An exosome concentration detection kit

Abstract

The application relates to the field of biotechnology, and discloses an exosome concentration detection kit, a detachable double-cassette main body, which comprises a cassette-type main body I and a cassette-type main body II; the cassette-type main body I is provided with valve gas transmission pipes on both sides, an internal waste liquid collecting bin and a collecting interface bin are arranged in the inner cavity, an optical detection window is detachably assembled on the top through fixing bolts, a chip card slot is arranged on the opposite surface of the main body II, and a fluorine rubber sealing ring is attached to the inner wall of the card slot; and a reagent bin fixing groove and a sample adding port are arranged on the top of the cassette-type main body II. The mechanical cooperation of the detachable double-cassette main body and the automatic fluid control equipment realizes the pre-packaging and fixing of all complex reagent pipelines in the cassette, the user does not need to manually pipette or connect any pipeline, only three actions of "adding a sample, putting the detachable double-cassette main body, and one-key starting" are needed, and the fluid control equipment can automatically complete puncture, sealing, fluid time sequence control and valve switching of up to 7-8 steps.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an exosome concentration detection kit. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells, and the molecular information they carry (proteins, nucleic acids, etc.) shows great potential in disease diagnosis. However, realizing their clinical application faces multiple challenges.

[0003] Although enzyme-linked immunosorbent assay (ELISA) methods are relatively simple to operate, the two-dimensional planar structure of traditional ELISA plates results in a limited surface area for antibody coating, which limits the capture capacity and detection dynamic range. The reaction depends on the passive diffusion of molecules and has a long incubation time. Traditional methods can usually only provide information on the "total amount" of exosomes and cannot further obtain information on the key molecules carried inside the exosomes on the same sample.

[0004] While emerging microfluidic chip technology has solved the above problems, it has also brought new application bottlenecks:

[0005] Microfluidic detection processes involve the sequential addition, incubation, and washing of up to 7-8 different reagents. Users need to manually connect the chip's multiple inlets to external reagent bottles, syringe pumps, and valve systems through complex tubing. This process is highly specialized, time-consuming, and prone to failure due to incorrect tubing connections or the introduction of air bubbles. Summary of the Invention

[0006] This invention provides an exosome concentration detection kit, which solves the problems mentioned in the background art.

[0007] This invention provides the following technical solution: an exosome concentration detection kit, comprising:

[0008] The detachable dual cartridge body includes a cartridge-type main body one and a cartridge-type main body two. The cartridge-type main body one is provided with valve gas transmission pipes on both sides, and has an internal waste liquid collection chamber and a collection interface chamber in its inner cavity. An optical detection window is detachably assembled on the top through fixing bolts. A chip slot is provided on the opposite side of the main body two, and a fluororubber sealing ring is pasted on the inner wall of the slot. The cartridge-type main body two has a reagent chamber fixing slot and a sample addition port on its top, and a reagent transmission pipe and a sample transmission pipe are embedded in its inner cavity. One end of the reagent transmission pipe is connected to the bottom puncture device.

[0009] The microfluidic chip, sealed and fitted into a chip slot, includes a chip body. The chip body has a capture and detection reaction chamber with a three-dimensional porous matrix fixed inside. A sample inlet and a reagent inlet are provided on one side of the chip, and a function switching module is provided on the other side. The function switching module is connected to the waste liquid outlet and the downstream analyte outlet through a diversion channel with a pressure valve.

[0010] A pre-packaged reagent assembly includes a reagent bag, which is fixed in a reagent compartment fixing groove and has a rubber sealing compartment at one end, which is coaxially aligned with the bottom puncture device.

[0011] The sample transfer tube is sealed to the sample inlet, the reagent transfer tube is sealed to the reagent inlet, the waste liquid outlet is connected to the internal waste liquid collection chamber, the downstream analyte outlet is connected to the collection interface chamber, and the valve gas transfer tube is connected to the gas pressure valve.

[0012] As a preferred technical solution of the present invention, the three-dimensional porous matrix is ​​an electrospun polystyrene membrane, which is fixed by the following process: soaking in 1% APTES ethanol solution and incubating at 37°C for 2 hours, rinsing with distilled water and then soaking in 2.5% glutaraldehyde aqueous solution and incubating at 25°C for 1 hour, and then fixing it to the inner wall of the capture and detection reaction chamber.

