Microfluidic device for exosome isolation and fully integrated nucleic acid / protein detection system

Abstract

The application discloses a microfluidic device for exosome separation and a fully integrated nucleic acid / protein detection system, comprising a microfluidic chip, wherein the microfluidic chip comprises a chip base, a separation module, a circulation pipeline module and a liquid discharge pipeline arranged on the chip base; the separation module comprises at least two separation cavities, a filter membrane is arranged in each separation cavity; the at least two separation cavities are sequentially connected in the order of the pore size of the filter membranes from large to small to form a graded filtration; a magnetic rotor capable of rotating under the action of external magnetic force is arranged in the separation cavity above the filter membrane; the liquid discharge pipeline is connected with the waste liquid outlet of the separation cavity with the smallest pore size of the filter membrane and is used for discharging waste liquid; the fluid in the separation cavity can be stirred through the rotation of the magnetic rotor above the filter membrane, so that the particles clogging the pores of the filter membrane are resuspended, the problem of easy clogging of the filter membrane is relieved, the exosomes to be recovered can also be resuspended, and the separation efficiency and recovery rate of the exosomes are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a microfluidic device for exosome separation and a fully integrated nucleic acid / protein detection system. BACKGROUND

[0002] Extracellular vesicles (such as exosomes) as an important medium for intercellular communication, their function in the organism gradually attracted the attention of researchers. They can regulate host-pathogen interactions, participate in the pathological process of various diseases such as infectious and inflammatory diseases, neurological diseases and cancer, and also play an important role in mediating intercellular communication in normal physiological processes.

[0003] In clinical medicine, extracellular vesicles also show great application potential. They contain rich biomarkers, which can be used to monitor clinical status, treatment response and disease progression, etc. In addition, due to their function of delivering biological molecules, extracellular vesicles are expected to develop into clinical drug delivery carriers.

[0004] Ultrafiltration is a common method for separating exosomes at present. The ultrafiltration process is usually carried out at room temperature, and the conditions are mild, without heating, so it will not cause damage to heat-sensitive biological molecules such as exosomes. The ultrafiltration process does not involve phase change, so the energy consumption is low, and no chemical reagents need to be added, which is a pollution-free and energy-saving separation technology. Ultrafiltration method only uses pressure as the driving force for membrane separation, so the separation device is simple, the process is short, the operation is simple, and it is easy to control and maintain. However, it also has the following disadvantages: first, the problem of filter clogging: during the extraction process, exosomes are easy to block the filter holes, which reduces the extraction efficiency of filtration, thereby reducing the separation efficiency; second, it is difficult to recover: during the elution process, exosomes attached to the filter membrane are difficult to recover, resulting in extraction loss and reducing the recovery rate.

[0005] Therefore, there is an urgent need for a microfluidic device for exosome separation and a fully integrated nucleic acid detection system to solve the above technical problems. SUMMARY

[0006] The present application aims to solve the above technical problems, that is, to solve the problem that the existing ultrafiltration method is easy to block the filter holes during the separation of exosomes, which reduces the extraction efficiency of filtration, thereby reducing the separation efficiency; and in the elution process, exosomes attached to the filter membrane are difficult to recover, resulting in extraction loss and reducing the recovery rate.

[0007] To this end, in a first aspect, the present application provides a microfluidic device for exosome separation, comprising a microfluidic chip, the microfluidic chip comprising a chip base, a separation module arranged on the chip base, a circulation pipeline module and a liquid discharge pipeline, the circulation pipeline module being connected with the separation module for forming circulation filtration, circulation cleaning and elution modes to finally separate exosomes, the separation module comprising at least one separation cavity, a filter membrane being arranged in each separation cavity, and the pore size of the filter membrane in each separation cavity being different, when the separation cavities are at least two, the separation cavities are sequentially connected in the order of the pore size of the filter membrane from large to small to form a graded filtration, a magnetic rotor capable of rotating under the action of external magnetic force being arranged in the separation cavity above the filter membrane, and the rotation axis of the magnetic rotor being parallel to the center line of the pore of the filter membrane, and the liquid discharge pipeline being connected with the waste liquid outlet of the separation cavity with the smallest pore size of the filter membrane for discharging waste liquid.

[0008] In the specific embodiment of the microfluidic device for exosome separation, the fluid flow above the filter membrane in the separation cavity during the circulation filtration, circulation cleaning and elution processes is perpendicular to the filtration direction of the filter membrane to form a tangential flow above the filter membrane; and / or

[0009] The microfluidic device for exosome separation further comprises a magnetic stirring driving unit, the magnetic stirring driving unit being located below the chip base, the magnetic stirring driving unit comprising a first support, a first driver, a first turntable and two magnets, the first driver being mounted on the first support, the first turntable being rotationally connected to the top end of the first support, the first driver being capable of rotating the first turntable, the first turntable being provided with magnets with two-pole magnetism, the magnets being capable of forming a magnetic field to drive the magnetic rotor to rotate during the rotation of the first turntable.

[0010] In the specific embodiment of the microfluidic device for exosome separation, the circulation pipeline module comprises a first conveying pipeline, a first return pipeline and a first circulation pipeline, one end of the first conveying pipeline being connected with the sample liquid inlet of the separation module and one end of the first return pipeline being connected with the sample liquid outlet of the separation module to form circulation filtration of sample liquid, the other end of the first conveying pipeline and the other end of the first return pipeline extending to the outside of the chip base for being inserted into a sample tube containing sample liquid, one end of the first circulation pipeline being in communication with the inlet of the separation cavity with the smallest pore size of the filter membrane, and the other end being in communication with the outlet of the separation cavity with the smallest pore size of the filter membrane for performing circulation filtration on the separated exosomes.

[0011] In the specific embodiment of the microfluidic device for exosome separation, the first circulation pipeline comprises a second conveying pipeline, a second return pipeline, a third return pipeline and a fourth return pipeline, one end of the second conveying pipeline is in communication with the inlet of the separation chamber with the smallest pore size of the filter membrane, one end of the second return pipeline is in communication with the outlet of the separation chamber with the smallest pore size of the filter membrane, the other end of the second return pipeline is in communication with one end of the fourth return pipeline through the third return pipeline, the other end of the fourth return pipeline is in communication with the other end of the second conveying pipeline, and the inlet and outlet of the separation chamber are both above the filter membrane in the separation chamber.

