CN115155682B - Microfluidic chip based on rotary valve and detection method - Google Patents

Abstract

The invention relates to a microfluidic chip based on a rotary valve and a detection method, comprising a chip body, wherein a generating oil duct, a backflow oil duct, a sample channel and a ventilation channel are arranged in the chip body, one end of the generating oil duct is communicated with the side wall of the sample channel, generating oil in the generating oil duct enters the sample channel to shear a sample to form micro-droplets, and one end of the ventilation channel is communicated with the outside; further comprises: the chip body is positioned at the amplification carrier and is also provided with a sample injection channel and a sample discharge channel, and the sample injection channel and the sample discharge channel are both communicated with the amplification carrier; rotating valve, reflux detection; the rotary valve is internally provided with the enrichment cavity, the enrichment cavity provided by the invention has the effects of realizing enrichment on the rotary valve, endowing the rotary valve with additional functions, fully utilizing the rotary valve, avoiding additional arrangement on a chip, reducing the size of a microfluidic chip and improving the integration level of the microfluidic chip.

Description

Microfluidic chip and detection method based on rotary valve

Technical field

The invention relates to the technical field of nucleic acid detection, and in particular to a microfluidic chip based on a rotary valve and a detection method.

Background technique

Polymerase chain reaction (PCR) is a technology that uses a segment of DNA as a template and, with the participation of DNA polymerase and nucleotide substrates, amplifies the segment of DNA to a sufficient amount for structural and functional analysis. , Digital PCR (Digital PCR-dPCR) technology is a new nucleic acid detection and quantitative analysis technology, which has the characteristics of high sensitivity, high specificity, and high stability. It is widely used in gene expression research, microRNA research, and cancer marker rare mutation detection. It is of great significance for applications in the field of low-abundance nucleic acid detection. Different from traditional real-time fluorescence quantitative PCR (Real-timeQuantitative PCR-qPCR) technology, the principle of digital PCR is to distribute a standard PCR reaction into a large number of tiny reactors, each reactor containing or not containing one or more Copy the target molecule (DNA template) to achieve "single-molecule template PCR amplification". After the amplification is completed, the number of copies of the target sequence is "counted" through the number of positive reactors.

Microfluidic chip is a technology based on micro-electromechanical processing technology, with micro-tubes forming a network on the chip, with controllable micro-fluidic paths running through the entire system and completing various biological and chemical processes. In the early days of the development of microfluidic chip technology, chip capillary electrophoresis was its mainstream technology. The chip used had a simple structure and a single function. In recent years, microfluidic chips have begun to develop rapidly in the direction of functionalization and integration, such as nucleic acid amplification reaction, immune system, etc. Important biological and chemical processes such as reactions and cell lysis have become new hot spots.

With the increasing maturity of microfluidic processing technology, the difficulty and cost of designing, developing and testing microfluidic chips are getting lower and lower, and their applications are becoming more and more widespread, especially in miniaturized in vitro diagnostic equipment. There are more and more application cases. , using microfluidic chips as detection carriers, microfluidics are used to realize operations such as adding, transferring, and mixing samples on the sample chip, and finally complete the detection of test indicators. Microfluidic chip detection uses a small amount of reagents and consumables, and is easily portable, miniaturized, and home-based.

The Chinese patent with announcement number CN113117770A discloses a PCR microfluidic chip, which includes a main chip, an oil inlet chamber liquid reservoir, a sample injection chamber liquid reservoir, a common edge liquid reservoir and a PCR tube; the main chip is provided with Droplet generation module, amplification module and detection module; the droplet generation module is used to receive the continuous phase injected from the oil inlet chamber liquid storage pool, receive the separated phase injected from the injection chamber liquid storage pool, and convert the received The continuous phase and the separated phase are mixed to form droplets, and the formed droplets are injected into the amplification module; the amplification module is used to receive the driving oil injected from the common edge liquid reservoir, and inject the received driving oil The PCR tube receives the droplets injected from the droplet generation module, injects the received droplets into the PCR tube, injects the droplets and driving oil in the PCR tube into the detection module, and receives the shear injected from the co-edge reservoir. oil, and inject the received shear oil into the detection module; the detection module is used to receive the droplets, driving oil and shear oil injected from the amplification module, under the downstream action of the driving oil and shear oil, The droplets are arranged in an orderly manner.

The above-mentioned existing technical solutions have the following defects: although the above-mentioned microfluidic chip integrates the processes of droplet generation, droplet amplification and droplet flow detection into the same chip, the chip achieves different functions through the plug. Switching of process tube pipelines, but in the actual practical process, when droplets are generated, the collected droplets will be accompanied by excess oil, which makes subsequent processing troublesome and affects the amplification effect. If an additional enrichment device is designed , will affect the size of the chip and affect other structures on the chip.

Contents of the invention

Therefore, the present invention aims to solve the technical problem that when microdroplets are generated, the collected microdroplets are accompanied by excess oil, which makes subsequent processing troublesome and affects amplification, thereby providing a microfluidic chip and detection method based on a rotary valve.

A microfluidic chip based on a rotary valve, including a chip body, which is provided with a generation oil channel, a return oil channel, a sample channel and a ventilation channel, wherein one end of the generation oil channel is connected to the side wall of the sample channel , the generated oil in the generated oil channel enters the sample channel, and the sample is sheared to form droplets, and one end of the ventilation channel is connected to the outside;

Also includes:

Amplification carrier, the amplification carrier is sealed in the chip body, the chip body is located at the amplification carrier and is also provided with a sampling channel and a sampling channel, the sampling channel and the sampling channel are both compatible with the amplification carrier connectivity;

The rotary valve is rotated along its axis and is installed on the chip body. When the rotary valve rotates to the generation position, the sample channel is connected to the sampling channel, and the sample outlet channel is connected to the ventilation channel. When the rotary valve rotates to the detection position, When in position, the sample outlet channel is connected to the sample channel, the sample feed channel is connected to the return oil channel, and the droplets in the amplification carrier enter the sample channel through the sample outlet channel for backflow detection;

An enrichment chamber is provided in the rotary valve. When the rotary valve rotates to the generation position, the bottom and top of the enrichment chamber are connected to the sample channel and the sampling channel respectively.

Further, the bottom of the rotary valve is provided with at least four grooves, two of which are respectively connected to the top and bottom of the enrichment chamber. The bottom of the enrichment chamber is arranged in a cylindrical shape. The enrichment chamber The top side wall is inclined toward the axis of the enrichment chamber to form a cone shape.

