CN115537288A - Microfluidic nucleic acid extraction detection box and detection system - Google Patents

Abstract

The invention provides a microfluidic nucleic acid extraction detection box and a detection system, and relates to the technical field of molecular detection. The method comprises the following steps: the sample injection device comprises an upper cover plate, a chip layer and a lower cover plate which are sequentially arranged from top to bottom, wherein a sample inlet is formed in the upper cover plate, and a sample cavity, a lysate storage cavity, a cleaning solution storage cavity, an eluent storage cavity, a mixing cavity, an amplification cavity, a buffer cavity, a rotary switching valve, a waste liquid cavity and a flow channel are formed in the chip layer. The rotary switching valve provided by the invention only relates to the connection of two paths of the amplification cavity, the extraction solution path and the waste liquid, so that only a tee joint is needed. The valve body provided by the invention is simple in structural design, and the cost of the chip and the detection box can be greatly reduced. In addition, the rotary switching valve is simplified in operation, the diversion and liquid separation efficiency is improved, the nucleic acid amplification and extraction efficiency can be accelerated, the nucleic acid extraction, amplification and detection are integrated, and the application prospect is good.

Description

Microfluidic nucleic acid extraction detection box and detection system

Technical Field

The invention relates to the technical field of molecular detection, in particular to a microfluidic nucleic acid extraction detection box and a detection system.

Background

The microfluidic chip is a technology which is based on a micro-electro-mechanical processing technology, forms a networked flow channel on substrates made of various materials such as silicon substrate, glass, plastic and the like, and is used for analysis and detection in the fields of biology, chemistry and the like. The microfluidic chip usually needs to integrate components such as micropumps and microvalves on the chip to realize the driving control of liquid. In recent years, due to the integratable characteristic of microfluidic chips, the realization of nucleic acid extraction, amplification and other functions on microfluidic chips has become a hot point of research in the microfluidic field.

In order to extract and amplify nucleic acid on the microfluidic chip, complex coordination such as combination of a micro valve and a pump is required. As described in CN107129939B patent, a rotary diversion valve, a groove for storing reagent, and a waste liquid pool are provided on the chip. The adsorption tank, the amplification tank, the weighing tank, the capture tank and other chambers are connected, the cracking tank, the cleaning tank, the elution tank and the waste liquid tank are communicated sequentially through the rotary flow guide valve according to the nucleic acid extraction process, and finally the amplification tank is communicated for amplification. This technical scheme involves the switching of selecting rotatory blast valve many times, and has higher to the operating accuracy requirement of blast valve, therefore holistic operation is comparatively complicated, and is consuming time higher, is unfavorable for the quick requirement of the ageing of micro-fluidic nucleic acid extraction. In addition, as the diversion valve is connected with a plurality of chambers, the design requirement on the diversion valve is higher, the design is more complex, for example, the content recorded in the patents CN110857743B and CN 112958173A, the design and the material use requirement of the rotary diversion valve are higher, a plurality of through holes and the use of materials are involved, and the cost of the microfluidic chip is increased.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a microfluidic nucleic acid extraction detection box and a detection system to solve the technical problems.

The invention is realized in the following way:

the invention provides a microfluidic nucleic acid extraction detection box, which comprises: the device comprises an upper cover plate, a chip layer and a lower cover plate which are sequentially arranged from top to bottom, wherein the upper cover plate is provided with a sample inlet, and the chip layer is provided with a sample cavity, a lysate storage cavity, a cleaning solution storage cavity, an eluent storage cavity, a mixing cavity, an amplification cavity, a buffer cavity, a rotary switching valve, a waste liquid cavity and a flow channel;

the sample cavity, the lysate storage cavity, the cleaning solution storage cavity and the eluent storage cavity are all communicated with the mixing cavity through microchannels, the mixing cavity is respectively communicated with the amplification cavity and the buffer cavity through two flow channels, liquid outlet ends of the two flow channels are both connected with the rotary switching valve, and the rotary switching valve is connected with the waste liquid cavity through microchannels so as to control the discharge of liquid in the mixing cavity; the buffer cavity is arranged on the mixing cavity and a flow passage of the rotary switching valve;

the waste liquid chamber is also provided with an external drive interface.

