Exosome isolation and detection device and method of use

Abstract

The application belongs to the field of exosome separation and detection, and relates to an exosome separation and detection device and a use method. In the separation stage, the conventional straight-through section is the narrowest to generate high shear viscoelastic force to make the particle size dependent migration, the largest expansion section is the widest to enlarge the displacement difference, the center flow channel is relatively wide to discharge large particles, the lateral flow channel is wide in the middle to collect exosomes into the liquid storage cavity, meanwhile, the first liquid flow control valve is closed to prevent high-speed fluid from directly entering the lysis module, so that the high shear flow field does not interfere with the subsequent biochemical reaction, after the liquid storage cavity collects enough volume, the first liquid flow control valve is opened, so that the exosome solution enters the lysis module in a low disturbance manner, the volume matching of continuous flow separation and batch lysis reaction is realized, after the lysis is completed, the second liquid flow control valve is opened to make the lysis liquid enter the detection module, and the time sequence closing and opening of the two valves also isolates the viscoelastic fluid and the detection reagent. The device realizes the compatibility of 95% separation efficiency of exosome separation, high-sensitivity miRNA detection in a single device.

Description

Technical Field

[0001] This invention belongs to the field of exosome isolation and detection, and relates to an exosome isolation and detection device and its usage method. Background Technology

[0002] Exosomes are heterogeneous, cell-derived lipid vesicles that carry genetic and proteomic information from their source cells. Studies have shown that the number of active exosomes released by tumor cells in the blood is far higher than that of normal cells; the expression levels of various miRNAs in tumor cell-derived exosomes differ from those in healthy individuals; and the phospholipid bilayer of exosomes protects internal nucleic acid substances from degradation. Therefore, exosome miRNAs are important biomarkers for liquid biopsies and have great potential applications in the early diagnosis, treatment, and prognosis of diseases such as cancer.

[0003] Currently, commonly used methods for exosome isolation and purification include ultracentrifugation (UC), immunoaffinity chromatography, polymer precipitation, nanofiltration, and size exclusion chromatography. These methods suffer from drawbacks such as reliance on immunolabeling or external physical fields, potential disruption of exosome structural integrity hindering downstream analysis, low purity, and long processing times. In recent years, the elastic lift generated by viscoelastic fluids on particles at high flow rates (…) , The particle size can be manipulated to achieve precise three-dimensional focusing, providing a new approach for exosome separation.

[0004] Currently, most methods for detecting miRNAs in exosomes involve amplification followed by detection. While this method can ensure detection sensitivity, it is cumbersome, time-consuming, has high background noise, and is prone to contamination. On the other hand, amplification-free techniques are difficult to detect low concentrations of targets.

[0005] Detection of characteristic miRNA molecules in exosomes requires three steps: 1) isolating tiny exosomes from body fluids; 2) lysing the exosome membrane to release its contents; and 3) detecting the expression of tumor marker miRNA molecules in exosomes. Current detection methods typically perform these steps independently, each requiring specialized equipment or kits. These methods are complex to perform manually, prone to sample contamination and loss, and have low detection efficiency.

[0006] To improve detection efficiency, several inventions have attempted to develop microfluidic chip devices that integrate exosome separation and detection. Patent application CN 109628277 A presents an integrated chip based on filtration, immunoassay, and nanopore detection technologies. However, its separation process relies on an external magnetic field, requiring multiple reagent additions and microvalve adjustments, making operation still complex. Patent application CN 110339874 A presents an integrated chip that can only detect the number of exosome particles, making it difficult to obtain their internal genetic and proteomics information. Patent application CN 111961584 A presents a functionally integrated microfluidic device, but the separation, lysis, and detection modules are independent and connected via conduits, still presenting problems of device complexity and contamination risk.

[0007] However, integrating viscoelastic fluid-based exosome separation and miRNA detection into a single device presents significant technical challenges. Maintaining the size-dependent elastic stress field during viscoelastic separation relies on high flow rates, while exosome lysis and detection require a low-disturbance reaction environment. Viscoelastic separation uses continuous flow, while miRNA quantification demands high consistency in reaction system volume and concentration. Furthermore, the high viscosity and small channel size of viscoelastic fluids result in high flow resistance, leading to flow field matching issues and fabrication difficulties. Additionally, direct introduction of viscoelastic fluid into the downstream reaction chamber may alter reagent composition, affecting enzyme activity and fluorescence signal stability.

