Exosome extraction device and detection method

Abstract

The invention relates to an exosome extraction device and a detection method, and relates to the technical field of biomedical detection.The exosome extraction device comprises a table plate, an extraction mechanism and a clamping mechanism, the extraction mechanism comprises a membrane bag, a filtered liquid collecting bottle, a sample bottle and a peristaltic pump which are arranged at the top of the table plate, and the top of the membrane bag is fixedly connected with a filtering end; the surface of the filter-out end is in threaded connection with a filter-out pipe, and the other end of the filter-out pipe is positioned in the filtered liquid collecting bottle. By arranging the membrane bag, the filter-out pipe, the liquid inlet pipe and the return pipe, the filter-out pipe can be in threaded connection with the filter-out end of the membrane bag, the liquid inlet pipe can be in threaded connection with the inlet end of the membrane bag, and the return pipe can be in threaded connection with the return end of the membrane bag through a connector, tight attachment can be achieved through rotation during follow-up connection, the flexibility of the pipeline is matched, and it is ensured that no liquid leaks at the connector; the purpose of avoiding sample loss caused by liquid leakage is achieved, the installation speed is increased, and the time is saved.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, and in particular to an exosome extraction device and detection method. Background Technology

[0002] Currently, exosomes are nanoscale vesicles secreted by cells that carry important biological information and have great potential in disease diagnosis and treatment.

[0003] Regarding the aforementioned technologies, current exosome research mainly focuses on three aspects: separation and purification, biomarker extraction, and functional analysis. Ultracentrifugation is currently the most commonly used separation method, but it suffers from time consumption and high equipment costs. If a filter membrane is used, and a peristaltic pump is used to power it, the liquid will pass through the filter membrane and then exit. Two bottles (sample bottle and filtrate collection bottle) are then used. The peristaltic pump keeps the liquid circulating, and particles with pore sizes smaller than the filter membrane will pass through the filter membrane and then enter the filtrate collection bottle. Finally, the number of growth factor proteins in each milliliter of the filtered liquid can be detected. This method is convenient, fast, and saves excessive costs. Summary of the Invention

[0004] 1. Technical problems to be solved The purpose of this application is to provide an exosome extraction device and detection method to solve the problems of time-consuming and high equipment cost of separation methods.

[0005] This application provides an exosome extraction device with the following technical solution: it includes a table, an extraction mechanism, and a clamping mechanism. The extraction mechanism includes a membrane pack, a filtrate collection bottle, a sample bottle, and a peristaltic pump, all mounted on the top of the table. A filter end is fixedly connected to the top of the membrane pack, and a filter tube is threadedly connected to the surface of the filter end. The other end of the filter tube is located inside the filtrate collection bottle. A reflux end and an inlet end are fixedly connected to the side of the membrane pack. A connector is threadedly connected to the surface of the reflux end, and a connecting tube is fixedly connected to the surface of the connector. A pressure indicator is inserted into the connecting tube. A connecting hole is opened on the side of the connector, and a reflux tube is inserted into the connecting hole. An inlet tube is threadedly connected to the surface of the inlet end. The other ends of the reflux tube and the inlet tube are located inside the sample bottle. The surface of the reflux tube is snapped into the inside of the peristaltic pump. By adopting the above technical solution, and by setting up a membrane pack, filter tube, inlet tube, and reflux tube, the filter tube can be threaded to the filter end of the membrane pack, the inlet tube to the inlet end of the membrane pack, and the reflux tube to the reflux end of the membrane pack via connectors. Subsequent connections can be made by rotation to achieve a tight fit. Combined with the flexibility of the tubing itself, this ensures no liquid leakage at the interfaces, thus avoiding sample loss due to leakage. It also improves installation speed and saves time. By setting up a peristaltic pump and reflux tube, the reflux tube can be snapped into the pump head of the peristaltic pump. After startup, the peristaltic pump generates power by squeezing the reflux tube, driving the liquid to be processed in the sample vial through the inlet tube into the membrane pack. After filtration, a portion of the liquid (including exudate) is filtered. The exosomes return to the sample bottle via the reflux end and reflux tube, forming a closed loop. Small molecule impurities enter the filtrate collection bottle through the filtration end and filtration tube. The circulating flow mode achieves the purpose of making the liquid flow parallel to the membrane surface (tangential flow filtration), reducing the deposition of exosomes on the membrane surface, reducing the membrane fouling rate, and improving the filtration efficiency. By setting a pressure indicator, it can be connected to the reflux pipeline through the connector to monitor the pressure changes in the circulation system in real time. If the membrane is blocked or the pipeline is bent, the pressure will rise sharply and trigger an abnormal reading on the indicator, allowing for timely shutdown and troubleshooting. This achieves the purpose of real-time pressure monitoring to avoid membrane damage caused by overpressure. Due to the implementation of the above device, a lot of costs can be reduced.

