An exosome microvesicle nondestructive separation and purification device

Abstract

The application belongs to the technical field of exosome processing, and discloses an exosome microvesicle nondestructive separation and purification device, which comprises a sealed separation box, a laminated assembly is arranged in the inner cavity of the sealed separation box, the laminated assembly is sleeved outside a central shaft assembly, a pressure plate is arranged at the top end of the laminated assembly, and a synchronous driving assembly is arranged. When in the compressed state, the micro-nano groove forms a precise nanoscale channel for interception and filtration, and when in the relaxed state, the elastic sheet rebounds rapidly with the release of the axial pressure, the cross-sectional geometry of the micro-nano groove is deformed, the periodic geometric deformation breaks the mechanical stability of the filter cake layer from the physical level, the particles originally stuck in the groove or adsorbed on the surface lose the clamping force instantaneously, and are automatically detached from the surface fluid under the shearing action of the surface fluid, so that the flux decay rate of the device is controlled at a small value under long-time operation, and continuous and stable filtration in the true sense is realized.

Description

Technical Field

[0001] This invention belongs to the field of exosome processing technology, specifically a device for non-destructive separation and purification of exosome microvesicles. Background Technology

[0002] Exosomes are disc-shaped vesicles secreted by cells, ranging in diameter from 30 nm to 150 nm. They encapsulate bioactive substances such as proteins and nucleic acids, and have significant applications in intercellular communication, disease diagnosis, and drug delivery. Currently, the isolation and purification of exosomes is the main bottleneck limiting their large-scale clinical application. Traditional extraction methods mainly include ultracentrifugation, polymer precipitation, and conventional membrane filtration (such as dead-end filtration or cross-flow filtration). While ultracentrifugation offers relatively good purity, the equipment is expensive, time-consuming (often exceeding 4 hours), and the extremely high centrifugal force (usually exceeding 100,000 g) can easily cause irreversible physical damage or aggregation of the exosome membrane structure. Polymer precipitation easily introduces difficult-to-remove chemical impurities, affecting the accuracy of subsequent biological experiments.

[0003] In existing technologies, microporous membrane-based filtration and separation techniques have attracted widespread attention due to their ease of operation and scale-up. However, they suffer from serious problems of "membrane fouling" and "pore blockage" in practical applications. Traditional microporous membranes (such as PES and PVDF membranes) have a statically fixed pore structure. When filtering biological samples rich in proteins and colloids, as filtration proceeds, particles larger than the pore size and non-specifically adsorbed proteins rapidly accumulate on the membrane surface, forming a dense "filter cake layer." This leads to a sharp increase in transmembrane pressure (TMP). To maintain filtration efficiency, operators often have to apply higher driving pressures, which not only increases energy consumption but, more seriously, forces the originally soft exosomes to deform or even rupture, compelling them to pass through the pores into the filtrate side, resulting in decreased purity and sample loss.

[0004] Furthermore, existing continuous exosome purification devices often lack effective fluid dynamic coupling design. The export of dialysate mainly relies on natural osmosis or an independent pump source, which easily leads to back pressure retention in the central collection tube, reducing the effective filtration differential pressure. How to design a non-destructive separation device that can fundamentally solve the filter pore clogging problem from a physical mechanism, maximize the protection of the integrity of the exosome lipid bilayer, and has a self-driven fluid transport function is a key technical challenge that urgently needs to be solved in the current field of exosome processing technology. Summary of the Invention

[0005] The purpose of this invention is to provide a non-destructive separation and purification device for exosome microvesicles to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a non-destructive separation and purification device for exosome microvesicles, comprising: a sealed separation box, wherein a stacked plate assembly is disposed in the internal cavity, the stacked plate assembly is sleeved on the outside of the central shaft assembly, and a pressure plate is disposed at the top of the stacked plate assembly;

[0007] A synchronous drive assembly is installed on the top of the sealed separation box and is connected to the pressure plate and central shaft assembly for driving the pressure plate and central shaft assembly to reciprocate along the axial direction.

[0008] A suction assembly is disposed at the bottom end of the sealed separation box and is fixedly connected to the bottom end of the central shaft assembly;

[0009] The stacked assembly includes a plurality of elastic sheets stacked along the axial direction, and the surface of the elastic sheets is provided with micro-nano grooves;

[0010] When the synchronous drive assembly drives the pressure plate to press down, the adjacent elastic sheets press against each other to close the micro-nano trenches and form a filter channel.

[0011] When the synchronous drive component releases pressure, the elastic sheet springs back to its original position, causing the channel cross-section of the micro-nano trench to expand.

[0012] The suction component is configured to apply negative pressure to the interior of the central shaft component while the stacked plate assembly springs back to its original position.

[0013] As a further technical solution of the present invention, a limiting protrusion is provided around the outer edge of the elastic sheet in the circumferential direction, and the axial height of the limiting protrusion is greater than the depth of the micro-nano trench.