[0013] As a preferred embodiment of the present invention, the reagent bag is a multilayer co-extruded film structure, pre-encapsulated with biotinylated CD63 antibody solution, streptavidin-HRP conjugate, and chemiluminescent substrate solution, and the rubber sealing chamber is made of nitrile rubber with a thickness of 3mm.

[0014] As a preferred embodiment of the present invention, the optical detection window is made of high-transmittance quartz glass, Dow Corning 734 silicone rubber sealant is applied to the fixing bolt connection, and the fluororubber sealing ring on the inner wall of the chip slot has a Shore hardness of 60±5.

[0015] An automated fluid control device for use with an exosome concentration detection kit, excluding an optical detection unit, includes:

[0016] a) Equipment compartment, with an air pump, lithium battery and control motherboard fixed inside, and a control screen fixed on the outer wall;

[0017] b) The top of the upright frame is fixed with drive motor one and drive motor two, and the output shafts of the motors are all connected to ball screws;

[0018] c) Telescopic pressure plate, connected to the lead screw of drive motor one, with a top pressure column and a top puncture device fixed at the bottom;

[0019] d) A gas transmission system, including a gas transmission main pipe, a first diversion valve, a diversion pipe, a second diversion valve, and a sealing seat, wherein the gas transmission main pipe is connected to a gas pump;

[0020] e) The control motherboard stores automation programs for controlling the drive motor, air pump, and diversion valve.

[0021] A method for detecting exosomes includes the following steps:

[0022] a) The sample to be tested is filtered through a 0.22μm filter membrane and then added to the sample inlet port of the cartridge-type main body two;

[0023] b) The cartridge-type main body one and the cartridge-type main body two are docked, a microfluidic chip is assembled, and an automated fluid control device is inserted;

[0024] c) Select puncture control and complete the puncture;

[0025] d) The device drives the sample and reagents into the microfluidic chip according to a preset time sequence to complete the exosome capture, incubation, washing, and chemiluminescence reaction;

[0026] e) Remove the detachable dual cartridge body, align the optical detection window with the external optical detection device, read the signal and calculate the exosome concentration;

[0027] f) Optional: Start the pyrolysis process, switch the gas pressure valve to the downstream analyte side, and collect the pyrolysis products.

[0028] The present invention has the following beneficial effects:

[0029] 1. This exosome concentration assay kit, through the mechanical cooperation of a detachable dual-cassette body and an automated fluid control device, pre-packages and fixes the complex reagent tubing inside the detachable dual-cassette body. Users do not need to manually pipette or connect any tubing throughout the process. They only need to perform three actions: "add sample, place in the detachable dual-cassette body, and start with one button." The fluid control device can automatically complete puncture, sealing, and up to 7-8 steps of fluid timing control and valve switching. This solves the bottleneck problems of the background technology, which is cumbersome, highly professional, time-consuming, and prone to detection failure due to incorrect insertion or air bubbles. It makes complex tests applicable to clinical departments and primary healthcare institutions.

[0030] 2. This exosome concentration detection kit uses a fluid control device that is solely responsible for fluid control, separating the expensive optical detection unit and reducing equipment costs. The optical detection window is designed so that the detachable dual cartridge body after the reaction is complete can be removed and directly placed into any existing chemiluminescence analyzer (ELISA reader) in the user's laboratory for reading. Through the decoupling strategy of "dedicated fluid control device + universal detection instrument", the customer avoids the need to repeatedly purchase the optical system, thus reducing the customer's procurement costs.

[0031] 3. This exosome concentration detection kit integrates a three-dimensional porous matrix in the capture and detection reaction chamber inside the microfluidic chip. Its specific surface area is hundreds of times that of traditional ELISA plates, which allows the capture antibodies to be covalently bonded at high density. This greatly improves the capture capacity and detection dynamic range of exosomes and solves the problem of low capture capacity in two-dimensional planes.

[0032] 4. This exosome concentration detection kit, through the setting of a function switching module, allows the fluid control device to control the valve to switch the flow path after completing chemiluminescence quantitative detection, pumping in lysis buffer to lyse the captured exosomes in situ, and collecting the nucleic acids or proteins inside from the downstream analyte outlet. This solves the problem of single information dimension in traditional methods and realizes one-stop "quantitative + qualitative" multidimensional analysis on the same sample. Attached Figure Description

[0033] Figure 1 This is a three-dimensional structural diagram of the present invention;

[0034] Figure 2 This is a schematic diagram of the chip slot structure of the present invention;

[0035] Figure 3 This is a schematic diagram of the fluid control device of the present invention;

[0036] Figure 4 This is a schematic diagram of the fluid control device of the present invention from another perspective;

[0037] Figure 5 This is a schematic diagram of the microfluidic chip mounting location structure of the present invention;

[0038] Figure 6 This is a schematic diagram of the bottom puncture device structure of the present invention;

[0039] Figure 7 This is a schematic diagram of the microfluidic chip structure of the present invention.