[0012] In the specific embodiment of the microfluidic device for exosome separation, the circulation pipeline module further comprises a cleaning input pipeline, a cleaning output pipeline and a fourth return pipeline, one end of the cleaning input pipeline is in communication with the other end of the second conveying pipeline, one end of the cleaning output pipeline is in communication with the other end of the second return pipeline through the fourth return pipeline, the other end of the cleaning input pipeline and the other end of the cleaning output pipeline both extend to the outside of the chip substrate, and the cleaning input pipeline, the second conveying pipeline, the second return pipeline, the fourth return pipeline and the cleaning output pipeline combine to form a circulating cleaning pipeline; and / or

[0013] The circulation pipeline module further comprises an elution input pipeline and an elution output pipeline, one end of the elution input pipeline is in communication with the other end of the second conveying pipeline, one end of the elution output pipeline is in communication with the second return pipeline through the third return pipeline, the other end of the elution input pipeline and the other end of the elution output pipeline both extend to the outside of the chip substrate, and the elution input pipeline, the second conveying pipeline, the second return pipeline, the third return pipeline and the elution output pipeline form an elution pipeline.

[0014] In the specific embodiment of the microfluidic device for exosome separation, the separation module comprises two separation chambers arranged in an up-down manner, two opposite side walls of the upper separation chamber are respectively provided with the sample liquid inlet and the sample liquid outlet for forming a tangential flow, one end of the sample liquid inlet is in communication with the first conveying pipeline, one end of the sample liquid outlet is in communication with the first return pipeline, one end of the second conveying pipeline is in communication with the inlet of the lower separation chamber, one end of the second return pipeline is in communication with the outlet of the lower separation chamber, and the inlet and the outlet of the lower separation chamber are respectively arranged on two opposite side walls of the separation chamber for forming a tangential flow.

[0015] In the specific embodiment of the microfluidic device for exosome separation, the chip base is provided with a mounting hole, an intermediate layer chip is fixed in the mounting hole, an upper layer chip is fixed at the top end of the mounting hole, and a lower layer chip is fixed at the bottom end of the mounting hole; the intermediate layer chip is provided with a first flow-through port; a cavity exists between the upper layer chip and the intermediate layer chip to form a separation cavity; a cavity exists between the intermediate layer chip and the lower layer chip to form another separation cavity; a filter membrane is fixed at the top end of the intermediate layer chip; another filter membrane is fixed at the top end of the lower layer chip; the waste liquid outlet is arranged on the side wall of the lower layer chip; and the inside of the lower layer chip is provided with a waste liquid cavity in communication with the waste liquid outlet so that the waste liquid generated after passing through the filter membrane enters the waste liquid cavity.

[0016] In the specific embodiment of the microfluidic device for exosome separation, a first hose section is arranged in the first return pipeline, a second hose section is arranged in the second conveying pipeline, and the first hose section and the second hose section are arranged in parallel in the same circular arc with different radii at the bottom of the chip base; and / or

[0017] A third hose section in the shape of a circular arc is arranged in the liquid discharge pipeline and at the bottom of the chip base; and / or

[0018] A sixth hose section is arranged in the fourth return pipeline, a fourth hose section parallel to the fourth hose section is arranged in the cleaning input pipeline and the cleaning output pipeline, and a fifth hose section parallel to the fifth hose section is arranged in the elution input pipeline and the elution output pipeline; and the fourth hose section, the fifth hose section, and the sixth hose section are arranged on the same circumferential track at the bottom of the chip base.

[0019] In the specific embodiment of the microfluidic device for exosome separation, the microfluidic device for exosome separation further comprises a support assembly, a first peristaltic pump unit, a second peristaltic pump unit and a fluid switching unit, the support assembly is used to install the microfluidic chip in a horizontal detachable fixed state, the first peristaltic pump unit is located below the chip base and can pressurize the first and second hose sections to make the fluid flow therein, the second peristaltic pump unit is located below the chip base and can pressurize the third hose section to make the waste liquid flow out of the drain pipeline, the fluid switching unit comprises a second support, a second driver, a second turntable and a lug, the second driver is installed on the second support, the second turntable is rotationally connected to the top end of the second support and is driven to rotate by the second driver, the top end edge part of the second turntable is fixed with two spaced lugs, and the two lugs can simultaneously close the fourth hose section and the fifth hose section or simultaneously close the fourth hose section and the fourth return pipeline or simultaneously close the fifth hose section and the fourth return pipeline through pressure sealing during rotation of the second turntable.

[0020] In a second aspect, the present application also provides a fully integrated nucleic acid / protein detection system, comprising an injection type nucleic acid / protein extraction unit, a detection system and the microfluidic device for exosome separation according to any one of the above technical solutions, the injection type nucleic acid / protein extraction unit injects the exosomes separated by the microfluidic device for exosome separation, and the injection type nucleic acid / protein extraction unit injects the exosomes into the detection system after lysis, nucleic acid / protein extraction and separation and purification.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] 1、The magnetic rotor is arranged above the filter membrane in each separation cavity, and the magnetic rotor can rotate above the filter membrane under the driving of the magnetic stirring driving unit to stir the fluid in the separation cavity, so that the particles blocking the pores of the filter membrane are resuspended, the problem of easy blocking of the filter membrane is alleviated, and the exosomes to be recovered are resuspended, and the separation efficiency and recovery rate of the exosomes are improved.

[0023] 2、The flow direction of the fluid in each separation cavity is perpendicular to the filtering direction of the filter membrane, so that the fluid forms a tangential flow relative to the filter membrane during transportation, which can also alleviate the blocking of the filter membrane to a certain extent and improve the separation efficiency and recovery rate of the exosomes.