[0006] Preferably, the clamping mechanism includes two vertical plates fixedly connected to the top of the tabletop, an electric push rod fixedly installed on the side of the vertical plates, a support frame fixedly connected to one end of the electric push rod, and several clamping plates fixedly connected to the side of the support frame. By adopting the above technical solution and setting up a clamping mechanism, the electric push rod can drive the support frame to move horizontally after receiving the command, and drive the clamping plate to move synchronously. The automatic fixing of the return pipe, inlet pipe and filter pipe can be completed without manual adjustment, so as to prevent the pipeline from shifting, bending or knotting due to vibration under the drive of the peristaltic pump, and to ensure the smooth flow of liquid circulation path.

[0007] Preferably, an L-shaped plate is fixedly connected to the top of the tabletop, and several storage boxes are fixedly connected to the side of the L-shaped plate. By adopting the above technical solution and setting up L-shaped plates and storage boxes, consumables can be stored in separate areas according to their type (such as different specifications of pipes, connectors, gaskets, sterile centrifuge tubes, labels, etc.), thus avoiding confusion in retrieval caused by scattered storage of consumables.

[0008] Preferably, an ultraviolet germicidal lamp is fixedly installed at the bottom of the L-shaped plate; By adopting the above technical solution and setting up ultraviolet germicidal lamps, the core components such as the table surface, membrane pack, sample bottle, filtrate collection bottle, pipeline and clamping mechanism can be irradiated and disinfected for 20-30 minutes before the extraction operation, achieving a broad-spectrum sterilization effect.

[0009] Preferably, the top of the tabletop is provided with a stainless steel layer, and the surface of the tabletop is provided with several rubber sleeves; By adopting the above technical solution and setting a stainless steel layer, it is possible to withstand repeated wiping with common cleaning agents such as alcohol and deionized water, and it is not easily corroded by biological liquids such as cell culture medium and buffer solution, thus improving cleaning efficiency. By setting rubber sleeves, which are placed at the four sharp corners, the purpose of protecting personnel is achieved.

[0010] Preferably, a support platform is fixedly connected to the bottom of the tabletop, and a plurality of support legs are fixedly connected to the bottom of the support platform; By adopting the above technical solution and setting up a support platform and support legs, the overall device can be provided with support.

[0011] Preferably, an RFID chip is fixedly installed on the surface of the membrane package, and a pressure sensor and a temperature sensor are provided on the side of the membrane package; By adopting the above technical solution and setting up RFID chips, key information of the membrane pack (such as molecular weight cutoff, membrane material, production batch, expiration date, maximum number of uses, sterilization status, etc.) can be quickly read and recorded using a dedicated reading device. Scanning the chip before extraction can automatically verify whether the membrane pack meets the requirements for exosome extraction (such as a 30-150nm cutoff range). After extraction, the data such as the number of uses and operation time in the chip are updated, achieving full life cycle tracking. By setting up pressure sensors, the filtration pressure inside the membrane pack (such as the pressure difference between the inlet and outlet) can be directly monitored. At the same time, the data can be transmitted to the device control system or display terminal in real time. When the pressure exceeds the safety threshold, the device will detect the pressure. When the pressure reaches a value (e.g., >0.3MPa), the system can automatically issue an early warning or pause the peristaltic pump to accurately detect abnormalities such as membrane blockage and tubing bends, thus avoiding membrane pore deformation (decreased exosome retention accuracy) or membrane rupture (sample loss) caused by high pressure. By setting a temperature sensor, the system can monitor the temperature of the liquid flowing through the membrane in real time, ensuring that the temperature is controlled within 2-8℃ (the optimal range for stable exosome activity). If the temperature exceeds the threshold (e.g., >10℃), the system can trigger an alarm, prompting the operator to adjust the temperature using an external temperature control device (e.g., an ice bath) to avoid exosome membrane protein denaturation and content degradation (e.g., mRNA degradation) caused by excessively high temperatures (e.g., room temperature >25℃).