[0040] In the diagram: 1. Cartridge-type main body one; 2. Cartridge-type main body two; 3. Optical detection window; 4. Fixing bolt; 5. Reagent compartment fixing slot; 6. Reagent bag; 7. Sample addition port; 8. Chip slot; 9. Microfluidic chip; 10. Internal waste liquid collection compartment; 11. Collection interface compartment; 12. Bottom puncture device; 13. Reagent transfer tube; 14. Sample transfer tube; 15. Fluid control device; 16. Valve gas transfer tube;

[0041] 51. Rubber-sealed compartment;

[0042] 901. Chip body; 902. Capture and detection reaction chamber; 903. Three-dimensional porous matrix; 904. Sample inlet; 905. Reagent inlet; 906. Fluid channel; 907. Function switching module; 908. Waste liquid outlet; 909. Downstream analyte outlet; 910. Pressure valve;

[0043] 1501. Equipment compartment; 1502. Air pump; 1503. Lithium battery; 1504. Control motherboard; 1505. Control screen; 1506. Stand; 1507. Drive motor one; 1508. Telescopic pressure plate; 1509. Top pressure column; 1510. Top puncture device; 1511. Gas transmission main pipe; 1512. Diverter valve one; 1513. Diverter pipe; 1514. Diverter valve two; 1515. Drive motor two; 1516. Sealing pressure plate; 1517. Sealing seat. Detailed Implementation

[0044] 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.

[0045] Please see Figure 1 - Figure 7 An exosome concentration detection kit includes a cartridge-type main body 1 and a cartridge-type main body 2. The cartridge-type main body 1 and the optical detection window 3 are fixedly assembled by fixing bolts 4. The optical detection window 3 is opened on the top of the side of the cartridge-type main body 1 close to the cartridge-type main body 2. Several reagent chamber fixing slots 5 are opened on the top of the side of the cartridge-type main body 2 away from the cartridge-type main body 1. A reagent bag 6 is provided on the inner wall of the reagent chamber fixing slot 5. A sample addition port 7 is opened on the top of the cartridge-type main body 2. Chip slots 8 are opened on the opposite sides of the cartridge-type main body 1 and the inner wall of the chip slot 8 is sealed with a microfluidic chip 9. An internal waste liquid collection chamber 10 and a collection interface chamber 11 are opened in the inner cavity of the cartridge-type main body 1. A reagent transfer tube 13 and a sample transfer tube 14 are embedded in the inner cavity of the cartridge-type main body 2.

[0046] Both sides of the cartridge-type main body 1 are connected to valve gas transmission pipes 16.

[0047] The outer wall of the cartridge-type main body 2 is also fitted with a fluid control device 15.

[0048] The reagent compartment fixing groove 5 is set at an angle. When the reagent bag 6 is placed on the reagent compartment fixing groove 5, the liquid inside the reagent bag 6 can flow to the chip card slot 8 side by gravity.

[0049] In a preferred embodiment: the microfluidic chip 9 includes a chip body 901, an inner cavity of which a capture and detection reaction chamber 902 is provided, and an inner cavity of the capture and detection reaction chamber 902 is provided with a three-dimensional porous matrix 903. A sample inlet 904 and a reagent inlet 905 are respectively provided on one side of the chip body 901. A fluid channel 906 is provided on the side of the sample inlet 904 and the reagent inlet 905 near the three-dimensional porous matrix 903, and the sample inlet 904 and the reagent inlet 905 are connected to the capture and detection reaction chamber 902 through the fluid channel 906. A function switching module 907 is provided on the other side of the chip body 901.

[0050] The function switching module 907 has two flow channels on the side away from the three-dimensional porous matrix 903;

[0051] Both of the two flow channels are equipped with air pressure valves 910 at the top. The two flow channels on the side of the function switching module 907 away from the three-dimensional porous matrix 903 are respectively connected to waste liquid outlet 908 and downstream analyte outlet 909.