[0008] Therefore, there is an urgent need for an integrated device that combines exosome separation and detection functions while ensuring exosome separation efficiency, miRNA detection sensitivity and accuracy. Summary of the Invention

[0009] The purpose of this invention is to solve the environmental conflict between high-flow-rate viscoelastic separation and low-disturbance biochemical reactions in the prior art, and to provide an exosome separation and detection device and its usage method.

[0010] To achieve the above objectives, the present invention employs the following technical solution: An exosome isolation and detection device includes an exosome isolation module, the output end of which is sequentially connected to the input end of an exosome lysis module and an exosome miRNA detection module, a first flow control valve is provided between the output end of the exosome isolation module and the input end of the exosome lysis module, and a second flow control valve is provided between the output end of the exosome lysis module and the input end of the exosome miRNA detection module. The exosome separation module includes a separation channel, which comprises a conventional straight section, an amplification and expansion section, and a diversion section connected in sequence. The inlet of the conventional straight section is connected to the sample inlet and the sheath fluid inlet, respectively. The diversion section includes a central channel and lateral channels located on both sides of the central channel. The outlet of the central channel is connected to the inlet of the waste liquid outlet, and the outlet of the lateral channels is connected in sequence to the liquid storage chamber and the inlet of the exosome lysis module. The cross-sectional area of ​​each section of the separated flow channel satisfies: Conventional straight section < lateral flow channel < central flow channel < enlarged expansion section.

[0011] A further improvement of the present invention is that: The amplified expansion section includes a diffusion triangle region and a rectifier rectangular region. The narrow end of the diffusion triangle region is connected to the conventional straight-through section, and the wide end of the diffusion triangle region is connected to the rectifier rectangular region. The two side walls of the diffusion triangle slope outward along the flow direction, forming a gradually expanding flow channel structure; The rectifier rectangular region has a constant width and length.

[0012] The cross-sectional area ratio of the conventional straight section, lateral flow channel, and central flow channel is 10:17:46.

[0013] Both the first and second flow control valves are steel needles, which are inserted vertically into the corresponding flow channels. The first flow control valve is used to close or open the flow channel between the liquid storage chamber and the exosome lysis module. The second flow control valve is used to close or open the flow channel between the exosome lysis module and the exosome miRNA detection module.

[0014] The outlet of the central flow channel is connected to the inlet of the serpentine channel, and the outlet of the serpentine channel is connected to the inlet of the waste liquid outlet.

[0015] The exosome lysis module includes lysis chambers and waste liquid chambers spaced apart; A connecting channel is provided between the pyrolysis chamber and the waste liquid chamber, and the connecting channel is located on one side of the upper port of the pyrolysis chamber.

[0016] Microcolumn arrays are installed at the inlets of both the sample inlet and the sheath fluid inlet.

[0017] A method of using the device described in this embodiment includes the following steps: Initially, both the first and second flow control valves are closed, and cell supernatant and sheath fluid are injected from the sample inlet and sheath fluid inlet, respectively. After the cell supernatant and sheath fluid merge, they enter the conventional straight section. With the assistance of the viscoelastic sheath fluid, all particles of all sizes in the cell supernatant undergo initial focusing on the side wall of the straight channel entrance. After passing through the end of the conventional straight section, the cell supernatant and sheath fluid enter the amplification and expansion section. The amplification and expansion section amplifies the lateral displacement difference of the particles, completing the lateral separation of particles of different sizes. Large-sized exosomes in the cell supernatant enter the waste liquid outlet through the central flow channel, while small-sized exosomes enter the liquid storage chamber through the lateral flow channels on both sides. Once the exosomes reach the preset capacity in the storage chamber, the first flow control valve is opened, and the exosomes begin to enter the exosome lysis module. After the exosomes are brought to a constant volume in the exosome lysis module, the second flow control valve is opened, and the exosomes enter the exosome miRNA detection module. After reacting with the pre-stored reagents in the exosome miRNA detection module, fluorescence determination is performed.