The rotary switching valve is designed in the liquid mixing chamber, so that the rotary valve does not need to be independently connected with all the chambers, and the flow guide direction of the rotary valve does not need to be frequently switched. Through the extraction detection box, the rotary switching valve provided by the invention only relates to the connection of two paths of an amplification cavity, an extraction solution path (including cracking, cleaning and elution) and waste liquid, so that only a tee joint is needed. The valve body provided by the invention is simple in structural design, and the cost of the chip and the detection box can be greatly reduced. In addition, the rotary switching valve is simplified in operation, the diversion and liquid separation efficiency is improved, the nucleic acid amplification and extraction efficiency can be accelerated, the nucleic acid extraction, amplification and detection are integrated, and the application prospect is good.

Specifically, the upper cover plate can be made of elastic plastics such as PDMS or the like, or ordinary plastic films and other materials, the upper cover plate is mainly used for sealing each cavity on the chip layer, and at least one sample inlet is arranged on the upper cover plate to facilitate sample injection. An elastic plastic such as one or more of Polydimethylsiloxane (PDMS), latex, thermoplastic rubber (TPR), thermoplastic elastomer (TPE), thermoplastic polyurethane elastomer rubber (TPU), silicone, and polyvinyl chloride (PVC).

The chip layer may be made of transparent plastic material, such as PC, PMMA, PDMS, ABS, etc. The part is used for packaging nucleic acid extraction reagents and is used as a carrier for nucleic acid extraction process and amplification process reactions.

The lower cover plate material can be made of plastic materials consistent with the chip layer material or different plastic materials, and the lower cover plate material is used for sealing the flow channel layer in the chip layer and providing a puncture structure (namely a bulge) and a valve body material accommodating cavity.

It should be noted that, in practical implementation, different processing and sealing manners can be selected according to the selection of different materials of each part. The upper cover plate can be, for example, a common plastic film, can be formed by selective punching or laser cutting, and can be sealed with the intermediate chip layer by selective double-sided adhesive tape. If the upper cover plate is made of elastic plastic, 3D printing, mold pouring or injection molding and other processes can be selected for processing, and the upper cover plate can be connected with the middle chip layer by gluing.

In particular, plasma coupling (Plasma) bonding sealing may be used when the upper cover plate and the middle chip layer are both made of PDMS. The intermediate level can be through mode machine-shaping such as 3D prints or moulds plastics, and lower floor's apron is because the structure is comparatively simple, removes that 3D prints and moulds plastics and also can be through modes such as CNC processing or mould pouring, like the lower apron of PC material is printed out to accessible 3D, the lower apron of CNC processing PMMA, or the PDMS apron of pouring takes SU-8 as the formpiston. The bonding of the lower cover plate and the middle layer can be sealed in an ultrasonic bonding mode and the like besides an adhesive bonding mode and a Plasma mode.

The nucleic acid extraction reagent used in the invention is pre-packaged in the corresponding chamber in a bubble cap mode, and comprises a lysate storage cavity, a cleaning solution storage cavity and an eluent storage cavity. When the device is used, the external reagent releasing module can be started, and the position of the lysate cavity is pressed, so that the bubble cap in the cavity is broken under the action of pressure and the protruding structure at the corresponding position of the bottom of the lower cover plate, and lysate, cleaning liquid or eluent is released.

The mixing cavity is used as the place for mixing reaction of various reagents and samples and nucleic acid extraction, the buffer cavity is used for buffering liquid in the mixing cavity, and the amplification cavity is used as the place for pre-packaging of amplification reagents and nucleic acid amplification and detection. The waste liquid cavity sucks waste liquid generated in the nucleic acid extraction providing process. Materials such as sponge which is easy to absorb water can be arranged according to needs to realize rapid water absorption.

An external driving interface is arranged, so that the extraction of the nucleic acid extraction reagent to the sample is realized through external driving, and the sample is distributed to enter an amplification area to realize amplification detection.

In a preferred embodiment of the present invention, an expansion valve is further disposed on the microchannel at the liquid inlet side of the mixing chamber. The expansion valve is normally opened, corresponding stress materials are contained in the valve chamber, and the valve can be closed to block the flow channel only after being stimulated by the outside. For example, the stressor material may be a heat-stressed expanded material, and the valve may be closed after the eluent is added, allowing liquid to enter the amplification chamber for amplification.

In a preferred embodiment of the present invention, an expansion valve is disposed on the flow channel of the amplification chamber near the mixing chamber. After the amplification sample is distributed, the position of the valve is locally heated to a certain temperature range, the expansion material expands to block the flow channel, and the amplification chamber forms a closed independent chamber for amplification reaction. The expansion material may be a foamed microsphere material or a composite of the foamed microsphere material and the elastic plastic.