[0012] Preferably, a sealing gasket is provided at the bottom of the pressure indicator; By adopting the above technical solution and setting a sealing gasket, the pressure indicator insertion interface is sealed.

[0013] This application also provides a detection method using the above-described exosome extraction device: S1: Preparation and Pretreatment Experimental material preparation: Take the sample to be tested, centrifuge at 3000×g for 10 minutes at 4℃ to remove cell debris and large particulate impurities, and retain the supernatant as the solution to be processed; Apparatus sterilization: Turn on the ultraviolet germicidal lamp at the bottom of the L-shaped plate to sterilize the table surface, membrane pack, sample bottles, filtrate collection bottles and tubing for 20-30 minutes. Membrane pack inspection: Scan the RFID chip on the surface of the membrane pack with an RFID chip reading device to confirm the molecular weight cutoff, batch number and expiration date of the membrane pack, and ensure that it meets the requirements for exosome extraction; S2: Device assembly and fixing Place the membrane pack, peristaltic pump, sample vial, and filtrate collection vial in their respective designated positions on the top of the table. Piping connection: First, thread one end of the filter tube to the filter end of the membrane pack, and extend the other end into the filtrate collection bottle. Then, connect one end of the inlet tube to the inlet end of the membrane pack, and extend the other end into the sample bottle. Next, connect the reflux tube to the reflux end of the membrane pack through the connection hole of the connector, and extend the other end into the sample bottle. Secure the surface of the reflux tube to the pump head of the peristaltic pump. Then, insert a pressure gauge into the connection tube of the connector to ensure that the sealing gasket fits tightly with the interface to prevent leakage. Finally, start the electric push rod of the clamping mechanism to move the support frame and the clamping plate until the clamping plate is close to the tube surface of the filter tube, inlet tube and reflux tube to complete the fixation and prevent the tube body from knotting or becoming messy. S3: Parameter Setting and Trial Run Pour the pretreated solution from S1 into the sample bottle, ensuring the liquid level does not exceed the inlet height of the reflux tube and the inlet tube. Set the initial flow rate of the peristaltic pump, start the peristaltic pump for 3-5 minutes of trial operation, and observe the liquid circulation status: confirm that the liquid flow in the return pipe and the inlet pipe is smooth, the pressure gauge reading is stable, liquid drips into the filtrate collection bottle from the filter pipe, and there is no leakage at any interface; S4: Cyclic Extraction and Process Monitoring Start the peristaltic pump and maintain the set flow rate for circulation filtration for 30-60 minutes. Real-time monitoring during the process: The filtration pressure and liquid temperature are recorded by the pressure and temperature sensors of the membrane pack, and the pressure changes in the pipeline are observed by the pressure indicator. If the pressure rises suddenly, the equipment is stopped to check whether the membrane pack is blocked or the pipeline is bent. Observe the amount of liquid in the filtrate collection bottle to determine the filtration efficiency of small molecule impurities; S5: End of extraction and sample collection When the liquid volume in the sample vial is concentrated to 1 / 5-1 / 10 of the initial volume, turn off the peristaltic pump and stop the circulation. Disconnect the reflux tube and the inlet tube, transfer the concentrate in the sample vial to a sterile centrifuge tube, and label the sample information; S6: Exosome Detection Concentration detection: Take 100 μL of concentrate and use nanoparticle tracking analysis technology to detect the number of exosomes per milliliter of concentrate; Biomarker detection: Take a portion of the concentrate and detect the content of exosome-specific biomarkers and target growth factors by Western blotting or ELISA to verify the purity and activity of exosomes; S7: Post-treatment and cleaning Disassemble all pipelines and treat the waste liquid in the filtrate collection bottle as biological waste; Rinse the sample vials, membrane packs, and tubing three times with deionized water, and after drying, store them in the L-shaped plate storage box. Simply turn off the UV germicidal lamp and record the device's usage status and the cumulative usage time of the membrane pack.