[0018] After the exosomes complete the volume adjustment and reaction in the exosome lysis module, the second liquid flow control valve is opened, including: The exosome lysis module includes lysis chambers and waste liquid chambers spaced apart, with a connecting groove between the lysis chambers and the waste liquid chambers located on one side of the upper port of the lysis chambers; When exosomes enter the lysis chamber, they react with the lysis buffer pre-stored in the lysis chamber, releasing exosome miRNA. Excess solution enters the waste liquid chamber through the connecting tank to achieve a constant volume. At this point, the second liquid flow control valve is opened, and the reaction solution in the lysis chamber enters the exosome miRNA detection module.

[0019] The injection rate of the sheath fluid is 1100~1300. The cell supernatant injection rate is 180~240. .

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses an exosome separation and detection device. The device constrains the cross-sectional areas of the conventional straight section, lateral flow channel, central flow channel, and amplified expansion section. During the separation stage, the narrowest conventional straight section generates high shear viscoelastic force, causing particle size-dependent migration. The widest amplified expansion section amplifies the displacement difference. The wider central flow channel discharges large particles, and the lateral flow channel, with its central width, collects exosomes into the storage chamber. Simultaneously, the first flow control valve is closed to prevent high-velocity fluid from directly entering the lysis module, avoiding interference from the high-shear flow field with subsequent biochemical reactions. After sufficient volume is collected in the storage chamber, the first flow control valve is opened, allowing the exosome solution to enter the lysis module with low disturbance, achieving volume matching between continuous flow separation and batch lysis reaction. After lysis, the second flow control valve is opened to allow the lysate to enter the detection module. The sequential closing and opening of the two valves also isolates the viscoelastic fluid from the detection reagents, preventing components such as PEO from affecting CRISPR enzyme activity. This device achieves 95% separation efficiency for exosome separation and high-sensitivity miRNA detection within a single device, simplifying operation and reducing sample transfer losses.

[0021] Furthermore, in this invention, the expansion section forms a gradually expanding structure by tilting the two side walls of the diffusion triangle outward along the flow direction. This can smoothly reduce the velocity gradient while maintaining a high flow velocity of the viscoelastic fluid, further amplifying the lateral displacement of particles of different sizes due to differences in elastic lift in the gradually expanding region. Then, the fluid is stabilized and shaped by a constant-width rectifying rectangular region, amplifying the difference in lateral displacement of the particles and ensuring that the particles are stable in position before entering the diversion section.

[0022] Furthermore, in this invention, the outlet of the central flow channel is connected to the inlet of the serpentine channel, the outlet of the serpentine channel is connected to the inlet of the waste liquid outlet, and the outlet of the central flow channel is connected to the serpentine channel. The meandering structure of the serpentine channel increases the flow resistance to compensate for the flow resistance difference between the central flow channel and the two lateral flow channels and their subsequent liquid storage chamber and pyrolysis module in the separation flow channel, thereby maintaining the stability of the lateral pressure field in the separation zone and ensuring the particle separation accuracy based on size.

[0023] Furthermore, in this invention, a connecting groove is provided on one side of the upper port of the lysis chamber, so that the exosome solution and lysis liquid injected into the lysis chamber can automatically overflow to the waste liquid chamber through the connecting groove after reaching a predetermined volume, thereby achieving constant volume of the reaction system. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the experimental apparatus and usage scenarios disclosed in the embodiments of the present invention; Figure 2 This is a schematic diagram of the chip structure disclosed in an embodiment of the present invention; Figure 3a This is a schematic diagram of the separation channel structure disclosed in an embodiment of the present invention; Figure 3b This is a schematic diagram illustrating the separation principle disclosed in an embodiment of the present invention; Figure 4 This is a schematic diagram of the detection principle and process disclosed in an embodiment of the present invention.