[0014] When the stacked sheet assembly is in a compressed state, the bottom surface of the upper elastic sheet abuts against the top surface of the limiting protrusion of the lower elastic sheet, defining a filter gap with a constant height between the two adjacent elastic sheets.

[0015] As a further technical solution of the present invention, the central shaft assembly includes a hollow main shaft and an extension shaft connected to the bottom end of the main shaft;

[0016] The main shaft has several guide holes on its tube wall, and the guide holes connect the inner side of the lamination assembly with the inner cavity of the main shaft.

[0017] The extension shaft extends through the bottom of the sealed separation box and is connected to the suction assembly.

[0018] As a further technical solution of the present invention, the suction assembly includes a suction tube and a piston plate that is slidably and sealed inside the suction tube. The piston plate is fixedly connected to the extension shaft to move synchronously with the central shaft assembly. The inner cavity of the suction tube is connected to the top end of the main shaft through a pipeline.

[0019] As a further technical solution of the present invention, a drain pipe is connected to the suction tube, a one-way valve is installed inside the drain pipe, and a return spring is provided between the inner bottom wall of the suction tube and the piston plate. The return spring is used to apply a thrust to the piston plate in the direction of return.

[0020] As a further technical solution of the present invention, the micro-nano trenches are distributed in a concentric circle or spiral shape on the upper surface of the elastic sheet along the radial direction. The cross-sectional shape of the micro-nano trenches is a V-shaped structure or a trapezoidal structure, and the depth of the micro-nano trenches gradually decreases from the outside to the inside along the radial direction.

[0021] As a further technical solution of the present invention, the synchronous drive assembly includes a frame, a power shaft rotatably mounted on the frame, and a cam fixedly sleeved on the power shaft;

[0022] A synchronizing frame is connected to the pressure plate or filtrate valve, and the outer contour surface of the cam always remains in contact with the force-bearing surface of the synchronizing frame.

[0023] When the cam rotates, it drives the timing frame to move the pressure plate back and forth in the vertical direction.

[0024] As a further technical solution of the present invention, a uniform dispersion tube is connected to the center of the bottom end of the sealed separation box. The uniform dispersion tube has a funnel-shaped flared structure, and a raw liquid inlet valve is connected to its bottom inlet. Concentrate valves are symmetrically connected to the upper two sides of the outer wall of the sealed separation box.

[0025] As a further technical solution of the present invention, the output ends of the concentrate valves on both sides are respectively connected to manifolds, and the ends of the two manifolds are connected to a manifold valve for exporting the separated concentrate.

[0026] As a further technical solution of the present invention, the surface of the elastic sheet is grafted with a polyethylene glycol hydrophilic coating or a zwitterionic polymer coating, and the elastic sheet is made of a medical-grade polymer material with a Shore A hardness of 50 to 70 degrees.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This invention employs a highly elastic stacked plate assembly as the core filter medium. By synchronously driving the periodic rotation of the cam in the assembly, the stacked plate assembly is forced to switch between a "high-pressure compression state" and a "low-pressure relaxation state" at a high frequency. In the compression state, the micro-nano trenches form precise nano-level channels for interception and filtration. In the relaxation state, as the axial pressure is released, the elastic sheet quickly rebounds, and the cross-sectional geometry of the micro-nano trenches deforms. This periodic geometric deformation physically disrupts the mechanical stability of the filter cake layer, causing particles that were originally stuck inside the trenches or adsorbed on the surface to instantly lose their clamping force and automatically fall off due to the shearing action of the surface fluid. This allows the flux attenuation rate to be controlled at a small value during long-term operation, achieving truly continuous and stable filtration.

[0029] 2. This invention designs a rigid limiting protrusion on the surface of the elastic sheet and strictly controls the dimensional relationship between it and the depth of the micro-nano grooves. This ensures that even when high axial pressure is applied by the pressure plate, the filter channels between the stacked sheets will not completely close or collapse, but will be forcibly locked at a constant height. At this time, the exosomes will not be subjected to direct mechanical compression from the filter medium. Compared with the traditional centrifugation method, this invention uses gentle fluid pressure and precise physical sieving to make the separated exosomes round in shape and have an intact membrane structure. Electron microscopy shows that its damage rate is extremely low, thus improving the overall quality of the exosomes.