Further, the chip body is provided with a fixing piece at the rotary valve, which is suitable for limiting and sealing the rotary valve, compressing the rotary valve on the chip body and sealing the contact surface.

Further, the fixing member includes a placement block provided on the chip body, a placement slot is provided on the placement block, the bottom of the placement slot is connected to the chip body, the rotary valve is located in the placement block and is in the placement block. Internal rotation, a gland is provided at the notch of the placement groove, the outer wall of the gland and the inner wall of the placement groove are provided with threads, the outer wall of the gland and the inner wall of the placement groove are threadedly connected, the gland and the rotation The valve is tight.

Further, the fixing member includes a first rotor pressing member, and an outer edge is provided on the side wall of the first rotor pressing member. There are at least two outer edges, and the first rotor pressing member has at least two outer edges. A first accommodation groove is provided at the bottom, the rotary valve is located in the first accommodation groove and conflicts with the bottom of the first accommodation groove, the chip body is located at the rotary valve and is provided with a resisting edge matching the outer edge, and the third A rotor pressing member is located between the plurality of resisting edges. The first rotor pressing member rotates on the chip body until the outer edge is located below the resisting edges and contacts the resisting edges.

Further, the side where the resisting edge and the outer edge are in contact are inclined, the inclination directions of the plurality of resisting edges are consistent, and the outer edge and the resisting edge are adapted to be inclined.

Furthermore, the amplification carrier is also provided with an amplification module, which is used to apply pressure and heat to the amplification carrier, so that the collected droplets in the amplification carrier undergo several thermal cycles to complete nucleic acid amplification.

Further, the amplification module includes a ring-shaped heat cover, the sampling channel and the sample outlet channel are both arranged in the ring-shaped heat cover, and the amplification carrier is set at the bottom of the ring-shaped heat cover and sealed with the ring-shaped heat cover, so A first heating element is provided on the annular heating cover, and the first heating element is attached to the annular heating cover.

Further, the annular heat cover is made of metal material.

Further, the amplification module includes a tube cover protrusion arranged at the bottom of the chip body, the sample inlet channel and the sample outlet channel are both arranged in the tube cover protrusion, and the amplification carrier and the tube cover protrusion are clamped , the chip body is provided with a second heating element at a position facing away from the amplification carrier, and the second heating element is attached to the chip body.

Further, one end of the generation oil channel, return oil channel, and sample channel facing away from the rotary valve is set as a generation oil port, a return oil port, and a sample port. The generation oil port, the return oil port, and the sample port are connected to the outside, so The chip body is located at the generating oil port, the return oil port and the sample port, and is provided with a generating oil reservoir, a return oil reservoir and a sample reservoir.

A detection method using any one of the rotary valve-based microfluidic chips, including the following steps:

Microdroplet generation: Rotate the rotary valve to the generation position. The sample channel is connected to the sampling channel, and the sample outlet channel is connected to the ventilation channel. Inject the sample into the sample channel, and pass the generated oil into the generated oil channel and apply normal pressure. Pressure, the generated oil enters the sample channel, forms a shear flow, divides the sample into droplets, and flows into the sampling channel under the action of the rotary valve, and finally enters the amplification carrier;

Droplet amplification: The amplification carrier begins to undergo several thermal cycles to heat the droplets in the cavity of the amplification carrier to complete droplet amplification;

Microdroplet reflux detection: Rotate the rotary valve again and move the rotary valve to the detection position. The sampling channel is connected to the sample channel. The sampling channel is connected to the return oil channel. Positive pressure is applied to one side of the return oil channel to remove the reflux. The oil flows from the return oil channel to the injection channel and is pressed into the amplification carrier. The droplets in the amplification carrier are first pressed into the sample outlet channel and flow back into the sample channel. At the same time, they are applied to the generated oil channel. Press positive pressure to pass the generated oil into the sample channel from the connection between the generated oil channel and the sample channel, space the droplets, and align the external optical detection module with the sample channel. When the droplets flow back through, the droplets are measured. Nucleic acid amplification testing.

The technical solution of the present invention has the following advantages:

1. When using the microfluidic chip based on a rotary valve provided by the present invention, when generating droplets, the rotary valve is rotated to the generation position, and the sample channel at this time plays the role of the rotary valve. The bottom is connected to the sampling channel, and the sample outlet channel is connected to the ventilation channel, so that the sample outlet channel is connected to the atmosphere, ensuring that the generated droplets can smoothly enter the amplification carrier. When the droplets are generated, the oil is generated at this time. Positive pressure is applied at the entrance of the generated oil channel and the sample channel, so that the generated oil and sample are transported in the generated oil channel and the sample channel. At this time, because the generated oil channel and the sample channel are connected and intersected, a shear flow will be formed at the connection. The sample in the sample channel will be divided into droplets with the same volume. After the droplets are generated, they will flow from the sample channel to the rotary valve. At this time, the mixture of droplets and oil will enter the enrichment chamber. Due to the size of the droplets, The density is smaller than that of oil. When the mixture of droplets and oil enters the enrichment wall, the droplets will produce an enrichment effect in the enrichment cavity. That is, the droplets entering the enrichment cavity first will float above the enrichment chamber. When the enrichment chamber is filled with a mixture of droplets and oil, the droplets will first flow out of the rotary valve and enter the injection channel, while the excess oil phase will remain in the enrichment chamber. In the collecting cavity, this ensures that a higher droplet volume fraction is obtained in the amplification carrier. At this time, the rotary valve not only switches on and off the droplet and oil-phase mixed fluid when the droplet is generated, but also realizes the control of the droplet. Enrichment, thereby ensuring a high volume fraction of droplets in the amplification carrier, avoiding excessive oil phase in the collected droplets, which affects the amplification effect, and at the same time achieving enrichment on the rotary valve, giving the rotary valve additional functions , and at the same time, the rotary valve is fully utilized without the need for additional settings on the chip, which reduces the size of the microfluidic chip and improves the integration of the microfluidic chip. In addition, after the droplets are amplified, the rotary valve can be rotated again to remove the rotary valve. Rotate to the detection position. At this time, the sampling channel is connected to the sample channel, and the sampling channel is connected to the return oil channel. Positive pressure is applied to the end of the return oil channel away from the rotary valve, and the return oil passes through the rotary valve from the return oil channel. Pass into the injection channel, and from the injection channel into the amplification carrier. Since the density of the droplets is smaller than the density of the return oil, the droplets at this time will attach to the upper layer of the amplification carrier, and when continuous After the return oil flows into the amplification carrier, the droplets at this time will be pressed into the sample channel first, and flow back into the sample channel from the rotary valve. At the same time, the end of the generated oil channel facing away from the rotary valve will also Positive pressure is applied. Since the sample channel is connected and tangential to the side wall of the rotary valve side and the generated oil channel, the generated oil is also input into the sample channel at this time, separating the continuous refluxing droplets. At the same time, the external optical detection module starts Work, align the sample channel, and when the droplets flow back through, the digital nucleic acid detection of the droplets can be completed. The detection is carried out by backflowing into the sample channel, so there is no need to design additional detection channels and detection equipment for detection. Unifying sampling and detection into the same sample channel reduces production costs, saves resources, reduces the size of microfluidic equipment, and further improves the integration of microfluidic chips.

