Platelet exosome multistage laminar flow separation device

Abstract

The present application relates to the technical field of platelet exosome separation, and discloses a platelet exosome multistage laminar flow separation device, which comprises a liquid storage tank, a sleeve frame is symmetrically sleeved outside the liquid storage tank, a laminar flow box for laminar flow is arranged below the liquid storage tank, a first separation cabin for realizing primary separation is arranged below the laminar flow box, a second separation cabin for realizing resonance separation is arranged below the first separation cabin, a shunt box for shunting is arranged below the second separation cabin, and a plurality of collection tubes are arranged below the shunt box in an array, the present application realizes stable rectification of a sample and sheath liquid through the laminar flow box, the inertial force and the Dean force are utilized to accurately grade microvesicles, exosomes and cell fragments in combination with the gradually deepening spiral groove structure of the conical core in the first separation cabin, and the second resonance separation is performed on the exosomes through the resonance screens with different amplitudes and phase differences arranged in the second separation cabin, so that the enrichment purity of the exosomes is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of platelet exosome separation technology, specifically to a multi-stage laminar flow separation device for platelet exosomes. Background Technology

[0002] Platelet exosomes, as important carriers of intercellular communication, carry various bioactive molecules such as proteins, lipids, and nucleic acids, showing great application potential in early disease diagnosis, targeted drug delivery, tissue regeneration and repair, and immune regulation. However, efficient and high-purity separation of platelet exosomes remains a core technological bottleneck restricting their clinical translation and industrial application. Currently, exosome separation mainly relies on methods such as ultracentrifugation, tangential flow filtration, immunoaffinity capture, and microfluidic chips. Among these, although ultracentrifugation is widely used, it is cumbersome and time-consuming, usually requiring multiple centrifugation steps lasting several hours or more, resulting in high equipment costs. Furthermore, the high centrifugal force can easily damage the exosome membrane structure, affecting its biological function, and it is difficult to remove microvesicles and protein aggregates of similar particle size. While tangential flow filtration can increase throughput, it suffers from limitations due to the distribution of membrane pore sizes. The existing methods suffer from several drawbacks. Firstly, they struggle to accurately distinguish between exosomes and small vesicles, resulting in poor homogeneity of the separated products. Secondly, while immunoaffinity assays utilize antibodies to specifically capture exosome surface markers, yielding high-purity exosomes, these methods are costly, require stringent detection conditions, and may leave residual labels that could affect downstream applications, hindering continuous, large-scale processing. Thirdly, while microfluidic chips exhibit excellent resolution and controllability at the microscale, most existing chip structures remain in the laboratory validation stage. These chips suffer from small channel sizes, limited flow rates, insufficient overall throughput, high processing precision requirements, and susceptibility to clogging, making them unsuitable for the large-volume, continuous sample introduction needs of clinical samples or industrial production. In summary, existing technologies generally suffer from common problems such as difficulty in balancing separation efficiency and purity, operational complexity, limited throughput, high cost, and inability to achieve continuous operation.

[0003] To this end, we propose a multi-stage laminar flow separation device for platelet exosomes. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-stage laminar flow separation device for platelet exosomes to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a multi-stage laminar flow separation device for platelet exosomes, comprising a storage tank, a connecting frame symmetrically sleeved on the outside of the storage tank, a laminar flow box for laminar flow disposed below the storage tank, a first separation chamber for primary separation disposed below the laminar flow box, a second separation chamber for resonant separation disposed below the first separation chamber, a diversion box for diverting flow disposed below the second separation chamber, and collection tubes arranged in an array below the diversion box.

[0006] Preferably, the interior of the storage tank is symmetrically equipped with inclined baffles. A first infusion tube and a second infusion tube are sequentially connected to the bottom of the storage tank. The first infusion tube is located between the two sets of baffles. There are two sets of the second infusion tube, both of which are bent and located below the two sets of baffles. The bottoms of the first and second infusion tubes are connected to the inner cavity of the laminar flow chamber. The baffles divide the interior of the storage tank into three parts. The sample is stored in the middle position, and the sheath fluid is stored in the two side positions. The sample is connected to the first infusion tube, and the sheath fluid is connected to the second infusion tube. The sample and the sheath fluid are transported to the interior of the laminar flow chamber through the first and second infusion tubes, respectively.