[0052] In a preferred embodiment: a rubber sealing chamber 51 is provided at one end of the reagent bag 6 near the cartridge body 1, a number of bottom puncture devices 12 are positioned corresponding to the rubber sealing chamber 51, a number of bottom puncture devices 12 are connected to one end of the reagent transfer tube 13, and the sample transfer tube 14 is connected to the sample addition port 7.

[0053] The outlets of reagent transfer tube 13 and sample transfer tube 14 on the side away from reagent chamber fixing slot 5 are located inside chip slot 8, and reagent transfer tube 13 and sample transfer tube 14 are sealed and abutted with sample inlet 904 and reagent inlet 905 by sealing rings. The three-dimensional porous matrix 903 corresponds to the position of optical detection window 3. Waste liquid outlet 908 and downstream analyte outlet 909 are sealed and abutted with internal waste liquid collection chamber 10 and collection interface chamber 11 by sealing rings, respectively.

[0054] In a preferred embodiment: the fluid control device 15 includes a device compartment 1501. An air pump 1502, a lithium battery 1503, and a control motherboard 1504 are fixedly mounted in the bottom cavity of the device compartment 1501. A control screen 1505 is fixedly mounted on the outer wall of the device compartment 1501. A stand 1506 is fixedly mounted on the top of the device compartment 1501. A drive motor 1507 and a drive motor 1515 are fixedly mounted on the top of the stand 1506. The output shafts of the drive motor 1507 and the drive motor 1515 are both fixedly mounted with lead screws. A telescopic pressure plate 1508 is provided on the inner wall of the stand 1506. The lead screw connected to the drive motor 1507 is threadedly connected to the inner wall of the telescopic pressure plate 1508. A sealing pressure plate 1516 is also provided on the top of the cartridge-type main body 2. The lead screw connected to the drive motor 1515 is threadedly connected to the inner wall of the sealing pressure plate 1516.

[0055] The top of the telescopic pressure plate 1508 is fixedly equipped with several top pressure columns 1509 corresponding to the rubber sealing chamber 51, and the bottom of each top pressure column 1509 is fixedly equipped with a top piercing device 1510.

[0056] A gas transmission main pipe 1511 is fixedly mounted on the top of the telescopic pressure plate 1508. Several diversion valves 1512 are fixedly mounted on the outer wall of the gas transmission main pipe 1511. The output ports of the diversion valves 1512 are respectively connected to several top puncture devices 1510. A diversion pipe 1513 is also connected to the outer wall of the gas transmission main pipe 1511. A diversion valve 1514 is connected to the end of the diversion pipe 1513. The output end of the diversion valve 1514 is connected to the sealing seat 1517. The input ends of the gas transmission main pipe 1511 and the valve gas transmission pipe 16 are both connected to the output end of the air pump 1502. The output ends of the two sets of valve gas transmission pipes 16 are both connected to two sets of air pressure valves 910 through valves.

[0057] The three-dimensional porous matrix 903 is an electrospun polystyrene membrane, which is fixed by the following process: soaking in 1% APTES ethanol solution and incubating at 37°C for 2 hours, rinsing with distilled water and then soaking in 2.5% glutaraldehyde aqueous solution and incubating at 25°C for 1 hour, and then fixing it to the inner wall of the capture and detection reaction chamber 902.

[0058] The reagent bag 6 has a multi-layer co-extruded film structure and is pre-encapsulated with biotinylated CD63 antibody solution, streptavidin-HRP conjugate, and chemiluminescent substrate solution. The rubber sealing chamber 51 is made of nitrile rubber with a thickness of 3mm.

[0059] The optical inspection window 3 is made of high-transmittance quartz glass. The connection of the fixing bolt 4 is coated with Dow Corning 734 silicone rubber sealant. The fluororubber sealing ring on the inner wall of the chip slot 8 has a Shore hardness of 60±5.

[0060] The control motherboard 1504 stores automation programs for controlling the drive motor, air pump 1502, and diversion valve; the fluid control device 15 has two puncture control schemes:

[0061] Option 1: The top pressure column 1509 integrates a pressure sensor, and the main control board 1504 controls the start and stop of the drive motor 1507 based on the puncture force value fed back by the pressure sensor.

[0062] Option 2: Without a pressure sensor, the control motherboard 1504 controls the puncture stroke by preset the running time of drive motor 1507. Drive motor 1507 is a stepper motor with a step angle of 1.8° and a ball screw with a lead of 2mm. The preset running time is 0.5 seconds, corresponding to a puncture stroke of 5mm.