[0026] The components are as follows: 1-Exosome separation module, 2-First flow control valve, 3-Exosome lysis module, 4-Second flow control valve, 5-Exosome miRNA detection module, 11-Sample inlet, 12-Sheath fluid inlet, 13-Separation channel, 14-Serpentine channel, 15-Reservoir chamber, 16-Waste outlet, 17-First channel, 18-Second channel, 31-Cycle chamber, 32-Waste chamber, 33-Connecting channel. 131-Conventional straight section, 132-Amplification expansion section, 133-Split section, 1331-Central channel, 1332-Side channel. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0028] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0029] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0030] In the description of the embodiments of the present invention, it should be noted that if terms such as "upper," "lower," "horizontal," or "inner" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of the invention is in use, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Furthermore, terms such as "first" and "second" are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0031] Furthermore, the use of the term "horizontal" does not imply that the component must be absolutely horizontal, but rather that it can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal than "vertical," and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0032] In the description of the embodiments of the present invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the present invention according to the specific circumstances.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings: This embodiment primarily addresses how to resolve the environmental conflicts between high-flow-rate viscoelastic separation and low-disturbance biochemical reactions, the volume control contradictions between continuous flow and constant-volume reactions, and the interference of viscoelastic fluids on downstream detection reagents in the integrated exosome separation and detection process. This aims to simplify operation and avoid sample contamination and loss while ensuring separation efficiency and detection sensitivity. Therefore, this embodiment discloses an exosome separation and detection device and its usage method, integrating exosome separation, lysis, and exosome miRNA detection processes, simplifying exosome miRNA detection operations, and promoting its clinical application in liquid biopsy.

[0034] In the separation phase, the separation principle disclosed in this embodiment is as follows: Cell supernatant and viscoelastic sheath fluid are simultaneously pumped into the chip inlet. After filtration, they converge through a cross-shaped flow channel and enter the separation direct current channel. With the assistance of the sheath fluid, all particles in the sample undergo initial focusing at the channel inlet sidewall. The viscoelastic sheath fluid has a high shear rate, and due to its shear-thinning rheological properties, the solution viscosity decreases, generating significant size-dependent elastic stress pointing towards the channel center on the particles in the sample. As a result, larger particles in the sample complete lateral migration more quickly. At the separation direct current channel outlet, the lateral displacement difference is amplified by the expansion zone, and the particles in the sample are separated laterally based on size. The larger particles exit the chip through the serpentine channel, while the smaller exosomes enter the subsequent chambers through the side channels. Specifically, in this embodiment, the small particle size entering the side channel 1332 is 150-200 nm, and the large particle size entering the central channel 1331 is 500-550 nm.

[0035] In the detection phase, the principle of exosome miRNA detection in this embodiment is as follows: Using CRISPR reaction: a crRNA for detecting target miRNA was designed and assembled with Cas13a enzyme to form a crRNA-guided, RNA-activated nuclease, which was then lyophilized and placed in the detection chamber as a reaction substrate. This nuclease can specifically recognize and activate the target miRNA, cleaving the reporter gene to separate the fluorescent group and the quenching group to generate fluorescence. The miRNA content was quantitatively analyzed by the relative quantitative analysis of the fluorescence gray value generated by the reaction.

[0036] See Figures 1 to 4 The following is a detailed description of this embodiment: See Figures 1 to 2 This embodiment discloses an exosome separation and detection device, including an exosome separation module 1. The output end of the exosome separation module 1 is sequentially connected to an exosome lysis module 3 and an exosome miRNA detection module 5. A first flow control valve 2 is provided between the output end of the exosome separation module 1 and the input end of the exosome lysis module 3, and a second flow control valve 4 is provided between the output end of the exosome lysis module 3 and the input end of the exosome miRNA detection module 5.

[0037] Furthermore, in this embodiment, the specific structure of the exosome separation module 1 is as follows: The exosome separation module 1 includes a sample inlet 11 and a sheath fluid inlet 12. Both the sample inlet 11 and the sheath fluid inlet 12 are equipped with microcolumn arrays. The sample inlet 11 is connected to a second flow channel 18 through a first flow channel 17. The second flow channel 18 is an annular flow channel that surrounds the periphery of the sheath fluid inlet 12. The sheath fluid inlet 12 is connected to the separation flow channel 13. The supernatant enters the first flow channel 17 and the second flow channel 18 after passing through the microcolumn array. After entering the second flow channel 18, it is divided into two paths and then mixed with the sheath fluid at the cross intersection before entering the separation flow channel 13.