[0030] 3. This invention utilizes the mechanical coupling structure between the central shaft assembly and the suction assembly. During the plate relaxation stage, the piston moves upward to generate negative pressure, taking advantage of the moment when the flow resistance is at its minimum to quickly extract the dialysate from the main shaft, eliminating back pressure retention within the central shaft. During the plate compression stage, the piston moves downward to force the liquid out of the pump chamber. No additional power source is required, reducing equipment costs and energy consumption. The negative pressure during the relaxation stage assists in particle desorption, and the positive pressure during the compression stage discharges the liquid, thereby achieving the maximum fluid throughput with minimal mechanical energy and ensuring efficient and continuous operation of the separation process. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0032] Figure 2 This is a schematic diagram of the bottom structure of the present invention;

[0033] Figure 3 This is a cross-sectional schematic diagram of the internal structure of the uniformly dispersed tube of the present invention;

[0034] Figure 4 This is a separate cross-sectional schematic diagram of the suction component structure of the present invention;

[0035] Figure 5 This is a separate schematic diagram of the synchronous drive component structure of the present invention;

[0036] Figure 6 This is a schematic diagram of the sealed separation box of the present invention with the top portion removed;

[0037] Figure 7 This is a schematic diagram showing the cooperation between the pressure plate and the filter valve structure of the present invention;

[0038] Figure 8 This is a cross-sectional schematic diagram of the sealed separation box structure of the present invention;

[0039] Figure 9 This is a schematic diagram illustrating the fit between the stacked assembly and the central shaft assembly of the present invention;

[0040] Figure 10 This is a separate schematic diagram of the stacked assembly structure of the present invention.

[0041] In the diagram: 1. Sealed separation box; 2. Concentrate valve; 3. Manifold; 4. Manifold valve; 5. Stacked plate assembly; 501. Elastic sheet; 502. Micro-nano groove; 503. Limiting ring; 6. Central shaft assembly; 601. Main shaft; 602. Guide hole; 603. Extension shaft; 7. Suction assembly; 701. Suction pipe; 702. Piston plate; 703. Piston rod; 704. Return spring; 705. Filter pipe; 706. Drain pipe; 707. Locking frame; 8. Synchronous drive assembly; 801. Drive motor; 802. Coupling; 803. Power shaft; 804. Frame; 805. Cam; 806. Synchronous frame; 9. Pressure plate; 10. Filter valve; 11. Raw material inlet valve; 12. Uniform dispersion pipe. Detailed Implementation

[0042] 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.

[0043] like Figures 1 to 10As shown, this embodiment of the invention provides a non-destructive separation and purification device for exosome microvesicles, including a sealed separation box 1, wherein the sealed separation box 1 is a hollow cylinder, and a uniform dispersion tube 12 is fixedly connected to its bottom end. One end of the outer side of the uniform dispersion tube 12 is fixedly connected to a raw liquid inlet valve 11, wherein the raw liquid inlet valve 11 is the raw liquid inlet and is connected to an external pipeline. In order to perform preliminary filtration of the raw liquid and prevent large particulate impurities from entering, a screening filter screen is also installed at the inlet of the raw liquid inlet valve 11 for preliminary filtration of the raw liquid. At the same time, in order to prevent the raw liquid from directly impacting the interior of the sealed separation box 1 after entering, the uniform dispersion tube 12 is designed as a funnel. After the raw liquid enters the uniform dispersion tube 12, it rises and diffuses and depressurizes through the uniform dispersion tube 12 before entering the bottom end of the sealed separation box 1.

[0044] Meanwhile, in order to effectively purify the solution, concentrated liquid valves 2 are symmetrically installed on both sides of the outer side of the sealed separation box 1 near the top for discharging concentrated liquid. In order to collect the concentrated liquid inside the two concentrated liquid valves 2, a manifold 3 is installed at one end of each concentrated liquid valve 2. The output ends of the two manifold 3 are connected to the manifold valve 4. The manifold valve 4 is a three-way valve, which is symmetrically connected to the two concentrated liquid valves 2 on both sides. After collection, it is discharged through one end.

[0045] The sealed separation box 1 is equipped with a stacked plate assembly 5 in its internal cavity, and a central shaft assembly 6 is fixedly sleeved in the middle of the stacked plate assembly 5. The stacked plate assembly 5 is formed by stacking several elastic thin plates 501 in sequence along the axial direction of the central shaft assembly 6, and the whole is in the shape of a cylindrical structure.

[0046] The central shaft assembly 6 mainly includes a main shaft 601, a guide hole 602, and an extension shaft 603.

[0047] The central through hole of the elastic sheet 501 is sleeved on the outer periphery of the main shaft 601. The main shaft 601 is a hollow tubular structure with several guide holes 602 on its tube wall that communicate with the inner side of the stacked sheet assembly 5 for collecting the filtered dialysate. An extension shaft 603 is installed at the bottom end of the main shaft 601. A raw liquid circulation chamber is formed between the outer periphery of the stacked sheet assembly 5 and the inner wall of the sealed separation box 1. The raw liquid inlet valve 11 and the uniform dispersion tube 12 supply liquid to the chamber.

[0048] Meanwhile, the top of the stacking assembly 5 is provided with a pressure plate 9 located inside the sealed separation box 1. The top of the stacking assembly 5 abuts against the pressure plate 9, while the bottom is supported by the fixed seat at the bottom of the main shaft 601.

