consumption. It can save reagent consumption, shorten extraction time, and reduce extraction costs.

[0133] Compared to Examples 3-7, Examples 1-2, by setting up the loading liquid box 40 and / or the eluent box 70, allow the loading liquid and eluent to be pre-embedded in the separation device, eliminating the need for operators to manually add various reagents and effectively avoiding human error. In use, simply puncture the loading liquid box 40 and the eluent box 70 to release the reagents, achieving fully automated exosome extraction.

[0134] Combination Figure 18 , Figure 20 and Figure 22 As can be seen, compared with the comparative example, the particle size distribution of Examples 1 and 3 is more concentrated, the number is larger, and the concentration is higher. Combination Figure 19 , Figure 21 and Figure 23 As can be seen, compared with the comparative example, in random field of view, the number of exosomes in Examples 1 and 3 is greater and their morphology is more complete.

[0135] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A separation device with a pre-embedded reagent lever release structure, characterized in that, It includes a rotation center (10) located on a base plate (460), a sample injection unit (21), a separation unit, and a collection unit; the sample injection unit (21) is used for the input and storage of sample liquid; the separation unit is connected downstream of the sample injection unit (21) and is used to capture or release the extracted target substance in the sample liquid; the collection unit is connected downstream of the separation unit and is used to collect the extracted target substance released by the separation unit. The separation unit includes a carrier storage tank (2201), a carrier binding tank (2202), and a carrier filtration tank (2203) disposed on the base plate (460) and distributed sequentially from the proximal end to the distal end of the rotation center (10). The carrier storage tank (2201) is used to add loading liquid containing a carrier capable of binding with the target analyte. The carrier binding tank (2202) is connected to the injection unit (21) and the carrier storage tank (2201). The eluent tank (2204) is used to add eluent capable of separating the target analyte from the carrier. The separation unit also includes an eluent tank (2204). The carrier filtration tank (2203) is connected to the carrier binding tank (2202) and the eluent tank (2204). The carrier filtration tank (2203) is provided with a carrier filter membrane (22034) for retaining the carrier. The carrier storage tank (2201) is equipped with a loading liquid box (40), and the bottom wall of the loading liquid box (40) has an eccentric loading liquid release hole (50); the carrier storage tank (2201) is equipped with a first support platform (100) and a first ejector platform (110) with their upper surfaces flush, and the loading liquid box (40) is placed on the first support platform (100) and the first ejector platform (110), the position of the first ejector platform (110) corresponding to the position of the loading liquid release hole (50); based on the The first support platform (100) acts as a lever at the fulcrum, and the first ejector pin platform (110) breaks the sealing film on the loading liquid release hole (50) of the loading liquid box (40), and the loading liquid in the loading liquid box (40) is released from the loading liquid release hole (50); the inner peripheral wall of the carrier storage pool (2201) is provided with a first convex wall (140), and the outer peripheral wall of the loading liquid box (40) is provided with a first concave wall (150) that is far away from the loading liquid release hole (50) and cooperates with the first convex wall (140). The eluent tank (2204) has the same structure as the carrier storage tank (2201). The eluent tank (2204) is provided with an eluent box (70), and the bottom wall of the eluent box (70) is provided with an eccentric eluent release hole (80). The eluent tank (2204) is provided with a second support platform (120) and a second ejector platform (130) with a flat upper surface. The eluent box (70) is placed on the second support platform (120) and the second ejector platform (130). The position of the second ejector platform (130) corresponds to the position of the eluent release hole (80). The inner peripheral wall of the eluent tank (2204) is provided with a second convex wall (160), and the outer peripheral wall of the eluent box (70) is provided with a second concave wall (170) that is away from the eluent release hole (80) and cooperates with the second convex wall (160). The separation device further includes a top plate (470) attached to the bottom plate (460), the top plate (470) having a loading fluid insertion hole (60) for the ejector pin to press down on the loading fluid box (40) and an eluent insertion hole (90) for the ejector pin to press down on the eluent box (70).

2. The separation device according to claim 1, wherein the upper surface of the first ejector plate (110) is provided with a seventh flow channel (380) for guiding flow, and the upper surface of the second ejector plate (130) is provided with an eighth flow channel (390) for guiding flow, for guiding flow after ejection.