[0038] See Figure 3a Furthermore, in this embodiment, the separation channel 13 includes a conventional straight section 131, an amplification and expansion section 132, and a diversion section 133 connected in sequence. The supernatant and sheath fluid are mixed and then enter the conventional straight section 131. The diversion section 133 includes a central channel 1331 and lateral channels 1332 located on both sides of the central channel 1331. The outlet of the central channel 1331 is connected to the inlet of the waste liquid outlet 16, and the outlet of the lateral channels 1332 is connected in sequence to the inlet of the storage chamber 15 and the exosome lysis module 3. The cross-sectional area of ​​each section of the separation channel 13 satisfies the following condition: conventional straight section 131 < lateral channels 1332 < central channel 1331 < amplification and expansion section 132.

[0039] Furthermore, in this embodiment, the total length of the separation channel 13 is 32. Width is 20 The width of the lateral flow channel 1332 is 34. The width of the central flow channel 1331 is 92. .

[0040] Furthermore, in this embodiment, the amplified expansion section 132 includes a diffusion triangle region and a rectifying rectangular region. The narrow end of the diffusion triangle region is connected to the conventional straight section 131, and the wide end of the diffusion triangle region is connected to the rectifying rectangular region. The two side walls of the diffusion triangle region are inclined outward along the flow direction to form a gradually expanding flow channel structure. The rectifying rectangular region has a constant width and length, which can stabilize the fluid and further amplify the particle displacement difference.

[0041] See Figure 3b The separation principle of this structure is as follows: After filtration, the sheath fluid and cell supernatant mix at the crossroads. With the assistance of the viscoelastic sheath fluid, particles of all sizes in the cell supernatant undergo initial focusing at the inlet sidewall of the conventional straight section 131. The formula for calculating the elastic lift force acting on particles in a viscoelastic fluid is:

[0042] in, The elastic lift coefficient, The particle diameter is for The model gives the first normal stress difference.

[0043] It is evident that the elastic stress is proportional to the cube of the particle size. Under the action of elastic stress, large particles migrate rapidly towards the center of the channel, while small particles migrate slowly. The amplified expansion section 132 at the outlet of the straight channel amplifies the lateral displacement difference of the particles, thereby completing the lateral separation of particles based on size. The exosomes are smaller in size and enter the subsequent chamber through the lateral flow channels 1332 on both sides. Other large particles in the cell supernatant flow out of the chip from the center of the channel through the central flow channel 1331 and the serpentine channel 14.

[0044] Furthermore, in this embodiment, the outlet of the central flow channel 1331 is connected to the inlet of the serpentine channel 14, and the outlet of the serpentine channel 14 is connected to the inlet of the waste liquid outlet 16. The function of the serpentine channel is to compensate for the flow resistance of the central flow channel at the end of the separation direct flow channel, so as to balance the flow resistance with the subsequent chambers and flow channels on both sides, and maintain the stability of the transverse pressure field of the separation zone.

[0045] Furthermore, in this embodiment, both the first flow control valve 2 and the second flow control valve 4 are steel needles, which are inserted vertically into the corresponding flow channels. The first flow control valve 2 is used to close or open the flow channel between the liquid storage chamber 15 and the exosome lysis module 3; the second flow control valve 4 is used to close or open the flow channel between the exosome lysis module 3 and the exosome miRNA detection module 5.

[0046] Among them, the diameter of the solid steel needle is 1-2 mm. Its bottom is inserted into the lower surface of the connecting channel, and the flow of liquid between the chambers is controlled by the vertical up-and-down movement of the steel needle relative to the chip. The liquid flow control valve separates the viscoelastic separation process, which depends on high shear rates, from the low-disturbance pyrolysis and detection reaction processes, avoiding interference from the high-velocity flow field to the biological reaction environment, and realizing the stable integration of multifunctional modules on the same chip.