[0063] In Scheme 1, the pressure sensor has a detection accuracy of ±0.01N, and the control motherboard 1504 has a preset upper limit of 5N and a lower limit of 1N for the puncture force.

[0064] In both Scheme 2 and Scheme 1, the output air pressure accuracy of air pump 1502 is 0.15±0.01MPa.

[0065] An exosome detection method, using a kit and a fluid control device, includes the following steps:

[0066] After the sample to be tested is filtered through a 0.22μm filter membrane, it is added to the sample addition port 7 of the cartridge-type main body 2.

[0067] b. The cartridge-type main body 1 is docked with the cartridge-type main body 2, and a microfluidic chip 9 is assembled and a fluid control device 15 is inserted.

[0068] c. Select a puncture control protocol:

[0069] Option 1: Start the high-precision program, drive motor 1507 to drive the top puncture device 1510 down, the pressure sensor feeds back the force value, the motor stops when the force value is between 1 and 5N, and the puncture is completed;

[0070] Option 2: Start the simplified procedure, drive motor 1507 for 0.5 seconds and then stop, the top puncture device 1510 descends 5mm to complete the puncture;

[0071] The device drives the sample and reagents into the microfluidic chip 9 according to a preset time sequence to complete the exosome capture, incubation, washing, and chemiluminescence reaction;

[0072] e. Remove the detachable dual cartridge body, align the optical detection window 3 with the external optical detection device, read the signal and calculate the exosome concentration;

[0073] f Optional: Start the pyrolysis program, switch the pressure valve 910 to the downstream analyte side, and collect the pyrolysis products.

[0074] Example 1:

[0075] The detachable dual cartridge body is made using a precision injection molding process;

[0076] Microfluidic chip 9 materials: PDMS layer thickness 2mm, glass substrate thickness 1mm

[0077] Fluid channel 906: 1 mm wide, 500 μm deep

[0078] Capture and Detection Reaction Chamber 902: 5 mm in diameter, 1 mm in depth, 100 μL in volume

[0079] Three-dimensional porous matrix 903: electrospun polystyrene membrane, pore size 0.2 μm, thickness 50 μm

[0080] Preparation process:

[0081] PDMS layer preparation: PDMS prepolymer and curing agent were mixed in a 10:1 ratio and vacuum degassed for 30 minutes.

[0082] The PDMS microfluidic layer is obtained by casting the PDMS microfluidic layer into a silicon mold prepared by photolithography, baking at 75°C for 2 hours to solidify, and then demolding.

[0083] Three-dimensional porous matrix 903 fixation: The electrospun membrane was immersed in 1% APTES ethanol solution and incubated at 37°C for 2 hours. It was then rinsed three times with distilled water, immersed in 2.5% glutaraldehyde aqueous solution, and incubated at 25°C for 1 hour. Finally, it was fixed on the inner wall of the reaction chamber.

[0084] Bonding process: The PDMS layer is bonded to the glass substrate via oxygen plasma treatment at a power of 100W for 30 seconds.

[0085] Immediately bonded after seconds, and baked at 80℃ for 1 hour to complete bonding.

[0086] Control program of fluid control device 15:

[0087] The automated program includes modules such as puncture control, fluid transfer timing control, and air pressure regulation.

[0088] Puncture control:

[0089] Option 1: The top pressure column 1509 integrates a pressure sensor, and the main control board 1504 controls the start and stop of the drive motor 1507 based on the puncture force value fed back by the pressure sensor.

[0090] Option 2: Without a pressure sensor, the control motherboard 1504 controls the puncture stroke by preset the running time of drive motor 1507. Drive motor 1507 is a stepper motor with a step angle of 1.8° and a ball screw with a lead of 2mm. The preset running time is 0.5 seconds, corresponding to a puncture stroke of 5mm.

[0091] In Scheme 1, the pressure sensor has a detection accuracy of ±0.01N, and the control motherboard 1504 has a preset upper limit of 5N and a lower limit of 1N for the puncture force.

[0092] In both Scheme 2 and Scheme 1, the output air pressure accuracy of air pump 1502 is 0.15±0.01MPa.