In a preferred embodiment of the present invention, the number of the amplification chambers is at least two, and a plurality of amplification chambers are arranged in parallel. For example, the number of amplification chambers is 2-10, such as 3, 5 or 8.

In an alternative embodiment, each amplification chamber is provided with a liquid inlet flow passage branch and a liquid outlet flow passage branch, the liquid outlet flow passage branch is communicated with the flow passage, and the liquid inlet flow passage branch is communicated with an expansion valve on the flow passage of the amplification chamber close to the mixing chamber.

In a preferred embodiment of the present invention, the external driving interface is connected to an external driving module, and the driving module is selected from a piston rod, a hydraulic piston, a piston ball, a pump, a pressing ball and a pressing film. The driving module can be a piston pump or a pneumatic pump and provides positive and negative pressure for driving liquid in the chip.

In a preferred embodiment of the present invention, a breathable water-stop film is further disposed on the external driving interface. Preventing excessive aspiration of the liquid.

In a preferred embodiment of the present invention, the lower cover plate has protrusions disposed below the lysate storage chamber, the cleaning solution storage chamber and the eluent storage chamber to pierce the bottom-sealed reagent films in the lysate storage chamber, the cleaning solution storage chamber and the eluent storage chamber. The shape of the protrusion includes, but is not limited to, an inverted triangular pyramid, a prism, and the like.

In an alternative embodiment, the number of the cleaning solution storage chambers is at least two. The number of the cleaning liquid storage chambers may be set as desired.

The invention also provides a microfluidic nucleic acid extraction and detection system which comprises the microfluidic nucleic acid extraction and detection box. The system can be matched with the micro-fluidic detection box structurally through each external module, so that the ordered release of reagents on a chip and the ordered driving of liquid can be realized, and the extraction, the amplification and the detection of nucleic acid can be carried out on a sample.

In a preferred embodiment of the present invention, the system further includes a reagent releasing module, a lysis assisting module, a liquid path driving module, a switching valve control module, a magnetic attraction movement module, a temperature control module, an expansion valve stimulation module, and a fluorescence detection module.

In an alternative embodiment, the lysis auxiliary module is an ultrasonic module or a magnetic stirring module, and the expansion valve stimulation module is an infrared laser module.

The invention also provides a use method of the microfluidic nucleic acid extraction detection kit, which comprises the following steps:

loading a sample into the detection box from the sample inlet, adjusting the rotary switching valve to enable the buffer cavity to be communicated with the waste liquid cavity, providing negative pressure under the action of external drive to enable lysate to flow into the mixing cavity from the lysate storage cavity, enabling the sample to enter the mixing cavity from the sample cavity, adsorbing nucleic acid through magnetic beads after lysis, and enabling the magnet to be close to the drive cavity;

starting the external driving module again, and sucking the waste liquid in the mixing cavity into the waste liquid cavity;

releasing the cleaning liquid in the cleaning liquid storage cavity, allowing the cleaning liquid to enter the mixing cavity for cleaning, and then discharging the cleaning liquid into the waste liquid cavity;

releasing the eluent in the eluent storage cavity to elute the magnetic beads, closing an expansion valve on a micro-channel at the liquid inlet side of the mixing cavity, adjusting the rotary switching valve to communicate the waste liquid cavity with the amplification cavity, and after elution is finished, driving the elution liquid to be distributed from the mixing cavity to the amplification cavity;

and closing the expansion valve on the flow channel of the amplification cavity, which is close to the mixing cavity, rotating the switching valve, and blocking the communication between the waste liquid cavity and the amplification cavity for amplification.

Compared with the prior art, the invention has the beneficial effects that:

the rotary switching valve is designed behind the liquid mixing chamber, so that the rotary valve does not need to be connected with all chambers independently, and the flow guide direction of the rotary valve does not need to be switched frequently. Through the extraction detection box, the rotary switching valve provided by the invention only relates to the connection of two paths of the amplification cavity, the extraction liquid path (including cracking, cleaning and elution) and the waste liquid, so that only a tee joint is needed. The valve body provided by the invention is simple in structural design, and the cost of the chip and the detection box can be greatly reduced. In addition, the rotary switching valve is simplified in operation, the diversion and liquid separation efficiency is improved, the nucleic acid amplification and extraction efficiency can be accelerated, the nucleic acid extraction, amplification and detection are integrated, and the application prospect is good.