[0014] 2. Beneficial effects In summary, this application includes at least one of the following beneficial technical effects: 1. This invention provides an exosome extraction device and detection method. By setting up a membrane pack, a filter tube, an inlet tube, and a reflux tube, the filter tube is connected to the filter end of the membrane pack, the inlet tube to the inlet end of the membrane pack, and the reflux tube to the reflux end of the membrane pack via connectors, all using threaded connections. Subsequent connections can be made by rotation to achieve a tight fit. Combined with the flexibility of the tubing itself, this ensures no liquid leakage at the interfaces, avoiding sample loss due to leakage, and improving installation speed, saving time. By setting up a peristaltic pump and a reflux tube, the reflux tube can be snapped into the pump head of the peristaltic pump. After startup, the peristaltic pump generates power by squeezing the reflux tube, driving the liquid to be processed in the sample vial through the inlet tube into the membrane pack. After filtration, part of the liquid is filtered out. The liquid (including exosomes) returns to the sample bottle through the reflux end and reflux tube, forming a closed loop. Small molecule impurities enter the filtrate collection bottle through the filtration end and filtration tube. The circulating flow mode achieves the purpose of making the liquid flow parallel to the membrane surface (tangential flow filtration), reducing the deposition of exosomes on the membrane surface, reducing the membrane fouling rate, and improving filtration efficiency. By setting a pressure indicator, it can be connected to the reflux pipeline through the connector to monitor the pressure changes in the circulation system in real time. If the membrane is blocked or the pipeline is bent, the pressure will rise sharply and trigger an abnormal reading on the indicator, allowing for timely shutdown and troubleshooting. This achieves the purpose of real-time pressure monitoring to avoid membrane damage caused by overpressure. Because of the above device, a lot of costs can be reduced. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the overall front view of the present invention; Figure 2 This is a front view schematic diagram of the extraction mechanism and clamping mechanism of the present invention; Figure 3 for Figure 2 Schematic diagram of the exploded structure of a mid-tangential flow membrane envelope; Figure 4 for Figure 3 Schematic diagram of the exploded side view of the mid-tangential flow membrane envelope; Figure 5 This is a side view of the clamping mechanism of the present invention. Figure 6 for Figure 1 A schematic diagram of the L-shaped plate from below; Figure 7 for Figure 4 Enlarged structural diagram at point A in the middle.

[0016] The components include: 1. Tabletop; 2. Extraction mechanism; 201. Membrane pack; 202. Filter tube; 203. Filtrate collection bottle; 204. Connector; 205. Pressure gauge; 206. Inlet tube; 207. Reflux tube; 208. Sample bottle; 209. Peristaltic pump; 210. Filter end; 211. Inlet end; 212. Reflux end; 213. Connecting tube; 214. Connecting hole; 3. Clamping mechanism; 301. Vertical plate; 302. Electric push rod; 303. Support frame; 304. Clamping plate; 4. L-shaped plate; 5. Storage box; 6. Stainless steel surface; 7. Rubber sleeve; 8. Support platform; 9. Support leg; 10. RFID chip; 11. Pressure sensor; 12. Temperature sensor; 13. Ultraviolet germicidal lamp; 14. Sealing gasket. Detailed Implementation

[0017] The following is in conjunction with the appendix Figure 1 - Appendix Figure 7 This application will be described in further detail below.

[0018] Example 1: An exosome extraction device, referring to... Figure 1 , Figure 2 , Figure 3 and Figure 4The system includes a table 1, an extraction mechanism 2, and a clamping mechanism 3. The extraction mechanism 2 includes a membrane pack 201, a filtrate collection bottle 203, a sample bottle 208, and a peristaltic pump 209, all mounted on the top of the table 1. A filter outlet 210 is fixedly connected to the top of the membrane pack 201, and a filter tube 202 is threadedly connected to the surface of the filter outlet 210. The other end of the filter tube 202 is located inside the filtrate collection bottle 203. A reflux end 212 and an inlet end 211 are fixedly connected to the side of the membrane pack 201. A connector 204 is threadedly connected to the surface of the reflux end 212, and a connecting tube 213 is fixedly connected to the surface of the connector 204. A pressure gauge 20 is inserted into the inside of the connecting tube 213. 5. A connection hole 214 is provided on the side of the connector 204. A reflux tube 207 is inserted into the connection hole 214. An inlet tube 206 is threadedly connected to the surface of the inlet end 211. The other end of the reflux tube 207 and the inlet tube 206 are located inside the sample vial 208. The surface of the reflux tube 207 is snapped into the inside of the peristaltic pump 209. By setting up the membrane pack 201, filter tube 202, inlet tube 206, and reflux tube 207, the filter tube 202 can be threadedly connected to the filter end 210 of the membrane pack 201, the inlet tube 206 to the inlet end 211 of the membrane pack 201, and the reflux tube 207 to the reflux end 212 of the membrane pack 201 via the connector 204. Subsequent... During connection, a tight fit can be achieved through rotation. Combined with the flexibility of the tubing itself, this ensures no liquid leakage at the interface, preventing sample loss due to leakage and improving installation speed, saving time. By setting up a peristaltic pump 209 and a return tube 207, the return tube 207 can be snapped into the pump head of the peristaltic pump 209. After startup, the peristaltic pump 209 generates power by squeezing the return tube 207, driving the liquid to be processed in the sample vial 208 through the inlet tube 206 into the membrane envelope 201. After filtration, part of the liquid (including exosomes) returns to the sample vial 208 through the return end 212 and the return tube 207, forming a closed loop. Small molecule impurities pass through the filter end... 210. The filtrate collection bottle 203 is entered through the filter tube 202. The circulating flow mode makes the liquid flow parallel to the membrane surface (tangential flow filtration), which reduces the deposition of exosomes on the membrane surface, reduces the membrane fouling rate, and improves the filtration efficiency. By setting a pressure indicator 205, it can be connected to the return pipe 207 through the connecting pipe 213 of the connector 204. Subsequently, the pressure changes in the circulation system are monitored in real time. If the membrane pack 201 is blocked or the pipeline is bent, the pressure will rise sharply and trigger the abnormal reading of the indicator. The machine can be stopped in time for troubleshooting. This achieves the purpose of real-time pressure monitoring to avoid membrane pack 201 damage due to overpressure. Because of the above device, a lot of costs can be reduced.