[0047] Furthermore, in this embodiment, the exosome lysis module 3 has the following structure: The exosome lysis module 3 includes lysis chambers 31 and waste liquid chambers 32 spaced apart. A connecting channel 33 is provided between the lysis chambers 31 and the waste liquid chamber 32. The connecting channel 33 is located on one side of the upper port of the lysis chamber 31, so that the reaction reagent in the lysis chamber 31 can overflow into the waste liquid chamber 32 after reaching a predetermined volume, thereby realizing the constant volume control of the reaction system and ensuring the accuracy and reproducibility of CRISPR reaction for relative quantitative analysis of miRNA.

[0048] Furthermore, in this embodiment, the exosome miRNA detection module 5 has a cone-shaped sidewall, and the internal diameter of the cavity gradually decreases from top to bottom. This allows for focusing of the fluorescent products of the CRISPR reaction, facilitating observation and improving the intensity and detectability of the fluorescence signal under low exosome concentration conditions, thereby reducing the detection limit of the target miRNA.

[0049] Furthermore, in this embodiment, the aperture of the micropillar array is 200~400 mm. It can initially filter large particulate impurities in sample solutions and sheath fluid.

[0050] Furthermore, in this embodiment, the bottom surfaces of the filter structure at the inlet, the separation channel, the chamber connecting channel, and each chamber are located on the same plane; the cross-sectional shape of each channel is rectangular, and the height is 50 mm. The depth of each chamber is 3. .

[0051] Furthermore, in this embodiment, the microfluidic device has multiple sets of parallel separation channels and chamber chains, which can simultaneously achieve multi-target joint detection.

[0052] Furthermore, in this embodiment, the microfluidic chip has a two-layer structure, with the upper layer consisting of microchannels and chambers, made of polydimethylsiloxane (PDMS) with a thickness of 3 mm. The lower layer is 1 layer thick. The glass slide serves as the chip substrate; the two materials can achieve good bonding.

[0053] See Figure 4 This embodiment also discloses a method for the isolation and detection of exosomes, characterized by comprising the following steps: In the initial state, both the first fluid flow control valve 2 and the second fluid flow control valve 4 are in the closed state.

[0054] Step 1: Inject cell supernatant and sheath fluid into sample inlet 11 and sheath fluid inlet 12 respectively; Step 2: After the cell supernatant and sheath fluid merge, they enter the conventional straight section 131. With the assistance of the viscoelastic sheath fluid, all particles of all sizes in the cell supernatant undergo initial focusing on the side wall of the straight channel entrance. Step 3: After passing through the end of the conventional straight section 131, the cell supernatant and sheath fluid enter the amplification and expansion section 132. The amplification and expansion section 132 amplifies the lateral displacement difference of the particles, completing the lateral separation of particles of different sizes. Large-sized exosomes in the cell supernatant enter the waste liquid outlet 16 through the central flow channel 1331, while small-sized exosomes enter the liquid storage chamber 15 through the lateral flow channels 1332 on both sides. Step 4: Once the exosomes have reached the preset capacity in the storage chamber 15, open the first flow control valve 2, and the exosomes enter the lysis chamber 31. Step 5: Exosomes begin to enter the exosome lysis module 3. The exosomes react with the lysis buffer pre-stored in the lysis chamber 31 to release exosome miRNA. Excess solution enters the waste liquid chamber 32 through the connecting tank 33 to achieve volume fixation. Step 6: After completing the volume adjustment, open the second liquid flow control valve 4, and the exosomes enter the exosome miRNA detection module 5. After reacting with the pre-stored reagents in the exosome miRNA detection module 5, fluorescence determination is performed.

[0055] Furthermore, in this step, the injection rate of the sheath fluid is 1100~1300. Sample injection rate 180~240 The sheath fluid was a 0.1–0.2% wt PEO solution. The lysis chamber volume was 50–70 mL. The volume of waste liquid chamber 32 is 30~40. The pyrolysis chamber 31 can overflow into the waste liquid chamber through the channel to achieve the volume adjustment and dilution of the reaction reagents.

