CN114247489A

CN114247489A - Microfluidic chip and exosome extraction method

Info

Publication number: CN114247489A
Application number: CN202111508045.6A
Authority: CN
Inventors: 董鸣; 王洪; 赵靓; 刘学宇
Original assignee: Guangzhou National Laboratory
Current assignee: Guangzhou National Laboratory
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2022-03-29
Anticipated expiration: 2041-12-10
Also published as: CN114247489B

Abstract

The invention relates to a micro-fluidic chip and an exosome extraction method. The micro-fluidic chip comprises a rotation center and at least one extraction mechanism, wherein the extraction mechanism comprises a sample injection unit, a capture unit and a collection unit, the collection unit is connected with the capture unit and is positioned at the downstream of the capture unit, the collection unit comprises a waste liquid pool, a target object pool and a three-way micro-channel, the waste liquid pool and the target object pool are communicated with a carrier filter pool through the three-way micro-channel, and when eluent flows into the three-way micro-channel, the pressure of a branch of the three-way micro-channel connected with the target object pool is lower than that of a branch of the three-way micro-channel connected with the waste liquid pool, so that the eluent containing the target object flows to the target pool. The loss amount is small when the micro-fluidic chip is used for extracting the target object.

Description

Microfluidic chip and exosome extraction method

Technical Field

The invention relates to the technical field of microfluidics, in particular to a microfluidic chip and an exosome extraction method.

Background

The exosome is an extracellular nanoscale vesicle formed by cells through an endocytosis-fusion-efflux process, the size of the vesicle is between 30nm and 150nm, and the content of the vesicle in blood is high. Exosomes have the properties and functions of transporting biologically active molecules, such as nucleic acids (mirnas), proteins and lipids, across the blood-brain barrier. The exosome is used as a carrier for communicating and transmitting substances among cells, plays a role in regulation in receptor cells, and plays a vital role in a series of physiological and pathological processes through research. At present, the exosomes are extracted mainly from body fluids such as blood, saliva, urine, cerebrospinal fluid, semen, saliva, pleural effusion, milk and the like.

The method for extracting the exosome mainly comprises the following steps: ultracentrifugation, density gradient centrifugation, polymer precipitation (PEG-base precipitation), ultrafiltration, magnetic bead immunization, and kit extraction. The ultracentrifugation method is the most common exosome purification means, and can separate vesicle particles with similar sizes by alternately carrying out low-speed centrifugation and high-speed centrifugation. The density gradient centrifugation method is to form a density stratum by ultracentrifugation and enrich exosomes for enrichment. The PEG-base precipitation method, which previously applied to the collection of viruses from samples such as serum, is now also used to precipitate exosomes, by exploiting the property of polyethylene glycol (PEG) to co-precipitate with hydrophobic proteins and lipid molecules, the principle of which may be related to competitive binding of free water molecules. The ultrafiltration centrifugation method is to use ultrafiltration membranes with different relative molecular masses to carry out selective separation. The magnetic bead immunization method is characterized in that specific markers (such as CD63 and CD9 protein) are arranged on the surface of the exosome, and the exosome can be adsorbed and separated after the exosome is incubated and combined with exosome vesicles by using magnetic beads coated with anti-marker antibodies.

However, the method for extracting exosomes by the above-mentioned exosome extraction method generally has the disadvantages of long time, complicated operation, large-scale instrument requirement, high cost and the like. Therefore, with the scientific progress, some microfluidic chips for realizing exosome extraction by adopting a microfluidic technology gradually appear. For example, the microfluidic chip in chinese patent CN 112517092A. When the microfluidic chips are used for extracting the exosomes, the operation is simple, multiple sample adding and sample changing are not needed, but the loss amount of the exosomes is high, and the amount of the harvested exosomes is low.

Disclosure of Invention

Based on this, it is necessary to provide a microfluidic chip capable of reducing the amount of exosome loss.

In addition, an exosome extraction method capable of reducing loss amount is also provided.

A microfluidic chip comprises a rotation center and at least one extraction mechanism, wherein the extraction mechanism comprises a sample introduction unit, a capture unit and a collection unit;

the sample introduction unit comprises a sample introduction pool and a separation pool, and the sample introduction pool is closer to the rotation center than the separation pool;

the capturing unit is connected with the sample injection unit and is positioned at the downstream of the sample injection unit, the capturing unit comprises an incubation pool, a carrier filtration pool, a loading liquid quantification pool and an eluent pool, the sample injection pool, the separation pool, the incubation pool and the carrier filtration pool are sequentially communicated, the loading liquid pool, the loading liquid quantification pool and the incubation pool are sequentially communicated, the eluent pool comprises an eluent storage cavity, an eluent quantification cavity, an eluent primary cavity and an eluent secondary cavity which are sequentially communicated, the eluent storage cavity is closer to the rotation center than the eluent quantification cavity, and the eluent secondary cavity is communicated with the carrier filtration pool;

the collecting unit is connected with the capturing unit and is positioned at the downstream of the capturing unit, the collecting unit comprises a waste liquid pool, a target object pool and a three-way micro-channel, and the waste liquid pool and the target object pool are communicated with the carrier filtering pool through the three-way micro-channel; when the eluent flows into the three-way micro-channel, the pressure of the branch of the three-way micro-channel connected with the target object pool is lower than that of the branch of the three-way micro-channel connected with the waste liquid pool, so that the eluent containing the target object flows to the target pool.

The research of the application shows that the traditional micro-fluidic chip utilizes the self offset of fluid in an acceleration state to complete liquid guiding, and realizes the control of different flow directions by changing the rotation direction, but the mode is easy to cause incomplete separation, and the waste liquid pool often contains more targets. Therefore, according to the micro-fluidic chip, when the eluent flows into the three-way micro-channel, the pressure of the branch of the three-way micro-channel connected with the target object pool is lower than that of the branch of the three-way micro-channel connected with the waste liquid pool, so that the eluent containing the target object flows to the target pool and does not enter the waste liquid pool, the target objects in the target object pool are more, the loss of the target object is reduced, and the yield of the target object is improved.

In one embodiment, the flow channel between the branch point of the three-way micro flow channel and the waste liquid pool is in an S-shaped bent structure.

In one embodiment, the separation cell comprises a first chamber, a second chamber and a connecting part for connecting the first chamber and the second chamber, the first chamber is communicated with the sample feeding cell, the connecting part is provided with a connecting channel for communicating the first chamber and the second chamber, and a micro channel for communicating the separation cell and the incubation cell is close to the connecting channel and has a depth smaller than that of the connecting channel.

In one embodiment, the microfluidic chip has a cover surface, a step structure is arranged in the connecting channel, the step structure is close to a microchannel communicating the separation tank and the incubation tank, and the surface of the step structure close to the cover surface is connected with the bottom surface of the microchannel communicating the separation tank and the incubation tank.