[0019] Please see Figure 1 and Figure 5The clamping mechanism 3 includes two vertical plates 301 fixedly connected to the top of the tabletop 1. An electric push rod 302 is fixedly mounted on the side of each vertical plate 301. One end of the electric push rod 302 is fixedly connected to a support frame 303. Several clamping plates 304 are fixedly connected to the side of the support frame 303. By setting the clamping mechanism 3, the electric push rod 302 can drive the support frame 303 to move horizontally after receiving a command, causing the clamping plates 304 to move synchronously. This allows for the clamping of the return pipe 207, the inlet pipe 206, and the filter pipe 207 without manual adjustment. The automatic fixing of 02 prevents the pipeline from shifting, bending, or knotting due to vibration driven by the peristaltic pump 209, ensuring unobstructed liquid circulation. An L-shaped plate 4 is fixedly connected to the top of the table 1, and several storage boxes 5 are fixedly connected to the side of the L-shaped plate 4. By setting up the L-shaped plate 4 and the storage boxes 5, the consumables can be stored in separate areas according to their type (such as different specifications of pipelines, connectors 204, sealing gaskets 14, sterile centrifuge tubes, labels, etc.), thus avoiding confusion caused by scattered storage of consumables.

[0020] Please see Figure 1 and Figure 6 An ultraviolet germicidal lamp 13 is fixedly installed at the bottom of the L-shaped plate 4. By setting the ultraviolet germicidal lamp 13, the core components such as the surface of the table plate 1, membrane pack 201, sample bottle 208, filtrate collection bottle 203, pipeline and clamping mechanism 3 can be irradiated and disinfected for 20-30 minutes before the extraction operation, achieving a broad-spectrum sterilization effect. The top of the table plate 1 is provided with a stainless steel layer 6, and the surface of the table plate 1 is provided with several rubber sleeves 7. By setting the stainless steel layer 6, it can withstand repeated wiping with common cleaning agents such as alcohol and deionized water, and is not easily corroded by biological liquids such as cell culture medium and buffer solution, thus improving cleaning efficiency. By setting the rubber sleeves 7, because they are placed at the four sharp corners, it achieves the purpose of protecting personnel.