In addition, the system can be matched with the microfluidic detection box structurally through various external modules, so that the ordered release of reagents on a chip and the ordered driving of liquid can be realized, and the extraction, the amplification and the detection of nucleic acid can be carried out on a sample.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic view of a microfluidic nucleic acid amplification cartridge;

FIG. 2 is a diagram of the explosion structure of the microfluidic nucleic acid extraction amplification cartridge;

FIG. 3 is a schematic diagram of a microfluidic nucleic acid extraction detection system;

FIG. 4 is a functional structure diagram of the micro-fluidic chip;

FIG. 5 is a front view of the micro-fluidic chip;

FIG. 6 is a schematic view A of a rotary switching valve flow path connection;

FIG. 7 is a schematic view B of the flow path connection of the switching valve;

FIG. 8 is a schematic view of an expansion valve opening;

fig. 9 is a schematic view of the expansion valve closing.

An icon: 100-chip layer; 101-rotary switching valve; 102-an expansion valve I; 103-expansion valve two; 104-a flow channel; 105 — an external drive interface; 110-a sample chamber; 120-a lysate storage chamber; 130-cleaning solution 1 cavity; 140-cleaning liquid 2 chamber; 150-an eluent storage chamber; 160-a mixing chamber; 170-an amplification chamber; 180-a buffer chamber; 190-waste chamber; 200-a lower cover plate; 300-upper cover plate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", "clockwise", "counterclockwise", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings or orientations or positional relationships that the products of the present invention usually place when in use, and are used only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the devices or elements referred to must have specific orientations, be constructed in specific orientations, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used solely to distinguish one from another, and are not to be construed as indicating or implying relative importance.

Furthermore, the terms "horizontal", "vertical", "overhang" and the like do not imply that the components are required to be absolutely horizontal or overhang, but may be slightly inclined. For example, "horizontal" merely means that the direction is more horizontal than "vertical" and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.

In the description of the present invention, it should also be noted that, unless otherwise explicitly stated or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.

Example 1

Referring to fig. 1 and 2, the present embodiment provides a microfluidic nucleic acid extraction and detection cassette, which includes an upper cover plate 300, a chip layer 100, and a lower cover plate 200 sequentially disposed from top to bottom. The upper cover plate 300 may be made of elastic plastic or common plastic film, which is mainly used for sealing the chambers on the chip layer 100. The chip layer 100 is used for packaging nucleic acid extraction reagents and is used as a carrier for nucleic acid extraction process and amplification process reactions. The lower cover plate 200 serves to seal the flow channel 104 layer within the chip layer 100 and to provide a piercing structure (i.e., protrusion) and valve body material receiving chamber.

Specifically, referring to fig. 1 and 2, the upper cover plate 300 is provided with a sample inlet.

As shown in fig. 4 and 5, the chip layer 100 is provided with a sample chamber 110, a lysate storage chamber 120, a wash solution storage chamber, an eluent storage chamber 150, a mixing chamber 160, an amplification chamber 170, a buffer chamber 180, a rotary switching valve 101, a waste liquid chamber 190, and a flow channel 104.

The material selection of the microfluidic nucleic acid extraction detection kit of the invention is as follows:

the upper cover plate 300 may be made of elastic plastic such as PDMS or the like, or a common plastic film or the like. The chip layer 100 may be made of transparent plastic material, such as PC, PMMA, PDMS, ABS, etc. The part is used for packaging nucleic acid extraction reagents and is used as a carrier for nucleic acid extraction process and amplification process reactions. The lower cover plate 200 may be made of a plastic material identical to the material of the chip layer 100, or may be made of a different plastic material, which is used for sealing the flow channel 104 layer in the chip layer 100 and providing a piercing structure and a valve material accommodating chamber.

