CN114269474A

CN114269474A - Microfluidic detection chip and application method thereof

Info

Publication number: CN114269474A
Application number: CN202080000131.3A
Authority: CN
Inventors: 胡涛; 袁春根; 崔皓辰
Original assignee: BOE Technology Group Co Ltd; Beijing BOE Health Technology Co Ld
Current assignee: BOE Technology Group Co Ltd; Beijing BOE Health Technology Co Ld
Priority date: 2020-02-14
Filing date: 2020-02-14
Publication date: 2022-04-01
Anticipated expiration: 2040-02-14
Also published as: CN114269474B; WO2021159521A1

Abstract

The embodiment of the disclosure provides a microfluidic detection chip and a use method thereof, wherein the microfluidic detection chip comprises: the liquid storage layer comprises a first liquid storage hole and a second liquid storage hole; a cap layer, comprising: a through hole corresponding to the first liquid storage hole and a sample adding hole corresponding to the second liquid storage hole; the bottom cover layer comprises a through hole corresponding to the first liquid storage hole; first sealing layer is located between stock solution layer and the bottom cap layer, includes: a first sealing structure configured to seal the first reservoir well; the micro flow channel layer is located between first sealing layer and the bottom cap layer, includes: a first opening corresponding to the first liquid storage hole, a second opening corresponding to the second liquid storage hole, and a first micro channel communicating the first opening and the second opening; the first micro-channel is positioned on one side of the micro-channel layer facing the bottom cover layer; the first elastic sealing layer is positioned on one side of the micro-flow channel layer, which is away from the bottom cover layer; and the second elastic sealing layer is positioned between the liquid storage layer and the top cover layer and comprises sample adding holes corresponding to the second liquid storage holes.

Description

Microfluidic detection chip and application method thereof

Technical Field

The disclosure relates to the technical field of biomedicine, in particular to a microfluidic detection chip and a using method thereof.

Background

The microfluidic technology is a technology for accurately controlling and controlling micro-scale fluid, and can integrate basic operation units such as samples, reaction, separation, detection and the like in the process of detection and analysis on a micro-nano-scale chip to automatically complete the whole analysis process. The microfluidic technology has the advantages of less sample consumption, high detection speed, simple and convenient operation, multifunctional integration, small volume, convenience in carrying and the like, and has great application potential in the fields of biology, chemistry, medicine and the like.

However, the microfluidic detection chip with a microstructure on the market at present needs to be manually mixed and the like, and cannot be used for mixing and the like a sample to be detected and a pre-stored liquid, so that the detection steps are complex and the detection speed is slow.

Disclosure of Invention

The utility model discloses a micro-fluidic detection chip who provides of implementing, wherein, includes:

a liquid storage layer comprising: a first reservoir configured to store a pre-sealed reagent, and a second reservoir configured to store a sample to be tested;

the top cover layer is positioned on one side of the liquid storage layer; the cap layer, comprising: the through hole corresponds to the first liquid storage hole, and the sampling hole corresponds to the second liquid storage hole;

the bottom cover layer is positioned on one side of the liquid storage layer, which is far away from the top cover layer; the bottom cap layer, comprising: the through hole corresponds to the first liquid storage hole in position;

the first sealing layer can be punctured and is positioned between the liquid storage layer and the bottom cover layer; the first sealing layer includes: a first sealing structure configured to seal the first reservoir well;

the micro-channel layer is positioned between the first sealing layer and the bottom cover layer; the microfluidic layer includes: the first opening hole corresponds to the position of the first liquid storage hole, the second opening hole corresponds to the position of the second liquid storage hole, and the first micro channel is communicated with the first opening hole and the second opening hole; the first micro-channel is positioned on one side of the micro-channel layer facing the bottom cover layer;

the first elastic sealing layer is positioned on one side, away from the bottom cover layer, of the micro-flow channel layer;

the second elastic sealing layer is positioned between the liquid storage layer and the top cover layer; the second elastomeric sealing layer comprising: and the sample adding hole corresponds to the second liquid storage hole in position.

Optionally, in an embodiment of the present disclosure, the first sealing layer further includes: a second sealing structure configured to seal the second reservoir well; the bottom cover layer is provided with a through hole corresponding to the second liquid storage hole.

Optionally, in an embodiment of the present disclosure, the method further includes: a second sealing layer that is pierceable;

the second sealing layer is positioned between the second elastic sealing layer and the liquid storage layer;

the second sealing layer comprising: a third sealing structure configured to seal the first reservoir well, and a fourth sealing structure configured to seal the second reservoir well.

Optionally, in an embodiment of the present disclosure, the liquid storage layer further includes: the waste liquid tank penetrates through the first drainage hole of the liquid storage layer, and a second micro-channel is communicated between the waste liquid tank and the first drainage hole;

the waste liquid groove and the second micro-channel are positioned on the surface of one side of the liquid storage layer facing the top cover layer;

the micro-channel layer further comprises: and the second drainage holes penetrate through the micro-channel layer and are communicated with the first drainage holes and the first micro-channel.

Optionally, in an embodiment of the present disclosure, the method further includes: an air pressure balancing hole;

the air pressure balance hole penetrates through the top cover layer and the second elastic sealing layer, the position of the air pressure balance hole corresponds to that of the waste liquid groove, and the air pressure balance hole is configured to communicate the waste liquid groove with the atmosphere.

Optionally, in an embodiment of the present disclosure, the method further includes: an adhesive layer between the microfluidic layer and the first sealing layer; the adhesive layer is configured to adhere the micro flow channel layer and the liquid storage layer.

Optionally, in an embodiment of the present disclosure, the liquid storage layer further includes: the air pressure adjusting hole penetrates through the liquid storage layer, and the third micro-channel is communicated with the second liquid storage hole and the air pressure adjusting hole;

the third micro-channel is positioned on the surface of one side of the liquid storage layer facing the bottom cover layer;

the first sealing layer further comprises: a fifth sealing structure configured to seal the air pressure adjusting hole;

and through holes corresponding to the positions of the air pressure adjusting holes are formed in the top cover layer, the micro flow channel layer, the first elastic sealing layer and the bottom cover layer.

Optionally, in an embodiment of the present disclosure, an edge of the air pressure adjusting hole on a side facing the bottom cover layer is stepped;

the fifth seal structure and a part of the adhesive layer are embedded in the stepped edge of the air pressure adjusting hole.

Optionally, in an embodiment of the present disclosure, the method further includes: a substrate layer located between the first elastomeric sealing layer and the bottom cap layer;

an antibody coating area is arranged on the surface of one side of the substrate layer close to the top cover layer;

the first elastomeric sealing layer comprising: a plurality of communicating pores corresponding to the positions of the antibody-coated regions;

the position of each communication hole corresponds to the position of the first microchannel.