[0056] The detection principle of this method is as follows: Using CRISPR reaction, a crRNA for detecting target miRNA was designed and assembled with cas13a enzyme to form a crRNA-guided, RNA-activated nuclease. This crRNA was then lyophilized and placed in the detection chamber as a reaction substrate. This nuclease can specifically recognize and activate the target miRNA, cleaving the reporter gene to separate fluorescent and quenched groups to generate fluorescence. The relative quantitative analysis of miRNA content is performed based on the fluorescence gray value generated by the reaction. Simultaneously, this device has multiple sets of separation channels and chamber chains connected in parallel, enabling simultaneous multi-target detection.

[0057] Furthermore, in this step, lysis buffer is pre-added to the lysis chamber 31, and CRISPR reagent is placed in the detection chamber for lyophilization.

[0058] The apparatus disclosed in this embodiment has the following advantages compared with the prior art: (1) This invention discloses a microfluidic device integrating exosome separation and detection. In use, sample cell supernatant and sheath fluid are pumped into the chip inlet via a syringe pump, lysis buffer is injected into the lysis chamber, and CRISPR reaction reagents are lyophilized and placed in the detection chamber. The flow control valve is switched on and off periodically, enabling continuous operation of exosome separation, lysis, and miRNA quantitative detection. The device is compact and easy to operate, avoiding sample loss and contamination risks caused by the dispersion and multiple transfers of separation and detection equipment in traditional methods. It lowers the technical threshold and has potential for liquid biopsy applications.

[0059] (2) The present invention uses a sample pump and a liquid flow control valve to precisely regulate the flow rate and injection time of the liquid in each channel, which can accurately control the separation, pyrolysis and detection process, and improve the stability, accuracy and efficiency of the system operation.

[0060] (3) This invention achieves stable collaborative operation of the high-shear separation zone and the low-disturbance detection zone within the same chip through a flow resistance compensation structure and a chamber-microvalve isolation design. Under integrated conditions, the exosome separation efficiency, recovery efficiency, and detection sensitivity remain stable, and there is no performance degradation due to structural integration.

[0061] (4) This invention discloses an exosome separation method based on viscoelastic fluid, achieving label-free, size-dependent, and highly efficient separation. It eliminates the need for antibodies, magnetic beads, or fluorescent labeling, avoiding complex pretreatment steps and the potential impact of exogenous labeling on exosome structure and function; it does not rely on external fields such as electric or magnetic fields, and can be completed using only a simple flow channel structure, which is beneficial for improving system stability and repeatability, reducing usage and maintenance costs, and facilitating integration and continuous operation; the exosome separation efficiency exceeds 90%, and the recovery efficiency reaches 85%~90%.

[0062] (5) This invention supports the lyophilization of CRISPR reaction reagents and their placement in the detection chamber for quantitative detection, enabling flexible analysis of exosome molecular information, meeting various detection needs, and improving the versatility and scalability of experiments; at the same time, multiple sets of structural chains on the chip are connected in parallel, enabling multi-target detection of exosome miRNAs, thereby improving diagnostic accuracy and having significant application scenarios in early cancer detection.

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An exosome separation and detection device, characterized in that, The system includes an exosome separation module (1), the output of which is sequentially connected to the input of an exosome lysis module (3) and an exosome miRNA detection module (5). A first flow control valve (2) is provided between the output of the exosome separation module (1) and the input of the exosome lysis module (3), and a second flow control valve (4) is provided between the output of the exosome lysis module (3) and the input of the exosome miRNA detection module (5). The exosome separation module (1) includes a separation channel (13), which includes a conventional straight section (131), an amplification expansion section (132), and a diversion section (133) connected in sequence. The inlet of the conventional straight section (131) is connected to the sample inlet (11) and the sheath fluid inlet (12), respectively. The diversion section (133) includes a central channel (1331) and lateral channels (1332) located on both sides of the central channel (1331). The outlet of the central channel (1331) is connected to the inlet of the waste liquid outlet (16), and the outlet of the lateral channel (1332) is connected in sequence to the liquid storage chamber (15) and the inlet of the exosome lysis module (3). The cross-sectional area of ​​each section of the separation channel (13) satisfies: Conventional straight section (131) < lateral flow channel (1332) < central flow channel (1331) < enlarged expansion section (132).