Different processing and sealing modes can be selected according to the selection of different materials of each part. If the upper cover plate 300 is a common plastic film, punching or laser cutting forming can be selected, and double-sided adhesive tape can be selected to seal with the middle chip layer 100; if the upper cover plate 300 is made of elastic plastic, 3D printing, mold casting, injection molding or other processes can be selected, and the upper cover plate can be connected with the middle chip layer 100 by gluing, and particularly, plasma coupling (Plasma) bonding sealing can be adopted when the upper cover plate 300 and the middle chip layer 100 are made of PDMS. The chip layer 100 can be formed by 3D printing or injection molding, and the lower cover plate has a simple structure, and can be formed by CNC processing or mold casting in addition to 3D printing and injection molding, for example, the lower cover plate 200 made of PC material can be printed by 3D, the lower cover plate 200 made of PMMA can be processed by CNC, or the PDMS cover plate can be cast by using SU-8 as a male mold. The bonding of the lower cover plate 200 and the chip layer 100 may be sealed by ultrasonic bonding or the like, in addition to adhesive bonding and Plasma bonding.

Referring to fig. 4 and 5, the chip layer 100 is a core part of the microfluidic nucleic acid extraction detection cartridge, and includes a sample chamber 110, a lysate storage chamber 120, a cleaning solution storage chamber, an eluent storage chamber 150, a mixing chamber 160, an amplification chamber 170, a buffer chamber 180, a rotary switching valve 101, a waste liquid chamber 190, and a flow channel 104. The chip is used for packaging the reagent chamber of the middle layer.

In this embodiment, two cleaning solution storage chambers, i.e., the cleaning solution 1 chamber 130 and the cleaning solution 2 chamber 140, are provided to facilitate better cleaning of magnetic beads.

The eluent storage chamber 150 is used to store eluent. Such as PBS.

The sample cavity 110, the lysate storage cavity 120, the cleaning solution 1 cavity 130, the cleaning solution 2 cavity 140 and the eluent storage cavity 150 are all communicated with the mixing cavity 160 through micro-channels, the mixing cavity 160 is respectively communicated with the amplification cavity 170 and the buffer cavity 180 through arranging two flow channels 104, liquid outlet ends of the two flow channels 104 are both connected with the rotary switching valve 101, and the rotary switching valve 101 is connected with the waste liquid cavity 190 through micro-channels to control the discharge of liquid in the mixing cavity 160; the buffer chamber 180 is disposed on the mixing chamber 160 and the flow passage 104 of the rotary switching valve 101.

The flow channels 104 may be equivalent to micro channels, and all have a function of conducting liquid.

The mixing chamber 160 is used as a place for mixing and reacting various reagents and samples and extracting nucleic acid, the buffer chamber 180 is used for buffering liquid in the mixing chamber 160, and the amplification chamber 170 is used as a place for pre-packaging amplification reagents and amplifying and detecting nucleic acid. The waste liquid chamber 190 sucks waste liquid generated in the nucleic acid extraction supply process. Materials such as sponge which is easy to absorb water can be arranged according to needs to realize rapid water absorption.

In order to control the flow of the fluid, a rotary switching valve 101 is provided at the outlet ends of the two flow passages 104, so that the flow path of the liquid is controlled by a three-way valve.

The micro-channel at the liquid inlet side of the mixing cavity 160 is also provided with a first expansion valve 102, and the flow channel 104 of the amplification cavity 170 close to the mixing cavity 160 is provided with a second expansion valve 103.

The expansion valve is normally open, the valve chamber contains the corresponding stress material, and only after being stimulated by the outside, the valve is closed to block the flow passage 104. For example, the stressor material may be a heat-stressed expanded material, and the first expansion valve 102 may be closed after the eluent is added, allowing liquid to enter the amplification chamber 170 for amplification. A schematic of expansion valve opening and closing is shown with reference to fig. 8 and 9. The lower substrate is provided with an expansion valve material accommodating groove, and the expansion material is thermally expandable and irreversible, for example, a composite of a foaming microsphere material and an elastic plastic thermally expands at a certain temperature, such as 120 ℃, to block the communication of the flow passage 104.

After the amplification sample is distributed, the position of the second expansion valve 103 is locally heated to a certain temperature range, the expansion material expands to block the flow passage 104, and the amplification cavity 170 forms a closed independent cavity for amplification reaction. The expansion material may be a foamed microsphere material or a composite of the foamed microsphere material and the elastic plastic.

The external driving interface 105 located at the waste liquid cavity 190 is connected with an external driving module, the driving module can be a piston pump or a pneumatic pump, positive and negative pressure is provided for driving liquid in the chip, and the driving liquid flows on the driving interface and is provided with a breathable water-stop film at the same time, so that excessive suction of the liquid is prevented.

The rotary switching valve 101 is controlled by an external switching module, and is connected with two channels 104 by rotating a certain angle.