[0024] 3. By the setting of the first peristaltic pump unit and the second peristaltic pump unit, one-way or multi-way fluid can be simultaneously driven and controlled by the aid of each hose segment designed on the microfluidic chip, and the fluid control efficiency is improved; by the setting of the fluid switching unit, the two bosses can directly press the fourth hose segment, the fifth hose segment and the fourth return pipeline during the rotation process, so as to realize the connection and cut-off of one-way or multi-way fluid controlled by one first driver at a time, and the working efficiency of the flow path switching is effectively improved without affecting the liquid path sealing of the microfluidic chip; the requirements of the full-closed and rapid separation of exosomes and the cleaning-free and disposable use of the microfluidic chip are met. BRIEF DESCRIPTION OF DRAWINGS

[0025] The preferred embodiments of the present application will be described below with reference to the accompanying drawings, in which:

[0026] Figure 1 is a front structure schematic diagram of the microfluidic chip provided by the present application;

[0027] Figure 2 is a back structure schematic diagram of the microfluidic chip provided by the present application;

[0028] Figure 3 is an exploded view of the microfluidic chip provided by the present application;

[0029] Figure 4 is an exosome separation principle schematic diagram of the microfluidic chip provided by the present application;

[0030] Figure 5 is a structure schematic diagram of the magnetic stirring driving unit;

[0031] Figure 6 is a flow path diagram for executing the cycle filtration of the present application;

[0032] Figure 7 is a flow path diagram for executing the cycle cleaning of the present application;

[0033] Figure 8 is a flow path diagram for executing the elution process of the present application;

[0034] Figure 9 is a structure schematic diagram of the second peristaltic pump unit;

[0035] Figure 10 is a structure schematic diagram of the first peristaltic pump unit;

[0036] Figure 11 is a structure schematic diagram of the fluid switching unit;

[0037] Figure 12 is a structure schematic diagram of the microfluidic chip installation and use.

[0038] LIST OF REFERENCE NUMERALS:

[0039] 1, chip base; 2, first conveying pipeline; 3, liquid discharge pipeline; 301, third hose section; 4, separation module; 41, upper layer chip; 42, magnetic rotor; 43, filter membrane; 44, lower layer chip; 45, middle layer chip; 5, first return pipeline; 51, first hose section; 6, second conveying pipeline; 61, second hose section; 7, second return pipeline; 71, second flow channel groove; 8, fourth return pipeline; 9, elution input pipeline; 10, elution output pipeline; 11, cleaning input pipeline; 12, cleaning output pipeline; 13, third return pipeline; 131, third flow channel groove; 14, fifth return pipeline; 141, fourth flow channel groove; 15, separation cavity; 16, waste liquid cavity; 17, first roller; 18, third driver; 19, third support; 20, protruding block; 21, second rotary disc; 22, second driver; 23, fourth driver; 24, second roller; 25, second rotary column; 26, magnet; 27, first driver; 28, first rotary disc; 29, fourth support; 30, second support; 31, first rotary column; 32, first flow channel groove; 33, support table; 34, supporting leg. DETAILED DESCRIPTION

[0040] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are some, but not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0041] In the description of the present application, it should be noted that the terms "upper", "lower", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the systems or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second" and the like are used to distinguish parts, and do not have special meanings unless otherwise stated.

[0042] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "setting", "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through intermediate medium, or the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0043] The present application relates to the field of biomedical technology, in particular to a microfluidic device for exosome separation and a full-integrated nucleic acid / protein detection system. The purpose is to solve the problem that in the process of separating exosomes by the existing ultrafiltration method, the exosomes are easy to block the filter holes, resulting in the decline of the filtration extraction efficiency, thereby reducing the separation efficiency; and in the elution process, the exosomes attached to the filter membrane are difficult to recover, thereby causing extraction loss and reducing the recovery rate. For this purpose, the microfluidic device for exosome separation provided by the present application comprises a microfluidic chip, the microfluidic chip comprises a chip base, a separation module, a circulation pipeline module and a liquid discharge pipeline arranged on the chip base, the circulation pipeline module is connected with the separation module for forming a circulation filtration, circulation cleaning and elution mode to finally separate the exosomes, the separation module comprises at least one separation cavity, one filter membrane is installed in each separation cavity, and the pore size of the filter membrane in each separation cavity is different, when there are at least two separation cavities, the separation cavities are sequentially connected in the order of the pore size of the filter membrane from large to small to form a fractional filtration, a magnetic rotor capable of rotating under the action of external magnetic force is arranged above the filter membrane in the separation cavity, and the rotation axis of the magnetic rotor is parallel to the center line of the filter membrane pore; the liquid discharge pipeline is connected with the waste liquid outlet of the separation cavity with the smallest filter membrane pore size for discharging waste liquid, and the flow direction of the fluid above the filter membrane in the separation cavity during the circulation filtration, circulation cleaning and elution process is perpendicular to the filtration direction of the filter membrane to form a tangential flow above the filter membrane; the present application arranges a magnetic rotor above the filter membrane in each separation cavity, which can rotate above the filter membrane under the driving of the magnetic stirring driving unit to stir the fluid in the separation cavity, thereby resuspending the particles blocking the filter membrane pores, relieving the problem of easy clogging of the filter membrane, and also resuspending the exosomes to be recovered, improving the separation efficiency and recovery rate of the exosomes; the flow direction of the fluid in each separation cavity is perpendicular to the filtration direction of the filter membrane, so that the fluid forms a tangential flow relative to the filter membrane during transportation, which also relieves the filter membrane clogging to some extent and improves the separation efficiency and recovery rate of the exosomes.

[0044] Next, the microfluidic device for exosome separation and the full-integrated nucleic acid / protein detection system provided by the embodiments of the present application will be described in detail in combination with the drawings.

[0045] Reference Figures 1-4The application provides a microfluidic device for separating exosomes, comprising a microfluidic chip, the microfluidic chip comprising a chip base 1, a separation module 4 arranged on the chip base 1, a circulating pipeline module and a liquid discharge pipeline 3, the circulating pipeline module being connected with the separation module 4 and used for forming a circulating filtration mode, a circulating cleaning mode and an elution mode to finally separate the exosomes, the separation module 4 comprising at least one separation cavity 15, one filter membrane 43 being arranged in each separation cavity 15, and the pore size of the filter membrane 43 in each separation cavity 15 being different, when the separation cavities are at least two, the separation cavities 15 are sequentially connected in the order of the pore size of the filter membrane 43 from large to small to form a fractional filtration, a magnetic rotor 42 capable of rotating under the action of external magnetic force being arranged in the separation cavity 15 above the filter membrane 43, and the liquid discharge pipeline 3 being connected with the waste liquid outlet of the separation cavity 15 with the smallest pore size of the filter membrane 43 and used for discharging waste liquid. In the application, the rotation axis of the magnetic rotor 42 can be parallel to the center line of the pore of the filter membrane 43.