In one embodiment, a slope structure is arranged in the connecting channel, the slope structure is close to a micro channel communicating the separation pool and the incubation pool, and a slope surface of the slope structure is connected with a bottom surface of the micro channel communicating the separation pool and the incubation pool.

In one embodiment, the incubation pool comprises a filter membrane chamber and a mixing chamber communicated with the filter membrane chamber, the filter membrane chamber is communicated with the separation pool, the loading liquid quantitative pool is communicated with the mixing chamber, the filter membrane chamber is provided with a first filter membrane structure, the first filter membrane structure comprises a first base frame matched with the filter membrane chamber and a first filter membrane positioned on the first base frame, the first filter membrane is close to a liquid inlet of the filter membrane chamber, and the first base frame is close to a liquid outlet of the filter membrane chamber;

and/or a second filter membrane structure is arranged in the carrier filter tank, the second filter membrane structure comprises a second base frame matched with the carrier filter tank and a second filter membrane positioned on the second base frame, the second filter membrane is close to the liquid inlet of the carrier filter tank, and the second base frame is close to the liquid outlet of the carrier filter tank.

In one embodiment, a flow assisting structure is arranged between the first base frame and the liquid outlet of the membrane filtering chamber and/or between the second base frame and the outlet of the carrier filtering tank, and the flow assisting structure is step-shaped or slope-shaped.

In one embodiment, at least one of the separation cell and the incubation cell, the loading liquid quantitative cell and the incubation cell, the incubation cell and the carrier filtration cell, the eluent reservoir and the eluent quantitative cavity, the eluent quantitative cavity and the eluent primary cavity, the eluent primary cavity and the eluent secondary cavity, and the eluent secondary cavity and the carrier filtration cell are in communication through a siphon flow channel.

In one embodiment, the width of the siphon flow channel is 150 to 500 μm, and the depth of the siphon flow channel is 100 to 500 μm.

In one embodiment, the width of the flow channel between the branch point of the three-way micro flow channel and the waste liquid pool is 0.5 mm-5 mm, and the depth is 150 μm-700 μm.

An exosome extraction method is used for extracting exosomes, and comprises the following steps:

adding raw materials containing exosomes into a sample injection pool;

and controlling the rotating speed of the microfluidic chip so that the exosomes in the raw material enter a target pool under the action of eluent after the raw material sequentially passes through the separation pool, the incubation pool and the carrier filter pool.

In one embodiment, the step of controlling the rotation speed of the microfluidic chip so that the exosomes in the raw material enter the target object pool under the action of the eluent after the raw material sequentially passes through the separation pool, the incubation pool and the carrier filtering pool comprises:

s1: controlling the microfluidic chip to centrifuge at a first rotation speed, so that the raw material flows into the separation pool from the sample injection pool, the loading liquid enters the loading quantitative pool from the loading pool, and the eluent enters the eluent quantitative cavity from the eluent storage cavity;

s2: after the microfluidic chip is kept stand for the first time, the microfluidic chip is controlled to be centrifuged at a second rotating speed, so that the raw material enters an incubation pool, the loading liquid enters the incubation pool from a loading quantitative pool, and the eluent enters an eluent primary cavity from an eluent quantitative cavity;

s3: controlling the microfluidic chip to rotate alternately between a third rotating speed and a fourth rotating speed so as to mix the raw materials and the loading liquid in the incubation pool;

s4: after standing the microfluidic chip for a second time, controlling the microfluidic chip to centrifuge at a fifth rotating speed, so that the liquid in the carrier filter tank enters a waste liquid tank, and the eluent enters an eluent secondary cavity from the eluent primary cavity;

s5: and after standing the micro-fluidic chip for a third time, controlling the micro-fluidic chip to centrifuge at a sixth rotating speed, so that the eluent in the eluent secondary cavity enters the carrier filter tank and then enters the target object tank together with the exosome.

Drawings

FIG. 1 is a diagram of a microfluidic chip (without a cover plate) according to an embodiment;

FIG. 2 is a cover plate of the microfluidic chip shown in FIG. 1;

FIG. 3 is a perspective view of the microfluidic chip shown in FIG. 1;

FIG. 4 is an enlarged view of portion A of the microfluidic chip shown in FIG. 1;

FIG. 5 is an enlarged view of the portion C of the microfluidic chip shown in FIG. 3;

FIG. 6 is a flow chart illustrating the assembly of the first filter structure of the microfluidic chip shown in FIG. 1;

FIG. 7 is an enlarged view of a portion of the microfluidic chip shown in FIG. 1;

FIG. 8 is a perspective view of another embodiment of a microfluidic chip (without a cover plate);

FIG. 9 is an NTA test chart of example 1;

FIGS. 10 to 11 are electron micrographs of exosomes of example 1;

FIG. 12 is an NTA test chart of comparative example 1;

fig. 13 to 14 are electron micrographs of exosomes of comparative example 1.

Reference numerals:

10. a microfluidic chip; 101. a center of rotation; 102. a base plate; 103. a cover plate; 102a, a covering surface; 111. a sample introduction pool; 112. a separation tank; 112a, a first chamber; 112b, a second chamber; 112c, connecting channels; 131. an incubation pool; 131a, a filter membrane chamber; 131b, a mixing chamber; 131c, a first filter membrane structure; 131d, a first base frame; 131e, a first filter membrane; 131f, a first fixing member; 132. a carrier filtration tank; 133. a loading liquid pool; 134. a liquid loading quantitative pool; 135. an eluent reservoir; 136. an eluent quantification cavity; 137. a primary cavity of eluent; 138. an eluent secondary cavity; 151. a waste liquid tank; 152. a target pool; 152a, an inlet section; 152b, a liquid collecting part; 153. a three-way microchannel; 161. an eluent waste pool; 162. a whole blood waste pool; 163. a loading liquid waste tank; 164. a weight-reducing balancing cavity; 103a and a sample inlet; 103b, an eluent sample adding port; 103c, a loading solution sample port; 103d, a target object liquid taking port; 103e, air holes.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may be present. When the terms "vertical," "horizontal," "left," "right," "upper," "lower," "inner," "outer," "bottom," and the like are used to indicate an orientation or positional relationship, it is for convenience of description only based on the orientation or positional relationship shown in the drawings, and it is not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the present application. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, in this context, the depth of each cavity, chamber, well and flow channel refers to the distance from the bottom of the corresponding element to the closure surface.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1 and 2, an embodiment of the present application provides a microfluidic chip 10, which includes at least one extraction mechanism and a rotation center 101, wherein the extraction mechanism includes a sample injection unit, a capture unit, and a collection unit.