[0007] Preferably, a manifold is fixedly installed in the middle of the laminar flow chamber cavity. The top of the manifold is inclined. A central limiting ring is fixedly installed at the center of the top of the manifold. A liquid outlet groove is opened through the bottom of the outer side of the central limiting ring. The liquid outlet groove is located on the lower side of the central limiting ring. A notch is pre-set on the lower side of the manifold. The first infusion pipe is connected to the inner side of the central limiting ring, and the second infusion pipe is connected to the outer side of the central limiting ring.

[0008] Preferably, a metal sintered plate is spaced apart below the manifold, and a guide bucket is provided below the metal sintered plate. The bottom of the guide bucket is connected to the inner cavity of the first separation chamber. In use, the sample and sheath fluid are transported to the inner and outer sides of the central limiting ring in the inner cavity of the laminar flow box through the first and second infusion pipes, respectively. Under the guidance of the manifold and the liquid outlet, the liquids are mixed. Then, the sample is transported to the top of the metal sintered plate through a pre-set notch at the bottom of the manifold, and then rectified by the metal sintered plate. The aperture of the metal sintered plate is 0.2 mm.

[0009] Preferably, a converging funnel is fixedly installed at the top of the first separation chamber cavity, and the converging funnel is connected to the guide funnel. An outer sleeve is fixedly installed at the bottom of the converging funnel, and the outer sleeve is trumpet-shaped. A conical core is sleeved inside the outer sleeve, and a support column extends from the bottom of the conical core and is fixedly connected to the bottom of the first separation chamber cavity. A spiral groove with inward indentation is opened on the outer side of the conical core. The depth of the spiral groove gradually decreases, specifically from 2 mm to 0.5 mm, and the pitch is 10 mm. At this time, after the mixed liquid flows through the converging funnel and the guide funnel to the space between the outer sleeve and the conical core, it is guided by the spiral groove and flows downward along the spiral groove, generating inertial force and Dean force, thereby separating the mixed solution into three layers. The fluid component located on the innermost layer is microvesicles, the fluid component located in the middle layer is exosomes and small vesicles, and the fluid component located on the outermost layer is platelets and cells.

[0010] Preferably, the bottom of the inner cavity of the first separation chamber is sequentially provided with a first annular groove, a second annular groove, and an annular plate. The three layers of fluid separated are transported to the first annular groove, the second annular groove, and the annular plate respectively under the action of inertia and Dean force, thereby achieving separation. The bottom of the first separation chamber is connected to a first discharge pipe, a second discharge pipe, and a third infusion pipe. The third infusion pipe is connected to the inner cavity of the second separation chamber. The third infusion pipe is located inside the first annular groove, the first discharge pipe is located between the first annular groove and the second annular groove, and the second discharge pipe is located between the second annular groove and the annular plate. The third infusion pipe, the first discharge pipe, and the second discharge pipe respectively transport microvesicles, exosomes, small vesicles, platelets, and cells. The first discharge pipe and the second discharge pipe directly discharge waste liquid, while the third infusion pipe transports microvesicles to the inner cavity of the second separation chamber.

[0011] Preferably, a flow guide plate is fixedly installed on one side of the inner cavity of the second separation chamber, and resonant screens are distributed at intervals on one side of the flow guide plate. The resonant screens 27 are arranged in five layers: the first layer has an amplitude of 0.5 mm and a wavelength of 2 mm; the second layer has a phase offset of 180°; the third layer has an amplitude of 0.3 mm; the fourth layer has a phase offset of 90°; and the fifth layer has an amplitude of 0.1 mm. The bottom of the inner cavity of the second separation chamber is connected to a bottom discharge pipe, which is located at the end of the resonant screen away from the flow guide plate. The outer side of the second separation chamber is connected to a side discharge pipe. The five sets of distributed resonant screens facilitate the separation of the mixture, thereby dividing the mixture into upper and lower layers. The upper layer is microvesicles, which are connected to the side discharge pipe and discharged as waste liquid through it. The lower layer is exosome enrichment liquid, which is transported to the inner cavity of the diversion box through the bottom discharge pipe.