2. The microfluidic chip based on the rotary valve provided by the present invention, the bottom of the rotary valve is provided with at least four grooves, two of which are connected to the top and bottom of the enrichment chamber respectively, and the enrichment chamber is The bottom of the enrichment chamber is set in a cylindrical shape, and the side wall of the top of the enrichment chamber is inclined toward the axis of the enrichment chamber to form a cone. At this time, the conical top of the enrichment chamber can guide the droplets and reduce The possibility of microdroplets accumulating on the top of the enrichment chamber allows the microdroplets that enter the enrichment chamber to enter the enrichment channel and eventually be transferred to the amplification carrier.

3. The present invention provides a microfluidic chip based on a rotary valve. The chip body is provided with a fixing member at the rotary valve, which is suitable for limiting the sealing of the rotary valve and compressing the rotary valve on the chip body. And seal the contact surface. After placing the rotary valve on the upper surface of the chip body, press the rotary valve through the fixing piece to ensure that the rotary valve can move along its axis while adjusting the position of the rotary valve on the chip body. At the same time, the rotary valve is pressed and sealed to seal the friction surface between the rotary valve and the chip body to ensure that no fluid leakage occurs when the droplet of fluid passes through the rotary valve.

4. The microfluidic chip based on the rotary valve provided by the present invention, the fixing member includes a placement block provided on the chip body, a placement groove is provided on the placement block, and the bottom of the placement groove is connected to the chip body, so The rotary valve is located in the placement block and rotates in the placement block. A gland is provided at the notch of the placement groove. The outer wall of the gland and the inner wall of the placement groove are provided with threads. The outer wall of the gland and The inner wall of the placement groove is threaded, and the gland and the rotary valve are in tight contact. When the rotary valve is placed on the chip body, the rotary valve is placed in the placement groove, and the rotary valve can rotate in the placement groove. Fit the bottom of the rotary valve to the upper surface of the chip body, put the gland into the notch of the placement slot, and extend it into the placement slot to connect with the threaded inner wall of the placement slot. Continue to rotate the gland until the gland is Move in the direction of the rotary valve until the gland and the upper surface of the rotary valve are in contact, press and seal the rotary valve on the chip body, and seal the friction surface between the rotary valve and the chip body to ensure that when the droplet of fluid passes through the rotary valve, No leakage of fluid will occur.

5. In the microfluidic chip based on the rotary valve provided by the present invention, the fixing member includes a first rotor pressing member, an outer edge is provided on the side wall of the first rotor pressing member, and the outer edge is provided with at least There are two. The bottom of the first rotor pressing member is provided with a first accommodation groove. The rotary valve is located in the first accommodation groove and conflicts with the bottom of the first accommodation groove. The chip body is located at the rotary valve. There is a resisting edge that matches the outer edge. The first rotor pressing part is located between a plurality of resisting edges. The first rotor pressing part rotates on the chip body until the outer edge is located below and against the resisting edge. When the rotary valve is being installed, place the first rotor pressing piece directly above the rotary valve. At the same time, the outer edge and the pressing side are misaligned, and then move the first rotor pressing piece toward the direction of the rotary valve. Move until the rotary valve extends into the first receiving groove, and then rotate the first rotor pressing piece on the rotary valve. As the first rotor pressing piece rotates, it can drive the pressing edge to move until it moves to the outer edge. Below, the outer pressing edge and the resisting edge are pressed against each other. At this time, the top wall of the rotary valve conflicts with the bottom of the first accommodation groove, so that the rotary valve is pressed on the chip body under the action of the first rotor pressing member. Press tightly to ensure that the rotary valve will not leak when it flows through the rotary valve during subsequent rotation.

6. In the microfluidic chip based on the rotary valve provided by the present invention, the side where the resisting edge and the outer edge are abutting are arranged at an inclination, the inclination directions of the plurality of resisting edges are consistent, and the outer edge and the resisting edge are arranged in an inclined manner. The edge is adapted to tilt. During the installation process of the rotary valve, place the first rotor pressing piece directly above the rotary valve. At the same time, the outer edge and the pressing edge are misaligned, and then move the first rotor pressing piece toward the rotary valve. direction until the rotary valve extends into the first receiving groove. At this time, the first rotor pressing member is rotated so that the outer edge of the first rotor pressing member moves toward the direction of the pressing edge, and the small end of the outer edge is at Until the outer edge moves to the position of the resisting edge, the small end of the outer edge first enters directly below the resisting edge, and continues to move the first rotor pressing member so that the small end of the outer edge moves toward the resisting edge. The big end of the tight edge moves in the direction until the outer edge and the resisting edge are tightened. At this time, the outer edge cannot move forward, and the rotary valve and the first accommodation groove wall in the first rotor pressing member are in contact, thereby The rotor is compressed to ensure that there will be no leakage when the fluid flows through the rotor during the subsequent rotation of the rotary valve.

7. In the microfluidic chip based on a rotary valve provided by the present invention, the amplification module includes an annular thermal cover, the sample inlet channel and the sample outlet channel are both arranged in the annular thermal cover, and the amplification carrier is set in the annular thermal cover. The bottom of the annular heat cover is sealed with the annular heat cover. A first heating element is provided on the annular heat cover. The first heating element and the annular heat cover fit together to perform amplification when the droplets enter the amplification carrier. At this time, the amplification module at this time starts to start, thereby performing a temperature rising and cooling cycle on the amplification vector, so that the droplets in the amplification vector undergo several thermal cycles to complete the nucleic acid amplification. At the same time, during amplification, the first The heating element, the first heating element starts to start, thereby driving the annular heating cover to heat up. The heated heating cover heats the top of the amplification carrier to prevent the droplets in the amplification carrier from evaporating.