Optionally, in an embodiment of the present disclosure, the substrate layer includes: the biosensor is positioned in the antibody coating area, and the coating layer is positioned on the surface of the biosensor;

the surface of the coating layer is coated with an antibody.

Optionally, in an embodiment of the present disclosure, the substrate layer is made of plastic, glass, or silicon material; or, the substrate layer is made of a printed circuit board.

Optionally, in an embodiment of the present disclosure, the substrate layer and the bottom cover layer are an integral structure.

Optionally, in an embodiment of the present disclosure, the sample application hole in the second elastic sealing layer is a flip-top sealing port.

Optionally, in an embodiment of the present disclosure, the top cover layer and the bottom cover layer are provided with a plurality of engaging structures on surfaces of sides facing the liquid storage layer;

the liquid storage layer is provided with a plurality of grooves which are respectively in one-to-one correspondence with the positions of the clamping structures;

clamping holes which are in one-to-one correspondence with the positions of the clamping structures are formed in each film layer between the clamping structures and the grooves;

the clamping structure is inserted into the corresponding groove through the corresponding clamping hole.

Correspondingly, the embodiment of the disclosure also provides a use method of the microfluidic detection chip, which includes:

adding the sample to be detected into the second liquid storage hole through the sample adding hole;

controlling the pre-sealed reagent in the first liquid storage hole to be injected into the first micro-channel;

and controlling the sample to be detected in the second liquid storage hole to be injected into the first micro-channel.

Optionally, in an embodiment of the present disclosure, the controlling injection of the pre-sealed reagent in the first reservoir hole into the first micro flow channel includes:

and injecting the pre-sealed reagent in the first liquid storage hole into the first micro-channel by squeezing the second elastic sealing layer at the position corresponding to the first liquid storage hole.

Optionally, in an embodiment of the present disclosure, the sample adding hole in the second elastic sealing layer is a flip-top sealing port;

after will examining the sample to be added in the second stock solution hole through the application of sample hole, control examine in the second stock solution hole examine the sample inject into before in the first microchannel, still include:

closing the flip-top sealable opening;

piercing the fifth seal structure;

and repeatedly extruding the second elastic sealing layer corresponding to the position of the second liquid storage hole for multiple times so as to uniformly mix the sample to be detected with the diluent prestored in the second liquid storage hole.

Optionally, in this disclosure, the controlling the sample to be detected in the second liquid storage hole to be injected into the first micro flow channel includes:

the second elastic sealing layer is arranged at the position corresponding to the air pressure adjusting hole through extrusion, so that the sample to be detected in the second liquid storage hole is injected into the first micro-channel.

Drawings

Fig. 1 is a schematic view of a layered structure of a microfluidic detection chip provided in an embodiment of the present disclosure;

fig. 2 is a schematic front view of a microfluidic detection chip according to an embodiment of the present disclosure;

FIG. 3 is a schematic diagram of a side view of a microfluidic detection chip for use in an embodiment of the disclosure;

fig. 4 is a second schematic side view of a microfluidic detection chip for use in embodiments of the present disclosure;

FIG. 5 is a schematic bottom view of a microfluidic detection chip for use with embodiments of the present disclosure;

FIG. 6 is a schematic structural diagram of a cap layer in an embodiment of the present disclosure;

FIG. 7 is a schematic structural view of a second elastomeric sealing layer in an embodiment of the present disclosure;

FIG. 8 is a schematic structural view of a second sealant layer in an embodiment of the present disclosure;

FIG. 9 is a schematic view of a liquid storage layer according to an embodiment of the disclosure;

FIG. 10 is a second schematic view illustrating a structure of a liquid storage layer according to an embodiment of the present disclosure;

FIG. 11 is a schematic structural view of a first sealant layer in an embodiment of the present disclosure;

FIG. 12 is a schematic structural view of an adhesive layer in an embodiment of the present disclosure;

FIG. 13 is a schematic view of a structure of a micro channel layer according to an embodiment of the disclosure;

FIG. 14 is a second schematic view illustrating a structure of a micro channel layer according to an embodiment of the disclosure;

FIG. 15 is a schematic structural view of a first elastomeric sealing layer in an embodiment of the present disclosure;

fig. 16 is one of schematic structural diagrams of a substrate layer in an embodiment of the disclosure;

fig. 17 is a second schematic structural view of a substrate layer in an embodiment of the disclosure;

FIG. 18 is a schematic structural diagram of a bottom cap layer in an embodiment of the disclosure;

FIG. 19 is a schematic cross-sectional view of a microfluidic detection chip of the embodiment of the present disclosure at the dashed line L in FIG. 2;

fig. 20 is a flowchart of a method for using the microfluidic detection chip according to an embodiment of the present disclosure.

Detailed Description

The embodiment of the invention provides a microfluidic detection chip and a using method thereof, aiming at the problems that the detection steps are complicated and the speed is low because the microfluidic detection chip needs manual mixing and other operations.

Fig. 1 is a schematic view of a layered structure of a microfluidic detection chip provided in an embodiment of the present disclosure, fig. 2 is a schematic view of a front structure of the microfluidic detection chip provided in an embodiment of the present disclosure, fig. 3 and fig. 4 are schematic views of a side structure of the microfluidic detection chip used in an embodiment of the present disclosure, fig. 5 is a schematic view of a bottom structure of the microfluidic detection chip used in an embodiment of the present disclosure, fig. 6 to fig. 18 are schematic views of specific structures of various film layers in the microfluidic detection chip in an embodiment of the present disclosure, and fig. 19 is a schematic cross-sectional view of the microfluidic detection chip in fig. 2 at a dotted line L.

The following describes in detail a specific embodiment of a microfluidic detection chip and a method for using the same according to an embodiment of the present invention with reference to the accompanying drawings. The thicknesses and shapes of the various film layers in the drawings are not to be considered true proportions, but are merely intended to illustrate the present invention.

The microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 1, includes:

referring to fig. 9 and 10, the liquid storage layer 4 includes: a first reservoir well (e.g., 401, 402, 403, 404 in the figure) configured to store a pre-sealed reagent, and a second reservoir well 405 configured to store a sample to be tested;

referring to fig. 2 and 6, the cap layer 1 is located on one side of the liquid storage layer 4; cap layer 1, comprising: through holes (101, 102, 103, 104) corresponding to the positions of the first liquid storage holes (401, 402, 403, 404), and a sample adding hole 105 corresponding to the position of the second liquid storage hole 405;

referring to fig. 5 and 18, the bottom cover layer 10 is located on the side of the liquid storage layer 4 away from the top cover layer 1; an underlayer 10, comprising: through holes (1001, 1002, 1003 and 1004) corresponding to the positions of the first liquid storage holes (401, 402, 403 and 404);

referring to fig. 11, the first sealing layer 5 is disposed between the liquid storage layer 4 and the bottom cover layer 10; a first sealing layer 5 comprising: a first sealing structure 501 configured to seal the first reservoir wells (401, 402, 403, 404);

referring to fig. 13 and 14, the micro flow channel layer 7 is located between the first sealing layer 5 and the bottom cover layer 10; the micro flow channel layer 7 includes:

first openings

701, 702, 703 and 704 corresponding to positions of the

first reservoirs

401, 402, 403 and 404, second openings 705 corresponding to positions of the second reservoirs 405, and first microchannels 707 connecting the

first openings

701, 702, 703 and 704 and the second openings 705; the first micro-channel 707 is located on the side of the micro-channel layer 7 facing the bottom cover layer 10;

referring to fig. 15, the first elastic sealing layer 8 is located on the side of the micro flow channel layer 8 close to the bottom cover layer 10;

referring to fig. 7, the second elastic sealing layer 2 is located between the liquid storage layer 4 and the top cover layer 1; a second elastomeric sealing layer 2 comprising: and the sampling hole 205 corresponds to the position of the second liquid storage hole 405.

The microfluidic detection chip provided by the embodiment of the disclosure can store the pre-sealed reagent in the first liquid storage hole by arranging the liquid storage layer, in the using process, the pre-sealed reagent stored in the first liquid storage hole can be injected into the first open pore in the microfluidic channel layer by puncturing the first sealing structure and extruding the first elastic sealing layer and the second elastic sealing layer, the sample to be detected can be injected into the second liquid storage hole by the top cover layer and the sample adding hole in the second elastic sealing layer, and the sample to be detected is controlled to be injected into the second open pore in the microfluidic channel layer, the first microfluidic channel is communicated with the first open pore and the second open pore, so that the uniform mixing of the sample to be detected and the pre-sealed reagent can be realized, the operations such as manual uniform mixing and the like are not needed, the operation is simple, the detection speed is high, the structure of the microfluidic detection chip is simple, the material cost and the manufacturing process cost are low, has the advantages of miniaturization, integration, automation and the like, and is convenient for large-scale production and application.

The microfluidic detection chip provided by the embodiment of the disclosure can accurately control and control the flow of the pre-sealed reagent and the sample to be detected by using the microfluidic technology, can integrate basic operation units such as the sample to be detected, reaction, waste liquid treatment and the like in the detection and analysis process into the microfluidic detection chip with a smaller size, has the advantages of less consumption of the sample to be detected, high detection speed, simple and convenient operation, multifunctional integration, small volume, convenience in carrying and the like, and has great application potential in the fields of biology, chemistry, medicine and the like. Specifically, the microfluidic detection chip has wide applicability, and can be adapted to various detection apparatuses such as an optical detection apparatus (e.g., absorbance detection, fluorescence detection, chemiluminescence detection, plasmon surface resonance detection, etc.), an electrochemical detection apparatus (e.g., current detection, potential detection, impedance detection, etc.), a giant magnetoresistance detection apparatus, and a piezoelectric detection apparatus.

The top cover layer 1 is provided with through

holes

101, 102, 103 and 104 corresponding to the positions of the first liquid storage holes 401, 402, 403 and 404 respectively so that the liquid injection pushing head can penetrate through the top cover layer 1 to press the second elastic sealing layer 2, and the top cover layer 1 is provided with a sample adding hole 105 corresponding to the position of the second liquid storage hole 405 so that a sample to be detected can be injected into the second liquid storage hole 405 through the sample adding hole 105. Specifically, the cap layer 1 may be made of polypropylene (PP), the cap layer 1 may have a length of about 65mm, a width of about 35mm, and a thickness of about 3mm, and the diameters of the through

holes

101, 102, 103, and 104 and the sample application hole 105 may be about 8 mm.

The bottom cover layer 10 is provided with through

holes

1001, 1002, 1003 and 1004 corresponding to the positions of the first liquid storage holes 401, 402, 403 and 404, respectively, so that an unsealing needle can penetrate through the through holes of the bottom cover layer 10 to puncture the first sealing layer 5, or an injection pushing head can penetrate through the bottom cover layer 10 to press the first elastic sealing layer 8, specifically, the bottom cover layer 10 can be made of polypropylene (PP) material, and the size of the bottom cover layer 10 can be set to be about 65mm long, about 35mm wide and about 3mm thick. The diameter of the through

holes

1001, 1002, 1003, 1004 may be set to about 8 mm.

The first liquid storage layer 401 in the liquid storage layer 4 may be used to store a pre-sealed reagent, and the first liquid storage layer 401 is sealed by the first sealing layer 5 and the second elastic sealing layer 2, in the embodiment of the present disclosure, four first liquid storage holes 401, 402, 403, and 404 are taken as an example, and in specific implementation, the number of the first liquid storage holes may be determined according to the type of the pre-sealed reagent that needs to be stored in advance, and the number of the first liquid storage holes is not limited here. In addition, in the embodiment of the present disclosure, the first liquid storage hole, the second liquid storage hole, and the through hole in the other film layer are illustrated as being circular, and when the embodiment is implemented specifically, the shapes of the first liquid storage hole, the second liquid storage hole, and the other through hole may be set according to actual situations, which is not limited herein.

Specifically, the liquid storage layer 4 may be made of an Acrylonitrile Butadiene Styrene (ABS) material, and the size of the liquid storage layer 4 may be about 65mm long, about 35mm wide, and about 8mm thick, and the diameter of the first

liquid storage hole

401, 402, 403, 404 may be about 8 mm.

It should be noted that, in the embodiment of the present disclosure, the first liquid storage hole and the second liquid storage hole in the liquid storage layer both have a function of temporarily storing liquid, the first liquid storage hole is used to store a pre-sealed reagent, and the second liquid storage hole can be used to store a diluent in advance, and can temporarily store a sample to be detected and the diluent, so that the sample to be detected and the diluent can be uniformly mixed.

In the embodiment of the present disclosure, the first sealing layer 5 may be made of a pierceable material such as an aluminum film, and the first sealing structure 501 in the first sealing layer 5 is used to seal the first liquid storage hole, so that the pre-sealing reagent can be sealed in the first liquid storage hole, one first sealing structure 501 may be used to seal each first liquid storage hole, or a plurality of first sealing structures 501 may be used to seal each first liquid storage hole, which is not limited herein. In order to ensure the sealing performance of the first sealing structure 501, the first sealing structure 501 is to be ensured to completely cover the first liquid storage hole, and the area of the first sealing structure 501 needs to be larger than that of the corresponding first liquid storage hole, or the area of the first sealing layer 5 may be set to be close to that of the liquid storage layer 4.

Above-mentioned first elastic sealing layer 8 can adopt the silica gel material, make first elastic sealing layer 8 have certain toughness, can annotate the extrusion of liquid pushing head or carry and draw down the effect and take place deformation, and, can not be annotated the liquid pushing head or unseal the needle puncture, can make and unseal the needle and puncture first sealing layer 5, and can change the inside atmospheric pressure of micro-fluidic detection chip, realize the flow control to pre-sealed reagent or the sample of waiting to examine, in addition, also can utilize the leakproofness of first elastic sealing layer 8, seal micro-flow channel layer 7. Specifically, the first resilient sealing layer 1 may be sized to be about 64mm long, about 34mm wide, and about 0.3mm thick, with the first resilient sealing layer 1 sized slightly smaller than the size of the bottom cover layer 10 so that the bottom cover layer 10 may completely cover the first resilient sealing layer 8.

Above-mentioned second elastic sealing layer 2 can adopt the silica gel material, make second elastic sealing layer 2 have certain toughness, can annotate the extrusion of liquid pusher or carry and draw down the effect and take place deformation, and, can not be annotated the liquid pusher or unseal the needle puncture, in order to change the inside atmospheric pressure of micro-fluidic detection chip, realize the flow control to pre-sealed reagent or waiting to examine the sample, concretely, the size of second elastic sealing layer 2 can set up to length about 64mm, width about 34mm, thickness about 0.3mm, the size of second elastic sealing layer 2 sets up to be less than the size of top cap layer 1 a little, thereby can make top cap layer 1 cover second elastic sealing layer 2 completely.

The

first openings

701, 702, 703 and 704 in the micro channel layer 7 correspond to the first liquid storage holes 401, 402, 403 and 404, the second opening 705 corresponds to the second liquid storage hole 405, and the

first openings

701, 702, 703 and 704 are communicated with the second opening 705 through the first micro channel 707, and after the first sealing layer 5 is punctured, the first openings are communicated with the corresponding first liquid storage holes, and the second openings are communicated with the corresponding second liquid storage holes, so that the liquid in the liquid storage layer 4 can be injected into the micro channel layer.

The micro channel layer 7 may be made of ABS (Acrylonitrile Butadiene Styrene) material, and the micro channel layer 7 may be sized to have a length of about 65mm, a width of about 35mm, and a thickness of about 2.5mm, and the

first openings

701, 702, 703, 704 and the second opening 705 may have a diameter of about 8 mm.

In the using process, an unsealing needle can be adopted to penetrate through the through hole and the first opening 701 in the bottom cover layer 10, puncture the first sealing structure 501 in the first sealing layer 5, then an injection pushing head is adopted to penetrate through the through hole in the top cover layer 1 to press the first elastic sealing layer 2, so that the pre-sealing reagent stored in the first liquid storage hole is injected into the first opening in the micro-flow channel, the sample to be detected can be injected into the second liquid storage hole through the sample adding holes in the top cover layer 1 and the second elastic sealing layer 2, and the sample to be detected is injected into the second opening 705 in the micro-flow channel layer 7, because the first opening and the second opening 705 are communicated through the first micro-flow channel 707, the mixing of the sample to be detected and the pre-sealing reagent can be realized, in the practical application process, the sequence of injecting the pre-sealing reagent stored in each first liquid storage hole into the micro-flow channel layer 7 can be controlled according to the practical requirements, and the speed of extruding the first elastic sealing layer or the second elastic sealing layer can be controlled, and the flow rate of the pre-sealed reagent of the sample to be detected is adjusted to finish corresponding detection.

In specific implementation, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 1, 11 and 18, the first sealing layer 5 may further include: a second sealing structure 502 configured to seal the second reservoir well; the bottom cover layer 10 is provided with a through hole 1005 corresponding to the second liquid storage hole.

Through set up second seal structure 502 in first sealing layer 5, can seal second stock solution hole, thereby can be in the second stock solution hole some diluent of prestore, can dilute the sample of examining of adding, in the use, will examine the sample and add the second stock solution hole in, and with the diluent mixing back, can adopt to open the needle and pass through-hole 1005 and the second through-hole on the bottom cap layer 10 and pierce second seal structure 502, so that examine after the mixing the sample and flow into in the micro flow channel layer 7.

When the method is specifically implemented, the volume of the diluent pre-existing in the second liquid storage hole can be set according to the concentration of the sample to be detected, so that the accurate dilution of the volume proportion can be realized, and the extra cost cannot be increased.

Further, in the above microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 1, fig. 8, and fig. 9, the microfluidic detection chip may further include: a second sealing layer 3 which can be punctured;

the second sealing layer 3 is positioned between the second elastic sealing layer 2 and the liquid storage layer 4;

a second sealant layer 3 comprising: a third sealing structure 301 configured to seal the

first reservoir wells

401, 402, 403, 404, and a fourth sealing structure 302 configured to seal the second reservoir well 405.

Through set up second sealing layer 3 in one side that stock solution layer 4 is close to top cap layer 1, can further seal first stock solution hole and second stock solution hole in the stock solution layer 4, make first stock solution hole and second stock solution hole under the effect of first sealing layer and second sealing layer, become inclosed storage structure, prevent that the reagent of storing in advance and the diluent in the stock solution layer 4 from spilling. Specifically, one third sealing structure 301 may be used to seal each first liquid storage hole, or a plurality of third sealing structures 301 may be used to seal each first liquid storage hole, which is not limited herein. Moreover, the area of the third sealing structure 301 needs to be ensured to completely cover the corresponding first liquid storage hole, the area of the fourth sealing structure 302 needs to be ensured to completely cover the corresponding second liquid storage hole, or the area of the second sealing layer 5 can be set to be close to the area of the liquid storage layer 4.

Specifically, the second sealing layer 3 may be made of a pierceable material such as an aluminum film, and during use, a unsealing needle may pierce the third sealing structure 301 through a through hole in the top cover layer 1, or a unsealing needle may pierce the fourth sealing structure 302 through a sample adding hole in the top cover layer 1.

In practical applications, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 9 and 10, the liquid storage layer 4 may further include: a waste liquid tank 407, a first drainage hole 408 penetrating through the liquid storage layer 4, and a second micro-channel 409 communicating between the waste liquid tank 407 and the first drainage hole 408;

the waste liquid tank 407 and the second micro flow channel 409 are positioned on the surface of the side of the liquid storage layer 4 facing the top cover layer 1;

referring to fig. 13 and 14, the micro flow channel layer 7 may further include: and a second drainage hole 708 penetrating through the micro flow channel layer 7, wherein the second drainage hole 708 is communicated with the first drainage hole 408 and the first micro flow channel 707.

As can be seen from a comparison of fig. 9 and 10, the reservoir 407 and the second microchannel 409 are groove-shaped and are located on the surface of the reservoir 4 facing the top cover layer 1, and the first drainage hole 408 penetrates through the reservoir 4, as can be seen from a comparison of fig. 13 and 14, the first microchannel 707 is located on the surface of the side of the microchannel layer 7 facing the bottom cover layer 10, and the second drainage hole 708 penetrates through the microchannel layer 7. The pre-sealed reagent injected into the

first openings

701, 702, 703 and 704 mixes with the reagent injected into the second opening 705 in the first microchannel 707 and reacts accordingly, and the waste liquid after the reaction flows into the second microchannel 409 through the second drainage hole 708 and the first drainage hole 408, and then flows into the waste liquid tank 407.

Specifically, the waste liquid tank 407 is illustrated as a rectangle, but in practical implementation, the waste liquid tank 407 may have another shape, for example, the waste liquid tank may have an irregular shape, and the volume of the waste liquid tank 407 may be set to 200uL or more, and the size of the second microchannel 409 may be set to be about 15mm long, about 300um wide, and about 200um deep. The diameter of the first conduction holes 408 may be set to about 1.5mm, and the diameter of the second conduction holes 708 may be set to about 1.5 mm.

In addition, as shown in fig. 6 and 7, the microfluidic detection chip provided in the embodiment of the present disclosure may further include: a gas pressure balance hole R;

the air pressure balance hole R penetrates the top cover layer 1 and the second elastic sealing layer 2, and the position of the air pressure balance hole R corresponds to the position of the waste liquid tank 407, and the air pressure balance hole R is configured to communicate the waste liquid tank 407 with the atmosphere.

The waste liquid tank 407 can be communicated with the atmosphere by arranging the air pressure balance hole R in the top cover layer 1 and the second elastic sealing layer 2, so that the air pressure balance inside the microfluidic detection chip is realized, and in the process of extruding the first elastic sealing layer and the second elastic sealing layer, the internal pressure can be released through the air pressure balance hole R, so that the liquid flow is realized.

Specifically, the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 1 and 12, may further include: an adhesive layer 6 between the micro flow channel layer 7 and the first sealing layer 5; the adhesive layer 6 is configured to adhere the micro flow channel 7 layer and the liquid storage layer 4.

The liquid storage layer 4 and the micro flow channel layer 7 can be bonded together by providing the adhesive layer 6, and the adhesive layer 6 is provided with through

holes

601, 602, 603, 604, 605 at positions corresponding to the first liquid storage hole and the second liquid storage hole, respectively, to ensure that the liquid in the liquid storage layer 4 can flow into the micro flow channel layer 7 through the adhesive layer 6. In order to allow the waste liquid to flow from the micro flow channel layer 7 into the waste liquid tank in the liquid storage layer 4, a through hole 608 corresponding to the first drainage hole is also provided in the adhesive layer 6.

Specifically, the adhesive layer 6 may be made of a glue material, and the coating range of the glue material may be set to be about 65mm long, about 35mm wide, about 0.3mm thick and about 0.3mm thick. The adhesive layer 6 may be made of a material having elastic expansion and contraction properties, the size of the adhesive layer 6 may be set to about 65mm in length, about 35mm in width, and about 0.3mm in thickness, the diameter of the through

holes

601, 602, 603, 604, 605 may be set to about 8mm, and the diameter of the through hole 608 may be set to about 1.5 mm.

In specific implementation, in the above microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 9 and 10, the liquid storage layer 4 further includes: an air pressure adjusting hole 406 penetrating the liquid storage layer 4, and a third micro channel 4010 communicating the second liquid storage hole 405 and the air pressure adjusting hole 406;

the third micro-channel 4010 is positioned on the surface of the liquid storage layer 4 facing to the bottom cover layer 10;

the first sealing layer 5 may further include: a fifth sealing structure 503 configured to seal the air pressure adjusting hole 406;

referring to fig. 6, 13, 15 and 18, the top cover layer 1, the micro channel layer 7, the first elastic sealing layer 8 and the bottom cover layer 10 are respectively provided with through

holes

106, 706, 806 and 1006 corresponding to the position of the air pressure adjusting hole 406, and specifically, the through

holes

106, 706, 806 and 1006 may be set to be about 8mm in size.

By providing the fifth sealing structure 503, the sealing performance of the air pressure adjusting hole 406 can be ensured before the sample to be tested in the second liquid storage hole 405 is controlled to be injected into the micro flow channel layer 7. The sample to be detected is injected into the second liquid storage hole 405, and after the sample to be detected and the diluent are mixed and diluted, the unsealing needle can penetrate through the bottom cover layer 10, the first elastic sealing layer 8 and the micro-flow channel layer 7 to puncture the fifth sealing structure 5, wherein the second elastic sealing layer 2 and the bonding layer 6 are not provided with through holes at the positions of the air pressure adjusting holes 406 and cannot be punctured, and subsequently, the second elastic sealing layer 2 can be extruded or pulled under the action of the liquid injection pushing head to change the air pressure in the air pressure adjusting holes 406, so that two or more than two kinds of liquid in the second liquid storage hole 405 are mixed uniformly.

After the fifth sealing structure 503 and the second sealing structure 502 are pierced, the air pressure inside the air pressure adjusting hole 406 may be changed by pressing the elastic sealing layer 2 or the adhesive layer 6, and the air pressure adjusting hole 406 and the second liquid storage hole 405 may be communicated through the third micro flow channel 4010, so that the air pressure in the second liquid storage hole 405 may be communicated through the air pressure adjusting hole 406, thereby achieving an effect of adjusting the air pressure in the second liquid storage hole 405 through the air pressure adjusting hole 406 to control the flow of the liquid in the second liquid storage hole 405.

Specifically, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 19, the edge of the air pressure adjusting hole 406 on the side facing the bottom cover layer 10 is stepped;

the fifth sealing structure 503 is embedded with a part of the adhesive layer 6 in the stepped edge of the air pressure-adjusting hole 406.

By setting the edge of the side of the air pressure adjusting hole 406 facing the bottom cover layer 10 to be in a step shape, and embedding the fifth sealing structure 503 and a part of the adhesive layer 6 into the edge of the step shape of the air pressure balancing hole 406, the air pressure adjusting hole 406 can be further sealed, and before controlling the sample to be detected in the second liquid storage hole 405 to be injected into the micro channel layer 7, the sealing performance of the air pressure adjusting hole 406 is ensured, and the diluent pre-sealed in the second liquid storage hole 305 is prevented from entering the air pressure adjusting hole 406. Also, the stepped edge of the air pressure adjusting hole 406 may be disposed flush with the inner surface of the third microchannel 4010. Specifically, the diameter of the side of the gas pressure adjusting hole 406 facing the bottom cover layer 10 may be set to about 11mm, and the depth of the stepped edge may be set to about 200 um.

In practical applications, the microfluidic detection chip provided in the embodiments of the present disclosure, as shown in fig. 1 and fig. 16, may further include: a substrate layer 9 located between the first elastic sealing layer 8 and the bottom cover layer 10;

the surface of the substrate layer 9 close to one side of the top cover layer 1 is provided with an antibody coating area;

referring also to fig. 15, the first elastomeric sealing layer 8 includes: a plurality of communicating holes 807 corresponding to the positions of the antibody-coated regions;

referring also to fig. 14, the position of each communication hole 807 corresponds to the position of the first micro flow channel 708.

After the pre-sealed reagent and the sample to be detected flow into the first micro flow channel 708 to be mixed, the pre-sealed reagent and the sample to be detected can flow into the antibody coating area of the substrate layer 9 through the communication holes 807 in the first elastic sealing layer 8 to generate antibody reaction, and the antibody coating area of the substrate layer 9 can be coated with antibodies for detecting multiple indexes, so that multi-index combined detection of a single sample to be detected is realized. Specifically, the area of the antibody-coated region in the above substrate layer 9 may be smaller than or equal to the area of all the communicating holes 807.

Specifically, the substrate layer 9 may be sized to be about 65mm long, about 35mm wide, and about 2.5mm thick. The substrate layer 9 is provided with through

holes

901, 902, 903, 904 corresponding to the positions of the first liquid storage holes, a through hole 905 corresponding to the position of the second liquid storage hole, and a through hole 906 corresponding to the position of the air pressure adjusting hole, and the diameters of the through

holes

901, 902, 903, 904, 905, 906 can be set to be about 8 mm.

In addition, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 17, the substrate layer 9 may include: a biosensor 906 located in the antibody coating region, a coating 907 located on the surface of the biosensor 906;

the surface of coating 907 is coated with antibodies.

It should be noted that in fig. 17, biosensor 906 and coating 907 are illustrated separately in order to illustrate the relative position relationship between biosensor 906 and coating 907, and in practical applications, coating 907 is coated on the surface of biosensor 906.

After the pre-sealing reagent and the sample to be detected flow into the first micro flow channel 708 to be mixed, the mixture can flow into the antibody coating area of the substrate layer 9 through the communication holes 807 in the first elastic sealing layer 8, and react with the antibody coated on the surface of the coating layer 907, and a detection signal is collected by the biosensor 906 and transmitted to a corresponding detection instrument.

The biosensor 906 is provided with a signal collecting area for collecting detection signals, the distribution of the signal collecting area can be point, line, point array, line array, or various regular and irregular shapes such as circle, square, diamond, etc., and the specific arrangement mode of the signal collecting area is not limited here.

Coating 907 or 907 may be coated on the biosensor 906, but not limited thereto.

Specifically, in the microfluidic detection chip provided by the embodiment of the present disclosure, the substrate layer may be made of plastic, glass, or silicon material; or the substrate layer is made of a printed circuit board.

For better coating the antibody, the substrate layer may be made of a plastic material capable of adsorbing the antibody, for example, a material with high light transmittance such as Polystyrene (PS) or polymethyl methacrylate (PMMA). And if the substrate layer is made of a printed circuit board, the biosensor can be directly integrated into the circuit of the printed circuit board.

In practical applications, in the microfluidic detection chip provided in the embodiment of the present disclosure, the substrate layer and the bottom cover layer may also be an integrated structure, and specifically, the antibody coating region of the substrate layer may be directly disposed at a corresponding position of the bottom cover layer to integrate the substrate layer into the bottom cover layer, so as to reduce one film layer, reduce the overall thickness of the microfluidic detection chip, and facilitate miniaturization of the microfluidic detection chip.

In practical applications, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 7, the sample application hole 205 in the second elastic sealing layer 2 is a flip-top sealing port.

As shown in the combined figure 2, the sample adding hole 105 of the top cover layer 1 can expose the sample adding hole 205, in the sample adding process, the cover of the sample adding hole 205 can be opened through the sample adding hole 105, the sample to be detected is injected into the second liquid storage hole through the

sample adding holes

105 and 205, the cover with the flip sealing opening can be closed after the injection is completed, the sample adding hole 205 still has sealing performance, and the sample to be detected in the second liquid storage hole can be uniformly mixed with diluent through extruding the position of the flip sealing opening of the second elastic sealing layer 2 subsequently.

In specific implementation, in the microfluidic detection chip provided in the embodiment of the present disclosure, as shown in fig. 1, fig. 6, fig. 9, fig. 10, and fig. 18, the top cover layer 1 and the bottom cover layer 10 are provided with a plurality of engaging structures T on the surfaces of the sides facing the liquid storage layer 4;

the liquid storage layer 4 is provided with a plurality of grooves U which are respectively in one-to-one correspondence with the positions of the clamping structures T;

in each film layer between the clamping structure T and the groove U, clamping holes V corresponding to the positions of the clamping structures T one to one are provided, for example, a plurality of clamping holes V are provided in the second elastic sealing layer 2 in fig. 7;

the clamping structure T is inserted into the corresponding groove U through the corresponding clamping hole V.

Specifically, the top cover layer 1, the second elastic sealing layer 2, the second sealing layer 3 and the liquid storage layer 4 may be extruded, so that the fastening structure T in the top cover layer 1 is inserted into each groove U at the corresponding position of the liquid storage layer 4 after passing through the fastening hole V in the second elastic sealing layer 2, thereby realizing tight bonding between the fastening structure T and the corresponding groove U, and thus the second elastic sealing layer 2 may be fixed between the top cover layer 1 and the liquid storage layer 4. When the second elastic sealing layer 2 is extruded, the top cover layer 1 can apply uniform pressure to the second elastic sealing layer 2, so that the second elastic sealing layer 2 is fixed above the liquid storage layer 4, and the position of the liquid storage push head cannot move due to the extrusion/lifting action of the liquid injection push head.

The bottom cover layer 10, the substrate layer 9, the first elastic sealing layer 8, the micro channel layer 7, the bonding layer 6 and the liquid storage layer 4 can be extruded, so that the clamping structure T in the bottom cover layer 10 penetrates through the substrate layer 9, the first elastic sealing layer 8, the micro channel layer 7 and the bonding layer 6 and then is embedded into each groove U at the corresponding position of the liquid storage layer 4, the tight bonding of the clamping structure T and the corresponding groove U is realized, and the first elastic sealing layer 8 can be fixed between the bottom cover layer 10 and the liquid storage layer 4. When the first elastic sealing layer 8 is extruded, the bottom cover layer 10 can apply uniform pressure to the first elastic sealing layer 8, so that the first elastic sealing layer 8 is fixed below the liquid storage layer 4, and the position of the liquid storage push head cannot move due to the extrusion/lifting action of the liquid injection push head.

In order to ensure the tightness and the sealing performance of the whole microfluidic detection chip, the height of the clamping structure T cannot be too high so as to prevent a gap from being formed between the top cover layer 1 and the liquid storage layer 4 or a gap from being formed between the bottom cover layer 10 and the liquid storage layer 4, and the height of the clamping structure T cannot be too low so as to prevent the clamping structure T from being inserted into the corresponding groove U.

Fig. 3 and 4 are side views of the microfluidic detection chip in two different directions in the embodiment of the present disclosure, and as is apparent from fig. 3 and 4, the whole microfluidic detection chip has better tightness.

Specifically, the groove U in the liquid storage layer 4 may be sized to be about 2mm long, about 1mm wide, and about 2mm deep.

In the embodiment of the present disclosure, in the top cap layer 1, the adhesive layer 6, the micro channel layer 7, the first elastic sealing layer 8, the substrate layer 9, and the bottom cap layer 10, each through hole corresponding to the first liquid storage hole, the second liquid storage hole, and the air pressure adjusting hole needs to satisfy the requirement that the unsealing needle or the liquid injection pusher can pass through without hindrance.

Based on the same inventive concept, the present disclosure also provides a method for using the above microfluidic detection chip, and as the principle of the method for solving the problems is similar to that of the above microfluidic detection chip, the implementation of the method for using the chip can refer to the above embodiment of the microfluidic detection chip, and repeated details are not repeated.

The application method of the microfluidic detection chip provided in the embodiments of the present disclosure, as shown in fig. 20, may include:

s100, adding a sample to be detected into the second liquid storage hole through the sample adding hole;

s200, controlling the pre-sealed reagent in the first liquid storage hole to be injected into the first micro-channel;

s300, controlling the sample to be detected in the second liquid storage hole to be injected into the first micro-channel.

The application method of the microfluidic detection chip provided by the embodiment of the disclosure can add the sample to be detected into the second liquid storage hole through the sample adding hole, and can respectively control the pre-sealed reagent in the first liquid storage hole and the sample to be detected in the second liquid storage hole to be injected into the first microchannel for mixing, so that the uniform mixing of the sample to be detected and the pre-sealed reagent is realized, the manual operations such as uniform mixing are not needed, the operation is simple, and the detection speed is high.

Referring to fig. 1 and 7, the sample application hole 205 in the second elastic sealing layer 2 may be a flip-top sealing port, and the step S100 may include: but open flip sealing port, adopt the pipettor to absorb a certain amount of sample of waiting to examine, puncture fourth seal structure 302, will examine the sample and pour into second stock solution hole 405 into.

Specifically, the step S200 may include:

and injecting the pre-sealed reagent in the first liquid storage hole into the first micro-channel by extruding the second elastic sealing layer corresponding to the position of the first liquid storage hole.

Specifically, taking the example of injecting the pre-sealed reagent in one of the first liquid storage holes into the first microchannel, for example, taking the example of injecting the pre-sealed reagent in the first liquid storage hole 401 into the first microchannel in fig. 9, after puncturing the first sealing structure and the third sealing structure with the unsealing needle, the unsealing needle is reset, and then the liquid injection pushing head is used to penetrate through the through hole of the top cover layer corresponding to the first liquid storage hole, and the internal pressure is changed by squeezing the second elastic sealing layer, so that the pre-sealed reagent is injected into the first microchannel through the first opening, specifically, the injection speed can be controlled not to exceed 5 mm/s.

In practical implementation, in the using method provided by the embodiment of the present disclosure, with reference to fig. 7, the sample adding hole 205 in the second elastic sealing layer 2 is a flip-top sealing port;

after the step S100 and before the step S300, the method may further include:

closing the flip-top sealing port;

piercing the fifth seal structure;

Specifically, after the flip-open sealing port is closed, an unsealing needle penetrates through the bottom cover layer, the substrate layer, the first elastic sealing layer and the micro-flow channel layer to puncture the fifth sealing structure, then the unsealing needle is reset, an injection pushing head penetrates through a sample adding hole of the top cover layer to extrude or lift the second elastic sealing layer, and the injection pushing head is controlled to move up and down for multiple times to uniformly mix a sample to be detected and diluent, for example, the injection pushing head can be controlled to move down for 5mm, then move up for 3mm and repeat for 40 times, and the reciprocating speed is not lower than 1 time/s, so that two or more than two liquids are uniformly distributed, and the uniform mixing operation is realized.

In a specific implementation, in the using method provided in the embodiment of the present disclosure, the step S300 may include:

the second elastic sealing layer corresponding to the position of the air pressure adjusting hole is extruded, so that the sample to be detected in the second liquid storage hole is injected into the first micro-channel.

Specifically, an unsealing needle is adopted to penetrate through a through hole corresponding to a second liquid storage hole in the bottom cover layer, the substrate layer, the micro-channel layer and the bonding layer so as to puncture the second sealing structure 502, then the unsealing needle is reset, an injection pushing head is adopted to penetrate through a through hole corresponding to an air pressure adjusting hole in the top cover layer so as to extrude the second elastic sealing layer, and a sample to be detected in the second liquid storage hole is injected into the micro-channel layer by changing the air pressure in the air pressure adjusting hole.

Then, the pre-sealed reagents in the other first reservoir wells may be injected into the microfluidic layer in the manner of the step S200, and the injection sequence of the pre-sealed reagents in the first reservoir wells may be determined according to actual needs.

The microfluidic detection chip and the use method thereof in the embodiment of the disclosure can store the pre-sealed reagent in the first liquid storage hole by arranging the liquid storage layer, can inject the pre-sealed reagent stored in the first liquid storage hole into the first open hole in the microfluidic channel layer by puncturing the first sealing structure and extruding the first elastic sealing layer and the second elastic sealing layer in the use process, can inject the sample to be detected into the second liquid storage hole by the top cover layer and the sample injection hole in the second elastic sealing layer, and control the sample to be detected to be injected into the second open hole in the microfluidic channel layer, wherein the first microfluidic channel is communicated with the first open hole and the second open hole, so that the uniform mixing of the sample to be detected and the pre-sealed reagent can be realized, the operations such as manual uniform mixing and the like are not needed, the operation is simple, the detection speed is high, the structure of the microfluidic detection chip is simple, the material cost and the manufacturing process cost are low, has the advantages of miniaturization, integration, automation and the like, and is convenient for large-scale production and application. And, through prestore diluent in the second stock solution hole to can realize waiting the operation such as dilution, mixing, ration of examining the sample to the singleness through the extrusion second elastic sealing layer, through the antibody parcel district setting on the base plate layer detect the antibody of a plurality of indexs, can realize the many indexs of the sample joint detection of waiting to the singleness.

While preferred embodiments of the present disclosure have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all alterations and modifications as fall within the scope of the disclosure.

It will be apparent to those skilled in the art that various changes and modifications may be made to the disclosed embodiments without departing from the spirit and scope of the disclosed embodiments. Thus, if such modifications and variations of the embodiments of the present disclosure fall within the scope of the claims of the present disclosure and their equivalents, the present disclosure is also intended to encompass such modifications and variations.

Claims

A microfluidic detection chip, comprising:

a liquid storage layer comprising: a first reservoir configured to store a pre-sealed reagent, and a second reservoir configured to store a sample to be tested;

the top cover layer is positioned on one side of the liquid storage layer; the cap layer, comprising: the through hole corresponds to the first liquid storage hole, and the sampling hole corresponds to the second liquid storage hole;

the bottom cover layer is positioned on one side of the liquid storage layer, which is far away from the top cover layer; the bottom cap layer, comprising: the through hole corresponds to the first liquid storage hole in position;

the first sealing layer can be punctured and is positioned between the liquid storage layer and the bottom cover layer; the first sealing layer includes: a first sealing structure configured to seal the first reservoir well;

the micro-channel layer is positioned between the first sealing layer and the bottom cover layer; the microfluidic layer includes: the first opening hole corresponds to the position of the first liquid storage hole, the second opening hole corresponds to the position of the second liquid storage hole, and the first micro channel is communicated with the first opening hole and the second opening hole; the first micro-channel is positioned on one side of the micro-channel layer facing the bottom cover layer;

the first elastic sealing layer is positioned on one side, close to the bottom cover layer, of the micro-flow channel layer;

the second elastic sealing layer is positioned between the liquid storage layer and the top cover layer; the second elastomeric sealing layer comprising: and the sample adding hole corresponds to the second liquid storage hole in position.
The microfluidic detection chip of claim 1, wherein the first sealing layer further comprises: a second sealing structure configured to seal the second reservoir well; the bottom cover layer is provided with a through hole corresponding to the second liquid storage hole.
The microfluidic detection chip of claim 1, further comprising: a second sealing layer that is pierceable;

the second sealing layer is positioned between the second elastic sealing layer and the liquid storage layer;

the second sealing layer comprising: a third sealing structure configured to seal the first reservoir well, and a fourth sealing structure configured to seal the second reservoir well.
The microfluidic detection chip of claim 1, wherein the liquid storage layer further comprises: the waste liquid tank penetrates through the first drainage hole of the liquid storage layer, and a second micro-channel is communicated between the waste liquid tank and the first drainage hole;

the waste liquid groove and the second micro-channel are positioned on the surface of one side of the liquid storage layer facing the top cover layer;

the micro-channel layer further comprises: and the second drainage holes penetrate through the micro-channel layer and are communicated with the first drainage holes and the first micro-channel.
The microfluidic detection chip of claim 4, further comprising: an air pressure balancing hole;

the air pressure balance hole penetrates through the top cover layer and the second elastic sealing layer, the position of the air pressure balance hole corresponds to that of the waste liquid groove, and the air pressure balance hole is configured to communicate the waste liquid groove with the atmosphere.
The microfluidic detection chip of claim 1, further comprising: an adhesive layer between the microfluidic layer and the first sealing layer; the adhesive layer is configured to adhere the micro flow channel layer and the liquid storage layer.
The microfluidic detection chip of claim 6, wherein the liquid storage layer further comprises: the air pressure adjusting hole penetrates through the liquid storage layer, and the third micro-channel is communicated with the second liquid storage hole and the air pressure adjusting hole;

the third micro-channel is positioned on the surface of one side of the liquid storage layer facing the bottom cover layer;

the first sealing layer further comprises: a fifth sealing structure configured to seal the air pressure adjusting hole;

and through holes corresponding to the positions of the air pressure adjusting holes are formed in the top cover layer, the micro flow channel layer, the first elastic sealing layer and the bottom cover layer.
The microfluidic detection chip according to claim 7, wherein the air pressure adjusting hole is stepped at an edge of a side facing the bottom cover layer;

the fifth seal structure and a part of the adhesive layer are embedded in the stepped edge of the air pressure adjusting hole.
The microfluidic detection chip of claim 1, further comprising: a substrate layer located between the first elastomeric sealing layer and the bottom cap layer;

an antibody coating area is arranged on the surface of one side of the substrate layer close to the top cover layer;

the first elastomeric sealing layer comprising: a plurality of communicating pores corresponding to the positions of the antibody-coated regions;

the position of each communication hole corresponds to the position of the first microchannel.
The microfluidic detection chip of claim 9, wherein the substrate layer comprises: the biosensor is positioned in the antibody coating area, and the coating layer is positioned on the surface of the biosensor;

the surface of the coating layer is coated with an antibody.
The microfluidic detection chip according to claim 9, wherein the substrate layer is made of plastic, glass or silicon material; or, the substrate layer is made of a printed circuit board.
The microfluidic detection chip of claim 9, wherein the substrate layer and the bottom cap layer are a unitary structure.
The microfluidic detection chip according to any one of claims 1 to 12, wherein the sample application hole in the second elastic sealing layer is a flip-top sealing port.
The microfluidic detection chip according to any one of claims 1 to 12, wherein the top cover layer and the bottom cover layer are provided with a plurality of clamping structures on the surfaces facing the side of the liquid storage layer;

the liquid storage layer is provided with a plurality of grooves which are respectively in one-to-one correspondence with the positions of the clamping structures;

clamping holes which are in one-to-one correspondence with the positions of the clamping structures are formed in each film layer between the clamping structures and the grooves;

the clamping structure is inserted into the corresponding groove through the corresponding clamping hole.
A method of using the microfluidic detection chip according to any one of claims 1 to 14, comprising:

adding the sample to be detected into the second liquid storage hole through the sample adding hole;

controlling the pre-sealed reagent in the first liquid storage hole to be injected into the first micro-channel;

and controlling the sample to be detected in the second liquid storage hole to be injected into the first micro-channel.
The method of use of claim 15, wherein the controlling injection of the pre-sealed reagent in the first reservoir well into the first microchannel comprises:

and injecting the pre-sealed reagent in the first liquid storage hole into the first micro-channel by squeezing the second elastic sealing layer at the position corresponding to the first liquid storage hole.
The use of claim 15, wherein the sample application aperture in the second elastomeric sealing layer is a flip-top sealable port;

after will examining the sample to be added in the second stock solution hole through the application of sample hole, control examine in the second stock solution hole examine the sample inject into before in the first microchannel, still include:

closing the flip-top sealable opening;

piercing the fifth seal structure;

and repeatedly extruding the second elastic sealing layer corresponding to the position of the second liquid storage hole for multiple times so as to uniformly mix the sample to be detected with the diluent prestored in the second liquid storage hole.
The use method of claim 17, wherein the controlling the injection of the sample to be detected in the second reservoir hole into the first micro flow channel comprises:

the second elastic sealing layer is arranged at the position corresponding to the air pressure adjusting hole through extrusion, so that the sample to be detected in the second liquid storage hole is injected into the first micro-channel.