Specifically, the microfluidic chip 10 includes a bottom plate 102 and a cover plate 103 covering the bottom plate 102, where the bottom plate 102 has a covering surface 102a, and the cover plate 103 covers the covering surface 102 a. The rotation center 101 and the extraction mechanism are provided on the base plate 102. In some embodiments, the material of the bottom plate 102 and the cover plate 103 is independently selected from at least one of polymethyl methacrylate (PMMA), Polycarbonate (PC), and polyvinyl chloride (PVC), respectively. In an alternative specific example, the material of both the base plate 102 and the cover plate 103 is PC. In some embodiments, the base plate 102 and the cover plate 103 are bonded by an adhesive (e.g., double-sided glue). In other embodiments, the base plate 102 and the cover plate 103 are bonded by thermocompression. In some embodiments, the base plate 102 is the same material as the cover plate 103. In other embodiments, the base plate 102 and the cover plate 103 are not the same material. In one embodiment, the cover plate 103 is a coated film having adhesive capability. The base plate 102 is bonded to the cover plate 103 by the adhesive force of the cover plate 103. Specifically, the cover plate 103 includes a base film and a glue layer on the base film, and the glue layer is close to the bottom plate 102. In an alternative specific example, the glue layer is a hydrophobic glue layer. In an alternative specific example, the surface of the flow path on the base plate 102, on which the liquid flows, is hydrophilically modified with a modifying agent. The flow path is hydrophilically treated to facilitate liquid flow. It is understood that in other embodiments, the materials of the base plate 102 and the cover plate 103 are not limited to those described above.

In one embodiment, the thickness of the base plate 102 is 5 mm. It is understood that the thickness of the base plate 102 is not limited thereto, and may be adjusted according to circumstances. In the illustrated embodiment, the base plate 102 has a disk shape and the cover plate 103 has a disk shape.

Referring to fig. 3 to 5, the sample injection unit is used for sample injection and preliminary purification. The sample introduction unit comprises a sample introduction pool 111 and a separation pool 112 communicated with the sample introduction pool 111, and the sample introduction pool 111 is closer to the rotation center 101 than the separation pool 112. The sample feeding pool 111 is used for feeding and storing raw materials; the separation tank 112 is used for preliminary purification of the raw material entering from the sample inlet tank 111. In some embodiments, the separation cell 112 includes a first chamber 112a, a second chamber 112b, and a connection for connecting the first chamber 112a and the second chamber 112 b. The first chamber 112a is close to the sample cell 111, the first chamber 112a is closer to the rotation center 101 than the second chamber 112b, and the first chamber 112a is communicated with the sample cell 111; the connecting portion has a connecting passage 112c for communicating the first chamber 112a with the second chamber 112 b. In one embodiment, the connecting channel is a groove.

In some embodiments, a flow aid is also disposed within the first chamber 112a, the flow aid being proximate to the connection. Through the arrangement of the flow aid, the raw materials can flow more smoothly to the second chamber 112b, and the separation is facilitated. Optionally, the flow aid is stepped or ramped.

In the illustrated embodiment, the sample inlet cell 111 is substantially in the shape of an arc belt, and is disposed around the rotation center 101; the separation tank 112 is substantially hourglass-shaped; the connecting channel 112c is substantially an elongated groove; the flow aid is approximately step-shaped, and the number of the steps is three. It is understood that, in other embodiments, the shapes of the sample inlet cell 111, the separation cell 112, the connection channel 112c, and the flow aid are not limited to the above, and may be other shapes.

The capture unit is connected with the sample injection unit and is positioned at the downstream of the sample injection unit. The capture unit captures the target and purifies the target by binding the carrier to the target to form a complex. Specifically, the capturing unit includes an incubation tank 131, a carrier filtration tank 132, a loading solution tank 133, a loading solution quantification tank 134, and an eluate tank.

The incubation pool 131 is located downstream of the separation pool 112 and is in communication with the separation pool 112 and the loading solution quantification pool 134, respectively, and is a place where the target is further purified and combined with its specific carrier to form a complex. In some embodiments, the microchannel that connects the separation cell 112 and the incubation cell 131 is close to the connection channel 112c, and the depth of the microchannel is smaller than the depth of the connection channel 112 c. By setting the depth of the micro flow channel for connecting the separation cell 112 and the incubation cell 131 to be smaller than the depth of the connecting channel 112c, the liquid in the second chamber 112b is not easy to enter the micro flow channel for connecting the separation cell 112 and the incubation cell 131 during backflow, thereby affecting the purification effect of the separation cell 112 and further improving the extraction amount of the target substance.

Taking the example of extracting exosomes from whole blood, after the whole blood enters the sample cell 111, plasma containing exosomes enters the first chamber 112a due to centrifugation and remains in the first chamber 112a, and blood cells enter the second chamber 112b through the connecting channel 112c under the centrifugation, so that the separation of the plasma and the blood cells is realized. The research of the application discovers that the blood cells in the second chamber of the traditional micro-fluidic chip are easy to flow back to the connecting channel in the process of reducing the centrifugal action to nothing and the subsequent process of not receiving the centrifugal action, and at the moment, the blood cells are easy to enter the incubation pool from the micro-channel because the depth of the micro-channel for communicating the separation pool and the incubation pool is the same as that of the connecting channel, so that the purification effect of the incubation pool and the combination of the exosome and the carrier are influenced, and the exosome extraction effect is further influenced. Therefore, in some embodiments of the present application, the depth of the micro channel connecting the separation cell 112 and the incubation cell 131 is set to be smaller than the depth of the connection channel 112c, so that the blood cells in the second chamber 112b are not easy to enter the micro channel connecting the separation cell 112 and the incubation cell 131 during the backflow, thereby reducing the influence of the backflow on the purification effect of the incubation cell 131 and increasing the extraction amount of the exosomes. In addition, since the depth of the micro flow channel that communicates the separation cell 112 with the incubation cell 131 is set to be smaller than the depth of the connection channel 112c, the plasma entering the incubation cell 131 is more easily filtered. In this case, it is not necessary to provide a chamber and a channel for storing a diluent for diluting the plasma on the microfluidic chip 10, and the design of the microfluidic chip 10 is simplified.

Further, in one embodiment, a step structure is provided in the connecting channel 112c, the step structure is close to the micro flow channel communicating the separation cell 112 and the incubation cell 131, and the surface of the step structure close to the covering surface 102a is connected to the bottom surface of the micro flow channel communicating the separation cell 112 and the incubation cell 131. A step structure is provided in the connection channel 112c, so that the connection channel 112c forms a first flow channel for the circulation of the component having a higher specific gravity and a second flow channel for the circulation of the component having a lower specific gravity (the depth of the first flow channel is greater than that of the second flow channel), thereby making it difficult for the component having a higher specific gravity to enter a microchannel that communicates the separation cell 112 and the incubation cell 131. Taking the whole blood extraction exosome as an example, when blood cells flow back from the second chamber 112b, the blood cells flow through the first flow channel formed by the step structure and the connecting channel 112c, and the blood plasma mainly flows through the second flow channel, so that the blood cells are not easy to enter the micro flow channel connecting the separation cell 112 and the incubation cell 131.