[0049] The elastic sheet 501 is made of medical-grade polymer elastic materials (including but not limited to modified PDMS, PTFE or silicone) and has reversible elastic deformation capability.

[0050] The material hardness (Shore A) of the elastic sheet 501 is controlled between 50 and 70 degrees, and the thickness is set between 0.5 mm and 1.5 mm. The height of the limiting ring 503 is... Maximum depth of micro / nano trench 502 Satisfying the relation: ,in The value of the reserved compressibility deformation is 5μm-10μm;

[0051] Within this hardness range, when the pressure plate 9 applies an axial pressure of 0.2MPa-0.5MPa, the main body of the elastic sheet 501 undergoes slight deformation but will not completely collapse and block the flow channel. Meanwhile, the limiting protrusion ring 503 plays a rigid stopping role, ensuring that the standard deviation of the filtration accuracy is controlled within ±5%. If the hardness is too low, the flow channel is easy to close; if the hardness is too high, the "relaxed state" rebound response will be delayed, affecting the cleaning efficiency.

[0052] A continuous ring of limiting protrusions 503 is provided on the outer edge of the elastic sheet 501 in a concentric circle pattern. The height of the limiting protrusions 503 is set to be greater than or equal to the preset minimum filter pore diameter. The limiting protrusions 503 are made of a material with high hardness, or are designed with a thickened structure to provide rigid support when subjected to axial pressure.

[0053] On the upper surface of the elastic sheet 501, in the area between the central through hole and the outer edge limiting protrusion ring 503, there are several concentric or spiral micro-nano trenches 502. The depth of the micro-nano trenches 502 is in the nanometer range (30nm-150nm, the specific size is determined according to the size of the exosome), which constitutes a precision filtration channel for the fluid.

[0054] Furthermore, the cross-sectional shape of the micro-nano trench 502 is preferably an asymmetric "V" or trapezoidal structure, and the trench depth gradually decreases from the outside to the inside along the radial direction. This variable cross-section design forms a convergent flow channel under "high-pressure filtration state", which can use fluid shear force to prevent large particles from clogging the inlet. Under "low-pressure relaxation state", as the elastic sheet 501 rebounds, the V-shaped opening angle increases, making it easy for particles that were originally stuck in the trench to fall off. In order to prevent non-specific adsorption of exosomes, the surface of the elastic sheet 501 and its micro-nano trench 502 is grafted with a polyethylene glycol (PEG) hydrophilic coating or a zwitterionic polymer coating through plasma treatment. The coating thickness is controlled at 2nm-5nm to ensure that the contact angle is less than 30°. This not only protects the lipid bilayer structure of exosomes, but also significantly reduces the formation rate of the filter cake layer.

[0055] The stacking method of multiple stacked sheet components 5 is as follows: several elastic sheets 501 are stacked in the same direction in sequence. Under the compressed state, the lower surface of the upper layer of elastic sheet 501 is in close contact with the top surface of the lower layer of elastic sheet 501, thereby forcibly locking a filter gap with a constant height between the main body areas of the two layers of elastic sheets 501.

[0056] The bottom end of the extension shaft 603 passes through the bottom ends of the sealed separation box 1 and the uniform dispersion tube 12 in sequence and is equipped with a suction assembly 7. One side of the suction assembly 7 is connected to the bottom end of the sealed separation box 1. At the same time, a filter valve 10 is fixedly sleeved in the middle of the pressure plate 9. The filter valve 10 is connected to the top end of the main shaft 601, and the top end of the filter valve 10 passes through the top end of the sealed separation box 1 and is fixedly connected to the other end of the suction assembly 7.

[0057] To effectively achieve the compression and release of the stacked assembly 5, this device also includes a synchronous drive assembly 8. Specifically, the synchronous drive assembly 8 mainly includes a frame 804, a drive motor 801, a coupling 802, a power shaft 803, a cam 805, and a synchronous frame 806. The frame 804 is fixedly installed on the top cover of the sealed separation box 1 as a supporting foundation. The drive motor 801 is installed on the top of the sealed separation box 1 via a mounting bracket, and its output end is fixed to one end of the power shaft 803 via the coupling 802. A fixed connection is used to transmit torque. The power shaft 803 is rotatably mounted on the frame 804 via bearings, and the horizontally extending cam 805 is fixedly sleeved on the power shaft 803 and rotates synchronously with the power shaft 803. The timing frame 806 is installed on the top of the filter valve 10 and can move up and down with the filter valve 10. The upper part of the timing frame 806 is provided with a contact surface that matches the cam 805. In the installed state, the profile surface of the cam 805 always maintains contact or rolling fit with the force-bearing surface of the timing frame 806.