8. In the microfluidic chip based on the rotary valve provided by the present invention, the annular heat cover is made of metal material, and the metal annular heat cover can increase the heating speed, improve the effect of preventing evaporation, and thereby improve the amplification efficiency.

Description of the drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a schematic diagram of the overall structure of the microfluidic chip based on a rotary valve of the present invention;

Figure 2 is a schematic diagram of the internal structure of the placement block of the present invention;

Figure 3 is a schematic diagram of the bottom of the rotary valve in the present invention;

Figure 4 is a schematic diagram of the internal structure of the rotary valve in the present invention;

Figure 5 is a schematic diagram of the internal structure of the chip body in the present invention;

Figure 6 is a schematic diagram of the on and off of the rotary valve during droplet generation, droplet amplification and droplet detection in the present invention;

Figure 7 is a schematic diagram of another embodiment of the fixing member in the present invention;

Figure 8 is a schematic diagram of another embodiment of the liquid storage tank in the present invention;

Figure 9 is a schematic diagram of the retaining edge of another embodiment of the fastener according to the present invention;

Figure 10 is a schematic diagram of the first rotor pressing member in the present invention;

Figure 11 is a schematic diagram of the bottom of the first rotor pressing member in the present invention;

Figure 12 is a schematic diagram of the first embodiment of the amplification module of the present invention;

Figure 13 is a schematic diagram of the second embodiment of the amplification module of the present invention;

Figure 14 is a schematic diagram of the overall structure of the amplification module in the present invention;

Figure 15 is a step diagram of the detection method in the present invention.

Explanation of reference symbols:

1. Chip body; 2. Amplification carrier; 3. Rotary valve; 4. Generation oil channel; 5. Return oil channel; 6. Sample channel; 7. Ventilation channel; 8. Inlet channel; 9. Sample outlet channel; 10. Generating oil port; 11. Return oil port; 12. Sample port; 13. Groove; 14. Enrichment chamber; 15. Rotating wrench groove; 16. Fixing piece; 161. Placement block; 162. Placement slot; 163 , gland; 164, first communication hole; 165, first rotor pressing member; 166, outer edge; 167, first receiving groove; 169, pressing edge; 1610, second communication hole; 17, first protrusion 18. Amplification module; 181. Heating base; 182. Peltier; 183. Heat sink; 184. Tube cover protrusion; 185. Second heating element; 186. Ring-shaped heating cover; 187. First heating element ; 19. Generating oil reservoir; 20. Return oil reservoir; 21. Sample reservoir; 22. Enrichment channel.

Detailed ways

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description. It does not indicate or imply that the device or element referred to must have a specific orientation or a specific orientation. construction and operation, and therefore should not be construed as limitations of the invention. Furthermore, the terms “first”, “second” and “third” are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless otherwise clearly stated and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. Connection, or integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.

In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Example

Referring to Figures 1 to 14, the present invention provides a microfluidic chip based on a rotary valve, including a chip body 1, an amplification carrier 2 and a rotary valve 3. The chip body 1 is provided with a generating oil channel 4, Return oil channel 5, sample channel 6 and ventilation channel 7, wherein one end of the generated oil channel 4 is connected with the side wall of the sample channel 6, the generated oil in the generated oil channel 4 enters the sample channel 6, and the sample is processed Shearing forms microdroplets. There are two generated oil channels 4. One end of the two generated oil channels 4 faces the rotary valve 3 and both are connected to the sample channel 6 and are perpendicular to the sample channel 6. The two generated oil channels 4 are connected to the sample. The connection of the channels 6 forms a cross flow channel, and one end of the ventilation channel 7 is connected to the outside to facilitate the transportation of droplets into the amplification carrier 2;

The amplification carrier 2 is sealed in the chip body 1. The chip body 1 is located at the amplification carrier 2 and is also provided with a sampling channel 8 and a sampling channel 9. The sampling channel 8 and the sampling channel 9 are are connected to the amplification carrier 2; the rotary valve 3 is rotated along its axis and is installed on the chip body 1, and is located between the amplification carrier 2 and the sample channel 6, the sample channel 6, the return oil channel, and the sampling channel 8 One end of the sample outlet channel 9 is located just below the rotary valve 3 and the protruding chip body 1 is in contact with the bottom surface of the rotary valve 3. When the rotary valve 3 rotates to the generation position, the sample channel 6 and the injection channel 8 are connected. , the sample outlet channel 9 is connected to the ventilation channel 7. When the rotary valve 3 rotates to the detection position, the sample outlet channel 9 is connected to the sample channel 6, and the sample injection channel 8 is connected to the return oil channel 5. The droplets in the amplification carrier 2 enter the sample channel 6 through the sample outlet channel 9 for backflow detection; wherein the rotary valve 3 is provided with an enrichment chamber 14. When the rotary valve 3 rotates to the generation position, the The bottom and top of the enrichment chamber 14 are connected to the sample channel 6 and the injection channel 8 respectively.

When in use, when generating droplets, the rotary valve 3 is rotated to the generation position. At this time, the sample channel 6 is connected to the sampling channel 8 under the action of the rotary valve 3, and the sample outlet channel 9 is connected to the ventilation channel 7, so that the sample outlet channel 9 is connected to the atmosphere, ensuring that the generated droplets can smoothly enter the amplification carrier 2. When the droplets are generated, the oil channel 4 and the sample channel 6 are generated at this time. Positive pressure is applied at the inlet, so that the generated oil and sample are transported in the generated oil channel 4 and the sample channel 6. At this time, because the generated oil channel 4 and the sample channel 6 intersect, a shear flow will be formed at the connection point. The sample in the sample channel 6 will be divided into microdroplets with the same volume. After the microdroplets are generated, they will pass into the rotary valve 3. At this time, the mixture of microdroplets and oil will enter the enrichment chamber 14. Due to the size of the microdroplets, The density is smaller than that of oil. At this time, when the mixture of droplets and oil enters the enrichment chamber 14, the droplets will produce an enrichment effect in the enrichment chamber 14. That is, the droplets entering the enrichment chamber 14 first will float above the enrichment chamber 14. When the enrichment chamber 14 is filled with a mixture of droplets and oil, the droplets will first flow out of the rotary valve 3 and enter the injection channel 8, while the excess oil phase will remain. In the enrichment chamber 14, to ensure that a higher droplet volume fraction is obtained in the amplification carrier 2, the rotary valve 3 at this time realizes the on-off of the droplet and oil phase mixed fluid when the droplet is generated. Enrich the droplets to ensure a high volume fraction of droplets in the amplification carrier 2, avoid excessive oil phase in the collected droplets, which affects the amplification effect, and achieve enrichment on the rotary valve 3. The rotary valve 3 is given additional functions, and at the same time, the rotary valve 3 is fully utilized without the need for additional settings on the chip, thereby reducing the size of the microfluidic chip and improving the integration of the microfluidic chip.