In an alternative specific example, the connecting channel 112c is a strip-shaped groove, the depth of the connecting channel 112c is 500 μm, and the width of the connecting channel 112c is 1500 μm; the height of the step structure was 200 μm and the width of the step structure was 750 μm. The height of the step structure refers to the distance from the side of the step structure close to the covering surface 102a to the bottom surface of the connecting channel 112 c; the width of the stepped structure refers to the length of the stepped structure in the width direction of the connection passage 112 c.

In another embodiment, a slope structure is provided in the connecting channel 112c, the slope structure is close to the microchannel communicating the separation cell 112 and the incubation cell 131, and the slope surface of the slope structure is connected with the bottom surface of the microchannel communicating the separation cell 112 and the incubation cell 131. The slope structure is arranged on the same principle as the step structure, and the distance from the bottom surface of the connecting channel 112c to the covering surface 102a is larger than the distance from the bottom surface of the microchannel for communicating the separation cell 112 and the incubation cell 131 to the covering surface 102a, so that the components with high specific gravity are not easy to enter the microchannel for communicating the separation cell 112 and the incubation cell 131 to influence the purification effect of the incubation cell 131.

In some embodiments, the incubation pool 131 comprises a filter chamber 131a and a mixing chamber 131b in communication with the filter chamber 131a, the filter chamber 131a being closer to the center of rotation 101 than the mixing chamber 131b, the filter chamber 131a being further from the center of rotation 101 than the first chamber 112 a; the filter membrane chamber 131a communicates with the separation tank 112. In one embodiment, the filter chamber 131a communicates with the separation tank 112 through a siphon flow path. The filter membrane chamber 131a is communicated with the separation pool 112 through a siphon flow channel, so that the liquid in the first chamber 112a can fill the siphon flow channel through capillary action after standing, and then the liquid in the first chamber 112a can enter the filter membrane chamber 131a under the centrifugal action, and the liquid in the first chamber 112a cannot enter the filter membrane chamber 131a when not standing. The siphon flow channel between the filter membrane chamber 131a and the separation tank 112 is arranged, so that the filter membrane chamber 131a and the separation tank 112 can realize fluid control without additionally arranging a capillary valve.

In the present embodiment, the width of the siphon flow channel is 150 to 500 μm, and the depth of the siphon flow channel is 100 to 500 μm. In an alternative specific example, the width of the siphon flow channel is 360 μm and the depth of the siphon flow channel is 300 μm. In some embodiments, the siphon flow path is U-shaped, and the siphon flow path is provided with a capillary valve. In other embodiments, the siphon flow path is V-shaped without capillary valves on the siphon flow path. When the capillary-free valve is used, the centrifugal rotating speed is controlled, so that the centripetal force applied to the liquid is greater than the capillary force, the liquid cannot flow into the siphon flow channel, and the effect of the capillary-free valve is realized. Compared with the U-shaped siphon flow channel, the capillary effect in the V-shaped siphon flow channel is weaker, so that the flow path control is more stable and reliable. Of course, reference is made herein to the dimensions of the siphon flow path and whether or not a capillary valve is provided. It is understood that in other embodiments, the filter membrane chamber 131a and the separation tank 112 can be communicated in other manners.

The filter membrane chamber 131a is provided with a first filter membrane structure 131 c. The first filter membrane structure 131c includes a first base frame 131d fitted to the filter membrane chamber 131a and a first filter membrane 131e mounted on the first base frame 131 d. The first filter membrane 131e is close to the liquid inlet of the filter membrane chamber 131a, and the first base frame 131d is close to the liquid outlet of the filter membrane chamber 131 a. The first filter 131e is used for further purification of the liquid in the first chamber 112 a. In some embodiments, the first filter membrane structure 131c is removably located within the filter membrane chamber 131 a. By detachably disposing the first filter membrane structure 131c in the filter membrane chamber 131a, the installation of the first filter membrane 131e is facilitated. In addition, the first base frame 131d is matched with the first filter membrane 131e, so that liquid can vertically pass through the membrane, and the use efficiency of the filter membrane is improved. In other embodiments, the first filter membrane structure 131c is integrally formed with the filter membrane chamber 131 a.

Referring to FIG. 6, in some embodiments, the first filter membrane structure 131c further includes a first fixing member 131f for fixing the first filter membrane 131e in the first base frame 131 d. In the illustrated embodiment, the first base frame 131d has a strip-shaped groove with a hole at the bottom, and the first fixing member 131f has a frame shape. The arrangement is such that when the liquid flows through the first filter membrane 131e, the first base frame 131d provides better support for the first filter membrane 131e, thereby further improving the use efficiency of the filter membrane; the components of the first filter membrane structure 131c are conveniently assembled when detachably connected. In use, the first filter membrane 131e is placed in the first base frame 131d, and then the first fixing member 131f is placed in the first base frame 131d to fix the first filter membrane 131e between the first fixing member 131f and the first base frame 131d, thereby forming the first filter membrane structure 131 c. It is understood that, in other embodiments, the shapes of the first base frame 131d and the first fixing member 131f are not limited to the above.

In one embodiment, the filter membrane chamber 131a is provided with a filter membrane groove for placing the first base frame 131d, the depth of the filter membrane groove is greater than that of the filter membrane chamber 131a, and the first filter membrane 131e is located in the filter membrane groove near the bottom side of the filter membrane chamber 131 a. The effective area of the first filter membrane 131e of the filter membrane chamber 131a can be larger, which is beneficial to filtration. Further, the first filter membrane structure 131c is detachably located in the filter membrane tank. It is understood that in other embodiments, the first base frame 131d may be omitted.

It should be noted that "the first base frame 131d matched with the filter chamber 131 a" means that the shape of the first base frame 131d is configured to be compatible with the filter chamber 131a, so that the first filter structure 131c is matched with the filter chamber 131a to perform a filtering function. The following is the same as the "second base frame matched to the carrier filtration tank 132". In an alternative embodiment, the first filter 131e has a pore size of 220 nm. It is understood that in other embodiments, the pore size of the first filter 131e can be selected according to the particular species to be removed.

Further, in some embodiments, a flow aid structure is further disposed between the first base frame 131d and the liquid outlet of the filter membrane chamber 131 a. A flow aid structure is arranged between the first base frame 131d and the liquid outlet of the filter membrane chamber 131a, so that the filtrate passing through the first filter membrane 131e can enter the mixing chamber 131b more smoothly. Optionally, the flow facilitating structure is adjacent to the mixing chamber 131b and is stepped or ramped. In the illustrated embodiment, the filter membrane chamber 131a has a gradually decreasing width on the side thereof adjacent to the mixing chamber 131 b. This arrangement facilitates the entry of liquid in the filter chamber 131a into the mixing chamber 131 b.