3. The separation device according to claim 1, wherein the collection unit comprises a target material collection tank (2301) and a waste liquid tank (2302) respectively connected to the carrier filter tank (2203), the target material collection tank (2301) being used to collect the extracted target material from the carrier filter tank (2203), and the waste liquid tank (2302) being used to collect the waste liquid from the carrier filter tank (2203).

4. The separation device according to claim 3, wherein the separation unit further comprises a centrifuge tank (430) disposed downstream of the carrier filter tank (2203), and a target material filter membrane (440) loaded with surfactant is provided between the centrifuge tank (430) and the target material collection tank (2301) for the extraction of target material.

5. The separation device according to claim 4, wherein the separation unit is detachably provided with a barrier (180) at the position of the carrier filter membrane (22034) to prevent fluid in the centrifuge (430) from flowing through the carrier filter membrane (22034); the separation device is provided with a placement port (190) for the barrier (180) to move in and out.

6. The separation device according to claim 1, characterized in that, The carrier filter tank (2203) has a carrier filter inlet (22031) and a carrier filter outlet (22032). A membrane placement groove is provided on the bottom wall between the carrier filter inlet (22031) and the carrier filter outlet (22032), and the carrier filter membrane (22034) is embedded in the membrane placement groove.

7. The separation device according to claim 1, wherein the separation unit further comprises a separation cell (2205), the sample injection unit (21) is connected to the carrier binding cell (2202) through the separation cell (2205), and the sample injection unit (21), the separation cell (2205) and the carrier binding cell (2202) are distributed sequentially from the proximal end to the distal end of the rotation center (10); The separation tank (2205) includes a first chamber (22051), a connecting part (22053), and a second chamber (22052). The first chamber (22051) is connected to the second chamber (22052) through the connecting part (22053), and the inner diameter of the connecting part (22053) is smaller than the inner diameters of the first chamber (22051) and the second chamber (22052).

8. The separation device according to claim 7, characterized in that, The separation unit further includes an impurity filtration tank (2206), the separation tank (2205) is connected to the carrier binding tank (2202), and the impurity filtration tank (2206) is provided with an impurity filter membrane (22064) for intercepting impurities. The sample injection unit (21) includes a sample inlet (2102) and a sample reservoir (2101). The sample inlet (2102) is connected to the sample reservoir (2101) to input sample solution into the sample reservoir (2101). The sample reservoir (2101) is used to store sample solution. The separation unit also includes an excess sample cell (2210), which is connected to one end of the separation cell (2205) near the rotation center (10) to collect excess sample solution during centrifugation after the sample solution is added to the sample cell (2101).

9. A method for extracting extracellular vesicles, characterized in that, Using the separation apparatus as described in any one of claims 1-8, the extraction method comprises the following steps: Add the sample solution to the injection unit (21); The rotation speed of the separation device is controlled so that the sample solution passes through the separation unit and the extracellular vesicles in the sample solution are captured; the extracellular vesicles captured by the separation unit are released into the collection unit. Stop rotating, press down the loading liquid box (40) through the loading liquid insertion hole (60) with the tool, and use the first support platform (100) as the fulcrum to pry the first ejector pin platform (110) to break the sealing membrane of the loading liquid box (40), so that the loading liquid is released into the carrier binding pool (2202) and mixed with the sample liquid for incubation; The rotation speed of the separation device is controlled to rotate in a high-low speed cycle, and the incubated liquid is filtered through the carrier filter tank (2203) to retain the carrier; Stop rotating, press down the eluent box (70) through the eluent insertion hole (90) with the tool, and use the second support platform (120) as a fulcrum to pry the second ejector platform (130) to break the sealing membrane of the eluent box (70), so that the eluent is released into the carrier filter pool (2203) to elute the target material bound to the carrier and collect it into the collection unit.

10. The extraction method according to claim 9, characterized in that, The carrier bonding tank (2202) is equipped with a stirring ball (30). In the step of controlling the rotation speed of the separation device to rotate at high and low speeds, the stirring ball (30) moves back and forth in the carrier bonding tank (2202) to stir the mixture. The high and low speed rotation is 2500-4000 rpm and 300-2000 rpm, and the number of alternations is 3-15 times.

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