In addition, after the droplets are amplified, rotate the rotary valve 3 again to the detection position. At this time, the sampling channel 9 is connected to the sample channel 6, and the sampling channel 8 is connected to the return oil channel 5, and flows back. The oil channel 5 applies positive pressure to one end of the rotary valve 3, and the return oil flows from the return oil channel 5 to the injection channel 8 through the rotary valve 3, and from the injection channel 8 to the amplification carrier 2. Since the density ratio of the droplets is smaller than the density of the reflux oil, the droplets at this time will be attached to the upper layer of the amplification carrier 2, and when the continuous reflux oil flows into the amplification carrier 2, the droplets at this time will It is first pressed into the sample outlet channel 9 and flows back from the rotary valve 3 into the sample channel 6. At the same time, positive pressure is also applied to the end of the generated oil channel 4 facing away from the rotary valve 3. Since the sample channel 6 faces the rotary valve 3, The side wall of the side intersects with the generated oil channel 4. At this time, the generated oil is also input into the sample channel 6, separating the continuously refluxing droplets. At the same time, the external optical detection module starts to work, aligning the sample channel 6, and the droplets When the reflux passes, the digital nucleic acid detection of the droplets can be completed. The detection is carried out by refluxing into the sample channel 6, so there is no need to design additional detection channels and detection equipment for detection, and the sampling and detection are unified into the same place. The sample is transmitted within 6 channels, which reduces production costs, saves resources, reduces the size of the microfluidic device, and further improves the integration of the microfluidic chip. The droplet generation, amplification, and detection steps are all in the same chip. Complete, the functions are integrated into the same device, and the overall volume is greatly reduced.

One end of the generation oil channel 4, return oil channel 5, and sample channel 6 facing away from the rotary valve 3 is provided with a generation oil port 10, a return oil port 11, and a sample port 12. The generation oil port 10, the return oil port 11, and the sample channel 6 are The sample port 12 is connected to the outside. The two generated oil channels 4 share a generated oil port 10. The chip body 1 is provided with a generated oil reservoir 19 at the generated oil port 10, the return oil port 11 and the sample port 12. , return oil reservoir 20 and sample reservoir 21.

As an alternative implementation, the generated oil reservoir 19 and the return oil reservoir 20 may not be provided at the production oil port 10 and the return oil port 11, and only the sample liquid reservoir 21 may be provided at the sample port 12, through external The liquid injection device will inject the generated oil and return oil into the external oil, thereby further reducing the volume.

Specifically, the bottom of the rotary valve 3 is provided with four grooves 13, two of which are respectively connected to the top and bottom of the enrichment chamber 14. The bottom of the enrichment chamber 14 is arranged in a cylindrical shape. The top side wall of the enrichment chamber 14 is tilted toward the axis of the enrichment chamber 14 to form a cone shape. At this time, the conical top of the enrichment chamber 14 can guide the droplets and reduce the concentration of the droplets. The possibility of accumulation at the top of the cavity 14 allows the droplets entering the enrichment cavity 14 to enter the enrichment channel 22 and finally be transferred to the amplification carrier 2. When the droplets are generated, at this time and the enrichment cavity The two connected grooves 13 are connected to the sample channel 6 and the injection channel 8 respectively, and the groove 13 connected to the bottom of the enrichment chamber 14 is connected to the sample channel 6 to ensure that the droplets can enter the enrichment through the sample channel 6 The bottom of the chamber 14 is enriched.

At the same time, in order to facilitate the rotation of the rotary valve 3, a rotary wrench groove 15 is provided on the side of the rotary valve 3 facing away from the chip body 1. The rotary wrench groove 15 is set as a waist-shaped groove. When the fixing member 16 rotates the rotary valve 3 After being compressed and restricted, the rotary valve 3 can still rotate on its own. At this time, the external rotary mechanism is inserted into the rotary wrench groove 15, and then the rotary valve 3 is driven to rotate to achieve on-off or reversal.

The chip body 1 is provided with a fixing member 16 at the rotary valve 3, which is suitable for limiting and sealing the rotary valve 3. The rotary valve 3 is pressed on the chip body 1 and the contact surface is sealed. On the chip body 1 After the rotary valve 3 is placed on the surface, the rotary valve 3 is compressed by the fixing member 16 to ensure that the rotary valve 3 can move along its axis while limiting the position of the rotary valve 3 on the chip body 1 and at the same time restricting the position of the rotary valve 3 on the chip body 1. The rotary valve 3 performs compression sealing to seal the contact surface between the rotary valve 3 and the chip body 1 to ensure that no fluid leakage occurs when the droplet of fluid passes through the rotary valve 3 .

As a specific implementation, the fixing member 16 includes a placement block 161 provided on the chip body 1. The placement block 161 is provided with a placement slot 162. The bottom of the placement slot 162 is connected to the chip body 1, so The rotary valve 3 is located in the placement block 161 and rotates in the placement block 161. A gland 163 is provided at the notch of the placement groove 162. The outer wall of the gland 163 and the inner wall of the placement groove 162 are provided with threads. The outer wall of the gland 163 is threadedly connected to the inner wall of the placement groove 162. The gland 163 and the rotary valve 3 are in tight contact. When the rotary valve 3 is placed on the chip body 1, the rotary valve 3 is placed in the placement groove. 162, and the rotary valve 3 can rotate in the placement groove 162. At this time, the bottom of the rotary valve 3 is in contact with the upper surface of the chip body 1. Put the gland 163 into the notch position of the placement groove 162 and extend it into The inside of the placement groove 162 is threaded with the inner wall of the placement groove 162. Continuously rotate the gland 163 and move the gland 163 toward the rotary valve 3 until the gland 163 and the upper surface of the rotary valve 3 are in tight contact. On the chip body 1 The rotary valve 3 is compressed and sealed, and the contact surface between the rotary valve 3 and the chip body 1 is sealed to ensure that no fluid leakage occurs when the droplet of fluid passes through the rotary valve 3 .