[0093] Fluid transport sequence: Sample driving gas pressure 0.15MPa for 10 seconds → Antibody reagent driving gas pressure 0.2MPa for 20 seconds → Washing solution driving gas pressure 0.18MPa (3 times, 5 seconds each time) → Substrate solution driving gas pressure 0.2MPa for 15 seconds.

[0094] Example 2: Exosome Detection Experiment

[0095] Experimental materials

[0096] (1) Sample: Serum sample from healthy individuals, stored at -80℃.

[0097] (2) Standards: Exosome standards of known concentrations 10^6-10^10 particles / mL.

[0098] (3) Reagents: biotinylated CD63 antibody, streptavidin-HRP conjugate, chemiluminescent substrate solution.

[0099] (4) Instruments: The reagent kit and fluid control device of this invention, ThermoMultiskan FC Chemistry

[0100] ELISA reader.

[0101] Experimental methods

[0102] (1) Sample pretreatment: Take 100 μL of serum sample and filter it with a 0.22 μm filter membrane to remove cell debris.

[0103] (2) Sample loading: Add the filtered sample to sample loading port 7.

[0104] (3) Equipment docking: Insert the detachable double cartridge body into the fluid control device 15 and start the detection program.

[0105] (4) Automatic reaction: The equipment automatically performs steps such as puncture, sample and reagent driving, and reaction.

[0106] (5) Signal detection: After the reaction is completed, the detachable dual cartridge body is transferred to a chemiluminescent microplate reader to read the RLU value.

[0107] (6) Calculation of results: Calculate the concentration of exosomes in the sample based on the standard curve.

[0108] Specifically

[0109] According to the testing procedure, the following reagents are packaged into reagent bags 6 made of flexible multilayer co-extruded film, each preferably having a volume of 500 μL, and a rubber sealing chamber 51 is installed:

[0110] Washing solution for reagent bag 1: PBST buffer containing 0.05% Tween-20.

[0111] Reagent bag 2 detects antibodies: 1 μg / mL of biotinylated CD63 antibody is dissolved in 1% BSA in PBST.

[0112] Reagent bag 3 signal amplification: Streptavidin-HRP conjugate diluted 1:5000 was dissolved in PBST with 1% BSA.

[0113] Reagent bag 4: Luminol / enhancer solution. Luminescent substrate A: luminol / enhancer solution.

[0114] Reagent bag 5 contains luminescent substrate B: hydrogen peroxide stabilized solution.

[0115] Reagent bag 6 lysis buffer: RIPA lysis buffer. Install and fix the prepared reagent bag 6 in the reagent compartment fixing slot 5 of the cartridge body 2, ensuring that the rubber sealing compartment 51 is aligned with the bottom puncture device 12.

[0116] Perform the following steps:

[0117] a. Sample loading: Take 100 μL of serum sample to be tested, filter it through a 0.22 μm filter membrane, and add it to the sample loading port 7 of the cartridge body 2.

[0118] b. Detachable Dual Chiller Body Loading: Connect chiller body 1 and chiller body 2, clamping the microfluidic chip 9 in the middle, and secure them with fixing bolts 4. Place the assembled detachable dual chiller body into the automated fluid control device 15.

[0119] c. Device startup: The user selects and starts the detection program on the control screen 1505.

[0120] Automatic sealing and puncture: Drive motor 1515 starts, driving the sealing plate 1516 to descend and press the sealing seat 1517 of the sample inlet port 7, achieving a pneumatic seal of the sample well. Drive motor 1507 starts, driving the telescopic plate 1508 to descend, causing the top puncture device 1510 to puncture the rubber sealing chambers 51 of all reagent bags 6. Simultaneously, the rubber sealing chambers 51 are pushed downwards, causing their bottom surfaces to be punctured by the bottom puncture device 12. This completes the establishment of the "gas path input" of all reagents from the top puncture device 1510 and the "liquid path output" from the bottom puncture device 12.

[0121] e-Automatic Fluid Timing Control: The main control board 1504 controls the air pump 1502 and each diversion valve 1512 and diversion valve 2 1514 according to a preset program, pumping fluid at a preferred flow rate of 5μL / min according to the following timing sequence:

[0122] Pumping sample: Pump 100 μL of sample continuously for 20 minutes.

[0123] First wash: Pump the washing solution into reagent bag 1 for 5 minutes.

[0124] Antibody incubation: Pump reagent bag 2 to detect antibodies for 15 minutes.

[0125] Second wash: Pump the washing solution into reagent bag 1 for 5 minutes.