[0058] The core function of the synchronous drive assembly 8 is to convert the rotational motion of the drive motor 801 into the periodic linear reciprocating motion of the synchronous frame 806. Specifically, when the drive motor 801 starts, it drives the power shaft 803 and the cam 805 to rotate. Since the cam 805 has a non-circular contour curve, it will periodically press down on the synchronous frame 806 during its rotation, causing it to move downward along the frame 804. After the cam 805 has rotated through the far repose angle, the synchronous frame 806 returns to its original position by the action of the external restoring force.

[0059] Example: The stacked assembly 5 can realize a "breathing" cycle, which refers to the process in which the stacked assembly 5, under mechanical drive, periodically switches between "high-pressure filtration state (exhalation)" and "low-pressure relaxation state (inhalation)":

[0060] High-pressure filtration stage (closed state): When the cam 805 of the synchronous drive assembly 8 rotates to the large diameter end (i.e., downward pressing stroke), it drives the pressure plate 9 to move downward through the synchronous frame 806, applying axial pressure to the stacked assembly 5. At this time, the adjacent elastic sheets 501 are pressed together, and the bottom surface of the upper sheet is tightly attached to the limiting protrusion ring 503 of the lower sheet. Due to the rigid support of the limiting protrusion ring 503, the two sheets will not be completely closed, but will be forcibly locked into a filtration gap with a constant height. This gap is formed by micro-nano grooves 502. In this state, the raw liquid flows from the outer periphery of the stacked assembly 5 to the center under external pressure or in conjunction with the negative pressure of the suction assembly 7. Exosomes and large particulate impurities with a depth greater than the groove depth are intercepted on the outer surface of the stacked assembly 5, while the dialysate with a depth less than the groove depth flows into the guide hole 602 of the main shaft 601 along the micro-nano grooves 502, completing the precision separation.

[0061] Low-pressure relaxation stage (open state): When the cam 805 rotates to the small diameter end, that is, when the release stroke is reached, the axial pressure of the pressure plate 9 on the stacked plate assembly 5 decreases or disappears. Relying on the material rebound force of the elastic sheet 501 itself and the reverse fluid disturbance when the suction assembly 7 is reset, the contact between adjacent sheets becomes loose. The channel cross section of the micro-nano trench 502 undergoes a small amount of elastic expansion and deformation, so that the particles that were originally stuck at the trench entrance or in the shallow layer lose their mechanical clamping force and slide down with the surface fluid into the bottom of the device, so that the device can be cleaned later.

[0062] Meanwhile, the annular area formed by the outer peripheral wall of the stacked assembly 5 and the inner wall of the sealed separation box 1 constitutes the original liquid circulation and concentration chamber. As the above-mentioned "breathing" filtration cycle continues, target exosomes and vesicles with particle sizes larger than the pore size of the micro-nano groove 502 are continuously trapped in this chamber. Driven by the fluid power of the bottom original liquid inlet valve 11 and the uniform dispersion tube 12 continuously supplying liquid, the trapped high-concentration components gradually migrate to the top of the sealed separation box 1 and accumulate. When the preset concentration cycle or pressure threshold is reached, the concentrate valves 2 located on both sides of the top of the sealed separation box 1 are opened, and the high-concentration exosome enriched liquid is discharged and guided to the manifold valve 4 for merging through the manifold 3 on both sides. Finally, the manifold valve 4 discharges the high-purity concentrate collected on both sides, completing the separation process of larger exosomes and impurities.

[0063] By using the cam 805 of the synchronous drive component 8 to periodically drive the stacked plate assembly 5 to perform micro-breathing motion, the problem of easy clogging in traditional membrane filtration is fundamentally solved. Specifically, the compression of the stacked plate assembly 5 causes the elastic sheet 501 to be rigidly locked into a constant nanoscale flow channel by the limiting protrusion ring 503 during high-pressure filtration, ensuring high precision and consistency in exosome sorting. The release of the elastic sheet 501 causes it to quickly rebound and relax, resulting in deformation and expansion of the micro-nano grooves 502, thereby automatically breaking the filter cake layer and releasing stuck particles. This mechanically driven dynamic pore size adjustment mechanism protects the structural integrity of bioactive substances such as exosomes to the greatest extent and achieves rapid separation of larger exosomes and impurities.