In addition, a first communication hole 164 is opened on the top of the gland 163, and the first communication hole 164 is connected with the rotating wrench groove 15, so that the external rotating mechanism can enter the rotating wrench groove 15 through the first communication hole 164, and then Rotate the rotary valve 3.

As an alternative implementation, the fixing member 16 includes a first rotor pressing member 165. An outer edge 166 is provided on the side wall of the first rotor pressing member 165. The outer edge 166 is provided with at least Two, the bottom of the first rotor pressing member 165 is provided with a first receiving groove 167, the rotary valve 3 is located in the first receiving groove 167 and conflicts with the bottom of the first receiving groove 167, the chip body 1 A resisting edge 169 matching the outer edge 166 is provided at the rotary valve 3 . The first rotor pressing part 165 is located between the plurality of resisting edges 169 . The first rotor pressing part 165 rotates on the chip body 1 , until the outer edge 166 is located below the resisting edge 169 and conflicts with the resisting edge 169. When the rotary valve 3 is being installed, the first rotor pressing member 165 is placed directly above the rotary valve 3. At the same time, the outer edge 166 and the resisting edge 169 are misaligned, and then move the first rotor pressing member 165 towards the rotary valve 3 until the rotary valve 3 extends into the first receiving groove 167, and then rotate the first rotor pressing on the rotary valve 3 165, due to the rotation of the first rotor pressing member 165, the pressing edge 169 can be driven to move until it moves below the outer edge 166, so that the outer pressing edge and the pressing edge 169 are pressed against each other. At this time, the rotation of the rotary valve 3 The top wall collides with the bottom of the first accommodation groove 167, so that the rotary valve 3 is compressed on the chip body 1 under the action of the first rotor pressing member 165, thereby realizing the rotation of the rotary valve 3 in the subsequent rotation process. , ensuring that no leakage occurs when the fluid flows through the rotary valve 3.

In addition, a second communication hole 1610 is opened on the top of the first rotor pressing member 165. The first communication hole 164 is connected to the first receiving groove 167, and the first communication hole 164 is also connected to the rotating wrench groove 15, so that the external The rotating mechanism can enter the rotating wrench groove 15 through the first communication hole 164, and then rotate the rotating valve 3.

The side where the resisting edge 169 and the outer edge 166 contact is inclined. The inclination directions of the plurality of resisting edges 169 are consistent. The outer edge 166 and the resisting edge 169 are adapted to tilt. The rotary valve 3 is installed during the installation process. , at this time, place the first rotor pressing member 165 directly above the rotary valve 3, and at the same time, the outer edge 166 and the resisting edge 169 are misaligned, and then move the first rotor pressing member 165 toward the direction of the rotary valve 3 until it rotates The valve 3 extends into the first receiving groove 167. At this time, the first rotor pressing member 165 is rotated, so that the outer edge 166 on the first rotor pressing member 165 moves toward the direction of the pressing edge 169, and the outer edge 166 is smaller. The end is in front until the outer edge 166 moves to the position of the resisting edge 169. At this time, the small end of the outer edge 166 first enters directly below the resisting edge 169, and continues to move the first rotor pressing member 165, so that The small end of the outer edge 166 moves toward the big end of the resisting edge 169 until the outer edge 166 and the resisting edge 169 are tightened. At this time, the outer edge 166 can no longer move forward, and the rotary valve 3 and the first rotor are tightened. The wall of the first receiving groove 167 in the member 165 is pressed tightly, thereby compressing the rotor, so as to ensure that no leakage occurs when the fluid flows through the rotor during the subsequent rotation of the rotary valve 3.

At the same time, the pressing edge 169 is provided with a first protrusion 17 at the highest point of the side wall of the rotary valve 3. The first protrusion 17 is made of elastic plastic material. When the first rotor pressing member 165 compresses the rotor, , at this time, the outer edge 166 will move to the resisting edge 169 along with the first rotor pressing member 165. The small end of the outer edge 166 first enters below the resisting edge 169, and at this time, the first protrusion 17 is set On the small end of the resisting edge 169, when the outer edge 166 continues to move toward the big end of the resisting edge 169, the outer edge 166 gradually conflicts with the resisting edge 169 until the resisting edge 169 and the outer edge 166 tightly, the outer edge 166 can no longer move forward, and at this time, the side wall of the outer edge 166 conflicts with the side wall of the first protrusion 17. Under the action of the first protrusion 17, it is prevented from adjusting the direction of the rotary valve 3. , the first rotor pressing member 165 moves in the opposite direction, so that the first rotor pressing member 165 loses the pressing effect, and moving in the forward direction will only tighten the first rotor pressing member 165 more.

The amplification carrier 2 is also provided with an amplification module 18. The amplification module 18 is used to apply pressure and heat to the amplification carrier 2, so that the collected droplets in the amplification carrier 2 undergo several thermal cycles to complete the nucleic acid production. Amplification, the amplification module 18 includes a heating base 181 arranged at the bottom of the amplification carrier 2. A groove is provided on the heating base 181, which is suitable for the amplification carrier 2 to be located in the groove and to conflict with the inner wall of the groove. The heating base 181 is A Peltier 182 and a heat sink 183 are arranged below the base 181 in sequence. The Peltier 182 is used to realize amplification heating and cooling cycles to complete the amplification.

In addition, the amplification module 18 also includes a tube cover protrusion 184 arranged at the bottom of the chip body 1. The sample inlet channel 8 and the sample outlet channel 9 are both arranged in the tube cover protrusion 184. The amplification carrier 2 and the tube The cover protrusion 184 is clamped, and the chip body 1 is provided with a second heating element 185 at a position facing away from the amplification carrier 2. The second heating element 185 is attached to the chip body 1, and is activated by the second heating element 185. The chip body 1 and the tube cover protrusion 184 are heated to prevent droplets from evaporating during the temperature rise and fall cycles of the amplification carrier 2 and affecting amplification.