Example 2

This example provides a microfluidic nucleic acid extraction detection system, which includes the microfluidic nucleic acid extraction detection cartridge of example 1 above. The system can be matched with the micro-fluidic detection box structurally through each external module, so that the ordered release of reagents on a chip and the ordered driving of liquid can be realized, and the extraction, the amplification and the detection of nucleic acid can be carried out on a sample.

The detection system further comprises a reagent release module, a lysis auxiliary module, a liquid path driving module, a switching valve control module, a magnetic attraction motion module, a temperature control module, an expansion valve stimulation module and a fluorescence detection module which are shown in fig. 3. The auxiliary module is an ultrasonic module or a magnetic stirring module, and the expansion valve stimulation module is an infrared laser module.

The invention takes the magnetic bead extraction method as the nucleic acid extraction process to extract the sample nucleic acid, and has wide applicability. The nucleic acid extraction process of the magnetic bead method generally includes lysis of the sample, nucleic acid adsorption, washing, and elution. The reagents used in the process are pre-packaged in each chamber by means of a bubble cap, and the nucleic acid amplification reagents are packaged in the amplification chamber 170 by means of freeze-dried balls or freeze-dried beads.

In combination with the magnetic bead extraction method, the invention provides a microfluidic nucleic acid extraction detection box and a use method of a detection system, and the use method comprises the following steps:

(1) The sample is added from the sample inlet, the external reagent releasing module is started, and the position of the lysate cavity is pressed, so that the bubble cap in the cavity is broken under the action of pressure and the protruding structure at the corresponding position at the bottom of the lower cover plate 200, and the lysate is released. The rotary switching valve 101 is controlled by the switching valve module to communicate the waste liquid cavity 190 pipeline and the buffer cavity 180 pipeline (shown in fig. 6), the external driving module provides negative pressure to drive the lysate and the sample to enter the mixing cavity 160 for mixing, the external starting chip starts the ultrasonic module outside the chip to fully crack the sample, and the nucleic acid in the sample is released by the lysate and is adsorbed by the magnetic beads in the lysate.

As shown in FIG. 6, the external valve body control module can be embedded into the linear groove above the valve body, and the valve body is rotated by a certain angle to drive the valve body to rotate, so that the external valve body control module is connected with the buffer liquid cavity or the PCR amplification cavity 170.

(2) The outside magnetism module of inhaling is close to mixing chamber 160 is started, and the drive module that starts once more absorbs waste liquid chamber 190 with mixing chamber 160's waste liquid, contains the sponge and absorbs the pad in the waste liquid chamber 190, can fully absorb the waste liquid.

(3) Releasing the cleaning solution 1, keeping the external magnetic module away, driving the cleaning solution 1 to enter the mixing chamber 160 to clean the magnetic beads, adjusting the magnetic module to be close to the mixing chamber 160, and starting the driving module to discharge and discharge the liquid to the waste liquid chamber 190; repeating the steps to carry out secondary cleaning.

(4) And the reagent releasing module is started again to release the eluent, the eluent is driven to the mixing cavity 160, the nucleic acid adsorbed on the magnetic beads is eluted, the magnetic beads are fixed by magnetic attraction, the laser module is started, the expansion valve I102 is heated to expand and close a flow channel between the reagent storage cavity and the mixing cavity 160, and the rotary valve is adjusted to the waste liquid cavity 190 to be communicated with the amplification cavity 170.

(5) After the elution is finished, the elution liquid is driven to be distributed from the mixing cavity 160 to the amplification cavity 170 to be combined with the freeze-drying reagent, the infrared laser module is started, the second expansion valve 103 is heated to block a communication pipeline between the mixing cavity 160 and the PCR amplification chamber, the switching valve 101 is rotated to block the communication between the waste liquid cavity 190 and the amplification cavity 170, a closed and independent area (shown in figure 7) is formed in the PCR amplification area, and the external temperature control module and the fluorescence detection module are started to perform PCR amplification and fluorescence signal detection on the nucleic acid.

According to the invention, through the matching of each external module and the structure on the microfluidic card box, the ordered release of the reagents on the chip and the ordered driving of the liquid can be realized, and the extraction, the amplification and the detection of the nucleic acid can be carried out on the sample.

The present invention has been described in terms of the preferred embodiment, and it is not intended to be limited to the embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A microfluidic nucleic acid extraction cartridge, comprising: the device comprises an upper cover plate, a chip layer and a lower cover plate which are sequentially arranged from top to bottom, wherein the upper cover plate is provided with a sample inlet, and the chip layer is provided with a sample cavity, a lysate storage cavity, a cleaning solution storage cavity, an eluent storage cavity, a mixing cavity, an amplification cavity, a buffer cavity, a rotary switching valve, a waste liquid cavity and a flow channel;

the sample cavity, the lysate storage cavity, the cleaning solution storage cavity and the eluent storage cavity are all communicated with the mixing cavity through microchannels, the mixing cavity is respectively communicated with the amplification cavity and the buffer cavity through two runners, liquid outlet ends of the two runners are all connected with the rotary switching valve, and the rotary switching valve is connected with the waste liquid cavity through microchannels to control the discharge of liquid in the mixing cavity; the buffer cavity is arranged on the mixing cavity and a flow channel of the rotary switching valve;

the waste liquid cavity is also provided with an external driving interface.

2. The microfluidic nucleic acid extraction and detection cartridge of claim 1, wherein the microchannel on the inlet side of the mixing chamber is further provided with an expansion valve.

3. The microfluidic nucleic acid extraction cartridge of claim 1, wherein an expansion valve is disposed on a flow channel of the amplification chamber adjacent to the mixing chamber.

4. The microfluidic nucleic acid extraction cartridge of claim 3, wherein the number of the amplification chambers is at least two, and a plurality of the amplification chambers are arranged in parallel;

preferably, every amplification chamber all is provided with feed liquor runner branch road and play liquid runner branch road, go out the liquid runner branch road with the runner intercommunication, feed liquor runner branch road with the amplification chamber is close to the expansion valve intercommunication on the runner of hybrid chamber.

5. The microfluidic nucleic acid extraction cartridge of claim 1, wherein the external driving interface is connected to an external driving module, and the driving module is selected from a piston rod, a hydraulic piston, a piston ball, a pump, a pressing ball, and a pressing membrane.

6. The microfluidic nucleic acid extraction test cassette of claim 1, wherein the external drive interface further comprises a gas-permeable water-stop film.

7. The microfluidic nucleic acid extraction detection kit of claim 1, wherein the lower cover plate is provided with protrusions below the lysate storage chamber, the cleaning solution storage chamber and the eluent storage chamber to puncture reagent films packaged at the bottom of the lysate storage chamber, the cleaning solution storage chamber and the eluent storage chamber;

preferably, the number of the cleaning solution storage chambers is at least two.

8. A microfluidic nucleic acid extraction detection system comprising the microfluidic nucleic acid extraction detection cartridge of any one of claims 1-7.

9. The microfluidic nucleic acid extraction and detection system of claim 8, further comprising a reagent release module, a lysis auxiliary module, a liquid path driving module, a switching valve control module, a magnetic attraction motion module, a temperature control module, an expansion valve stimulation module, and a fluorescence detection module;

preferably, the cracking auxiliary module is an ultrasonic module or a magnetic stirring module, and the expansion valve stimulation module is an infrared laser module.

10. A method of using the microfluidic nucleic acid extraction cartridge of any one of claims 1-7, comprising the steps of:

loading a sample into the detection box from the sample inlet, adjusting the rotary switching valve to enable the buffer cavity to be communicated with the waste liquid cavity, providing negative pressure under the action of external drive to enable lysate to flow into the mixing cavity from the lysate storage cavity, enabling the sample to enter the mixing cavity from the sample cavity, adsorbing nucleic acid through magnetic beads after lysis, and enabling the magnet to be close to the drive cavity;

starting the external driving module again, and sucking the waste liquid in the mixing cavity into the waste liquid cavity;

releasing the cleaning liquid in the cleaning liquid storage cavity, allowing the cleaning liquid to enter the mixing cavity for cleaning, and then discharging the cleaning liquid into the waste liquid cavity;

releasing the eluent in the eluent storage cavity to elute the magnetic beads, closing an expansion valve on a micro-channel on the liquid inlet side of the mixing cavity, adjusting the rotary switching valve to communicate the waste liquid cavity with the amplification cavity, and driving the elution liquid to be distributed from the mixing cavity to the amplification cavity after the elution is finished;

and closing the expansion valve on the flow channel of the amplification cavity, which is close to the mixing cavity, rotating the switching valve, and blocking the communication between the waste liquid cavity and the amplification cavity for amplification.

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