[0046] In one embodiment, referring to Figure 4 , the fluid flow above the filter membrane 43 in the separation cavity 15 during the circulating filtration, the circulating cleaning and the elution process is perpendicular to the filtration direction of the filter membrane 43 to form a tangential flow above the filter membrane 43.

[0047] In the above embodiment, the filter membranes 43 in each separation cavity 15 have different pore sizes, and the fluid sequentially passes through the filter membranes 43 with different sizes to realize fractional filtration, so that the required exosomes can be filtered out. During the filtration process, the magnetic rotor 42 is driven to rotate to stir the fluid, so that the particles blocking the pores of the filter membrane 43 are resuspended, the problem of easy blocking of the filter membrane 43 is alleviated, and the exosomes to be recovered are also resuspended, so that the separation efficiency and recovery rate of the exosomes are improved. The fluid transportation in each separation cavity 15 is a tangential flow, which can wash away the particles blocking the pores of the filter membrane 43, also can alleviate the blocking of the filter membrane 43 to a certain extent, and improve the separation efficiency and recovery rate of the exosomes.

[0048] In one embodiment, referring to Figures 3-4 , the separation module 4 comprises two separation cavities 15 arranged in an up-down distribution, used for forming two-stage filtration, two opposite side walls of the upper separation cavity 15 are respectively provided with a sample liquid inlet and a sample liquid outlet for forming a tangential flow, the sample liquid inlet is connected with one end of the first conveying pipeline 2, the sample liquid outlet is connected with one end of the first backflow pipeline 5, the inlet of the lower separation cavity 15 is connected with one end of the second conveying pipeline 6, the outlet of the lower separation cavity 15 is connected with one end of the second backflow pipeline 7, and the inlet and the outlet of the lower separation cavity 15 are arranged on two opposite side walls of the separation cavity 15 for forming a tangential flow.

[0049] Specifically, the chip base 1 is provided with a mounting hole, the mounting hole is fixed with an intermediate layer chip 45, the top end of the mounting hole is fixed with an upper layer chip 41, the bottom end of the mounting hole is fixed with a lower layer chip 44, the intermediate layer chip 45 is provided with a first flow-through port, there is a cavity between the upper layer chip 41 and the intermediate layer chip 45 to form a separate cavity 15, there is a cavity between the intermediate layer chip 45 and the lower layer chip 44 to form another separate cavity 15, the two separate cavities 15 are communicated through the first flow-through port, the top end of the intermediate layer chip 45 is fixed with a filter membrane 43, the top end of the lower layer chip 44 is fixed with another filter membrane 43, a waste liquid outlet is arranged on the side wall of the lower layer chip 44, and the inside of the lower layer chip 44 is provided with a waste liquid cavity 16 communicated with the waste liquid outlet to enable the waste liquid generated after passing through the filter membrane 43 to enter the waste liquid cavity 16. The sample liquid outlet and the sample liquid inlet are both arranged on the side wall in the mounting hole. The magnetic rotor is placed in the separate cavity and can rotate under the action of the magnetic force driving unit and can be suspended in the separate cavity during rotation.

[0050] More specifically, the lower layer chip 44 includes a substrate and a cover plate, the substrate is provided with a second flow-through port, the cover plate is fixed on the bottom end of the substrate by bonding, there is a waste liquid cavity 16 communicated with the second flow-through port between the cover plate and the substrate, and one corner end of the substrate is provided with a waste liquid outlet communicated with the waste liquid cavity 16.

[0051] When the separation module 4 filters, the sample liquid enters the upper separate cavity 15 through the sample liquid inlet and is discharged through the sample liquid outlet, a tangential flow is formed in this flow process, the sample liquid is filtered by the filter membrane 43 during the flow process, the filtered fluid enters the lower separate cavity 15, and then is filtered by the filter membrane 43 in the separate cavity 15 again, the fluid remaining between the two filter membranes 43 is the required exosome, the filtered waste liquid enters the waste liquid cavity 16 through the second flow-through port and is discharged into the liquid discharge pipeline 3 through the waste liquid outlet, realizing the operation of separating the exosome through two-stage filtration, the exosome between the two filter membranes 43 circulates in the first circulation pipeline to filter out other small particles in the exosome, improving the separation purity of the exosome, and the tangential flow is used to alleviate the problem of blockage of the filter membrane 43 hole.

[0052] It should be noted that the number of separation cavities 15 corresponds to the number of filter membranes 43 one by one, and the number of separation cavities 15 is not specifically limited in the present application, which can be two to form two-stage filtration, or one or three or four, and can be flexibly set according to actual needs. For example, if the separation cavities are three and arranged in sequence from top to bottom, a third separation cavity is stacked above the two-stage filtration described above, the pore size of the filter membrane in the third separation cavity is the largest, and then arranged in sequence from top to bottom. At this time, the sample liquid first enters the uppermost separation cavity, and the fluid after the large impurities are removed by the first-stage filtration enters the middle-layer separation cavity for the second-stage filtration, and the fluid after the second-stage filtration enters the lower separation cavity for the third-stage filtration. The fluid remaining after the first-stage and second-stage filtration is returned to the sample liquid, thereby achieving the purpose of circulating filtration. The microfluidic device can also be connected in series, and by selecting a more fine filter membrane pore size, the 20nm-200nm exosomes recovered in the first stage can be recovered more finely. For example, two filter membranes with pore sizes of 50nm and 150nm are combined to perform secondary filtration on the 20nm-200nm exosomes recovered in the first stage, so that exosomes with a size of 50nm-150nm can be recovered.