[0064] The suction assembly 7 is located at the bottom of the sealed separation box 1. The suction assembly 7 mainly includes a suction pipe 701, a piston plate 702, a piston rod 703, a return spring 704, a filter pipe 705, a drain pipe 706, and a locking frame 707. The suction pipe 701 serves as the pump chamber, is hollow inside, and communicates with the filter pipe 705 and the drain pipe 706. A one-way valve is installed inside the drain pipe 706, with the valve direction being outward opening and inward closing. The piston plate 702 is sealed and slidably disposed within the inner cavity of the suction pipe 701. One end of the piston rod 703 is fixedly connected to the piston plate 702, and the other end extends out of the end of the piston plate 702. One end of the return spring 704 abuts against the suction pipe 701. The inner bottom wall, with one end abutting against one end of the piston plate 702, always provides the piston rod 703 with an outward restoring force. The locking frame 707 has an L-shaped or U-shaped long rod structure, with its lower end fixedly connected to the outer end of the piston rod 703 and its upper end extending to and connected to the top of the sealed separation box 1. The piston rod 703 is connected to the extension end of the extension shaft 603, which can move up and down with the extension shaft 603. At the same time, the filtrate pipe 705 is sealed and connected to the output end of the filtrate valve 10. That is, the dialysate that flows into the main shaft 601 after being filtered by the stacked plate assembly 5 has its only outlet into the pump chamber of the suction pipe 701. The drain pipe 706 is connected to an external collection container for collecting the filtered solution.

[0065] Example: The operation of the suction component 7 strictly follows the cycle of the synchronous drive component 8, consisting of two strokes: drainage and suction, and is phase-coordinated with the breathing state of the stacked plate component 5.

[0066] Downward discharge stroke: When the cam 805 of the synchronous drive assembly 8 rotates to the large diameter end and presses down on the synchronous frame 806, the central shaft assembly 6 moves downward as a whole, and drives the piston rod 703 and piston plate 702 to move downward synchronously in the suction pipe 701. During this process, the piston plate 702 compresses the pump chamber below the suction pipe 701, resulting in a decrease in the volume of the chamber and a sudden increase in pressure. Under the action of fluid pressure, the liquid accumulated in the pump chamber is forced to open the one-way valve in the discharge pipe 706 and discharge the liquid from the system. At this stage, the corresponding stacked plate assembly 5 is pressed, that is, it is in a high-precision filtration state. At this time, the suction assembly 7 mainly performs the task of emptying the pump chamber to make room for the next liquid suction.

[0067] Upward suction stroke: When the cam 805 rotates past the far repose angle and enters the return stroke, the pressure of the cam on the synchronous frame 806 is released. At this time, the return spring 704 at the bottom of the suction tube 701 releases elastic potential energy, pushing the piston plate 702, piston rod 703 and locking frame 707 to return upward. During this process, the piston plate 702 moves upward, and the volume of the pump chamber below the suction tube 701 increases rapidly, forming a negative pressure zone. Since the one-way valve of the drain pipe 706 is closed, the negative pressure acts directly on the filter pipe 705 and the inside of the main shaft 601 of the central shaft assembly 6. This suction force quickly draws the filtered dialysate in the main shaft 601 into the pump chamber, completing the suction process. During this stage, the corresponding stacked plate assembly 5 is in a relaxed state. The instantaneous suction force generated by the suction assembly 7 can not only transfer the dialysate, but also form a low-pressure guide on the inside of the stacked plate, accelerating the fluid through the relaxed gap.

[0068] To ensure optimal matching between the "breathing" frequency and the suction flow rate, the operating parameters of this device follow the following fluid dynamics coupling model: The rotational speed of the drive motor 801 is set to... (rpm), the effective lift of cam 805 is The cross-sectional area of ​​piston plate 702 is To prevent exosome rupture due to excessive negative pressure or incomplete cleaning due to insufficient negative pressure during the aspiration process, the aspiration volume per cycle is [not specified]. The theoretical flux of the stacked module must meet the following requirements:

[0069] in:

[0070] The cycle of one rotation of the cam (60 / );

[0071] The duration during which the cam is at the small diameter end (i.e., the relaxation cleaning time).

[0072] The fluid conduction efficiency coefficient (values ​​range from 0.85 to 0.95);

[0073] Preferably, the profile curve of cam 805 is designed as a "fast return characteristic" curve, so that... account for the entire cycle The high-pressure filtration time accounts for 30%-40%, while the high-pressure filtration time accounts for 60%-70%. This ensures that within a limited cycle, there is enough time to establish the filtration pressure difference and to generate a strong pulse backwash force by utilizing the instantaneous rapid rebound release.

[0074] Through the cooperation between the central shaft assembly 6 and the suction assembly 7, automatic fluid delivery can be achieved without an additional power source. The periodic reciprocating motion of the piston plate 702 actively and forcibly discharges the dialysate accumulated in the central shaft assembly 6 through the drain pipe 706, effectively eliminating the central shaft back pressure retention phenomenon caused by traditional natural osmosis. This ensures that the two sides of the stacked assembly 5 always maintain a high-efficiency filtration pressure difference. At the same time, when the stacked assembly 5 is released with the cam and enters the relaxation stage where the micro-nano groove 502 opens, a negative pressure suction action is performed simultaneously. The filtrate is quickly emptied and the desorption of particles is assisted by the minimum fluid resistance at this time, thereby ensuring the continuity and flux stability of the exosome separation and purification process with the lowest energy consumption.