[0126] Signal amplification: The signal from the pumped reagent bag 3 is amplified and lasts for 10 minutes.

[0127] Third wash: Pump the washing solution into reagent bag 1 for 5 minutes.

[0128] Chemiluminescence: Reagent bags 4 and 5 simultaneously pump luminescent substrates A / B, causing them to mix in the fluid channel 906 of the microfluidic chip 9 and flow through the capture and detection reaction chamber 902.

[0129] f. Reading: During or after step e7, the user removes the entire detachable dual cartridge body from the fluid control device 15 and places it in an external chemiluminescent microplate reader such as ThermoMultiskanFC, aligning the optical detection window 3 with the detector and reading the chemiluminescent signal RLU value.

[0130] g. Optional Multidimensional Analysis: If multidimensional analysis is required, the user can return the device to the fluid control unit 15. The fluid control unit 15 initiates the pyrolysis procedure: the gas pump 1502 pressurizes the gas pressure valve 910 through the valve gas transfer pipe 16, closes the channel to the waste liquid outlet 908, and opens the channel to the downstream analyte outlet 909. Subsequently, the device pumps the pyrolysis solution from the reagent bag 6, and the pyrolysis products flow out from the outlet of the collection interface chamber 11, which is collected by the user using centrifuge tubes.

[0131] The function switching module 907 is located on the microfluidic chip 9, downstream of the capture and detection reaction chamber 902.

[0132] The function switching module 907 acts as a fluid switch.

[0133] In the default detection state: the pneumatic valve 910 at the waste liquid outlet 908 is not activated, the pneumatic valve 910 at the downstream analyte outlet 909 is not activated, and the fluid such as washing liquid or chemiluminescent substrate liquid flows out of the capture and detection reaction chamber 902 and is guided to the waste liquid outlet 908 and enters the internal waste liquid collection chamber 10.

[0134] During the switching collection state: As described in Example 2(g), when it is necessary to collect downstream analytes such as pyrolysis products, the air pump 1502 in the fluid control device 15 pressurizes the air pressure valve 910 located at the waste liquid outlet 908 through the valve gas transmission pipe 16. The air pressure causes the air pressure valve 910 to deform or move, thereby closing the passage to the waste liquid outlet 908 and opening the passage to the downstream analyte outlet 909. At this time, the subsequently pumped liquid, such as pyrolysis liquid, will flow out from the downstream analyte outlet 909 and enter the collection interface chamber 11 for collection.

[0135] The fluid control device 15 achieves puncture and sealing through two independent drive motors 1507 and 1515 and a ball screw.

[0136] Sealing: Drive motor 2 1515 drives the sealing pressure plate 1516 to descend, pressing the "seal seat 1517" of the "sample addition port 7" on the cartridge body 2 to achieve pneumatic sealing of the sample well.

[0137] Puncture: Drive motor 1507 drives telescopic pressure plate 1508 to descend. This telescopic pressure plate 1508 drives the top puncture device 1510 to puncture the rubber sealing chamber 51 at the top of the reagent bag 6; simultaneously, the top pressure column 1509 on the telescopic pressure plate 1508 pushes the reagent bag 6 downward, causing its bottom surface to be punctured by the bottom puncture device 12. This completes the establishment of the "top gas path input" and "bottom liquid path output".

[0138] Fluid timing control: The control board 1504 of the equipment stores the automation program. It controls the air pump 1502 to generate air pressure, and distributes this air pressure through the gas transmission main pipe 1511 and multiple diversion valves 1512 and 1514.

[0139] Pumping fluid: The main board controls the diversion valve 1512 to pump gas into the top of a specific reagent bag 6 according to the preset timing sequence as described in Example 2(e). The gas pressure is used to force the liquid in the bag out from the bottom puncture device 12 and send it into the microfluidic chip 9 through the reagent transfer tube 13.

[0140] Switching valves: The main board controls the air pump 1502 and the diversion valve 1514 to supply air to the air pressure valve 910 on the microfluidic chip 9 through the valve gas transmission pipe 16, so as to achieve flow path switching.

[0141] 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.