[0012] Preferably, a vortex plate is fixedly installed on the inner side of the diversion box. The vortex plate facilitates the increase of the vortex degree of the exosome enrichment liquid delivered to the inner side of the diversion box. A chassis is fixedly installed at the bottom of the diversion box, and a drain pipe is connected to the bottom array of the chassis. The drain pipe is connected to the collection pipe.

[0013] Preferably, a collection tray is fixedly installed at the bottom of the collection tube, and a drive motor is fixedly installed at the center of the bottom of the collection tray. The drive motor drives the collection tray and the collection tube to rotate, thereby alternately collecting the exosome enrichment fluid discharged from the drain tube.

[0014] Preferably, a mounting bracket is fixedly installed at the bottom of the drive motor, and the top of the mounting bracket is fixedly connected to the bottom of the distributor box.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Stable rectification of sample and sheath fluid is achieved through a laminar flow chamber. Combined with the gradually varying depth spiral groove structure of the cone core in the first separation chamber, microvesicles, exosomes, and cell debris are precisely graded using inertial force and Dean force. Then, secondary resonant separation is performed through a multi-layered resonant sieve with differentiated amplitude and phase in the second separation chamber, which significantly improves the enrichment purity of exosomes and effectively solves the problem of difficulty in accurately distinguishing components with similar particle sizes in existing technologies.

[0016] 2. The device adopts a multi-stage series and continuous flow design, requiring no manual intervention from sample injection into the storage tank to collection in the split box. It is equipped with a rotating collection tube to achieve alternating collection, making it suitable for processing large-volume samples. It overcomes the limitations of traditional ultracentrifugation, such as cumbersome operation and low throughput of microfluidic chips, and meets the needs of clinical and industrial-scale separation.

[0017] 3. The entire process uses physical separation to avoid damage to the vesicle structure caused by the high shear force of ultracentrifugation. At the same time, the resonant sieve operates under low amplitude conditions, which effectively maintains the integrity of exosomes and provides high-quality samples for subsequent functional research and application.

[0018] 4. The separation modules at each level are directly connected by pipelines, resulting in a compact overall structure. All functional components work together, eliminating the need for frequent replacement of consumables or complex debugging, significantly reducing the difficulty of operation and equipment maintenance costs. Compared with the immune affinity method, it has higher economic efficiency and repeatability. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a cross-sectional view of the internal structure of the liquid storage tank of the present invention; Figure 3 This is a cross-sectional view of the internal structure of the laminar flow box of the present invention; Figure 4 This is a cross-sectional view of the connection structure of the busbar of the present invention; Figure 5 This is a cross-sectional view of the internal structure of the first separation chamber of the present invention; Figure 6 This is a cross-sectional view of the internal structure of the outer sleeve of the present invention; Figure 7 This is a cross-sectional view of the internal structure of the second separation chamber of the present invention; Figure 8 This is a cross-sectional view of the internal structure of the diversion box of the present invention; Figure 9 This is an exploded view of the connection structure of the collection tray of the present invention.