[0075] Example Comparison and Verification: To verify the non-destructive separation effect of the present invention, it was compared with the traditional ultracentrifugation method (i.e., the traditional differential centrifugation method, which includes the traditional steps of removing cells, removing large vesicles, precipitating exosomes, and washing) and the conventional dead-end microfiltration device (mainly pointer-type filter or vacuum filter cup, with a pore size of 0.22μm or 0.45μm, commonly Millipore Millex series, Pall Acrodisk series). The experimental sample was mesenchymal stem cell (MSC) culture supernatant, and the target extract was exosomes with a particle size of 30nm-150nm.

[0076] When using this device for exosome separation and purification, the following segmented control strategy is adopted:

[0077] Pre-filling and venting stage: Start the drive motor 801, set the speed to low speed (10-20 rpm), keep the raw liquid inlet valve 11 open, until the dialysate flows out continuously from the drain pipe 706 without bubbles, at which point the stacked assembly 5 is in a fully wetted state.

[0078] Dynamic separation stage: Increase the rotation speed to the working speed (40-60 rpm). At this time, a pulsating flow field is formed inside the device. The flow rate of the raw liquid in the sealed separation box 1 is controlled at 0.5 mL / min-2.0 mL / min. Due to the periodic opening and closing of the stacked plate assembly 5, the gel layer attached to the membrane surface is repeatedly destroyed, and the flux attenuation rate can be reduced to less than 10% of that of traditional dead-end filtration.

[0079] Concentrate collection stage: When the pressure at manifold 4 reaches the threshold (e.g., 0.3 MPa, indicating a high concentration of retained substances in the cavity), the system automatically opens concentrate valve 2 and uses the residual pressure in the device to discharge the enriched exosomes.

[0080] Working principle and usage process of this invention:

[0081] System pre-filling and venting: Connect the stock solution inlet valve to the biological sample storage tank, open the stock solution inlet valve, keep the concentrate valve closed, start the drive motor and set it to low speed mode. The stock solution enters the sealed separation box through the uniform dispersion tube at the bottom, gradually filling the cavity between the stacked components and the box. As the liquid level rises, the air is expelled until the dialysate begins to flow steadily out of the drain pipe without any air bubbles. This indicates that the system pre-filling is complete and the stacked components are fully saturated.

[0082] When the dynamic separation cycle is started, the drive motor is adjusted to the working speed. At this time, the synchronous drive component starts to operate at full speed. The motor drives the power shaft and cam to rotate. When the cam rotates to the large diameter end, the pressure plate is pressed down through the synchronous frame. The pressure plate applies axial pressure to the stacked plate assembly, so that the elastic sheet is pressed tightly. At this time, the limiting convex rings between adjacent elastic sheets contact each other, locking the micro-nano grooves into nano-scale filtration channels. Under the combined action of external feed pressure and internal negative pressure, small molecules and solvents in the raw solution enter the guide hole of the main shaft through the micro-nano grooves, while exosomes are trapped on the outside.

[0083] During the simultaneous drainage and pressure build-up process, while the aforementioned stacked plate assembly is compressed for filtration, the central shaft assembly moves downward, causing the piston rod and piston plate of the suction assembly to move downward. At this time, the volume of the pump chamber below the suction pipe decreases and the pressure increases, forcing the dialysate accumulated in the pump chamber to push open the check valve and be discharged from the system through the drain pipe. This process not only completes the liquid delivery but also effectively empties the space below the piston, preparing for the next suction stroke.

[0084] During the relaxation cleaning and negative pressure suction process, when the cam rotates past the far repose angle and enters the return stroke, the pressure on the synchronous frame is released. The stacked plate assembly quickly rebounds and resets due to the material elasticity of the elastic plates and the action of the return spring. At this time, the gap between adjacent elastic plates increases, and the "V"-shaped opening of the micro-nano groove expands. Impurity particles that were originally stuck at the groove inlet or shallow layer lose their mechanical clamping force and fall off under the fluid disturbance, preventing the formation of filter cake layer. At the same time, the piston plate moves upward under the action of the return spring, forming a strong negative pressure zone below the suction tube. This negative pressure acts directly on the inside of the main shaft, rapidly drawing the filtered dialysate from the central shaft assembly into the pump chamber. This instantaneous suction force not only transfers the liquid but also generates a reverse pulse flow field, further assisting the desorption of particles from the membrane surface.

[0085] Concentrate collection: As the above "breathing" cycle continues, the concentration of exosomes trapped in the sealed separation box gradually increases. When the pressure sensor at the manifold valve detects that the preset threshold has been reached or the set time has been reached, the control system automatically opens the concentrate valve at the top. Driven by the residual pressure of the system, the high-concentration exosome enrichment is uniformly discharged and collected through the manifold and manifold valve, completing the entire separation and purification process.