The loading liquid reservoir 133 is used for containing a loading liquid (the loading liquid is a solution containing a carrier capable of specifically binding to the target). A loading liquid quantifying tank 134 is located downstream of the loading liquid tank 133 for quantifying the loading liquid. The loading liquid quantitative pool 134 is farther from the rotation center 101 than the loading liquid pool 133. The loading solution quantifying pool 134 is communicated with the incubation pool 131, so that the quantified loading solution can enter the incubation pool 131 to be combined with the target. Specifically, the loading liquid quantitative pool 134 communicates with the mixing chamber 131b, and the loading liquid quantitative pool 134 is closer to the rotation center 101 than the mixing chamber 131 b. In one embodiment, the loading solution quantitative reservoir 134 is in communication with the mixing chamber 131b through a siphon flow path.

A carrier filtration tank 132 is located downstream of the incubation tank 131 for filtering the carriers. Specifically, the carrier filtration tank 132 communicates with the mixing chamber 131 b. In one embodiment, a siphon flow path is provided between the carrier filtration tank 132 and the mixing chamber 131 b. In the illustrated embodiment, the mixing chamber 131b, the siphon flow path, the mixed liquid flow path, and the carrier filtering tank 132 are sequentially communicated. In some embodiments, a second filter membrane structure is disposed within the carrier filtration tank 132, the second filter membrane structure including a second base frame that mates with the carrier filtration tank 132 and a second filter membrane disposed on the second base frame. The second filter membrane is close to the liquid inlet of the carrier filter tank 132, and the second base frame is close to the liquid outlet of the carrier filter tank 132. The second base frame is arranged to facilitate the liquid to flow vertically through the membrane. The second filter membrane structure is used to intercept the carrier. In one embodiment, the pore size of the second filter is 2 μm to 4 μm. It will be appreciated that in other embodiments, the pore size of the second filter may be selected according to the particular species to be removed. In some embodiments, the second filter membrane structure is removably located within the carrier filter basin 132. By removably arranging the second filter membrane structure within the carrier filter basin 132, installation of the second filter membrane is facilitated. In other embodiments, the second filter membrane structure is integrally formed with the carrier filter basin 132.

In some embodiments, the second filter membrane structure further comprises a second fixing member for fixing the second filter membrane in the second base frame. In one embodiment, the second base frame is in the shape of a strip-shaped groove with a hole at the bottom, and the second fixing piece is in the shape of a frame. Due to the arrangement, when the liquid flows through the second filter membrane, the second base frame provides better support for the second filter membrane, so that the use efficiency of the filter membrane is further improved; and each part of the second filter membrane structure is convenient and fast to assemble when being detachably connected. When the filter membrane fixing device is used, the second filter membrane is firstly placed in the second base frame, and then the second fixing piece is placed in the second base frame so as to fix the second filter membrane between the second fixing piece and the second base frame, so that a second filter membrane structure is formed. It is to be understood that, in other embodiments, the shapes of the second base frame and the second fixing member are not limited to the above.

Further, a flow-aid structure is also provided between the second base frame and the outlet of the carrier filtration tank 132. A flow aid structure is arranged between the second base frame and the outlet of the carrier filter tank 132 to facilitate the filtrate of the carrier filter tank 132 to flow downstream. Optionally, the flow-assisting structure is adjacent to the three-way microchannel 153, and is stepped or sloped.

The eluent pool comprises an eluent storage cavity 135, an eluent quantitative cavity 136, an eluent primary cavity 137 and an eluent secondary cavity 138 which are sequentially communicated, the eluent storage cavity 135 is close to the rotating center 101, and the eluent secondary cavity 138 is communicated with the carrier filtering pool 132. The eluent storage cavity 135 is used for containing eluent; the eluent quantification cavity 136 is used for quantifying eluent; the eluent primary cavity 137 is used for containing quantitative eluent; the eluent secondary chamber 138 is adapted to receive eluent from the eluent primary chamber 137. By arranging the eluent pool as above, the eluent can be released at a designated time period when the microfluidic chip 10 is used to perform the function of separating the carrier from the target. In some embodiments, the eluent reservoir 135 and the eluent quantification chamber 136, the eluent quantification chamber 136 and the eluent primary chamber 137, the eluent primary chamber 137 and the eluent secondary chamber 138, and the eluent secondary chamber 138 and the carrier filter 132 are in communication via siphon flow channels. In the illustrated embodiment, the siphon channels are all V-shaped. Of course, in other embodiments, the communication of the eluent reservoir 135 with the eluent quantification chamber 136, the eluent quantification chamber 136 with the eluent primary chamber 137, the eluent primary chamber 137 with the eluent secondary chamber 138, and the eluent secondary chamber 138 with the carrier filter cell 132 is not limited to siphon flow channels.

It will be appreciated that in other embodiments, the eluent reservoir may be augmented or augmented with chambers to achieve control of eluent transport in addition to the illustrated embodiments. For example, as the number of raw material purifications increases or decreases based on the illustrated embodiment, the chamber of the eluent pool also increases or decreases accordingly, so that when the eluent reaches the carrier filtration pool 132, the liquid without the mixed liquid reaches the waste liquid pool 151 and the complex is trapped in the carrier filtration pool 132.

Referring to fig. 7, the collecting unit is connected to and located downstream of the capturing unit, and the collecting unit is mainly used for collecting the target object. The collection unit comprises a waste liquid pool 151, a target object pool 152 and a three-way micro-channel 153, wherein the waste liquid pool 151 and the target object pool 152 are communicated with the carrier filter pool 132 through the three-way micro-channel 153. The waste liquid tank 151 is used for storing filtrate formed when the composite formed by the target object and the carrier is filtered by the carrier filtering tank 132; the target reservoir 152 is used for collecting the eluent containing the target; the three-way micro-channel 153 is used for the carrier filter 132 to respectively communicate with the waste liquid tank 151 and the target object tank 152. The research of the application shows that the traditional micro-fluidic chip utilizes the self offset of fluid in an acceleration state to complete liquid guiding, and realizes the control of different flow directions by changing the rotation direction, but the mode is easy to cause incomplete separation, and the waste liquid pool often contains more targets. Therefore, according to the present invention, after the waste liquid tank 151 is filled with waste liquid without containing eluent, when eluent flows into the three-way micro channel 153, the pressure of the branch of the three-way micro channel 153 connected to the target tank 152 is lower than the pressure of the branch of the three-way micro channel 153 connected to the waste liquid tank 151, so that eluent containing target flows to the target tank without entering the waste liquid tank 151, and thus more target substances are contained in the target tank 152, the loss of the target substances is reduced, and the yield of the target substances is increased. In some embodiments, when the eluent flows into the three-way microchannel 153, the pressure of the branch of the three-way microchannel 153 connected to the target cell 152 is lower than the pressure of the branch of the three-way microchannel 153 connected to the waste liquid cell 151, and this is achieved by the absence of the air holes 103e in the waste liquid cell 151. The carrier filtration tank 132 fills the waste liquid tank 151 with a filtrate for filtering a complex formed by combining the target with the carrier. At this time, the eluent containing the target cannot enter the waste liquid pool 151 and the target pool 152.