[0020] Legend In the diagram: 1. Storage tank; 2. Connecting frame; 3. Baffle plate; 4. First infusion pipe; 5. Second infusion pipe; 6. Laminar flow box; 7. Manifold plate; 8. Central limiting ring; 9. Discharge trough; 10. Metal sintered plate; 11. Guide bucket; 12. First separation chamber; 13. Converging bucket; 14. Outer sleeve; 15. Conical core; 16. Spiral groove; 17. First annular groove; 18. Second annular groove; 19. Annular plate; 20. First discharge pipe; 21. Second discharge pipe; 22. Second separation chamber; 23. Third infusion pipe; 24. Guide pad plate; 25. Bottom discharge pipe; 26. Side discharge pipe; 27. Resonant screen; 28. Diverter box; 29. ​​Vortex plate; 30. Chassis; 31. Discharge pipe; 32. Collection pipe; 33. Collection tray; 34. Drive motor; 35. Mounting frame. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Please see Figure 1 - Figure 9 As shown, the present invention provides a technical solution: a multi-stage laminar flow separation device for platelet exosomes, including a storage tank 1, a connecting frame 2 symmetrically sleeved on the outside of the storage tank 1, a laminar flow box 6 for laminar flow is arranged below the storage tank 1, a first separation chamber 12 for primary separation is arranged below the laminar flow box 6, a second separation chamber 22 for resonant separation is arranged below the first separation chamber 12, a diversion box 28 for diversion is arranged below the second separation chamber 22, and collection tubes 32 are arrayed below the diversion box 28.

[0023] like Figure 2 , Figure 3 and Figure 4 As shown; the interior of the storage tank 1 is symmetrically equipped with inclined baffles 3. The bottom of the storage tank 1 is connected to a first infusion pipe 4 and a second infusion pipe 5. The first infusion pipe 4 is located between two sets of baffles 3. There are two sets of second infusion pipes 5, both of which are bent and located below the two sets of baffles 3. The bottoms of the first infusion pipe 4 and the second infusion pipe 5 are connected to the inner cavity of the laminar flow chamber 6. The baffles 3 divide the interior of the storage tank 1 into three parts. The sample is stored in the middle position, and the sheath fluid is stored in the two sides. The sample is connected to the first infusion pipe 4, and the sheath fluid is connected to the second infusion pipe 5. The sample and the sheath fluid are transported to the interior of the laminar flow chamber 6 through the first infusion pipe 4 and the second infusion pipe 5, respectively.

[0024] Furthermore, a manifold 7 is fixedly installed in the middle of the inner cavity of the laminar flow box 6. The top of the manifold 7 is inclined. A central limiting ring 8 is fixedly installed at the center of the top of the manifold 7. A liquid outlet groove 9 is opened through the bottom of the outer side of the central limiting ring 8. The liquid outlet groove 9 is located on the lower side of the central limiting ring 8. A notch is pre-set on the lower side of the manifold 7. The first infusion pipe 4 is connected to the inner side of the central limiting ring 8, and the second infusion pipe 5 is connected to the outer side of the central limiting ring 8.

[0025] Furthermore, a metal sintered plate 10 is spaced below the manifold 7, and a guide bucket 11 is located below the metal sintered plate 10. The bottom of the guide bucket 11 is connected to the inner cavity of the first separation chamber 12. In use, the sample and sheath fluid are transported to the inner and outer sides of the central limiting ring 8 located in the inner cavity of the laminar flow box 6 through the first infusion pipe 4 and the second infusion pipe 5, respectively. Under the guidance of the manifold 7 and the liquid outlet trough 9, the liquid is mixed. Then, it is transported to the top of the metal sintered plate 10 through the pre-set notch at the bottom of the manifold 7, and then rectified by the metal sintered plate 10. The aperture of the metal sintered plate 10 is 0.2 mm.

[0026] like Figure 5 and Figure 6As shown; a converging bucket 13 is fixedly installed at the top of the inner cavity of the first separation chamber 12, and the converging bucket 13 is connected to the guide bucket 11. An outer sleeve 14 is fixedly installed at the bottom of the converging bucket 13, and the outer sleeve 14 is flared. A conical core 15 is sleeved inside the outer sleeve 14, and a support column extends from the bottom of the conical core 15 and is fixedly connected to the bottom of the inner cavity of the first separation chamber 12. A spiral groove 16 that is recessed inward is opened on the outer side of the conical core 15, and the depth of the spiral groove 16 gradually becomes shallower. The volume gradually decreases from 2mm to 0.5mm, and the pitch is 10mm. At this time, the mixture flows through the converging funnel 13 and the guiding funnel 11 to the outer sleeve 14 and the conical core 15, and is guided by the spiral groove 16 and flows downward along the spiral groove 16, generating inertial force and Dean force, which in turn separates the mixed solution into three layers. The fluid component on the innermost layer is microvesicles, the fluid component in the middle layer is exosomes and small vesicles, and the fluid component on the outermost layer is platelets and cells.