[0053] In one embodiment, referring to Figures 1-3 , the circulation pipeline module includes a first conveying pipeline 2, a first return pipeline 5, and a first circulation pipeline. One end of the first conveying pipeline 2 is connected with the sample liquid inlet of the separation module 4, and one end of the first return pipeline 5 is connected with the sample liquid outlet of the separation module 4 to form circulating filtration of the sample liquid. The other end of the first conveying pipeline 2 and the other end of the first return pipeline 5 extend to the outside of the chip substrate 1 for insertion into a sample tube containing the sample liquid. One end of the first circulation pipeline is in communication with the inlet of the separation cavity 15 with the smallest pore size of the filter membrane 43, and the other end is in communication with the outlet of the separation cavity 15 with the smallest pore size of the filter membrane 43 for circulating filtration of the exosomes separated. When performing circulating filtration, the other end of the first conveying pipeline 2 and the other end of the first return pipeline 5 are inserted into the same sample tube to form circulating filtration of the sample liquid.

[0054] Specifically, as Figure 1 shown, the first flow channel and the second flow channel are respectively arranged at the bottom of the chip substrate 1. The first conveying pipeline 2 is in communication with the sample liquid inlet through the first flow channel, and the first return pipeline 5 is in communication with the sample liquid outlet through the second flow channel. The first flow channel and the second flow channel are respectively located on the two sides of the separation module 4, which is conducive to forming a tangential flow. The first flow channel and the second flow channel each include a first flow channel groove 32 and a first flow channel plate (the first flow channel plate is not shown in the figure in order to show the first flow channel groove 32). The first flow channel groove 32 is opened at the bottom of the chip substrate 1, and the first flow channel plate seals the first flow channel groove 32 to form the corresponding first flow channel or second flow channel.

[0055] Specifically, referring to Figures 1-3 and Figure 6 , the first circulation pipeline comprises the second delivery pipeline 6, the second return pipeline 7, the third return pipeline 13 and the fourth return pipeline 8, one end of the second delivery pipeline 6 is in communication with the inlet of the separation cavity 15 with the smallest pore size of the filter membrane 43, one end of the second return pipeline 7 is in communication with the outlet of the separation cavity 15 with the smallest pore size of the filter membrane 43, the other end of the second return pipeline 7 is in communication with one end of the fourth return pipeline 8 through the third return pipeline 13, the other end of the fourth return pipeline 8 is in communication with the other end of the second delivery pipeline 6, and the inlet and outlet of the separation cavity 15 are both above the filter membrane 43 located therein. The separation cavity 15 with the smallest pore size of the filter membrane 43 is used to separate the desired exosomes. The circulation filtration flow path is as shown in Figure 6 .

[0056] In the above embodiment, the filtered exosomes can flow in the first circulation pipeline, the exosomes can be filtered to remove the particles that do not meet the requirements, the separation and purification degree of the exosomes is improved, and the exosomes flow in circulation, form a tangential flow above the filter membrane 43, alleviate the problem of clogging of the pores of the filter membrane 43, and improve the separation efficiency and recovery rate of the exosomes.

[0057] In one embodiment, referring to Figures 1-3 and Figure 7 , the circulation pipeline module further comprises a cleaning input pipeline 11, a cleaning output pipeline 12 and a fourth return pipeline 14, one end of the cleaning input pipeline 11 is in communication with the other end of the second delivery pipeline 6, one end of the cleaning output pipeline 12 is in communication with the other end of the second return pipeline 7 through the fourth return pipeline 14, the other end of the cleaning input pipeline 11 and the other end of the cleaning output pipeline 12 both extend to the outside of the chip substrate 1, and the cleaning input pipeline 11, the second delivery pipeline 6, the second return pipeline 7, the fourth return pipeline 14 and the cleaning output pipeline 12 combine to form a circulation cleaning pipeline. The other end of the cleaning input pipeline 11 and the other end of the cleaning output pipeline 12 are both inserted into the cleaning liquid to form a circulation cleaning of the cleaning liquid. The cleaning liquid enters the second delivery pipeline 6 through the cleaning input pipeline 11, is delivered to the separation cavity 15 where the exosomes are separated through the second delivery pipeline 6, the cleaning liquid cleans the exosomes, the small particle impurities in the exosomes are filtered into the waste liquid cavity 16 through the filter membrane 43 and are discharged through the discharge pipeline 3, and the cleaning liquid that passes through the filter membrane 43 is returned to the original cleaning liquid through the second return pipeline 7, the fourth return pipeline 14 and the cleaning output pipeline 12, so as to achieve the purpose of cleaning and removing impurities. The cleaning liquid flow path is as shown in Figure 7 .

[0058] In one embodiment, referring to Figures 1-3 and Figure 8, the circulating pipeline module further comprises an elution input pipeline 9 and an elution output pipeline 10, one end of the elution input pipeline 9 is communicated with the other end of the second conveying pipeline 6, one end of the elution output pipeline 10 is communicated with the second backflow pipeline 7 through the third backflow pipeline 13, the other end of the elution input pipeline 9 and the other end of the elution output pipeline 10 are both extended to the outside of the chip substrate 1, and the elution input pipeline 9, the second conveying pipeline 6, the second backflow pipeline 7, the third backflow pipeline 13 and the elution output pipeline 10 form an elution pipeline. The other end of the elution input pipeline 9 and the other end of the elution output pipeline 10 are both inserted into the elution liquid to form an elution mode. The elution liquid is conveyed into the second conveying pipeline 6 through the elution input pipeline 9, enters the cavity where the separated exosomes are located through the second conveying pipeline 6, and elutes the exosomes in the separation cavity 15 and the exosomes adhered to the filter membrane 43 and flows back to the elution liquid through the second backflow pipeline 7, the third backflow pipeline 13 and the elution output pipeline 10, and the elution is repeated to elute all the exosomes, and the elution liquid flow path is as shown in Figure 8 In the present application, the circulating filtration operation is first performed, then the circulating cleaning operation is performed, and finally the elution operation is performed.