[0021] Please see Figure 1 , Figure 3 and Figure 7The bottom of the tabletop 1 is fixedly connected to a support platform 8, and the bottom of the support platform 8 is fixedly connected to several support legs 9. By setting the support platform 8 and the support legs 9, the overall device is provided with support force. An RFID chip 10 is fixedly installed on the surface of the membrane pack 201. A pressure sensor 11 and a temperature sensor 12 are set on the side of the membrane pack 201. By setting the RFID chip 10, key information of the membrane pack 201 (such as molecular weight cutoff, membrane material, production batch, expiration date, maximum number of uses, sterilization status, etc.) can be quickly read and recorded by a dedicated reading device. Scanning the chip before extraction can automatically verify whether the membrane pack 201 meets the requirements for exosome extraction (such as the 30-150nm cutoff range). After extraction, the data such as the number of uses and operation time in the chip are updated to achieve the effect of full life cycle tracking. By setting the pressure sensor 11, the filtration pressure inside the membrane pack 201 (such as the pressure difference between the inlet end 211 and the outlet end) can be directly monitored. Data can be transmitted to the device control system or display terminal in real time. When the pressure exceeds the safety threshold (e.g., >0.3MPa), the system can automatically issue an early warning or stop the peristaltic pump 209 to accurately detect abnormalities such as blockage of the membrane capsule 201 or bending of the pipeline, thus avoiding membrane pore deformation (decreased exosome retention accuracy) or membrane capsule 201 rupture (sample loss) caused by high pressure. By setting a temperature sensor 12, the temperature of the liquid flowing through the membrane capsule 201 can be monitored in real time to ensure that the temperature is controlled at 2-8℃ (the optimal range for stable exosome activity). If the temperature exceeds the threshold (e.g., >10℃), the system can trigger an alarm to prompt the operator to adjust the temperature using an external temperature control device (e.g., an ice bath) to avoid exosome membrane protein denaturation and content degradation (e.g., mRNA degradation) caused by excessively high temperature (e.g., room temperature >25℃). A sealing gasket 14 is set at the bottom of the pressure indicator 205 to seal the interface of the pressure indicator 205.

[0022] This application also provides a detection method using the above-described exosome extraction device: S1: Preparation and Pretreatment Experimental material preparation: Take the sample to be tested (such as stem cell culture medium), centrifuge at 3000×g for 10 minutes at 4℃ to remove cell debris and large particulate impurities, and retain the supernatant as the solution to be processed; Device disinfection: Turn on the ultraviolet germicidal lamp 13 at the bottom of the L-shaped plate 4 to sterilize the surface of the table 1, membrane pack 201, sample bottle 208, filtrate collection bottle 203 and pipeline for 20-30 minutes. Inspection of membrane package 201: Scan the RFID chip 10 on the surface of membrane package 201 using the RFID chip 10 reading device to confirm the molecular weight cutoff (e.g., 30-150nm), batch number, and expiration date of membrane package 201 to ensure compliance with exosome extraction requirements; S2: Device assembly and fixing Place the membrane pack 201, peristaltic pump 209, sample bottle 208, and filtrate collection bottle 203 in the designated positions on the top of the table 1; Piping Connections: First, thread one end of the filter tube 202 to the filter outlet 210 of the membrane pack 201, and extend the other end into the filtrate collection bottle 203. Then, connect one end of the inlet tube 206 to the inlet 211 of the membrane pack 201, and extend the other end into the sample bottle 208. Next, connect the reflux tube 207 to the reflux outlet 212 of the membrane pack 201 through the connection hole 214 of the connector 204, and extend the other end into the sample bottle 208. The surface is snapped into the pump head of the peristaltic pump 209. Then, a pressure indicator 205 is inserted into the connecting pipe 213 of the connector 204 to ensure that the sealing gasket 14 fits tightly with the interface to prevent leakage. Finally, the electric push rod 302 of the clamping mechanism 3 is activated, so that the support frame 303 drives the clamping plate 304 to move until the clamping plate 304 is tightly attached to the pipe surface of the filter pipe 202, the inlet pipe 206 and the return pipe 207 to complete the fixation and prevent the pipe body from being knotted or messy. S3: Parameter Setting and Trial Run Pour the pretreated liquid in S1 into sample bottle 208, ensuring the liquid level does not exceed the inlet height of reflux tube 207 and inlet tube 206. Set the initial flow rate of the peristaltic pump 209 (e.g., 10-20 mL / min), start the peristaltic pump 209 for 3-5 minutes of trial operation, and observe the liquid circulation status: confirm that the liquid flow in the return pipe 207 and the inlet pipe 206 is smooth, the pressure indicator 205 reading is stable (the initial pressure is generally ≤0.1 MPa), liquid drips from the filter pipe 202 into the filtrate collection bottle 203, and there is no leakage at any interface; S4: Cyclic Extraction and Process Monitoring Start the peristaltic pump 209 and maintain the set flow rate for circulation filtration for 30-60 minutes. Real-time monitoring during the process: The filtration pressure (avoiding exceeding 0.3MPa to prevent membrane 201 from breaking) and liquid temperature (controlled at 2-8℃ to protect exosome activity) are recorded by the pressure sensor 11 and temperature sensor 12 of the membrane pack 201. The pressure change in the pipeline is observed by the pressure indicator 205. If the pressure rises suddenly, the equipment is stopped to check whether the membrane pack 201 is blocked or the pipeline is bent. Observe the liquid volume in the filtrate collection bottle 203 to determine the filtration efficiency of small molecule impurities (such as free proteins and salts); S5: End of extraction and sample collection When the liquid volume in sample vial 208 is concentrated to 1 / 5 to 1 / 10 of the initial volume, turn off peristaltic pump 209 to stop circulation; Disassemble the reflux tube 207 and the inlet tube 206, transfer the concentrate (containing exosomes) in the sample bottle 208 to a sterile centrifuge tube, and label the sample information (such as extraction time, membrane pack 201 batch). S6: Exosome Detection Concentration detection: Take 100 μL of concentrate and use nanoparticle tracking analysis (NTA) technology to detect the number of exosomes per milliliter of concentrate; Biomarker detection: Take a portion of the concentrate and detect the content of exosome-specific biomarkers (such as CD63 and CD9) and target growth factors (such as VEGF and EGF) by Western blotting or ELISA to verify the purity and activity of exosomes. S7: Post-treatment and cleaning Disassemble all pipelines and treat the waste liquid in the filtrate collection bottle 203 as biological waste; Rinse sample vials 208, membrane packs 201 and tubing three times with deionized water, and after drying, store them in storage box 5 of L-shaped plate 4; Turn off the ultraviolet germicidal lamp 13, record the device's usage status and the cumulative usage time of the membrane pack 201 (data can be updated via the RFID chip 10).