[0027] Furthermore, the bottom of the inner cavity of the first separation chamber 12 is sequentially provided with a first annular groove 17, a second annular groove 18, and an annular plate 19. The separated three layers of fluid are transported to the first annular groove 17, the second annular groove 18, and the annular plate 19 respectively under the action of inertia and Dean's force, thereby achieving separation. The bottom of the first separation chamber 12 is connected to a first discharge pipe 20, a second discharge pipe 21, and a third infusion pipe 23. The third infusion pipe 23 is connected to the inner cavity of the second separation chamber 22. The tube 23 is located inside the first annular groove 17. The first discharge tube 20 is located between the first annular groove 17 and the second annular groove 18. The second discharge tube 21 is located between the second annular groove 18 and the annular plate 19. The third infusion tube 23, the first discharge tube 20 and the second discharge tube 21 respectively transport microvesicles, exosomes and small vesicles, as well as platelets and cells. The first discharge tube 20 and the second discharge tube 21 directly discharge waste liquid. The third infusion tube 23 transports microvesicles to the inner cavity of the second separation chamber 22.

[0028] like Figure 7 As shown; a flow guide plate 24 is fixedly installed on one side of the inner cavity of the second separation chamber 22. Resonant screens 27 are distributed at intervals on one side of the flow guide plate 24. The resonant screens 27 are arranged in five layers: the first layer has an amplitude of 0.5 mm and a wavelength of 2 mm; the second layer has a phase offset of 180°; the third layer has an amplitude of 0.3 mm; the fourth layer has a phase offset of 90°; and the fifth layer has an amplitude of 0.1 mm. The bottom of the inner cavity of the second separation chamber 22 is connected to a bottom discharge pipe 25, which is located at the end of the resonant screen 27 away from the flow guide plate 24. The outer side of the second separation chamber 22 is connected to a side discharge pipe 26. The five sets of distributed resonant screens 27 facilitate the separation of the mixture, thereby dividing the mixture into upper and lower layers. The upper layer is microvesicles, which are connected to the side discharge pipe 26 and discharged as waste liquid through it. The lower layer is exosome enrichment liquid, which is transported to the inner cavity of the diversion box 28 through the bottom discharge pipe 25.

[0029] like Figure 8 ,and Figure 9 As shown; a vortex plate 29 is fixedly installed on the inner side of the diversion box 28. The vortex plate 29 facilitates the increase of the vortex degree of the exosome enrichment liquid delivered to the inner side of the diversion box 28. A chassis 30 is fixedly installed at the bottom of the diversion box 28, and a drain pipe 31 is connected to the bottom array of the chassis 30, and the drain pipe 31 is connected to the collection pipe 32.

[0030] Furthermore, a collection tray 33 is fixedly installed at the bottom of the collection tube 32, and a drive motor 34 is fixedly installed at the center of the bottom of the collection tray 33. The drive motor 34 drives the collection tray 33 and the collection tube 32 to rotate, thereby alternately collecting the exosome enrichment fluid discharged from the drain tube 31.

[0031] Furthermore, a mounting bracket 35 is fixedly installed on the bottom of the drive motor 34, and the top of the mounting bracket 35 is fixedly connected to the bottom of the distributor box 28.

[0032] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0033] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multi-stage laminar flow separation device for platelet exosome, comprising a liquid storage tank (1), characterized in that: The storage tank (1) is symmetrically fitted with a connecting frame (2). A laminar flow box (6) for laminar flow is provided below the storage tank (1). A first separation chamber (12) for primary separation is provided below the laminar flow box (6). A second separation chamber (22) for resonant separation is provided below the first separation chamber (12). A diversion box (28) for diversion is provided below the second separation chamber (22). A collection pipe (32) is arranged in an array below the diversion box (28).