[0059] In the above embodiment, referring to Figure 2 The second backflow pipeline 7 comprises a second flow channel groove 71 and a second flow channel plate, the second flow channel groove 71 is opened at the bottom of the chip substrate 1, and the second flow channel plate seals the second flow channel groove 71 to form the second backflow pipeline 7. The third backflow pipeline 13 and the fourth backflow pipeline 14 are both arranged at the top end of the chip substrate 1, the third backflow pipeline 13 comprises a third flow channel groove 131 and a third flow channel plate, the fourth backflow pipeline 14 comprises a fourth flow channel groove 141 and a fourth flow channel plate, the third flow channel groove 131 and the fourth flow channel groove 141 are symmetrically arranged at the top end of the chip substrate 1, and the third flow channel plate and the fourth flow channel plate are both connected with the chip substrate 1 to seal the corresponding third flow channel groove 131 and fourth flow channel groove 141. Three-way pipelines, four-way pipelines and straight-through pipelines are opened at the bottom of the chip substrate 1, the three-way pipelines are used for respectively communicating with the third backflow pipeline 13, the elution output pipeline 10 and the fourth backflow pipeline 8. The four-way pipelines are used for respectively communicating with the fourth backflow pipeline 8, the second conveying pipeline 6, the cleaning input pipeline 11 and the elution input pipeline 9. The fourth backflow pipeline 14 is communicated with the cleaning output pipeline 12 at the bottom of the chip substrate 1 through the straight-through pipeline.

[0060] In one embodiment, a first soft pipe section 51 is arranged in the first backflow pipeline 5, and a second soft pipe section 61 is arranged in the second conveying pipeline 6, and the first soft pipe section 51 and the second soft pipe section 61 are distributed in the bottom of the chip substrate 1 in the same center circular arc with different radii. As shown in Figure 2As shown, the first hose section 51 is located outside the second hose section 61, and the first hose section 51 and the second hose section 61 are fixed at the bottom of the chip substrate 1 near one corner end.

[0061] In one embodiment, a third hose section 301 in an arc shape is arranged in the drain pipeline 3 and at the bottom of the chip substrate 1. As shown, Figure 2 As shown, the third hose section 301 is arranged diagonally to the first hose section 51.

[0062] In one embodiment, a sixth hose section is arranged in the fourth return pipeline 8, a fourth hose section parallel to each other is arranged in the cleaning input pipeline 11 and the cleaning output pipeline 12, and a fifth hose section parallel to each other is arranged in the elution input pipeline 9 and the elution output pipeline 10. The fourth hose section, the fifth hose section and the sixth hose section are located on the same circumferential track at the bottom of the chip substrate 1. As shown, Figure 2 As shown, the fourth hose section, the fifth hose section and the fourth return pipeline 8 are diagonally distributed on the chip substrate 1 with the separation module 4.

[0063] In one embodiment, referring to Figure 5 The microfluidic device for exosome separation further comprises a magnetic stirring driving unit, which is located below the chip substrate 1. The magnetic stirring driving unit comprises a first support, a first driver 27, a first rotating disc 28 and two magnets 26. The first driver 27 is installed on the first support, and the first rotating disc 28 is rotationally connected to the top end of the first support. The first driver 27 drives the first rotating disc 28 to rotate. The first rotating disc 28 is provided with the magnets 26 with two-pole magnetism. The magnets 26 can form a magnetic field to drive the magnetic rotor 42 to rotate in the process of following the first rotating disc 28 to rotate. In the present application, the magnets can be two, which are symmetrically distributed as shown. Figure 5 As shown, the two magnets are symmetrically distributed. It can also be a bar-shaped magnet with two poles, which are all within the protection scope of the present application.

[0064] In one embodiment, referring to Figures 9-11, the microfluidic device for exosome separation further comprises a support assembly, a first peristaltic pump unit, a second peristaltic pump unit and a fluid switching unit, the support assembly is used for mounting the microfluidic chip so that it is in a horizontal detachable fixed state, the first peristaltic pump unit is located below the chip base 1 and can press the first hose section 51 and the second hose section 61 to make the fluid in them flow, the second peristaltic pump unit is located below the chip base 1 and can press the third hose section 301 to make the waste liquid in it discharge from the liquid discharge pipeline 3, the fluid switching unit comprises a second support 30, a second driver 22, a second rotating disc 21 and a lug 20, the second driver 22 is mounted on the second support 30, the second rotating disc 21 is rotationally connected to the top end of the second support 30 and is driven to rotate by the second driver 22, the top end edge part of the second rotating disc 21 is fixed with two spaced lugs 20, the two lugs 20 can simultaneously close the fourth hose section and the fifth hose section, or simultaneously close the fourth hose section and the fourth return pipeline 8, or simultaneously close the fifth hose section and the fourth return pipeline 8 by pressing during the rotation of the second rotating disc 21. The fourth section pipe section and the fifth hose section are closed when the sample liquid is filtered, that is, the circulating filtering operation is performed; the fourth hose section and the fourth return pipeline 14 are closed when the circulating cleaning is performed; the fifth hose section and the fourth return pipeline 8 are closed when the elution is performed. The two end sidewalls of the lug 20 are inclined surfaces which play a transition guiding role.

[0065] Specifically, referring to Figure 9 , the first peristaltic pump unit comprises a third support 19, a third driver 18 and a first rotating column 31, the third driver 18 is fixed on the third support 19, the first rotating column 31 is rotationally connected to the top end of the third support 19 and is driven to rotate by the third driver 18, the first rotating column 31 is fixed with four first support shafts which are equally spaced and circumferentially distributed, two first rollers 17 which are parallelly distributed are rotationally connected to the first support shafts, one of the first rollers 17 is used for extruding the first hose section 51, and the other first roller 17 is used for extruding the second hose section 61. The third driver 18 drives the first rotating column 31 to rotate, thereby driving the first roller 17 to rotate, the first roller 17 extrudes the corresponding first hose section 51 or the second hose section 61 in the rotating process, thereby realizing the delivery of the fluid.