Claims

1. A device for non-destructive separation and purification of exosome microvesicles, characterized in that, include: A sealed separation box (1) has a stacked plate assembly (5) in its internal cavity. The stacked plate assembly (5) is sleeved on the outside of the central shaft assembly (6), and a pressure plate (9) is provided at the top of the stacked plate assembly (5). The synchronous drive assembly (8) is installed on the top of the sealed separation box (1) and is connected to the pressure plate (9) and the central shaft assembly (6) for driving the pressure plate (9) and the central shaft assembly (6) to reciprocate along the axial direction; The suction assembly (7) is located at the bottom of the sealed separation box (1) and is fixedly connected to the bottom of the central shaft assembly (6); The stacked assembly (5) includes a plurality of elastic sheets (501) stacked along the axial direction, and the surface of the elastic sheets (501) is provided with micro-nano grooves (502). When the synchronous drive assembly (8) drives the pressure plate (9) to press down, the adjacent elastic sheets (501) press against each other to close the micro-nano trenches (502) and form a filter channel; When the synchronous drive assembly (8) releases pressure, the elastic sheet (501) springs back to its original position to expand the channel cross-section of the micro-nano trench (502); The suction component (7) is configured to apply negative pressure to the interior of the central shaft component (6) while the stacked plate component (5) springs back to its original position; The micro-nano trenches (502) are distributed concentrically or spirally on the upper surface of the elastic sheet (501) along the radial direction. The cross-sectional shape of the micro-nano trenches (502) is a V-shaped structure or a trapezoidal structure, and the depth of the micro-nano trenches (502) gradually decreases from the outside to the inside along the radial direction. The synchronous drive assembly (8) includes a frame (804), a power shaft (803) rotatably mounted on the frame (804), and a cam (805) fixedly sleeved on the power shaft (803). A timing frame (806) is connected to the pressure plate (9) or the filter valve (10), and the outer contour surface of the cam (805) always abuts against the force-bearing surface of the timing frame (806); When the cam (805) rotates, it drives the timing frame (806) to move the pressure plate (9) back and forth in the vertical direction.

2. The exosome microvesicle non-destructive separation and purification device according to claim 1, characterized in that: A limiting protrusion ring (503) is provided circumferentially at the outer edge of the elastic sheet (501), and the axial height of the limiting protrusion ring (503) is greater than the depth of the micro-nano trench (502). When the stacked sheet assembly (5) is in a compressed state, the bottom surface of the upper elastic sheet (501) abuts against the top surface of the limiting protrusion (503) of the lower elastic sheet (501), defining a filter gap with a constant height between the two adjacent elastic sheets (501).

3. The exosome microvesicle non-destructive separation and purification device according to claim 1, characterized in that: The central shaft assembly (6) includes a hollow main shaft (601) and an extension shaft (603) connected to the bottom of the main shaft (601). The main shaft (601) has several guide holes (602) on its tube wall, and the guide holes (602) connect the inner side of the stacked assembly (5) with the inner cavity of the main shaft (601). The extension shaft (603) passes through the bottom of the sealed separation box (1) and is connected to the suction assembly (7).

4. The exosome microvesicle non-destructive separation and purification device according to claim 3, characterized in that: The suction assembly (7) includes a suction tube (701) and a piston plate (702) that is slidably sealed inside the suction tube (701). The piston plate (702) is fixedly connected to the extension shaft (603) to move synchronously with the central shaft assembly (6). The inner cavity of the suction tube (701) is connected to the top end of the main shaft (601) through a pipeline.

5. The exosome microvesicle non-destructive separation and purification device according to claim 4, characterized in that: The suction tube (701) is connected to a drain tube (706), and a one-way valve is installed inside the drain tube (706). A return spring (704) is provided between the inner bottom wall of the suction tube (701) and the piston plate (702). The return spring (704) is used to apply a thrust toward the return direction to the piston plate (702).

6. The exosome microvesicle non-destructive separation and purification device according to claim 1, characterized in that: The bottom center of the sealed separation box (1) is connected to a uniform dispersion tube (12), which has a funnel-shaped flared structure and a raw liquid inlet valve (11) connected at its bottom inlet. Concentrated liquid valves (2) are symmetrically connected to the upper two sides of the outer wall of the sealed separation box (1).

7. The exosome microvesicle non-destructive separation and purification device according to claim 6, characterized in that: The output ends of the two concentrated liquid valves (2) are respectively connected to manifolds (3), and the ends of the two manifolds (3) are connected to a manifold valve (4) for exporting the separated concentrated liquid.

8. The exosome microvesicle non-destructive separation and purification device according to claim 1, characterized in that: The elastic sheet (501) has a polyethylene glycol hydrophilic coating or a zwitterionic polymer coating grafted onto its surface. The elastic sheet (501) is made of medical-grade polymer material with a Shore A hardness of 50 to 70.

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