2. The platelet exosome multi-stage laminar flow separation device according to claim 1, characterized in that: The storage tank (1) is symmetrically equipped with inclined partitions (3). The storage tank (1) is connected to a first infusion pipe (4) and a second infusion pipe (5) in sequence. The first infusion pipe (4) is located between the two sets of partitions (3). There are two sets of the second infusion pipe (5), both of which are bent and located below the two sets of partitions (3). The bottoms of the first infusion pipe (4) and the second infusion pipe (5) are connected to the inner cavity of the laminar flow box (6).

3. The platelet exosome multi-stage laminar flow separation device according to claim 2, characterized in that: A manifold (7) is fixedly installed in the middle of the inner cavity of the laminar flow box (6). The top of the manifold (7) is inclined. A central limiting ring (8) is fixedly installed at the center of the top of the manifold (7). A liquid outlet groove (9) is opened through the bottom of the outer side of the central limiting ring (8).

4. The platelet exosome multi-stage laminar flow separation device according to claim 3, characterized in that: Metal sintered plates (10) are spaced apart below the manifold (7), and a guide bucket (11) is provided below the metal sintered plates (10), and the bottom of the guide bucket (11) is connected to the inner cavity of the first separation chamber (12).

5. The platelet exosome multi-stage laminar flow separation device according to claim 4, characterized in that: A flow-gathering bucket (13) is fixedly installed at the top of the inner cavity of the first separation chamber (12), and the flow-gathering bucket (13) is connected to the flow-guiding bucket (11). An outer sleeve (14) is fixedly installed at the bottom of the flow-gathering bucket (13). A conical core (15) is sleeved inside the outer sleeve (14), and a support column extends from the bottom of the conical core (15) and is fixedly connected to the bottom of the inner cavity of the first separation chamber (12). A spiral groove (16) is provided on the outer side of the conical core (15) that is recessed inward.

6. The platelet exosome multi-stage laminar flow separation device according to claim 5, characterized in that: The bottom of the inner cavity of the first separation chamber (12) is provided with a first annular groove (17), a second annular groove (18) and an annular plate (19) in sequence. The bottom of the first separation chamber (12) is connected to a first discharge pipe (20), a second discharge pipe (21) and a third infusion pipe (23). The third infusion pipe (23) is connected to the inner cavity of the second separation chamber (22).

7. The platelet exosome multi-stage laminar flow separation device according to claim 6, characterized in that: A flow guide plate (24) is fixedly installed on one side of the inner cavity of the second separation chamber (22). Resonant screens (27) are distributed at intervals on one side of the flow guide plate (24). A bottom discharge pipe (25) is connected to the bottom of the inner cavity of the second separation chamber (22). The bottom discharge pipe (25) is located at the end of the resonant screen (27) away from the flow guide plate (24). A side discharge pipe (26) is connected to the outside of the second separation chamber (22).

8. The platelet exosome multistage laminar flow separation device according to claim 7, characterized in that: A vortex plate (29) is fixedly installed on the inner side of the diversion box (28), and a chassis (30) is fixedly installed on the bottom of the diversion box (28). A drain pipe (31) is connected to the bottom array of the chassis (30), and the drain pipe (31) is connected to the collection pipe (32).

9. The platelet exosome multi-stage laminar flow separation device according to claim 8, characterized in that: A collection tray (33) is fixedly installed at the bottom of the collection tube (32), and a drive motor (34) is fixedly installed at the center of the bottom of the collection tray (33).

10. The platelet exosome multistage laminar flow separation device according to claim 9, characterized in that: The bottom of the drive motor (34) is fixedly mounted with a mounting bracket (35), and the top of the mounting bracket (35) is fixedly connected to the bottom of the distributor box (28).

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