2. The exosome separation and detection device according to claim 1, characterized in that, The amplified expansion section (132) includes a diffusion triangle region and a rectifier rectangular region. The narrow end of the diffusion triangle region is connected to the conventional straight section (131), and the wide end of the diffusion triangle region is connected to the rectifier rectangular region. The two side walls of the diffusion triangle slope outward along the flow direction, forming a gradually expanding flow channel structure; The rectifier rectangular region has a constant width and length.

3. The exosome separation and detection device according to claim 1, characterized in that, The cross-sectional area ratio of the conventional straight section (131), the lateral flow channel (1332), and the central flow channel (1331) is 10:17:

46.

4. The exosome separation and detection device according to claim 1, characterized in that, The first flow control valve (2) and the second flow control valve (4) are both steel needles. The steel needles are inserted into the corresponding flow channels in a vertical direction. The first flow control valve (2) is used to close or open the flow channel between the liquid storage chamber (15) and the exosome lysis module (3). The second flow control valve (4) is used to close or open the flow channel between the exosome lysis module (3) and the exosome miRNA detection module (5).

5. The exosome separation and detection device according to claim 1, characterized in that, The outlet of the central flow channel (1331) is connected to the inlet of the serpentine channel (14), and the outlet of the serpentine channel (14) is connected to the inlet of the waste liquid outlet (16).

6. The exosome separation and detection device according to claim 1, characterized in that, The exosome lysis module (3) includes lysis chambers (31) and waste liquid chambers (32) spaced apart. A connecting channel (33) is provided between the pyrolysis chamber (31) and the waste liquid chamber (32), and the connecting channel (33) is located on one side of the upper port of the pyrolysis chamber (31).

7. The exosome separation and detection device according to claim 1, characterized in that, Microcolumn arrays are provided at the inlets of both the sample inlet (11) and the sheath fluid inlet (12).

8. A method of using the device according to claim 1, characterized in that, Includes the following steps: In the initial state, the first flow control valve (2) and the second flow control valve (4) are both closed, and cell supernatant and sheath fluid are injected from the sample inlet (11) and the sheath fluid inlet (12) respectively. After the cell supernatant and sheath fluid merge, they enter the conventional straight section (131). With the assistance of the viscoelastic sheath fluid, all particles of all sizes in the cell supernatant undergo initial focusing on the side wall of the straight channel entrance. After passing through the end of the conventional straight section (131), the cell supernatant and sheath fluid enter the amplification and expansion section (132). The amplification and expansion section (132) amplifies the lateral displacement difference of the particles, completing the lateral separation of particles of different sizes. Large-sized exosomes in the cell supernatant enter the waste liquid outlet (16) through the central flow channel (1331), while small-sized exosomes enter the liquid storage chamber (15) through the lateral flow channels (1332) on both sides. Once the exosomes reach the preset capacity in the storage chamber (15), the first liquid flow control valve (2) is opened, and the exosomes begin to enter the exosome lysis module (3). After the exosomes complete the volume adjustment and reaction in the exosome lysis module (3), the second liquid flow control valve (4) is opened, and the exosomes enter the exosome miRNA detection module (5). After reacting with the reagents pre-stored in the exosome miRNA detection module (5), fluorescence determination is performed.

9. The method of using the exosome separation and detection device according to claim 8, characterized in that, After the exosomes are brought to a constant volume in the exosome lysis module (3), the second flow control valve (4) is opened, including: The exosome lysis module (3) includes lysis chambers (31) and waste liquid chambers (32) spaced apart, and a connecting groove (33) is provided between the lysis chambers (31) and the waste liquid chambers (32), and the connecting groove (33) is located on one side of the upper port of the lysis chamber (31). When the exosomes enter the lysis chamber (31), the exosomes react with the lysis buffer pre-stored in the lysis chamber (31) to release exosome miRNA. The excess solution enters the waste liquid chamber (32) through the connecting tank (33) to achieve a constant volume. At this time, the second liquid flow control valve (4) is opened, and the reaction solution in the lysis chamber (31) enters the exosome miRNA detection module (5).

10. The method of using the exosome separation and detection device according to claim 8, characterized in that, The injection rate of the sheath fluid is 1100~1300. The cell supernatant injection rate is 180~240. .

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