Further, the flow channel between the branch point of the three-way micro flow channel 153 and the waste liquid tank 151 is in an S-shaped bent structure. By setting the flow channel between the branch point of the three-way micro flow channel 153 and the waste liquid pool 151 to be an S-shaped bent structure, the length of the flow channel is increased, and the mixing degree of the eluent entering the waste liquid pool 151 is reduced. As the waste liquid pool 151 is filled with the waste liquid, the eluent containing the target is more difficult to enter the waste liquid pool 151, and even if the eluent containing the target enters the flow channel, the amount thereof is extremely small. In one embodiment, the channel between the branch point of the three-way microchannel 153 and the waste reservoir 151 has a width of 0.5mm to 1.5mm and a depth of 150 μm to 700 μm. In an alternative specific example, the channel between the branch point of the three-way microchannel 153 and the waste liquid tank 151 has a width of 1mm and a depth of 500 μm.

In some embodiments, the branch of the three-way microchannel 153 connected to the carrier-filter 132 and the branch of the three-way microchannel 153 connected to the target cell 152 have an angle (α) of 30 ° to 90 °. In an alternative embodiment, the branch of the three-way microchannel 153 connected to the carrier-filter cell 132 is at an angle of 60 ° to the branch of the three-way microchannel 153 connected to the target cell 152. In the illustrated embodiment, the target reservoir 152 and the branch of the three-way microchannel 153 connected to the target reservoir 152 are located on the right side of the branch of the three-way microchannel 153 connected to the waste reservoir 151. It is understood that in other embodiments, the target reservoir 152 and the branch of the three-way microchannel 153 connected to the target reservoir 152 may also be located to the left of the branch of the three-way microchannel 153 connected to the waste reservoir 151.

Referring to FIG. 3, in some embodiments, the depth of each portion of the pool of target objects 152 is substantially the same.

Referring to fig. 8, in other embodiments, the target reservoir 152 includes an inlet portion 152a and a liquid collection portion 152b in communication with the inlet portion, the inlet portion 152a is in communication with the three-way microchannel 153, and the liquid collection portion 152b is deeper than the inlet portion 152 a. The target is communicated through the three-way microchannel 153, and the inlet part 152a reaches the liquid collecting part 152b, and the liquid collecting part 152b collects the target. The depth of the liquid collecting portion 152b is set to be deeper than the inlet portion 152a, so that the target can be taken out from the target tank 152. In the embodiment of fig. 8, the liquid collecting portion 152b is substantially cylindrical. It is understood that in other embodiments, the shape of the liquid trap 152b is not limited to a cylindrical shape, but may be other shapes.

In some embodiments, the microfluidic chip 10 further includes an eluent waste reservoir 161, a whole blood waste reservoir 162, and a loading solution waste reservoir 163. The eluent waste reservoir 161 is in communication with the eluent quantification chamber 136 and is adapted to hold an eluent in excess of the capacity of the eluent quantification chamber 136. A whole blood waste reservoir 162 communicates with the first chamber 112a for holding material in excess of the capacity of the first chamber 112 a. The loading liquid waste tank 163 is communicated with the loading liquid quantitative chamber, and is used for containing the loading liquid exceeding the capacity of the loading liquid quantitative chamber. Further, the waste eluent pool 161 is communicated with the eluent quantifying cavity 136 by adopting a wide-mouth flow channel, so that the inaccurate eluent quantification caused by a siphon effect is avoided. The whole blood waste reservoir 162 communicates with the first chamber 112a using a wide-mouth flow path to avoid a siphon effect and to allow less than theoretical amount of raw material for separation. The loading liquid waste tank 163 is communicated with the loading liquid quantitative cavity by adopting a wide-mouth flow channel so as to avoid the siphon effect and cause inaccurate loading liquid quantification. In an alternative embodiment, the wide flow channel has a width of 2mm and a depth of 500 μm.

In some embodiments, the microfluidic chip 10 further comprises a de-balancing chamber 164. The micro-fluidic chip 10 can be rotated in a centrifuge after sample loading by the arrangement of the de-weighting balancing cavity 164. In the illustrated embodiment, there are multiple weight-reducing trim cavities 164.

In the illustrated embodiment, the cover plate 103 is provided with a sample inlet 103a, an eluent sample inlet 103b, a loading solution sample inlet 103c, a target solution taking inlet 103d, and an air hole. The arrangement of the air holes enables the fluid to flow more smoothly when the fluid needs to flow. It will be appreciated that in other embodiments, eluent sample port 103b and loading solution sample port 103c can be omitted.

In the illustrated embodiment, the extraction mechanism is one. It will be appreciated that in other embodiments, the extraction mechanism may also be multiple.

In addition, an embodiment of the present application further provides an extraction method of exosomes, which uses the microfluidic chip 10 of any of the above embodiments to perform extraction.

In some embodiments, the above extraction method comprises the steps of: adding the raw material containing exosomes to a sample injection cell 111; and controlling the rotation speed of the microfluidic chip 10 so that the exosomes in the raw material enter the target cell under the action of the eluent after the raw material sequentially passes through the separation cell 112, the incubation cell 131 and the carrier filtration cell 132.

In one embodiment, the raw material is added in an amount of 250. mu.L to 550. mu.L. For example 500. mu.L. In some embodiments, the eluent is not predisposed in eluent reservoir 135; the loading liquid is not preset in the loading liquid bath 133. In use, an appropriate amount (e.g., 100 μ L to 250 μ L) of eluent is added to the eluent reservoir 135; an appropriate amount (e.g., 130. mu.L to 250. mu.L) of the loading liquid is added to the loading liquid bath 133. Of course, when the chip is already pre-loaded with the relevant reagents, no further addition is necessary.

In some embodiments, the step of controlling the rotation speed of the microfluidic chip 10 so that the exosomes in the raw material enter the target cell under the action of the eluent after the raw material passes through the separation cell 112, the incubation cell 131 and the carrier filtration cell 132 in sequence comprises:

s1: the micro-fluidic chip 10 is controlled to centrifuge at the first rotation speed, so that the raw material flows into the separation cell 112 from the sample injection cell 111, the loading liquid enters the loading quantitative cell from the loading cell, and the eluent enters the eluent quantitative cavity 136 from the eluent storage cavity 135.

After the first rotation speed centrifugation is finished, the raw material flows into the separation cell 112 from the sample inlet cell 111, wherein the plasma enters the first chamber 112a, the blood cells enter the second chamber 112b, and the redundant blood enters the whole blood waste cell 162; the loading liquid enters the loading quantitative pool from the loading pool, and the redundant loading liquid enters the loading liquid waste pool 163; the eluent flows from the eluent reservoir 135 to the eluent quantification chamber 136 and excess eluent flows to the eluent waste reservoir 161.