[0023] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Identical components are represented by the same reference numerals. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. An exosome extraction device, comprising a table (1), an extraction mechanism (2), and a clamping mechanism (3), characterized in that: The extraction mechanism (2) includes a membrane pack (201), a filtrate collection bottle (203), a sample bottle (208), and a peristaltic pump (209) mounted on the top of the table (1). A filter outlet (210) is fixedly connected to the top of the membrane pack (201), and a filter tube (202) is threaded onto the surface of the filter outlet (210). The other end of the filter tube (202) is located inside the filtrate collection bottle (203). A reflux end (212) and an inlet end (211) are fixedly connected to the side of the membrane pack (201), and a connector (…) is threaded onto the surface of the reflux end (212). 204), the surface of the connector (204) is fixedly connected to the connecting tube (213), the inside of the connecting tube (213) is inserted with a pressure indicator (205), the side of the connector (204) is provided with a connecting hole (214), the inside of the connecting hole (214) is inserted with a return tube (207), the surface of the inlet end (211) is threadedly connected with a liquid inlet tube (206), the other end of the return tube (207) and the liquid inlet tube (206) are located inside the sample bottle (208), and the surface of the return tube (207) is snapped into the inside of the peristaltic pump (209).

2. The exosome extraction device according to claim 1, characterized in that: The clamping mechanism (3) includes two vertical plates (301) fixedly connected to the top of the table (1). An electric push rod (302) is fixedly installed on the side of the vertical plate (301). A support frame (303) is fixedly connected to one end of the electric push rod (302). Several clamping plates (304) are fixedly connected to the side of the support frame (303).

3. The exosome extraction device according to claim 1, characterized in that: The top of the tabletop (1) is fixedly connected to an L-shaped plate (4), and the sides of the L-shaped plate (4) are fixedly connected to several storage boxes (5).

4. The exosome extraction device according to claim 3, characterized in that: An ultraviolet germicidal lamp (13) is fixedly installed at the bottom of the L-shaped plate (4).

5. The exosome extraction device according to claim 1, characterized in that: The top of the tabletop (1) is provided with a stainless steel layer (6), and the surface of the tabletop (1) is provided with several rubber sleeves (7).

6. The exosome extraction device according to claim 1, characterized in that: The bottom of the tabletop (1) is fixedly connected to a support platform (8), and the bottom of the support platform (8) is fixedly connected to several support legs (9).

7. The exosome extraction device according to claim 1, characterized in that: An RFID chip (10) is fixedly installed on the surface of the membrane package (201), and a pressure sensor (11) and a temperature sensor (12) are provided on the side of the membrane package (201).

8. An exosome extraction device according to claim 1, characterized in that: The pressure indicator (205) is provided with a sealing gasket (14) at its bottom.