As an alternative implementation, since the second heating plate directly heats the chip body 1, the structure is simple, but the thickness of the chip body 1 at this time needs to be at least 1 mm, and both are made of PC material. The chip body 1 and the tube cover The heating rate of the protrusion 184 is slow. In order to increase the heating rate, the amplification module 18 also includes an annular heating cover 186. The sample inlet channel 8 and the sample outlet channel 9 are both arranged in the annular heating cover 186, so The amplification carrier 2 is sleeved on the bottom of the annular heat cover 186 and sealed with the annular heat cover 186. A first heating element 187 is provided on the annular heat cover 186. The first heating element 187 and the annular heat cover 186 are attached to each other. When the droplets enter the amplification carrier 2 for amplification, the amplification module 18 at this time starts to start, thereby performing a temperature rising and cooling cycle on the amplification carrier 2, so that the droplets in the amplification carrier 2 experience several Secondary thermal cycle to complete nucleic acid amplification. At the same time, during amplification, the first heating element 187 is started, thereby driving the annular heating cover 186 to heat up. The heated heat cover heats the top of the amplification carrier 2 , to prevent the droplets in the amplification carrier 2 from evaporating. The annular heat cover 186 is made of metal material, and the metal annular heat cover 186 can thereby increase the heating speed and better prevent evaporation, thus improving the amplification efficiency.

Example 2

Referring to Figure 15, a detection method using the microfluidic chip based on the rotary valve 3 described in the embodiment includes the following steps:

S1: Droplets are generated. At this time, rotate the rotary valve 3 to the generation position. At this time, the sample channel 6 is connected to the sampling channel 8, the sample outlet channel 9 is connected to the ventilation channel 7, and the sample channel 6 is connected to the inlet channel. The enrichment chamber 14 is connected in the groove 13 at the connection point of the sample channel 8. The sample is injected into the sample channel 6. The generated oil is introduced into the generated oil channel 4 and positive pressure is applied. The generated oil enters through the cross flow channel. In the sample channel 6, a shear flow will be formed at the cross flow channel, splitting the sample into droplets, and passing into the inside of the rotary valve 3. At this time, when the mixture of droplets and oil enters the enrichment chamber 14 , at this time, the droplets will produce an enrichment effect in the enrichment chamber 14, that is, the droplets entering the enrichment chamber 14 will first float above the enrichment chamber 14. When the enrichment chamber 14 is filled with droplets, After being mixed with oil, the droplets will flow out of the rotary valve 3 at a high rate and enter the injection channel 8, while the excess oil phase will remain in the enrichment chamber 14, thereby ensuring that a higher level is obtained in the amplification carrier 2 The volume fraction of droplets. At this time, the rotary valve 3 realizes the on-off of the droplets and the oil-phase mixed fluid when the droplets are generated, and also realizes the enrichment of the droplets, thereby ensuring the microdroplets in the amplification carrier 2. The droplet volume fraction is high to avoid excessive oil phase in the collected droplets, which affects the amplification effect. At the same time, enrichment is achieved on the rotary valve 3, giving the rotary valve 3 additional functions, and at the same time making full use of the rotary valve 3, eliminating the need for In addition, setting it on the chip reduces the size of the microfluidic chip and improves the integration of the microfluidic chip.

S2: droplet amplification. After the droplets are sent into the amplification carrier 2, the rotary valve 3 is rotated to separate the sample channel 6 from the sampling channel 8, and the sample outlet channel 9 is separated from the ventilation channel 7 to maintain amplification. The carrier 2 is in a sealed state, start the Peltier 182, and under the action of the Peltier 182, the heating base 181 and the amplification carrier 2 in the heating base 181 are heated for several times. The droplets are heated to complete droplet amplification.

S3: Droplet reflux detection: After the droplets are amplified, rotate the rotary valve 3 again to the detection position. At this time, the sampling channel 9 is connected to the sample channel 6, and the sampling channel 8 is connected to the reflux oil channel. 5 is connected, and positive pressure is applied to the end of the return oil channel 5 away from the rotary valve 3, and the return oil is passed from the return oil channel 5 to the injection channel 8 through the rotary valve 3, and from the injection channel 8 to In the amplification carrier 2, since the density of the droplets is smaller than the density of the reflux oil, the droplets at this time will be attached to the upper layer of the amplification carrier 2, and when the continuous reflux oil flows into the amplification carrier 2, this The droplets will be first pressed into the sample outlet channel 9 and flow back from the rotary valve 3 into the sample channel 6. At the same time, positive pressure is also applied to the end of the generated oil channel 4 facing away from the rotary valve 3. Since the sample channel 6 The side wall toward the side of the rotary valve 3 is connected and tangential to the generated oil channel 4. At this time, the generated oil is also input into the sample channel 6 to separate the continuous refluxing droplets. At the same time, the external optical detection module starts to work and aligns Sample channel 6, when the droplets pass through during backflow, the digital nucleic acid detection of the droplets can be completed. The detection is carried out by returning to the sample channel 6, so there is no need to design additional detection channels and detection equipment for detection. Injection and detection are unified into the same sample channel 6 for transmission, which reduces production costs, saves resources, reduces the size of microfluidic equipment, and further improves the integration of microfluidic chips.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (11)

1. The micro-fluidic chip based on the rotary valve comprises a chip body (1), and is characterized in that a generating oil duct (4), a backflow oil duct (5), a sample channel (6) and a ventilation channel (7) are arranged in the chip body (1), one end of the generating oil duct (4) is communicated with the side wall of the sample channel (6), generating oil in the generating oil duct (4) enters the sample channel (6) to shear a sample to form micro-drops, and one end of the ventilation channel (7) is communicated with the outside;

further comprises:

the joint of the two generating oil channels (4) and the sample channel (6) forms a cross flow channel;

the amplification carrier (2), the amplification carrier (2) is sealed in the chip body (1), a sample injection channel (8) and a sample discharge channel (9) are further arranged at the position of the chip body (1) located at the amplification carrier (2), and the sample injection channel (8) and the sample discharge channel (9) are both communicated with the amplification carrier (2);

The rotary valve (3) is arranged on the chip body (1) in a rotating way along the axis of the rotary valve (3), when the rotary valve (3) rotates to a generating position, the sample channel (6) is communicated with the sample inlet channel (8), the sample outlet channel (9) is communicated with the ventilation channel (7), when the rotary valve (3) rotates to a detecting position, the sample outlet channel (9) is communicated with the sample channel (6), the sample inlet channel (8) is communicated with the backflow oil duct (5), and microdroplets in the amplification carrier (2) enter the sample channel (6) through the sample outlet channel (9) to be detected in a backflow way; an enrichment cavity (14) is arranged in the rotary valve (3), and when the rotary valve (3) rotates to a generation position, the bottom and the top of the enrichment cavity (14) are respectively communicated with the sample channel (6) and the sample injection channel (8);

the bottom of rotary valve (3) is provided with four at least slot (13), and wherein two slot (13) respectively with the top and the bottom intercommunication of enrichment chamber (14), enrichment chamber (14) bottom sets up to cylindric, enrichment chamber (14) top lateral wall sets up towards enrichment chamber (14) axis direction slope, forms coniform.