In one embodiment, the first rotation speed is 2500rpm to 5500rpm, and the corresponding centrifugation time is 120s to 480 s.

S2: after the microfluidic chip 10 is kept still for the first time, the microfluidic chip 10 is controlled to be centrifuged at the second rotating speed, so that the raw material enters the incubation pool 131, the loading liquid enters the incubation pool 131 from the loading quantitative pool, and the eluent enters the eluent primary cavity 137 from the eluent quantitative cavity 136.

After the microfluidic chip 10 is stood for the first time, the plasma in the first chamber 112a enters a siphon flow channel between the separation pool 112 and the filter membrane chamber 131a through capillary action, the loading liquid in the loading liquid quantification pool 134 enters a siphon flow channel between the loading liquid quantification pool 134 and the mixing chamber 131b through capillary action, the eluent in the eluent quantification chamber 136 enters a siphon flow channel between the eluent quantification chamber 136 and the eluent primary chamber 137 through capillary action, and conditions are provided for the plasma to enter the filter membrane chamber 131a, the loading liquid to enter the mixing chamber 131b and the eluent to enter the eluent primary chamber 137 (if the siphon flow channel between the separation pool 112 and the filter membrane chamber 131a is empty, the liquid in the separation pool 112 is difficult to enter the filter membrane chamber 131a even if the centrifugal speed is high, if the siphon flow channel between the loading liquid quantification pool 134 and the mixing chamber 131b is empty, the liquid in the loading liquid quantification pool 134 also enters the mixing chamber 131b, and if the siphon flow channel between the eluent quantification chamber 136 a and the eluent primary chamber 137 is empty The siphon flow channel of (a) is not filled with liquid, the liquid in the eluent quantifying cavity 136 is difficult to enter the eluent primary cavity 137). Then under the action of the second rotation speed, the plasma in the first chamber 112a enters the filter membrane chamber 131a and is filtered by the first filter membrane 131e, and the filtrate enters the mixing chamber 131 b; the loading liquid in the loading liquid quantitative pool 134 enters the mixing chamber 131 b; the eluent in the eluent quantification chamber 136 enters the eluent primary chamber 137. Optionally, the first time is 5s to 50 s. The second rotating speed is 2000-4500 r/min, and the corresponding centrifugal time is 15-120 s.

S3: the microfluidic chip 10 is controlled to alternately operate between the third rotation speed and the fourth rotation speed to mix the raw material and the loading solution in the sub-incubation pool 131.

Optionally, the third rotation speed is 2500-4000 rpm, and the corresponding centrifugation time is 3-20 s. The fourth rotating speed is 300-2000 r/min, and the corresponding centrifugal time T5 is 3-20 s. Of course, the direction of the alternate rotation is not particularly limited, and may be the same direction or the same direction. Alternatively, the number of alternate runs is 3 to 15.

S4: and after standing the microfluidic chip 10 for a second time, controlling the microfluidic chip 10 to centrifuge at a fifth rotation speed, so that the liquid in the carrier filter 132 passes through the second filter membrane and then enters the waste liquid tank 151, and the eluent enters the eluent secondary chamber 138 from the eluent primary chamber 137.

After the microfluidic chip 10 is stood still for the second time, the mixture in the mixing chamber enters the siphon flow channel between the carrier filter 132 and the mixing chamber 131b through capillary action, so that conditions are provided for the mixture in the mixing chamber 131b to enter the carrier filter 132; the eluent in the primary eluent chamber 137 enters a siphon flow channel between the primary eluent chamber 137 and the secondary eluent chamber 138 by capillary action, providing conditions for the eluent to enter the secondary eluent chamber 138. Then under the action of a fifth rotating speed, the liquid in the carrier filtering tank 132 enters the waste liquid tank 151, and the complex is intercepted in the carrier filtering tank 132; the eluent in the eluent primary chamber 137 enters the eluent secondary chamber 138.

Optionally, the second time is between 5s and 50 s. The fifth rotating speed is 2000-5000 r/min, and the corresponding centrifugal time is 20-120 s.

S5: and after the micro-fluidic chip 10 is kept stand for the third time, the micro-fluidic chip 10 is controlled to be centrifuged at the sixth rotating speed, so that the eluent in the eluent secondary cavity 138 enters the carrier filter tank 132 and then carries the exosome to enter the target object tank 152.

After the microfluidic chip 10 is stood still for the third time, the eluent in the eluent secondary cavity 138 enters the capillary flow channel between the eluent secondary cavity 138 and the carrier filter 132 through the capillary action, so as to provide conditions for the eluent to enter the carrier filter 132. Then under the action of the sixth rotation speed, the eluent in the eluent secondary cavity 138 enters the carrier filtering pool 132 and then acts on the complex to divide the complex into a carrier and exosomes, wherein the carrier is trapped in the carrier filtering pool 132, and the exosomes enter the target object pool 152 along with the eluent.

Optionally, the third time is 5s to 40 s. The sixth rotating speed is 2500-6500 r/min, and the corresponding centrifugal time is 30-120 s.

The extraction method of the exosome adopts the microfluidic chip 10 for extraction, only one sample is added in the whole extraction process, and the used reagent amount is small; the chip automatically carries out the operation steps of whole blood separation, exosome loading, exosome elution, waste liquid separation and the like, the overall extraction time of the chip can be as low as 5min at least, and the extraction can be finished only by 13min (the traditional method generally needs 3h20min) at most; compared with the method for extracting the exosome by adopting the microfluidic chip in CN112517092A, the method for extracting the exosome by adopting the microfluidic chip 10 has the advantages that the concentration of the exosome is improved by about 36.7%, and the obtained exosome is larger in quantity. In addition, compared with the method for extracting the exosome by adopting the microfluidic chip in CN112517092A, when the microfluidic chip 10 is adopted to extract the exosome, the centrifugal speed is lower (the centrifugal speed of eluent and loading liquid entering a corresponding quantitative cavity and whole blood entering a separation pool is reduced from 4000 rpm-7000 rpm to 2500 rpm-5500 rpm; the centrifugal speed of plasma and loading liquid entering an incubation pool and eluent entering a primary cavity of the eluent is reduced from 3000 rpm-5000 rpm to 2000 rpm-4500 rpm; and the centrifugal speed of the eluent entering a target pool after entering a carrier filter pool is reduced from 7000 rpm-9000 rpm to 2500 rpm-6500 rpm), so that the safety is better.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

The structure of the microfluidic chip adopted in this embodiment is shown in fig. 1, wherein some parameters of the microfluidic chip of this embodiment are as follows: a loading solution quantification pool (120 mu L) and an eluent quantification cavity (200 mu L).