[0066] Specifically, referring to Figure 10 , the second peristaltic pump unit comprises a fourth support 29, a fourth driver 23 and a second rotating column 25, the fourth driver 23 is fixed on the fourth support 29, the second rotating column 25 is rotationally connected to the top end of the fourth support 29 and is driven to rotate by the fourth driver 23, the second rotating column is fixed with four second support shafts which are equally spaced and circumferentially distributed, a second roller 24 is rotationally connected to the second support shafts, the second roller 24 can extrude the corresponding third hose section 301 in the rotating process, thereby realizing the discharge of the waste liquid from the liquid discharge pipeline 3.

[0067] In the present application, a plurality of grooves are opened at the bottom of the chip substrate 1 for mounting each pipeline in the circulation pipeline module. The grooves where the first hose section 51, the second hose section 61 and the third hose section 301 are located are annular structures. The bottom of the chip substrate 1 in the area where the fourth hose section, the sixth hose section and the fifth hose section are located is provided with an annular groove for inserting the protrusion 20 in the fluid switching unit and being able to rotate along the annular groove. Two first rollers 17 are mounted on each first supporting shaft, achieving the purpose of simultaneous control of two-way fluid transportation. Through the arrangement of the first peristaltic pump unit, the second peristaltic pump unit and the fluid switching unit, one-way or multi-way fluid can be simultaneously driven and controlled by means of each hose section designed on the microfluidic chip, improving the fluid control efficiency. Through the arrangement of the fluid switching unit, the two protrusions can directly press the fourth hose section, the fifth hose section and the fourth return pipeline 8 during rotation, realizing that one first driver 27 controls one-way or multi-way fluid communication and cut-off at a time, improving the working efficiency of flow path switching while not affecting the liquid path sealing of the microfluidic chip; meeting the requirements of full-closed and rapid separation of exosomes and one-time use of the microfluidic chip.

[0068] In the above embodiment, the support assembly includes a support table 33 and a supporting leg 34, a plurality of supporting legs 34 are fixed on the support table 33, the first peristaltic pump unit, the second peristaltic pump unit, the magnetic stirring driving unit and the fluid switching unit are arranged on the support table 33, the supporting leg 34 is provided with a first connecting hole, the chip substrate 1 is provided with a second connecting hole corresponding to the first connecting hole, when the chip substrate 1 is placed on the support table, the first connecting hole and the second connecting hole can be connected by a screw to realize detachable fixing of the chip substrate 1 on the supporting leg, when the chip substrate is installed, the first roller 17, the second roller 24 and the protrusion 20 are in contact with the corresponding hose section to realize the corresponding function. When the microfluidic chip is used, only the screw needs to be disassembled to replace a new microfluidic chip, see Figure 12 .

[0069] It should be noted that although the above uses a screw to detachably and fixedly connect the chip substrate 1 and the support table, this is only an example, and other structures can also be used, such as a cover plate arranged directly above the support table, the chip substrate 1 is fixed by extrusion through the cover plate, the cover plate can be driven by a telescopic device to move up and down to separate or press the chip substrate 1, which is also within the protection scope of the present application.

[0070] In the present application, the first driver 27, the second driver 22 and the third driver 18 can be motors or rudders. With part of the hose in the microfluidic chip, one-way or multi-way pumping fluid is realized by the pressure separation of the roller and the hose.

[0071] In addition, in the detailed description of the present application, the pore size of the two filter membranes corresponding to the two separate cavities can also be changed as needed to adapt to the requirements of different separation and recovery objects, such as selecting the pore sizes of the two filter membranes to be 20 nm and 200 nm when separating and recovering exosomes, selecting the pore sizes of the two filter membranes to be 1 μm and 50 μm when separating and recovering tissue cells, selecting the pore sizes of the two filter membranes to be 100 nm and 50 μm when separating and recovering pathogenic bacteria, and the like; the two filter membranes of the separation cavity can also be simplified to one filter membrane as needed, and the pore size of the different filter membranes is selected to adapt to the requirements of different separation and recovery objects, such as selecting the pore size of the first filter membrane to be 1 μm when separating and recovering objects larger than 1 μm, and removing the second filter membrane.

[0072] On the other hand, the present application also provides a fully integrated nucleic acid / protein detection system, comprising an injection type nucleic acid / protein extraction unit, a detection system, and the microfluidic device for exosome separation of any one of the above technical solutions, the injection type nucleic acid / protein extraction unit injects the exosomes separated by the microfluidic device for exosome separation, the injection type nucleic acid / protein extraction unit performs lysis, nucleic acid / protein extraction, and separation and purification on the exosomes, and then injects the exosomes into the detection system for detection to give a medical molecular diagnostic result of the exosomes.

[0073] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the protection scope of the technical solutions of the embodiments of the present application.

Claims

1. A microfluidic device for exosome isolation, characterized by, The microfluidic chip comprises a chip base, a separation module arranged on the chip base, a circulation pipeline module and a liquid discharge pipeline, the circulation pipeline module is connected with the separation module to form a circulation filtration, circulation cleaning and elution mode to finally separate exosomes, the separation module comprises at least one separation cavity, a filter membrane is arranged in each separation cavity, and the pore size of the filter membrane in each separation cavity is different, when the separation cavities are at least two, the separation cavities are sequentially connected in the order of the pore size of the filter membrane from large to small to form a graded filtration, a magnetic rotor capable of rotating under the action of external magnetic force is arranged in the separation cavity above the filter membrane, and the liquid discharge pipeline is connected with the waste liquid outlet of the separation cavity with the smallest pore size of the filter membrane to discharge waste liquid. During the circulation filtration, circulation cleaning and elution process, the fluid flow direction above the filter membrane in the separation cavity is perpendicular to the filtration direction of the filter membrane to form a tangential flow above the filter membrane. The microfluidic device for separating exosomes further comprises a magnetic stirring driving unit, the magnetic stirring driving unit is located below the chip base, the magnetic stirring driving unit comprises a first support, a first driver, a first turntable and two magnets, the first driver is arranged on the first support, the first turntable is rotatably connected to the top end of the first support, the first driver drives the first turntable to rotate, the first turntable is provided with magnets with two-pole magnetism, and the magnets can drive the magnetic rotor to rotate in the magnetic field during the rotation of the first turntable. The circulation pipeline module comprises a first conveying pipeline, a first return pipeline and a first circulation pipeline, one end of the first conveying pipeline is connected with the sample liquid inlet of the separation module, one end of the first return pipeline is connected with the sample liquid outlet of the separation module to form a circulation filtration of sample liquid, the other end of the first conveying pipeline and the other end of the first return pipeline extend to the outside of the chip base to be inserted into a sample tube containing sample liquid, one end of the first circulation pipeline is connected with the inlet of the separation cavity with the smallest pore size of the filter membrane, and the other end is connected with the outlet of the separation cavity with the smallest pore size of the filter membrane to perform a circulation filtration on the separated exosomes.