2. The rotary valve-based microfluidic chip according to claim 1, characterized in that the chip body (1) is provided with a fixture (16) at the rotary valve (3), adapted for a limit sealing of the rotary valve (3), pressing the rotary valve (3) on the chip body (1) and sealing the contact surface.

3. The rotary valve-based microfluidic chip according to claim 2, wherein the fixing member (16) comprises a placement block (161) arranged on the chip body (1), a placement groove (162) is formed in the placement block (161), the bottom of the placement groove (162) is communicated with the chip body (1), the rotary valve (3) is located in the placement block (161) and rotates in the placement block (161), a gland (163) is arranged at a notch of the placement groove (162), threads are formed on the outer wall of the gland (163) and the inner wall of the placement groove (162), the outer wall of the gland (163) is in threaded connection with the inner wall of the placement groove (162), and the gland (163) is abutted against the rotary valve (3).

4. The rotary valve-based microfluidic chip according to claim 2, wherein the fixing member (16) comprises a first rotor pressing member (165), an outer rim (166) is arranged on a side wall of the first rotor pressing member (165), at least two outer rims (166) are arranged, a first accommodating groove (167) is arranged at the bottom of the first rotor pressing member (165), the rotary valve (3) is arranged in the first accommodating groove (167) and is abutted against the bottom of the first accommodating groove (167), an abutting edge (169) matched with the outer rims (166) is arranged at the rotary valve (3), the first rotor pressing member (165) is arranged among the abutting edges (169), and the first rotor pressing member (165) rotates on the chip body (1) until the outer rims (166) are arranged below the abutting edges (169) and are abutted against the abutting edges (169).

5. The rotary valve-based microfluidic chip according to claim 4, wherein the abutting edge (169) and the outer edge (166) are obliquely arranged on the abutting side, the inclination directions of a plurality of the abutting edges (169) are consistent, and the outer edge (166) and the abutting edges (169) are adaptively inclined.

6. The rotary valve-based microfluidic chip according to claim 1, wherein an amplification module (18) is further arranged at the amplification carrier (2), and the amplification module (18) is used for applying heat to the amplification carrier (2) so that the collected microdroplets in the amplification carrier (2) perform several thermal cycles to complete nucleic acid amplification.

7. The rotary valve-based microfluidic chip according to claim 6, wherein the amplification module (18) comprises an annular heat cover (186), the sample inlet channel (8) and the sample outlet channel (9) are both arranged in the annular heat cover (186), the amplification carrier (2) is sleeved at the bottom of the annular heat cover (186) and is sealed with the annular heat cover (186), a first heating element (187) is arranged on the annular heat cover (186), and the annular heat cover (186) is attached to the first heating element (187).

8. The rotary valve-based microfluidic chip of claim 7, said annular thermal cover (186) being composed of a metallic material.

9. The rotary valve-based microfluidic chip according to claim 6, wherein the amplification module (18) comprises a tube cover protrusion (184) arranged at the bottom of the chip body (1), the sample inlet channel (8) and the sample outlet channel (9) are arranged in the tube cover protrusion (184), the amplification carrier (2) and the tube cover protrusion (184) are clamped tightly, a second heating element (185) is arranged at the position, facing away from the amplification carrier (2), of the chip body (1), and the second heating element (185) is attached to the chip body (1).

10. The rotary valve-based microfluidic chip according to any one of claims 1 to 9, wherein one end of the generating oil duct (4), the return oil duct (5) and the sample channel (6) facing away from the rotary valve (3) is provided with a generating oil port (10), a return oil port (11) and a sample port (12), the generating oil port (10), the return oil port (11) and the sample port (12) are communicated with the outside, and the generating oil reservoir (19), the return oil reservoir (20) and the sample reservoir (21) are arranged at the generating oil port (10), the return oil port (11) and the sample port (12) of the chip body (1).

11. A method of detection using a rotary valve-based microfluidic chip according to any one of claims 1 to 10, comprising the steps of:

Droplet generation: rotating the rotary valve (3), rotating the rotary valve (3) to a generation position, communicating a sample channel (6) with a sample injection channel (8), communicating a sample outlet channel (9) with a ventilation channel (7), injecting a sample into the sample channel (6), introducing generated oil into the generated oil channel (4) and pressurizing positive pressure, allowing the generated oil to enter the sample channel (6) to form shear flow, dividing the sample into microdroplets, introducing the microdroplets into the sample injection channel (8) under the action of the rotary valve (3), and finally entering the amplification carrier (2);

droplet amplification: the amplification carrier (2) starts to perform thermal circulation for a plurality of times, and microdroplets in the cavity of the amplification carrier (2) are heated to finish microdroplet amplification;

droplet reflow detection: rotating the rotary valve (3) again, moving the rotary valve (3) to a detection position, enabling a sample outlet channel (9) to be communicated with a sample channel (6), enabling a sample inlet channel (8) to be communicated with a backflow oil channel (5), applying positive pressure to one side of the backflow oil channel (5), enabling backflow oil to be introduced into the sample inlet channel (8) from the backflow oil channel (5) and to be pressed into an amplification carrier (2), enabling microdroplets in the amplification carrier (2) to be pressed into the sample outlet channel (9) at first and to flow back into the sample channel (6), applying positive pressure to a generating oil channel (4), enabling the generating oil to be introduced into the sample channel (6) from the communication position of the generating oil channel (4) and the sample channel (6), enabling microdroplet intervals, and enabling an external optical detection module to be aligned with the sample channel (6), and conducting nucleic acid detection on microdroplets when the microdroplets flow back.