The depth of the connecting channel is 500 μm, and the width of the connecting channel is 1500 μm; the height of the step structure was 200 μm and the width of the step structure was 750 μm. The width of the V-shaped siphon flow channel is 360 mu m, and the depth of the V-shaped siphon flow channel is 300 mu m.

In this example, whole blood was used to extract exosomes, and the specific process parameters are shown in table 1.

TABLE 1

After measurement, 200 mu L of eluent is eluted, and about 190 mu L of exosome collecting liquid is finally obtained in the target object pool; the collection was subjected to NTA (nanopartile Tracking analysis) to follow the exosomes, as shown in FIG. 9, with an average particle size around 93.7nm and an exosome concentration of about 4.1E +6 Particles/mL. The collected liquid was examined by electron microscopy (SEM) and the results are shown in FIGS. 10 and 11.

Example 2

The structure of the microfluidic chip used in this example is substantially the same as that of example 1, except that the volume of the eluent quantification cell of the microfluidic chip used in this example is 250 μ L, and the parameters in the extraction method are different, and the parameters of the extraction method of this example are shown in table 2.

TABLE 2

After measurement, 250 mu L of eluent is eluted, and about 240 mu L of exosome collecting liquid is finally obtained in the target object pool; the collection was subjected to NTA (nanopartile Tracking analysis) to follow the exosomes, with an average particle size around 103.5nm and an exosome concentration of about 4.0E +6 Particles/mL.

Example 3

The structure of the microfluidic chip used in this example is substantially the same as that of example 1, except that the volume of the eluent quantification cell of the microfluidic chip used in this example is 250 μ L, and the parameters in the extraction method are different, and the parameters of the extraction method of this example are shown in table 3.

TABLE 3

After measurement, 250 mu L of eluent is eluted, and about 240 mu L of exosome collecting liquid is finally obtained in the target object pool; the sample solution was subjected to NTA (nanoparticle Tracking analysis) to track exosomes with average particle size around 100.9nm and exosome concentration of about 3.7E +6 Particles/mL.

Comparative example 1

The method adopts ultra-separation to extract exosomes in plasma, and comprises the following specific steps:

1. 200. mu.L of plasma was diluted with 10ml of PBS and centrifuged at 220g and 4 ℃ for 10min in T1, and the supernatant was collected.

2. The supernatant obtained in step 1 was centrifuged at 2000g at 4 ℃ for 20min, T2 was collected.

3. The supernatant obtained in step 2 was centrifuged at 15000g and 4 ℃ for 30min at T3, and the supernatant was collected.

4. The supernatant obtained in step 3 was filtered through a 0.22 μm syringe filter to remove particles having a diameter of 220nm or more.

5. The supernatant from step 4 was transferred to an ultracentrifuge tube and centrifuged at 120000g at 4 ℃ for 70min T4.

6. The supernatant was discarded, an appropriate amount of PBS (100 μ L) was added to resuspend the exosome pellet, and step 5 was repeated (T5 ═ 70 min).

7. The supernatant was discarded, and an appropriate amount of PBS (100. mu.L) was added to resuspend the exosome pellet to obtain an exosome solution.

The test results for exosomes collected in the comparative example are as follows:

the resulting collection was subjected to NTA (nanopartile Tracking analysis) to track exosomes, and as shown in FIG. 12, the exosome concentration was about 2.3E +6Particles/mL at a particle size of about 133.9 nm. The collected liquid was examined by electron microscopy (SEM), and the results are shown in FIGS. 13 and 14.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims

1. The microfluidic chip is characterized by comprising a rotation center and at least one extraction mechanism, wherein the extraction mechanism comprises a sample introduction unit, a capture unit and a collection unit;

2. The microfluidic chip according to claim 1, wherein the flow channel between the branch point of the three-way micro-channel and the waste liquid pool is in an S-shaped bent structure.

3. The microfluidic chip according to claim 1, wherein the separation cell comprises a first chamber, a second chamber and a connecting portion for connecting the first chamber and the second chamber, the first chamber is communicated with the sample injection cell, the connecting portion has a connecting channel for communicating the first chamber and the second chamber, and a microchannel for communicating the separation cell and the incubation cell is close to the connecting channel and has a depth smaller than that of the connecting channel.

4. The microfluidic chip according to claim 3, wherein the microfluidic chip has a capping surface, a step structure is disposed in the connecting channel, the step structure is adjacent to the microchannel communicating the separation chamber and the incubation chamber, and a surface of the step structure adjacent to the capping surface is connected to a bottom surface of the microchannel communicating the separation chamber and the incubation chamber.

5. The microfluidic chip according to claim 3, wherein a slope structure is disposed in the connecting channel, the slope structure is close to the micro channel connecting the separation pool and the incubation pool, and a slope surface of the slope structure is connected to a bottom surface of the micro channel connecting the separation pool and the incubation pool.

6. The microfluidic chip according to claim 1, wherein the incubation pool comprises a filter membrane chamber and a mixing chamber in communication with the filter membrane chamber, the filter membrane chamber is in communication with the separation pool, the loading liquid quantification pool is in communication with the mixing chamber, the filter membrane chamber is provided with a first filter membrane structure, the first filter membrane structure comprises a first base frame matched with the filter membrane chamber and a first filter membrane positioned on the first base frame, the first filter membrane is close to a liquid inlet of the filter membrane chamber, and the first base frame is close to a liquid outlet of the filter membrane chamber;

7. The microfluidic chip according to claim 6, wherein a flow assisting structure is disposed between the first base frame and the liquid outlet of the membrane filtering chamber and/or between the second base frame and the outlet of the carrier filtering tank, and the flow assisting structure is stepped or sloped.

8. The microfluidic chip according to claim 1, wherein at least one of the separation pool and the incubation pool, the loading liquid quantitative pool and the incubation pool, the incubation pool and the carrier filtration pool, the eluent reservoir and the eluent quantitative cavity, the eluent quantitative cavity and the eluent primary cavity, the eluent primary cavity and the eluent secondary cavity, and the eluent secondary cavity and the carrier filtration pool are in communication via a siphon flow channel.

9. The microfluidic chip according to claim 8, wherein the width of the siphon flow channel is 150 μm to 500 μm, and the depth of the siphon flow channel is 100 μm to 500 μm.

10. The microfluidic chip according to any one of claims 1 to 9, wherein the channel between the branch point of the three-way microchannel and the waste liquid tank has a width of 0.5mm to 1.5mm and a depth of 150 μm to 700 μm.

11. An exosome extraction method, characterized in that the microfluidic chip of any one of claims 1 to 10 is used for extraction, and the extraction method comprises the following steps:

adding raw materials containing exosomes into a sample injection pool;

12. The extraction method according to claim 11, wherein the step of controlling the rotation speed of the microfluidic chip so that the exosomes in the raw material enter the target object pool under the action of the eluent after the raw material passes through the separation pool, the incubation pool and the carrier filtering pool in sequence comprises: