CN116925906A

CN116925906A - Microfluidic detection chip, automatic extraction detection analysis equipment and method

Info

Publication number: CN116925906A
Application number: CN202310998478.7A
Authority: CN
Inventors: 方彬彬; 江浴沂; 刘南
Original assignee: Tongguan Shanghai Biotechnology Co ltd
Current assignee: Tongguan Shanghai Biotechnology Co ltd
Priority date: 2023-08-09
Filing date: 2023-08-09
Publication date: 2023-10-24

Abstract

The micro-fluidic detection chip comprises a substrate, a film layer and a switching valve, wherein the film layer and the substrate are positioned on the upper surface and the lower surface of the substrate, a plurality of micro-channels and a plurality of chambers are defined by the film layer and the substrate, the switching valve and the vesicle are used for storing reagents, the switching valve is rotatably arranged on the valve chamber and comprises a plurality of communication grooves, the butt joint position of the substrate and the switching valve forms the valve chamber, the valve chamber comprises a plurality of cavities, each cavity is communicated with the vesicle and the chambers through each micro-channel, the communication grooves switch the communication state with each cavity by virtue of the rotation angle of the switching valve, and further realize communication or sealing among the micro-channels communicated with each chamber and/or the vesicle, and further switch the flow path of fluid so as to integrate a plurality of detection links. An automatic extraction detection analysis device and method are also provided.

Description

Microfluidic detection chip, automatic extraction detection analysis equipment and method

Technical Field

The invention relates to the field of nucleic acid detection biochemical analysis instruments, in particular to a micro-reagent reaction device for microfluidic liquid separation.

Background

Conventional PCR instruments generally employ PCR reaction tubes as containers for reagent reaction systems, and typically include single tube, 8-bar, 96-well plate PCR plates, and the like. In the conventional nucleic acid detection method, the reaction reagent generally needs to be subjected to sample pretreatment independently, and the steps include sample mixing, reagent addition, sample transfer and the like. The human operation factors in the process influence the purity and efficiency of nucleic acid extraction, and the consistency and repeatability influence the experimental result; the problems of uneven liquid separation of the nucleic acid reaction reagent, liquid reflux, large consumption of the reaction reagent, long reaction time and high cost exist.

Compared with the traditional container for PCR reaction reagent, the microfluidic chip can perform biological and chemical reactions within a tiny volume range, and has the advantages of small volume of required reagent, high detection sensitivity, high reaction speed, high reaction efficiency, easiness in integration and the like. The current nucleic acid detection method mainly comprises the steps of manually processing samples and then using a PCR instrument to carry out nucleic acid analysis. The manual operation causes the pretreatment of the sample and the extraction of the nucleic acid to be different, and can influence the detection accuracy. Furthermore, manual operation limits the throughput of the test and the location of the test, which can be risky.

In view of this, it is necessary to provide a fully automatic apparatus integrating the steps of nucleic acid mixing, extraction, transfer, testing, etc.

Disclosure of Invention

The invention aims to provide a microfluidic detection chip which can automatically integrate a plurality of links required by detection.

The microfluidic detection chip for achieving the purpose comprises a substrate and film layers positioned on the upper surface and the lower surface of the substrate, wherein the film layers and the substrate define a plurality of micro channels and a plurality of chambers, and the chip further comprises a switching valve; the chip further comprises vesicles for storing reagents; a valve chamber is formed at the butt joint position of the base plate and the switching valve, a plurality of cavities are included in the valve chamber, and each cavity is communicated with the vesicle and the chamber through each micro-channel; the switching valve is rotatably arranged on the valve chamber and comprises a plurality of communication grooves; the communication groove switches the communication state with each cavity by virtue of the rotation angle of the switching valve, so that the communication or the sealing between the chambers and/or the micro-channels of the vesicles is realized, and the flow path of the fluid is switched, so that a plurality of detection links are integrated.

In one or more embodiments, a portion of the bore is disposed not in communication with the communication groove at a portion of the rotation angle of the switching valve, and a portion of the bore is disposed in communication with the communication groove at a portion of the rotation angle of the switching valve at all times.

In one or more embodiments, the first part of the cavity encloses a region, the second part of the cavity is located in or/and outside the region, one part of the communicating groove is used for communicating two or more of the first part of the cavity, and the other part of the communicating groove is used for communicating one or more of the first part of the cavity and one or more of the second part of the cavity.

In one or more embodiments, the microfluidic detection chip includes a plurality of switching valves and a plurality of valve chambers, each of which is selectively or partially or fully in openable and closable communication with the switching valve in each of the valve chambers through the micro flow channel.

In one or more embodiments, the vesicles are disposed outside the membrane layer and are fluidly connected to the microchannel by a lancing device; the lancing device comprises a protruding lancing and a concave space, the micro flow channel is communicated with the concave space, the vesicle is borne in the concave space, the vesicle comprises a shell membrane and an accommodating space surrounded by the shell membrane, the accommodating space is used for loading reagent, the shell membrane is in pre-contact with the protruding lancing without lancing, and is in contact with the protruding lancing and is punctured when the shell membrane is subjected to external pressure, so that the reagent flows to the concave space and the micro flow channel.

In one or more embodiments, the chamber includes a nucleic acid extraction chamber, a PCR amplification reagent chamber, a waste liquid chamber, a quantification chamber, and a plurality of PCR reaction chambers, the nucleic acid extraction chamber pre-embeds a nucleic acid adsorption membrane, the PCR amplification reagent chamber pre-embeds a PCR amplification reagent, the waste liquid chamber is used for collecting waste liquid, each of the PCR reaction chambers pre-embeds a primer probe, and the quantification chamber is used for volumetric quantification.

In one or more embodiments, the switching valve includes a valve body on which the communication groove is provided, and a valve cover for fixing the valve body in a valve chamber, the valve body including an elastic portion for maintaining a conforming seal with the valve chamber by virtue of an elastic force generated by self deformation.

The invention further aims to provide automatic extraction detection analysis equipment which comprises the microfluidic detection chip, an optical detection device, a PCR amplification device, an ultrasonic mixing device, a first driving device, a second driving device, a third driving device, a fourth driving device and a fifth driving device, wherein the PCR amplification device is used for amplifying substances to be detected in the microfluidic detection chip; the ultrasonic mixing device is used for carrying out vibration mixing on the PCR amplification reagent cavity of the microfluidic detection chip; the first driving device is used for driving the switching valve to rotate and controlling the switching angle and the residence time of the switching valve; the second driving device is used for pressing the vesicles to break the vesicles; the third driving device is used for moving the microfluidic detection chip to a specific position in the analysis equipment; the fourth driving device is used for providing driving force for the fluid flow of the microfluidic detection chip; and the fifth driving device is used for driving the optical detection device to move so that the optical detection device can optically detect the specific part of the reaction cavity of the microfluidic detection chip.

It is still another object of the present invention to provide an automatic extraction detection analysis method for detection analysis using the microfluidic detection chip described above, the method comprising: rotating the control valve to enable the control valve to be in a first type of angle, enabling the microfluidic detection chip to sample, and guiding a sample to flow through a cavity until an object to be detected in the sample is adsorbed; rotating the control valve to enable the control valve to be in a second class angle, and guiding various cleaning solutions to flow through a cavity adsorbed with the object to be detected, and cleaning and/or eluting the object to be detected until the nucleic acid adsorbate is obtained; rotating the control valve to make the control valve at a third type of angle, introducing air pressure or hydraulic pressure, flushing the nucleic acid adsorbate into a chamber with a PCR amplification reagent for amplification reaction, and generating an object to be detected; and (3) rotating the control valve to enable the control valve to be in a fourth type of angle, introducing air pressure or hydraulic pressure, and sending the object to be detected into a PCR reaction chamber with a primer probe.

In one or more embodiments, the control valve is rotated to a fifth type of angle to block the microchannels at both ends of a chamber.

According to the microfluidic detection chip, the communicating groove and the pore cavity structure are arranged, the communicating groove switches the communicating state with each pore cavity by means of the rotation angle of the switching valve, so that the communication or sealing of each cavity and/or vesicle with the switching valve is realized, and the flow path of fluid is switched, so that a plurality of detection links are integrated, such as sample injection, cleaning, extraction and amplification links are integrated in one device, and errors possibly caused by artificial transfer are reduced. The vesicle can reduce the reagent packaged in the chip, and can provide power through extrusion, thereby effectively reducing the complexity of production and design.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description in conjunction with the accompanying drawings and embodiments, in which:

FIG. 1 is a schematic diagram of a portion of one embodiment of a microfluidic detection chip;

FIG. 2 is a schematic diagram of one embodiment of a film and a substrate;

3A-3B are schematic structural views of one embodiment of a valve cartridge;

FIG. 4 is a schematic view of one embodiment of a bore distribution structure within a valve cavity;

FIG. 5 is a schematic diagram of one embodiment of a vesicle;

FIG. 6 is a schematic view of an embodiment of a lancing device;

FIG. 7 is a schematic view of an embodiment of a valve cover;

FIG. 8 is a schematic diagram of the structural distribution of one embodiment of a microfluidic detection chip;

FIG. 9A is a fluid path profile of an automated extraction detection analysis method at step one;

FIG. 9B is a graph of the fluid path profile of the automatic extraction detection analysis method at step two;

FIG. 9C is a graph of the fluid path profile of the automated extraction detection analysis method at step three;

FIG. 9D is a graph of the fluid path profile of the automated extraction detection analysis method at step four;

FIG. 9E is a graph of the fluid path profile at step five in the automated extraction detection analysis method;

FIG. 9F is a graph of the fluid path profile at step six in the automated extraction detection analysis method;

FIG. 9G is a graph of the fluid path profile at step seven in the automated extraction detection analysis method;

FIG. 9H is a graph of the fluid path profile at step eight in the automated extraction detection analysis method;

FIG. 9I is a graph of the fluid path profile at step nine in the automated extraction detection analysis method;

FIG. 9J is a fluid path profile for one embodiment of an automated extraction detection analysis method at step ten;

FIG. 9K is a fluid path profile of another embodiment of an automated extraction detection analysis method at step ten;

fig. 9L is a distribution diagram of the fluid path at the tenth step in the automatic extraction detection analysis method.

Sign mark description

5. Micro-channel

6. Chamber chamber

20. Switching valve

21. First switching valve

22. Second switching valve

23. Input terminal

24. Communication groove

25. Bore cavity

27. Valve chamber

28. Valve core

30. Valve cover

40. Vesicle with a membrane

41. First vesicle

42. Second vesicle

43. Third vesicle

44. Fourth vesicle

100. Microfluidic detection chip

110. Lower film

111. An outlet

112 Inlet pipeline of PCR reaction cavity

113 Outlet pipeline of PCR reaction cavity

120. Film coating

130. Nucleic acid adsorption membrane

140. Freeze-dried beads

150. Waterproof breathable film

160. Primer probe

200. Substrate board

201. Liquid inlet cavity

202. Air inlet

204. Nucleic acid extraction chamber

205. Quantitative cavity

207. First ventilation hole

208 PCR reaction chamber

209 PCR amplification reagent chamber

210. Waste liquid cavity

211. Second ventilation hole

212. Puncturing device

213. Liquid storage cavity

214. Third ventilation hole

241. First communicating groove

242. Second communicating groove

243. Third communicating groove

244. Fourth communicating groove

281. Upper valve core

282. Lower valve core

283. Raised column

284. Concave groove

401. Shell membrane

402. Accommodation space

2122. Protruding thorn

2123. Concave space

Detailed Description

The present invention will be further described with reference to specific embodiments and drawings, in which more details are set forth in the following description in order to provide a thorough understanding of the present invention, but it will be apparent that the present invention can be embodied in many other forms than described herein, and that those skilled in the art may make similar generalizations and deductions depending on the actual application without departing from the spirit of the present invention, and therefore should not be construed to limit the scope of the present invention in terms of the content of this specific embodiment.

It is noted that these and other figures are merely examples, which are not drawn to scale and should not be construed as limiting the scope of the invention as it is actually claimed.

The structure of the microfluidic detection chip is understood in conjunction with fig. 1, 2 and 8 and includes a substrate 200 and film layers on upper and lower surfaces of the substrate 200, the film layers and the substrate 200 defining a plurality of micro channels 5 and a plurality of chambers 6. In the embodiment shown in fig. 2, the film layer includes an upper film 120 and a lower film 110, and the substrate 200 is positioned between the upper film 120 and the lower film 110.

In some embodiments, chamber 6 includes a nucleic acid extraction chamber 204, a PCR amplification reagent chamber 209, a waste chamber 210, a quantification chamber 205, a reservoir chamber 213, a plurality of PCR reaction chambers 208, and the like. The nucleic acid extraction chamber 204 is embedded with the nucleic acid adsorption film 130, the nucleic acid adsorption film 130 can be used for adsorbing substances such as RNA in a sample, impurities can be washed away by a lysate or alcohol without washing away the RNA, and the RNA can be washed away by EB liquid. Preferably, the nucleic acid adsorbing membrane 130 is immobilized within the nucleic acid extraction chamber 204 and cannot shake or move within the chamber.

PCR amplification reagents are pre-embedded in the PCR amplification reagent chamber 209, and the PCR amplification reagents can exist in the form of freeze-dried beads 140. The freeze-dried beads can be stored at normal temperature, can only stay in the PCR amplification reagent cavity, can not move into other pipelines and cavities, and can be rapidly dissolved after encountering water. The waste chamber 210 is used to collect waste, and preferably the waste chamber 210 is in communication with the atmosphere and liquid cannot flow out of the chip. The primer probes 160 are pre-buried in each PCR reaction cavity 208, and the primer probes 160 can be stored at normal temperature and can only stay in the PCR reaction cavities 208 without moving into other pipelines and cavities, and can be quickly dissolved after encountering water. The liquid storage cavity 213 is used for collecting excessive liquid and is used as a buffer transit space between the chambers.

The chip further comprises a switching valve 20, as will be appreciated with reference to fig. 1-4, 7, the abutment of the base plate 200 with the switching valve 20 forms a valve chamber 27, the switching valve 20 being rotatably arranged within the valve chamber 27. The switching valve includes a plurality of communication grooves 24, and the communication grooves 24 change angles following the rotation of the switching valve 20. In some embodiments, the switching valve 20 includes a valve body 28 and a valve cover 30, the valve body 28 is provided with a communication groove 24, and the valve cover 30 fixes the valve body 28 in the valve chamber 27, for example, by welding the valve body to the outer periphery of the valve chamber by ultrasonic welding. The valve chamber 27 includes a plurality of bores 25 therein, each bore 25 being in communication with one of the chambers 6 through one of the microchannels 5, the communication groove 24 being located above the bore 25. The communication groove 24 switches the communication state with each bore 25 by the rotation angle of the switching valve 20. When the communicating groove 24 rotates to above the pair of cavities 25, the two cavities 25 are communicated, so that the communication or the sealing of each cavity 6 is realized, and the flow paths of the fluid are switched, so that a plurality of detection links are integrated.

P0-P8 in FIG. 4 refer to a circle of bores 25. As the switching valve 20 is at the rotation angle shown in fig. 4, the first communicating groove 241 connects the bores P4 and P2, the second communicating groove 242 communicates the bores P5 and P6, the third communicating groove 243 communicates the bores P7 and P0, and the fourth communicating groove 244 communicates the bores P8 and P1, respectively. When the angle of the switching valve 20 is changed, each communication groove 24 switches the cavity 25 communicated with the communication groove, and switching of the fluid flow paths is further realized.

In some embodiments, the valve core 28 includes an elastic portion for maintaining a conforming seal with the valve chamber by virtue of an elastic force generated by self-deformation. The valve cap 30 presses the valve body 28 and elastically deforms the valve body 28, and the valve body 28 maintains sealing engagement with the valve chamber without leakage by virtue of elastic force generated by self deformation.

In the embodiment shown in fig. 3A, the valve core 28 includes an upper valve core 281 and a lower valve core 282, and the lower valve core 282 is an elastic portion, and is deformed to some extent after being enclosed in the valve cover 30. The upper valve core 281 is provided with an input end 23, and the input end 23 is used for receiving external torque to enable the switching valve 20 to rotate along with the input end, for example, a stepping motor is used for realizing rotation of a specific angle. The material of the upper spool 281 is preferably of high hardness, and a material with little elastic deformation is selected to achieve the mating of the input 23 with an external input so that the spool rotates with the input.

The upper spool 281 and the lower spool 282 may be connected by means such as welding, in the embodiment shown in fig. 3A, the upper spool 281 includes a boss post 283, the lower spool 282 includes a recess 284 that mates with the boss post 283, and the positional interface between the upper spool 281 and the lower spool 282 is accomplished, and the two are held stationary by welding, as shown in fig. 3B.

In some embodiments, a portion of the bore 25 is disposed not in communication with the communication groove 24 at a portion of the rotation angle of the switching valve 20, and a portion of the bore 25 is disposed always in communication with the communication groove 24 at a portion of the rotation angle of the switching valve 20. For example, the first partial bore defines an enclosed region, such as a circle, and the second partial bore is located within or/and outside the enclosed region, with one portion of the communication slot for communicating with two or more of the first partial bores and another portion of the communication slot for communicating with one or more of the first partial bores and one or more of the second partial bores.

As will be appreciated in connection with fig. 4, the cavity P0 is located inside the circular area C surrounded by the other cavities, by providing a channel extending in the radial direction of the valve body, such as the third communication groove 243, the switching valve 20 can be made to maintain communication of the cavity P0 with one of the communication grooves 24 all the time at a plurality of rotation angles, and the third communication groove 243 can communicate the cavity P0 with any one of the cavities P1 to P8 outside a plurality of times. It is also possible to provide an arc-shaped channel such as the first communicating groove 241 such that the arc-shaped channel avoids some of the bores, and if the upper side of the bore P3 is not in contact with any communicating groove, the bore P3 is blocked by the body of the valve cartridge, and the micro flow channel communicating with the bore P3 is in a blocked state.

The arrangement of the communication groove not only realizes the connection of 45-degree adjacent channels, but also realizes the connection of 90-degree channels, and is also connected with the central hole channel, so that a plurality of flexible sites are provided, and the chip is convenient to realize an integrated operation flow.

It will be appreciated by those skilled in the art that the channel shape of the communication channel includes, but is not limited to, the shape shown in the drawings, and that other shapes of communication channels capable of performing the above functions are applicable to the present disclosure, nor are the number of bores limited to the examples shown in the drawings, but are determined by the staff according to the specific test procedure.

For more complicated and precise measurement, the switching valve 20 may be provided in plural, and each chamber may be selectively or partially or entirely in openable and closable communication with the switching valve in each valve chamber 27 through a microchannel. As shown in fig. 8, the microfluidic detection chip includes a first switching valve 21, a second switching valve 22, and two valve chambers 27 corresponding thereto, respectively, and the two switching valves may have the same structure or may have different structures.

In some embodiments, the chip further comprises a liquid inlet chamber 201 and an air inlet 202, which are respectively communicated with the cavity 25 of the valve chamber where the first switching valve 21 is located through the micro-channel 5, and a waste liquid chamber 210 is communicated with the cavity 25 of the valve chamber where the second switching valve 22 is located. The air inlet 202 is used for supplying air pressure for driving fluid to flow, and the liquid inlet cavity 201 is used for supplying a sample. The air inlet 202 is externally connected with an air source and can provide pressure.

Preferably, the air inlet 202 has a sealing ring to prevent air leakage.

The flow of fluid in each flow path is driven either pneumatically or hydraulically. Care is taken to balance the pressure in the flow path. To solve this problem, in some embodiments, the chip further includes a plurality of ventilation holes, and the waterproof ventilation film 150 is attached to the surface in the ventilation holes, and the waterproof ventilation film 150 can only pass through air, and cannot pass through liquid. The air holes are communicated with the atmosphere outside the microfluidic detection chip and the micro-channels 5 and/or the chambers 6 and/or the cavities 25, and the positions of the air holes on the microfluidic detection chip can be arranged on each flow path of the fluid so as to balance the air pressure in each flow path. As shown in fig. 8, the second ventilation holes 211 are communicated with the waste liquid cavity 210, so that the liquid flowing into the waste liquid cavity can realize pressure balance and pressure relief effects through the second ventilation holes 211 at the tail end; the third ventilation holes 214 are communicated with the liquid storage cavity 213, so that the problem that fluid cannot continue to flow due to the fact that the pressure in the flow path is high is avoided. The vent may also be in communication with a bore 25 of the chamber in which the switching valve 20 is located, such as the first vent 207.

The chip further comprises vesicles 40 for storing reagents, wherein the vesicles 40 are connected with the cavities 25 in the valve chamber 27 through the micro-channels 5, and the communication between the liquid in each vesicle and each cavity is realized by adjusting the rotation angle of the switching valve. The vesicle 40 is used to store reagents required for extracting nucleic acids. In the embodiment shown in FIG. 8, a plurality of vesicles 40 each store a different nucleic acid reagent, such as a plurality of wash lysates or alcohol. The vesicles 40 may be powered by being compressed, and are simple and readily available. In addition, the vesicle is positioned outside the chip, and the reagent is encapsulated in the vesicle without being encapsulated in a cavity, so that the complexity of the microfluidic chip can be reduced.

Further, in some embodiments, the vesicles 40 are disposed outside the membrane layer, fluidly connected to the microchannels by the lancing device 212. The lancing device 212, as shown in FIG. 6, includes a protruding lance 2122 and a recessed space 2123, and the microchannel 5 communicates with the recessed space 2123 and the vesicle 40 is supported in the recessed space 2123. As shown in fig. 5, the vesicle 40 includes a shell membrane 401 and a housing space 402 surrounded by the shell membrane 401, the housing space 402 storing a liquid reagent. The case membrane 401 is pre-contacted with the protruding piercing 2122 without piercing, and is contacted with the protruding piercing 2122 and pierced when being subjected to external pressure, so that the reagent in the accommodation space 402 flows to the micro flow channel 5 and continuously flows therein by means of hydraulic pressure. The external force to which the vesicle 40 is subjected may be a downward force, such as from top to bottom, to force the shell membrane 401 into contact with the protruding burr 2122. By squeezing the vesicle, the shell membrane 401 is pierced by the protruding stings, so that the reagents inside the vesicle flow along the depressions into the micro-channels.

Preferably, the pre-packaged reagents in the vesicles can all be stored at normal temperature. In some embodiments, at least one vesicle 40 is in communication with the air inlet 202, the dosing chamber 205, the PCR amplification reagent chamber 209, the PCR reaction chamber 208, and the air vent via the switching valve 20 to perform a washing function. When the liquid flows into one cavity after the vesicle is extruded and another liquid is needed to be used for cleaning, the liquid which is retained in the pipeline before is cleaned by the cleaning liquid in the vesicle, and then the liquid is used for cleaning.

In combination with the above description of the microfluidic detection chip, an automatic extraction detection analysis method can be understood, and the method uses the automatic extraction detection analysis device to perform detection. The method comprises the following steps: rotating the control valve to make the control valve at a first type angle, so that the microfluidic detection chip samples, and guiding a sample to flow through a cavity until an object to be detected in the sample is adsorbed; rotating the control valve to enable the control valve to be in a second class angle, and guiding various cleaning liquids to flow through the cavity adsorbed with the object to be detected, so as to clean the object to be detected until the nucleic acid adsorbate is obtained; rotating the control valve to make the control valve at a third type of angle, introducing air pressure or hydraulic pressure, flushing the nucleic acid adsorbate into a chamber with a PCR amplification reagent for amplification reaction, and generating an object to be detected; and (3) rotating the control valve to enable the control valve to be at a fourth type of angle, introducing air pressure or hydraulic pressure, and sending the object to be detected into a PCR reaction chamber with a primer probe and detecting the object by an optical detection device.

In some embodiments, the control valve may be turned to a fifth angle to block the micro-channels at two ends of a chamber, thereby closing the flow path.

For example, the microfluidic detection chip is injected, the control valve is rotated to be in a first type of angle, and the sample is guided to flow through a chamber until the object to be detected is adsorbed. Rotating the control valve to enable the control valve to be in a first type and a second type, guiding a first cleaning solution to flow through the chamber of the object to be detected, cleaning the object to be detected, and removing salt ions, cell fragments, proteins and the like in the object to be detected; or the control valve is rotated to be in a second type of angle, and a second cleaning solution is guided to flow through the chamber of the object to be detected, so that the object to be detected is cleaned, and salt ions, proteins and the like in the object to be detected are removed; or rotating the control valve to make the control valve be positioned at a third group of angles of a second class, wherein the first cleaning liquid, the second cleaning liquid and the like are in the nucleic acid adsorption film; and rotating the control valve to enable the control valve to be in a fourth second-class angle, guiding the eluent to flow through the chamber of the object to be detected, cleaning the object to be detected, and eluting the nucleic acid from the nucleic acid adsorbate. And (3) rotating the control valve to enable the control valve to be at a third type of angle, introducing air pressure or hydraulic pressure to flush the nucleic acid mixed solution in the quantitative cavity to the freeze-drying cavity PCR amplification reagent cavity, and uniformly mixing the nucleic acid mixed solution with the PCR amplification reagent in the freeze-drying cavity PCR amplification reagent cavity to perform amplification reaction. The control valve is rotated to be in a fourth type of angle, and air pressure or hydraulic pressure is introduced to drive liquid in the PCR amplification reagent cavity of the freeze-drying cavity to enter the PCR reaction cavity of the reaction cavity. The control valve is rotated to enable the control valve to be in a fifth angle, micro-channels at two ends of the PCR reaction cavity of the reaction cavity are blocked, sealing is achieved, and liquid in the reaction cavity after heating is prevented from flowing into other micro-channels, so that PCR amplification is prevented from being influenced.

The use of one embodiment of the method is described in more detail below in conjunction with fig. 9A-9H.

First, as shown in FIG. 9A, the first switching valve 21 and the second switching valve 22 are rotated to communicate the third communication groove 243 of the first switching valve 21 with the cavities P0 and P4, and further to communicate the liquid inlet chamber 201, the third communication groove 243 and the nucleic acid extraction chamber 204; the third communication groove 243 of the second switching valve 22 communicates with the bores P0, P4, and further communicates the nucleic acid extraction chamber 204, the third communication groove 243 of the second switching valve 22, and the waste liquid chamber 210.

By means of the first switching valve 21 and the second switching valve 22, after the sample enters from the liquid inlet chamber 201, it flows into the cavity P4 along the micro channel 5, then enters the cavity P0 through the third communicating groove 243, and then flows out of the third communicating groove 243 along the micro channel 5 to the nucleic acid extraction chamber 204, and the nucleic acid substance in the sample can be adsorbed by the nucleic acid adsorbing membrane 130 of the nucleic acid extraction chamber 204. After the sample is adsorbed, the rest liquid flows into the cavity P4 of the second switching valve 22 through the outlet of the nucleic acid extraction cavity 204, then enters the cavity P0 through the third communication groove 243, then flows into the waste liquid cavity 210 along the micro-channel 5, the waste liquid cavity 210 is communicated with the second air holes 211, and the sample liquid finishes flowing from the liquid inlet cavity 201 into the nucleic acid extraction cavity 204 and the waste liquid cavity 210, so that the adsorption of nucleic acid substances is realized.

Preferably, the liquid is required to slowly pass through the nucleic acid adsorbing membrane 130, and the whole liquid after passing through the membrane flows into the waste liquid chamber 210. At this time, the nucleic acid in the sample solution remaining in the tube is adsorbed by the nucleic acid adsorbing membrane.

Step two as shown in fig. 9B is then performed, rotating only the first switching valve 21 without rotating the second switching valve 22. The third communication groove 243 of the first switching valve 21 communicates with the cavities P0 and P5, the cavity P5 is connected to the first vesicle 41, and the cavity P0 is connected to the inlet of the nucleic acid extraction chamber 204. After the first vesicle 41 is pierced, the first cleaning solution in the storage space 402 flows into the cavity 25 and the third communicating groove in this order through the microchannel 5, flows into the nucleic acid isolation chamber 204, and passes through the nucleic acid adsorbing membrane 130 of the nucleic acid isolation chamber 204. The first cleaning solution in the first vesicle 41 is used to clean the nucleic acid adsorbing membrane, so that salt ions, cell debris, proteins, etc. on the nucleic acid adsorbing membrane 130 can be removed, and the reagent flowing through the nucleic acid adsorbing membrane 130 continues to flow into the waste liquid chamber 210 through the micro flow channel and the valve chamber of the second switching valve 22.

Preferably, the liquid needs to slowly pass through the membrane, and all the liquid after passing through the membrane flows into the waste liquid chamber 210. At this time, the first cleaning liquid remains in the nucleic acid extraction chamber and the tube.

Then, as shown in FIG. 9C, the first switching valve 21 is rotated continuously, the angle of the second switching valve 22 is kept constant, the third communication groove 243 of the first switching valve 21 is connected to the cavities P0, P6, and the nucleic acid extraction chamber 204 and the waste liquid chamber 210 are communicated through the second switching valve. The second washing solution in the second vesicle 42 is used to wash the nucleic acid adsorbing membrane 130 of the nucleic acid extracting chamber 204, and to remove salt ions, proteins, and the like on the nucleic acid adsorbing membrane 130 again. The reagent flowing through the nucleic acid adsorbing membrane 130 continues to flow into the waste liquid chamber 210 through the micro flow channel and the valve chamber of the second switching valve 22.

Preferably, the liquid needs to be passed through the membrane at a medium speed, and the whole liquid after passing through the membrane flows into the waste liquid chamber 210. At this time, the second washing liquid remains in the nucleic acid extraction chamber and the tube.

Subsequently, as shown in FIG. 9D, the first switching valve 21 is continuously rotated, the angle of the second switching valve 22 is kept constant, the third communication groove 243 of the first switching valve 21 is connected to the bores P0, P7, and the nucleic acid extraction chamber 204 and the waste liquid chamber 210 are communicated through the second switching valve. The third vesicle 43 is pressed into the third washing liquid, enters the nucleic acid extraction chamber 204 along the micro flow channel, and finally flows into the waste liquid chamber 201. The third washing liquid may be a high-concentration alcohol for washing the first washing liquid, the second washing liquid, etc. in the nucleic acid-adsorbing film.

Preferably, the liquid needs to be quickly subjected to membrane filtration, and all the liquid after membrane filtration flows into the waste liquid chamber 210. At this time, the third cleaning solution remains in the nucleic acid extraction chamber and the line.

Then, step five shown in fig. 9E is performed, the first switching valve 21 is rotated, and the angle of the second switching valve 22 is kept unchanged. The third communication groove 243 of the first switching valve 21 is connected to the bores P0, P3. The nucleic acid extraction chamber 204 is heated, air is introduced into the air inlet 202, and alcohol, water, etc. in the nucleic acid extraction chamber are discharged to the waste liquid chamber 210, so that no liquid remains in the nucleic acid extraction chamber and the pipeline.

Subsequently, as shown in fig. 9F, the first switching valve 21 is continuously rotated, and the first communication groove 241 of the first switching valve 21 is communicated with the bores P8, P0. The fourth vesicle 44 is pressed outward, so that the fourth vesicle 44 is pierced by the protrusion, and the eluent in the fourth vesicle 44 flows along the micro flow channel to the nucleic acid extraction chamber 204. The eluent stays in the nucleic acid extraction chamber 204 for a period of time such that the nucleic acid adsorption membrane 130 is sufficiently immersed in the eluent to elute the nucleic acid substances on the nucleic acid adsorption membrane, thereby obtaining purified nucleic acids.

Preferably, the flow of fluid through the second switching valve 22 may be stopped by controlling the valve and the driving means, without continuing to flow to the waste chamber 210.

Step seven as shown in fig. 9G is then performed, rotating the first switching valve 21 and the second switching valve 22. The third communication groove 243 of the first switching valve 21 is connected to the bores P0, P3 to communicate the air inlet 202 with the nucleic acid extraction chamber 204. The fourth communication groove 244 of the second switching valve 222 is connected to P4 and P5 to connect the quantitative chamber 205 and the outlet of the nucleic acid extraction chamber 204. The second communication groove 242 of the first switching valve 21 is connected with the bores P1, P2 to connect the outlet of the dosing chamber 205 with the liquid storage chamber 213. Air is introduced into the air inlet 202, and purified nucleic acid in the nucleic acid isolation chamber 204 is introduced into the quantitative chamber 205.

Preferably, the flow of liquid to the dosing chamber 205 to the valve 21 is stopped and no flow to the reservoir 213 is required. There is no reservoir which would flow along the line into the valve 22 if there were an excess of liquid, as a preventive mechanism.

Step eight as shown in fig. 9H is performed to rotate the first switching valve 21 and the second switching valve 22. The first communication groove 241 of the first switching valve 21 is connected to the bores P1, P3 to communicate the air inlet 202 with the dosing chamber 205. The second communication groove 242 of the second switching valve 22 is communicated with P5 and P6 to communicate the quantitative chamber 205 and the PCR amplification reagent chamber 209. The gas inlet 202 is filled with gas, and the purified nucleic acid in the quantitative chamber 205 is pressed into the PCR amplification reagent chamber 209.

Preferably, the liquid of the dosing chamber flows entirely into the PCR amplification reagent chamber 209, but cannot exceed the volume of the PCR amplification reagent chamber to avoid flowing into the waste chamber 210.

Step nine shown in fig. 9I is then performed to rotate the first switching valve 21 and the second switching valve 22. The third communication groove 243 of the first switching valve 21 is connected to the cavities P8, P0 to communicate the fourth vesicle 44 with the nucleic acid extraction chamber 204 again. The first communication groove 241 of the second switching valve 222 is connected to P4 and P6 to communicate the outlet of the nucleic acid extracting chamber 204 with the inlet of the PCR amplification reagent chamber 209. The second communication groove 242 is connected with P7 and P8 to communicate the outlet of the PCR amplification reagent chamber 209 with the first vent 207.

The fourth vesicle 44 contains an eluent, the eluent enters the nucleic acid extraction chamber 204 along the micro flow channel, and the eluent stays in the nucleic acid extraction chamber for a period of time, so that the eluent is fully contacted with the nucleic acid adsorption membrane 130, and the nucleic acid substances on the nucleic acid adsorption membrane are eluted again, so that the purified nucleic acid is obtained. The fourth vesicle 44 is again pressed, and the purified nucleic acid in the nucleic acid extraction chamber is pressed into and fills the PCR amplification reagent chamber 209. At this time, the ultrasonic vibration device may be started so that the premixed RT-PCR reaction system in the PCR amplification reagent chamber is mixed.

Preferably, all of the liquid in the nucleic acid extraction chamber is pressed into the PCR amplification reagent chamber.

Step ten shown in fig. 9J is performed to rotate the first switching valve 21 and the second switching valve 22. The second communication groove 242 of the first switching valve 21 is connected to the bores P8, P1 to communicate the fourth bladder 44 with the dosing chamber 205. The fourth communication groove 244 of the second switching valve 22 is connected to the cavities P5 and P6 to communicate the quantitative chamber 205 with the PCR amplification reagent chamber 209, and the first communication groove 241 of the second switching valve 22 is connected to the cavities P7 and P1 to communicate the PCR amplification reagent chamber 209 with the PCR reaction chamber 208. The inlets of the plurality of PCR reaction chambers 208 are adjacent to chamber P1 and the outlets 111 are connected to chamber P2.

The fourth vesicle 11 is pressed, and the reaction liquid which is uniformly mixed in the PCR amplification reagent chambers is pressed into and fills each PCR reaction chamber. At this time, the sum of the liquid volume in the quantitative chamber 205, the liquid volumes of the pipelines at both ends of the quantitative chamber 205, the liquid volumes of the nucleic acid extraction chamber, and the liquid volumes of the pipelines at both ends of the nucleic acid extraction chamber 204 is equal to the sum of the liquid volumes in the PCR amplification reagent chamber 209 and the liquid volumes of the pipelines at both ends of the PCR amplification reagent chamber 209, and is also equal to the sum of the liquid volumes in the PCR reaction chamber 208 and the liquid volumes of the pipelines at both ends of the PCR reaction chamber 208.

The above-described manner is achieved by squeezing the vesicles, which in another embodiment may also be pneumatically driven. As shown in fig. 9K, the first communication groove 241 of the first switching valve 21 communicates with the cavities P3, P1 to communicate the air inlet 202 with the quantitative chamber 205, the fourth communication groove 244 of the second switching valve communicates with the cavities P5, P6 to communicate the quantitative chamber 205 with the PCR amplification reagent chamber 209, and the first communication groove 241 of the second switching valve 22 communicates with the cavities P7, P1 to communicate the PCR amplification reagent chamber 209 with the PCR reaction chamber 208. The inlets of the plurality of PCR reaction chambers 208 are adjacent to chamber P1 and the outlets 111 are connected to chamber P2. The air inlet 202 is filled with air, and the uniformly mixed reaction liquid in the PCR amplification reagent chambers is pressed into and fills each PCR reaction chamber in an air pressure driving mode.

Finally, step eleven is carried out to seal the valve. The second communicating groove 242 of the first switching valve 21 is communicated with the cavities P3 and P2, so that the air inlet 202 is communicated with the third ventilation port 214 through the liquid storage cavity 213, and the microfluidic chip is separated from the air inlet device. The fourth communicating groove 244 of the second switching valve 22 communicates with the cavity P1 and the cavity P2, but does not communicate with each other, and at this time, the PCR reaction cavity inlet pipeline 112 and the PCR reaction cavity outlet pipeline 113 respectively communicate with the cavity P1 and the cavity P2, so as to achieve a sealing effect, and prevent the liquid in the reaction cavity 208 from flowing into other micro channels after heating, thereby affecting PCR amplification.

Therefore, through the angle control of the first switching valve 21 and the second switching valve 22, the integrated automation of a plurality of links of reagent automatic sample injection, extraction, cleaning, reaction and detection can be realized, the liquid backflow can be effectively avoided by means of the design of the cavity and the connecting groove, and the method has the advantages of high flux, low cost, easy operation and high accuracy, and solves the problems of large consumption of the reaction reagent and long reaction time. Can realize the automatic extraction and detection of various pathogenic nucleic acids such as coronavirus, influenza virus, adenovirus and the like.

The microfluidic detection chip adopts a 6-color optical system, different primer probes are pre-embedded in 12 holes, 72 virus positives can be detected in total, 72 items can be detected at one time, and multi-item detection is realized; the method has the advantages that the method is also provided with a trace reaction system, in some embodiments, the reaction system can reach 2.8ul, the method is accurate and trace, the use of reagents is effectively saved, the method can be particularly used in the aspect of sample precious detection links, such as criminal investigation detection, and the obtained samples are extremely rare, so that the trace detection can be used for detecting results normally.

In combination with the above description of the microfluidic detection chip, it may also be understood that an automatic extraction detection analysis apparatus includes an optical detection device, a PCR amplification device, a first driving device, a second driving device, a third driving device, a fourth driving device, and a fifth driving device. The PCR amplification device is used for amplifying substances to be detected in the microfluidic detection chip, the first driving device is used for driving the switching valve to rotate and controlling the switching angle and the residence time of the switching valve, the second driving device is used for applying pressure to the vesicle to enable the vesicle to be broken, the third driving device is used for moving the microfluidic detection chip to a specific position in the analysis equipment, the fourth driving device is used for providing driving force for fluid flow of the microfluidic detection chip, and the fourth driving device can be a gas compression device for example so as to press gas into a gas inlet of the microfluidic detection chip. And the fifth driving device is used for driving the optical detection device to move so that the optical detection device can carry out optical detection on the specific part of the reaction cavity of the microfluidic detection chip to obtain a detection result.

In some embodiments, the apparatus further comprises an ultrasonic mixing device for vibration mixing of the PCR amplification reagent chambers of the microfluidic detection chip.

In some embodiments, the automatic extraction detection analysis device further comprises a main control module, wherein the main control module is in signal connection with the optical detection device, the PCR amplification device, the first driving device, the second driving device, the third driving device, the fourth driving device and the fifth driving device, and each of the first driving device, the second driving device, the third driving device, the fourth driving device and the fifth driving device further comprises an independent auxiliary control module, and each independent auxiliary control module receives the instruction of the main control module and independently controls the actuation of each driving device.

Specifically, the first driving device comprises a first independent auxiliary control module and a driving piece, and the driving piece is used for driving each switching valve to rotate under the instruction of the first independent auxiliary control module and controlling the switching angle of each switching valve. The second driving device comprises a second independent auxiliary control module and a driving piece, wherein the driving piece is used for pressing the vesicle under the instruction of the second independent auxiliary control module. The third driving device comprises a third independent auxiliary control module and a driving piece, wherein the driving piece is used for carrying the micro-fluidic chip to a specific position in the instrument under the instruction of the third independent auxiliary control module. The fourth driving device comprises a fourth independent auxiliary control module and a driving piece, and the driving piece is used for pressing the microfluidic detection chip on the PCR amplification device under the instruction of the fourth independent auxiliary control module. The fifth driving device comprises a fifth independent auxiliary control module and a driving piece, wherein the driving piece is used for driving the optical fiber of the optical detection device to reciprocate under the instruction of the fifth independent auxiliary control module, and the optical fiber can vertically align each reaction cavity PCR reaction cavity of the microfluidic detection chip.

The gas compression device comprises a gas compression independent auxiliary control module and a driving piece, wherein the driving piece is used for pressing gas into the gas inlet of the microfluidic detection chip under the instruction of the gas compression independent auxiliary control module. The ultrasonic mixing device comprises an ultrasonic independent auxiliary control module and a driving piece, wherein the driving piece is used for vibrating and mixing the freeze-drying cavity PCR amplification reagent cavity of the microfluidic detection chip under the instruction of the ultrasonic independent auxiliary control module, the PCR amplification device circularly heats the reaction cavity PCR reaction cavity area, the optical detection device emits light with specific wavelength, the reaction cavity PCR reaction cavity of the microfluidic detection chip is irradiated by the optical fiber, and fluorescence generated by excitation after the irradiation of the reaction cavity PCR reaction cavity reagent is recycled into the optical module by the optical fiber. The optical detection device converts the optical signal into an electrical signal and then obtains an experimental result through a software algorithm.

The automatic extraction detection analysis equipment can realize closed full-automatic reagent sample addition, full-automatic nucleic acid extraction, automatic mixing of reaction reagents and high-precision liquid separation of a plurality of PCR reaction holes, effectively solves the problem of low detection flux, can realize rapid automatic high-precision liquid separation, and improves the reaction speed. In addition, the simultaneous detection of 72 different molecular targets is realized through the light of a plurality of reaction holes and a plurality of reaction wavelengths, and the multiplex nucleic acid detection is realized.

It should be noted that, the foregoing descriptions of the words "first" and "second" are used to define the components, and are merely for convenience of distinguishing the corresponding components, and the words do not have special meaning unless otherwise stated, so they are not to be construed as limiting the scope of the application.

Meanwhile, the present application uses specific words to describe embodiments of the present application. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic is associated with at least one embodiment of the application. Thus, it should be emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various positions in this specification are not necessarily referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the application may be combined as suitable.

While the application has been described in terms of preferred embodiments, it is not intended to be limiting, but rather to the application, as will occur to those skilled in the art, without departing from the spirit and scope of the application. Therefore, any modification, equivalent variation and modification of the above embodiments according to the technical substance of the present application fall within the protection scope defined by the claims of the present application.

Claims

1. The microfluidic detection chip comprises a substrate and a film layer positioned on the upper surface and the lower surface of the substrate, wherein the film layer and the substrate define a plurality of micro channels and a plurality of chambers, and the chip further comprises a switching valve;

it is characterized in that the method comprises the steps of,

the chip further comprises vesicles for storing reagents;

a valve chamber is formed at the butt joint position of the base plate and the switching valve, a plurality of cavities are included in the valve chamber, and each cavity is communicated with the vesicle and the chamber through each micro-channel;

the switching valve is rotatably arranged on the valve chamber and comprises a plurality of communication grooves;

the communication groove switches the communication state with each cavity by virtue of the rotation angle of the switching valve, so that the communication or the sealing between the chambers and/or the micro-channels of the vesicles is realized, and the flow path of the fluid is switched, so that a plurality of detection links are integrated.

2. The microfluidic detection chip according to claim 1, wherein a part of the cavity is provided so as not to communicate with the communication groove at a part of the rotation angle of the switching valve, and a part of the cavity is provided so as to always communicate with the communication groove at a part of the rotation angle of the switching valve.

3. The microfluidic chip according to claim 2, wherein the first part of the cavities define a region, the second part of the cavities are located in or/and outside the region, one part of the communication grooves are used for communicating two or more of the first part of the cavities, and the other part of the communication grooves are used for communicating one or more of the first part of the cavities and one or more of the second part of the cavities.

4. The microfluidic detection chip according to any one of claim 1, wherein the microfluidic detection chip comprises a plurality of switching valves and a plurality of valve chambers, each of the chambers being selectively or partially or fully in openable and closable communication with the switching valve in each of the valve chambers via the microchannel.

5. The microfluidic detection chip according to claim 1, wherein the vesicles are disposed outside the membrane layer and are fluidically connected to the microchannels by a puncturing device; the puncturing device comprises a protruding puncture and a concave space, the micro-flow channel is communicated with the concave space, the vesicle is loaded in the concave space,

the vesicle includes a housing membrane and a housing space surrounded by the housing membrane, the housing space is used for loading a reagent, the housing membrane is in pre-contact with the protruding thorns without puncturing, and is arranged to be in contact with the protruding thorns and punctured when the housing membrane is subjected to external pressure, so that the reagent flows to the recessed space and the micro flow channel.

6. The microfluidic chip according to claim 1, wherein the chamber comprises a nucleic acid extraction chamber, a PCR amplification reagent chamber, a waste liquid chamber, a quantitative chamber and a plurality of PCR reaction chambers, wherein the nucleic acid extraction chamber is embedded with a nucleic acid adsorption film, the PCR amplification reagent chamber is embedded with a PCR amplification reagent, the waste liquid chamber is used for collecting waste liquid, the PCR reaction chambers are embedded with primer probes, and the quantitative chamber is used for volumetric quantification.

7. The microfluidic detection chip according to claim 1, wherein the switching valve comprises a valve core and a valve cover, the valve core is provided with the communication groove, the valve cover is used for fixing the valve core in a valve chamber, the valve core comprises an elastic part, and the elastic part is used for keeping fit and sealing with the valve chamber by virtue of elastic force generated by self deformation.

8. An automatic extraction detection analysis apparatus, characterized by comprising:

the microfluidic detection chip according to any one of claims 1-7;

the ultrasonic mixing device is used for vibrating and mixing the PCR amplification reagent cavity of the microfluidic detection chip;

an optical detection device;

the PCR amplification device is used for realizing amplification of substances to be detected in the microfluidic detection chip;

The first driving device is used for driving the switching valve to rotate and controlling the switching angle and the residence time of the switching valve;

a second driving means for rupturing the vesicles;

the third driving device is used for moving the microfluidic detection chip to a specific position in the analysis equipment;

the fourth driving device is used for providing driving force for the fluid flow of the microfluidic detection chip; and

and the fifth driving device is used for driving the optical detection device to move so that the optical detection device can optically detect the specific part of the microfluidic detection chip.

9. An automatic extraction detection analysis method, characterized in that the method uses the microfluidic detection chip according to any one of claims 1 to 7 for detection analysis, the method comprising:

rotating the control valve to enable the control valve to be in a first type of angle, enabling the microfluidic detection chip to sample, and guiding a sample to flow through a cavity until an object to be detected in the sample is adsorbed;

rotating the control valve to enable the control valve to be in a second class angle, and guiding various cleaning solutions to flow through a cavity adsorbed with the object to be detected, and cleaning and/or eluting the object to be detected until the nucleic acid adsorbate is obtained;

Rotating the control valve to make the control valve at a third type of angle, introducing air pressure or hydraulic pressure, flushing the nucleic acid adsorbate into a chamber with a PCR amplification reagent for amplification reaction, and generating an object to be detected;

and (3) rotating the control valve to enable the control valve to be in a fourth type of angle, introducing air pressure or hydraulic pressure, and sending the object to be detected into a PCR reaction chamber with a primer probe.

10. The automated extraction detection assay of claim 9 wherein the control valve is rotated to a fifth type of angle to block the microchannels at both ends of a chamber.