2. The microfluidic device for exosome isolation according to claim 1, wherein, The first circulation pipeline comprises a second conveying pipeline, a second return pipeline, a third return pipeline and a fourth return pipeline, one end of the second conveying pipeline is connected with the inlet of the separation cavity with the smallest pore size of the filter membrane, one end of the second return pipeline is connected with the outlet of the separation cavity with the smallest pore size of the filter membrane, the other end of the second return pipeline is connected with one end of the fourth return pipeline through the third return pipeline, the other end of the fourth return pipeline is connected with the other end of the second conveying pipeline, and the inlet and outlet of the separation cavity are located above the filter membrane in the separation cavity.

3. The microfluidic device for exosome isolation of claim 2, wherein, The circulation pipeline module further comprises a cleaning input pipeline, a cleaning output pipeline and a first reflux pipeline, one end of the cleaning input pipeline is communicated with the other end of the second conveying pipeline, one end of the cleaning output pipeline is communicated with the other end of the second reflux pipeline through the first reflux pipeline, the other end of the cleaning input pipeline and the other end of the cleaning output pipeline both extend to the outside of the chip substrate, and the cleaning input pipeline, the second conveying pipeline, the second reflux pipeline, the first reflux pipeline and the cleaning output pipeline combine to form a circulating cleaning pipeline; The circulation pipeline module further comprises an elution input pipeline and an elution output pipeline, one end of the elution input pipeline is communicated with the other end of the second conveying pipeline, one end of the elution output pipeline is communicated with the second reflux pipeline through a third reflux pipeline, the other end of the elution input pipeline and the other end of the elution output pipeline both extend to the outside of the chip substrate, and the elution input pipeline, the second conveying pipeline, the second reflux pipeline, the third reflux pipeline and the elution output pipeline form an elution pipeline.

4. The microfluidic device for exosome isolation of claim 2, wherein, The separation module comprises two separation cavities arranged in an up-down manner, two opposite side walls of the upper separation cavity are respectively provided with the sample liquid inlet and the sample liquid outlet for forming a tangential flow, one end of the sample liquid inlet is communicated with the first conveying pipeline, and one end of the sample liquid outlet is communicated with the first reflux pipeline; one end of the second conveying pipeline is communicated with the inlet of the lower separation cavity, and one end of the second reflux pipeline is communicated with the outlet of the lower separation cavity; the inlet and the outlet of the lower separation cavity are arranged on two opposite side walls of the lower separation cavity for forming a tangential flow.

5. The microfluidic device for exosome isolation of claim 4, wherein, The chip substrate is provided with a mounting hole, an intermediate layer chip is fixed in the mounting hole, an upper layer chip is fixed at the top end of the mounting hole, and a lower layer chip is fixed at the bottom end of the mounting hole; the intermediate layer chip is provided with a first flow-through opening, a cavity is formed between the upper layer chip and the intermediate layer chip to form a separation cavity, and a cavity is formed between the intermediate layer chip and the lower layer chip to form another separation cavity; a filter membrane is fixed at the top end of the intermediate layer chip, another filter membrane is fixed at the top end of the lower layer chip, the waste liquid outlet is arranged on the side wall of the lower layer chip, and a waste liquid cavity is arranged in the lower layer chip and communicated with the waste liquid outlet so that the waste liquid generated after passing through the filter membrane enters the waste liquid cavity.

6. The microfluidic device for exosome isolation of claim 3, wherein, A first hose section is arranged in the first reflux pipeline, and a second hose section is arranged in the second conveying pipeline; the first hose section and the second hose section are arranged in parallel in the same circular arc with different radii and the same center at the bottom of the chip substrate; A third hose section in the shape of a circular arc is arranged in the liquid discharge pipeline and at the bottom of the chip substrate. The fourth return pipeline is provided with a sixth hose section, the cleaning input pipeline and the cleaning output pipeline are each provided with a fourth hose section parallel to each other, the elution input pipeline and the elution output pipeline are each provided with a fifth hose section parallel to each other, and the fourth hose section, the fifth hose section and the sixth hose section are located on the same circumferential track at the bottom of the chip substrate.

7. The microfluidic device for exosome isolation of claim 6, wherein, The microfluidic device for exosome separation further comprises a support assembly, a first peristaltic pump unit, a second peristaltic pump unit and a fluid switching unit, the support assembly is used for mounting the microfluidic chip so that it is in a horizontal detachable fixed state, the first peristaltic pump unit is located below the chip substrate and can pressurize the first hose section and the second hose section to make the fluid therein flow, the second peristaltic pump unit is located below the chip substrate and can pressurize the third hose section to make the waste liquid therein discharge from the liquid discharge pipeline, the fluid switching unit comprises a second support, a second driver, a second turntable and a lug, the second driver is mounted on the second support, the second turntable is rotationally connected to the top end of the second support and is driven to rotate by the second driver, the top end edge part of the second turntable is fixed with two spaced lugs, and the two lugs can simultaneously close the fourth hose section and the fifth hose section, or simultaneously close the fourth hose section and the fourth return pipeline, or simultaneously close the fifth hose section and the fourth return pipeline by pressurization during rotation of the second turntable.

8. A fully integrated nucleic acid / protein detection device, characterized in that, The microfluidic device for exosome separation according to any one of claims 1-7, an injection nucleic acid protein extraction unit, a detection system and the microfluidic device for exosome separation, the exosomes separated by the microfluidic device for exosome separation are injected into the injection nucleic acid protein extraction unit, the exosomes are lysed, nucleic acid protein extraction and separation and purification in the injection nucleic acid protein extraction unit, and then injected into the detection system for detection.

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