[0142] 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. An exosome concentration detection kit, characterized in that, Include: The detachable dual cartridge body includes a cartridge-type main body one and a cartridge-type main body two. The cartridge-type main body one is provided with valve gas transmission pipes on both sides, and has an internal waste liquid collection chamber and a collection interface chamber in its inner cavity. An optical detection window is detachably assembled on the top through fixing bolts. A chip slot is provided on the opposite side of the main body two, and a fluororubber sealing ring is pasted on the inner wall of the slot. The cartridge-type main body two has a reagent chamber fixing slot and a sample addition port on its top, and a reagent transmission pipe and a sample transmission pipe are embedded in its inner cavity. One end of the reagent transmission pipe is connected to the bottom puncture device. The microfluidic chip, sealed and fitted into a chip slot, includes a chip body. The chip body has a capture and detection reaction chamber with a three-dimensional porous matrix fixed inside. A sample inlet and a reagent inlet are provided on one side of the chip, and a function switching module is provided on the other side. The function switching module is connected to the waste liquid outlet and the downstream analyte outlet through a diversion channel with a pressure valve. A pre-packaged reagent assembly includes a reagent bag, which is fixed in a reagent compartment fixing groove and has a rubber sealing compartment at one end, which is coaxially aligned with the bottom puncture device. The sample transfer tube is sealed to the sample inlet, the reagent transfer tube is sealed to the reagent inlet, the waste liquid outlet is connected to the internal waste liquid collection chamber, the downstream analyte outlet is connected to the collection interface chamber, and the valve gas transfer tube is connected to the gas pressure valve. The function switching module is located on the microfluidic chip, downstream of the capture and detection reaction chamber; The function switching module acts as a fluid switch; When downstream analytes need to be collected, the air pump in the fluid control equipment pressurizes the air pressure valve located at the waste liquid outlet through the valve gas transmission pipe. The air pressure causes the air pressure valve to deform or move, thereby closing the channel to the waste liquid outlet and opening the channel to the downstream analyte outlet.

2. The exosome concentration detection kit according to claim 1, characterized in that, The three-dimensional porous matrix is ​​an electrospun polystyrene membrane, which is fixed by the following process: soaking in 1% APTES ethanol solution and incubating at 37°C for 2 hours, rinsing with distilled water and then soaking in 2.5% glutaraldehyde aqueous solution and incubating at 25°C for 1 hour, and then fixing it to the inner wall of the capture and detection reaction chamber.

3. The exosome concentration detection kit according to claim 1, characterized in that, The reagent bag has a multi-layer co-extruded film structure and is pre-encapsulated with biotinylated CD63 antibody solution, streptavidin-HRP conjugate, and chemiluminescent substrate solution. The rubber sealing chamber is made of nitrile rubber with a thickness of 3 mm.

4. The exosome concentration detection kit according to claim 1, characterized in that, The optical detection window is made of high-transmittance quartz glass, and Dow Corning 734 silicone rubber sealant is applied to the fixing bolt connection. The fluororubber sealing ring on the inner wall of the chip slot has a Shore hardness of 60±5.

5. A fluid control device for an exosome concentration detection kit according to any one of claims 1 to 4, characterized in that It does not include an optical detection unit, but includes: a) Equipment compartment, with an air pump, lithium battery and control motherboard fixed inside, and a control screen fixed on the outer wall; b) The top of the upright frame is fixed with drive motor one and drive motor two, and the output shafts of the motors are all connected to ball screws; c) Telescopic pressure plate, connected to the lead screw of drive motor one, with a top pressure column and a top puncture device fixed at the bottom; d) A gas transmission system, including a gas transmission main pipe, a first diversion valve, a diversion pipe, a second diversion valve, and a sealing seat, wherein the gas transmission main pipe is connected to a gas pump; e) The control motherboard stores an automation program for controlling the drive motor, air pump, and diversion valve one and diversion valve two.

6. A method for detecting exosomes, characterized in that, Using the kit of claim 3 and the device of claim 5, the steps include: a) The sample to be tested is filtered through a 0.22μm filter membrane and then added to the sample inlet port of the cartridge-type main body two; b) The cartridge-type main body one and the cartridge-type main body two are docked, a microfluidic chip is assembled, and an automated fluid control device is inserted; c) Select puncture control and complete the puncture; d) The device drives the sample and reagents into the microfluidic chip according to a preset time sequence to complete the exosome capture, incubation, washing, and chemiluminescence reaction; e) Remove the detachable dual cartridge body, align the optical detection window with the external optical detection device, read the signal and calculate the exosome concentration; f) Optional: Start the pyrolysis process, switch the gas pressure valve to the downstream analyte side, and collect the pyrolysis products.

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