9. A detection method using an exosome extraction device as described in claim 1, characterized in that... Includes the following steps: S1: Preparation and Pretreatment Experimental material preparation: Take the sample to be tested, centrifuge at 3000×g for 10 minutes at 4℃ to remove cell debris and large particulate impurities, and retain the supernatant as the solution to be processed; Disinfection of the apparatus: Turn on the ultraviolet germicidal lamp (13) at the bottom of the L-shaped plate (4) to sterilize the surface of the table (1), membrane pack (201), sample bottle (208), filtrate collection bottle (203) and pipeline for 20-30 minutes; Membrane pack (201) inspection: Scan the RFID chip (10) on the surface of the membrane pack (201) using the RFID chip (10) reading device to confirm the molecular weight cutoff, batch and expiration date of the membrane pack (201) to ensure that it meets the requirements for exosome extraction; S2: Device assembly and fixing Place the membrane pack (201), peristaltic pump (209), sample bottle (208), and filtrate collection bottle (203) at the designated positions on the top of the table (1); Piping Connection: First, thread one end of the filter tube (202) to the filter end (210) of the membrane pack (201), and extend the other end into the filtrate collection bottle (203). Then, connect one end of the inlet tube (206) to the inlet end (211) of the membrane pack (201), and extend the other end into the sample bottle (208). Subsequently, connect the reflux tube (207) to the reflux end (212) of the membrane pack (201) through the connection hole (214) of the connector (204), and extend the other end into the sample bottle (208). Finally, connect the reflux tube (207) to the reflux end (212) of the membrane pack (201) through the connection hole (214) of the connector (204), and extend the other end into the sample bottle (208). 7) The surface is snapped into the pump head of the peristaltic pump (209), and then a pressure indicator (205) is inserted into the connecting pipe (213) of the connector (204) to ensure that the sealing gasket (14) fits tightly with the interface to prevent leakage. Finally, the electric push rod (302) of the clamping mechanism (3) is activated to make the support frame (303) drive the clamping plate (304) to move until the clamping plate (304) is in close contact with the pipe surface of the filter pipe (202), the inlet pipe (206) and the return pipe (207) to complete the fixation and prevent the pipe body from being knotted or messy. S3: Parameter Setting and Trial Run Pour the pretreated liquid in S1 into the sample bottle (208), and the liquid level should not exceed the inlet height of the reflux tube (207) and the inlet tube (206); Set the initial flow rate of the peristaltic pump (209), start the peristaltic pump (209) for 3-5 minutes of trial operation, and observe the liquid circulation status: confirm that the liquid flow in the return pipe (207) and the inlet pipe (206) is smooth, the pressure indicator (205) reading is stable, liquid drips into the filtrate collection bottle (203) from the filter pipe (202), and there is no leakage at any interface; S4: Cyclic Extraction and Process Monitoring Start the peristaltic pump (209) and maintain the set flow rate for circulation filtration for 30-60 minutes. Real-time monitoring during the process: The filtration pressure and liquid temperature are recorded by the pressure sensor (11) and temperature sensor (12) of the membrane pack (201). The pressure change in the pipeline is observed by the pressure indicator (205). If the pressure rises suddenly, the equipment is paused to check whether the membrane pack (201) is blocked or the pipeline is bent. Observe the amount of liquid in the filtrate collection bottle (203) to determine the filtration efficiency of small molecule impurities; S5: End of extraction and sample collection When the liquid volume in the sample vial (208) is concentrated to 1 / 5-1 / 10 of the initial volume, the peristaltic pump (209) is turned off to stop the circulation; Disconnect the reflux tube (207) and the inlet tube (206), transfer the concentrate in the sample bottle (208) to a sterile centrifuge tube, and label the sample information; S6: Exosome Detection Concentration detection: Take 100 μL of concentrate and use nanoparticle tracking analysis technology to detect the number of exosomes per milliliter of concentrate; Biomarker detection: Take a portion of the concentrate and detect the content of exosome-specific biomarkers and target growth factors by Western blotting or ELISA to verify the purity and activity of exosomes; S7: Post-treatment and cleaning Disassemble all pipelines and treat the waste liquid in the filtrate collection bottle (203) as biological waste; Rinse the sample bottle (208), membrane pack (201) and tubing three times with deionized water, and after drying, store them in the storage box (5) of the L-shaped plate (4); Turn off the ultraviolet germicidal lamp (13), and record the device usage status and the cumulative usage time of the membrane pack (201).

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