CN115155682A - Micro-fluidic chip based on rotary valve and detection method - Google Patents
Micro-fluidic chip based on rotary valve and detection method Download PDFInfo
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- CN115155682A CN115155682A CN202210771245.9A CN202210771245A CN115155682A CN 115155682 A CN115155682 A CN 115155682A CN 202210771245 A CN202210771245 A CN 202210771245A CN 115155682 A CN115155682 A CN 115155682A
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
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- B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
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Abstract
The invention relates to a micro-fluidic chip based on a rotary valve and a detection method, wherein the micro-fluidic chip comprises a chip body, wherein a generation oil duct, a return oil duct, a sample channel and a ventilation channel are arranged in the chip body, one end of the generation oil duct is communicated with the side wall of the sample channel, generation oil in the generation 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 comprising: the chip body is also provided with a sample inlet channel and a sample outlet channel at the position of the amplification carrier, and the sample inlet channel and the sample outlet channel are both communicated with the amplification carrier; rotating the valve to perform backflow detection; the rotary valve has the advantages that the enrichment is realized on the rotary valve, the rotary valve is endowed with additional functions, the rotary valve is fully utilized, the arrangement on a chip is not needed, the size of a microfluidic chip is reduced, and the integration level of the microfluidic chip is improved.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a micro-fluidic chip based on a rotary valve and a detection method.
Background
Polymerase Chain Reaction (PCR) is a technology for amplifying a section of DNA to a sufficient quantity under the participation of DNA polymerase and nucleotide substrates by using the section of DNA as a template so as to carry out structural and functional analysis, and a Digital PCR (Digital PCR-dPCR) technology is a novel nucleic acid detection and quantitative analysis technology, has the characteristics of high sensitivity, high specificity and high stability, and has important significance in the application of low-abundance nucleic acid detection fields of gene expression research, microRNA research, cancer marker rare mutation detection and the like. Different from the traditional Real-time Quantitative PCR (Real-time Quantitative PCR-qPCR) technology, the digital PCR has the principle that a standard PCR reaction is distributed into a large number of tiny reactors, each reactor contains or does not contain one or more copies of target molecules (DNA templates), so that single-molecule template PCR amplification is realized, and after the amplification is finished, the copy number of a target sequence is counted through the number of positive reactors.
The micro-fluidic chip is based on micro-electro-mechanical processing technology, and a network is formed on the chip by micro-pipelines, so that the controllable micro-channels penetrate through the whole system and complete various biological and chemical processes. In the early development stage of the microfluidic chip technology, chip capillary electrophoresis is the mainstream technology, and the used chip has a simple structure and a single function; in recent years, micro-fluidic chips have been rapidly developed toward functionalization and integration, and important biological and chemical processes such as nucleic acid amplification reaction, immune reaction, cell lysis, etc. become new hot spots.
With the increasing maturity of microfluidic processing technology, the difficulty and cost of microfluidic chip design, development and test are lower and lower, the application is wider and wider, and especially, the application cases in miniaturized in-vitro diagnostic equipment are more and more, the microfluidic chip is used as a detection carrier, operations such as sample adding, transferring, mixing and the like on a sample sheet are realized through microfluidic, and the detection of a test index is finally completed. The micro-fluidic chip has the advantages of small amount of used reagent consumables, easy realization, portability, miniaturization and family.
Chinese patent No. CN113117770A discloses a PCR microfluidic chip, which comprises a main chip, an oil inlet cavity liquid storage tank, a sample inlet cavity liquid storage tank, a common side liquid storage tank and a PCR tube; the main piece is provided with a droplet generation module, an amplification module and a detection module; the droplet generation module is used for receiving the continuous phase injected from the oil inlet cavity liquid storage tank, receiving the separated phase injected from the sample inlet cavity liquid storage tank, mixing the received continuous phase and the received separated phase to form droplets, and injecting the formed droplets into the amplification module; the amplification module is used for receiving the driving oil injected from the common-side liquid storage tank, injecting the received driving oil into the PCR tube, receiving the microdroplets injected from the microdroplet generation module, injecting the received microdroplets into the PCR tube, injecting the microdroplets and the driving oil in the PCR tube into the detection module, receiving the shearing oil injected from the common-side liquid storage tank, and injecting the received shearing oil into the detection module; the detection module is used for receiving the micro-droplets, the driving oil and the shearing oil injected from the amplification module, and under the downstream action of the driving oil and the shearing oil, the micro-droplets are arranged in order.
The above prior art solutions have the following drawbacks: although the above-mentioned microfluidic chip integrates the processes of droplet generation, droplet amplification and droplet flow detection into the same chip, and the chip switches different flow channel pipelines through the plugs, in a practical process, when droplets are generated, the collected droplets may be accompanied by excessive oil, and the subsequent processing is troublesome, and the amplification effect is affected, while if an enrichment device is additionally designed, the size of the chip may be affected, and other structures on the chip may be affected.
Disclosure of Invention
Therefore, the invention provides a micro-fluidic chip based on a rotary valve and a detection method, aiming at solving the technical problems that when the micro-droplets are generated, the collected micro-droplets are accompanied with excessive oil, the subsequent treatment is troublesome, and the amplification is influenced.
A micro-fluidic chip based on a rotary valve comprises a chip body, wherein a generation oil channel, a return oil channel, a sample channel and a ventilation channel are arranged in the chip body, one end of the generation oil channel is communicated with the side wall of the sample channel, generation oil in the generation oil channel 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 comprising:
the amplification carrier is sealed in the chip body, a sample inlet channel and a sample outlet channel are also arranged at the amplification carrier of the chip body, and the sample inlet channel and the sample outlet channel are both communicated with the amplification carrier;
the rotary valve is rotationally arranged on the chip body along the axis of the rotary valve, when the rotary valve rotates to a generation position, the sample channel is communicated with the sample feeding channel, the sample discharging channel is communicated with the ventilation channel, when the rotary valve rotates to a detection position, the sample discharging channel is communicated with the sample channel, the sample feeding channel is communicated with the return oil channel, and droplets in the amplification carrier enter the sample channel through the sample discharging channel for return detection;
wherein be provided with the enrichment chamber in the rotary valve, when the rotary valve rotated to and produced the position, the bottom and the top in enrichment chamber communicate with sample channel and advance a kind passageway respectively.
Furthermore, the bottom of rotary valve is provided with four at least slots, wherein two the slot respectively with enrichment chamber's top and bottom intercommunication, enrichment chamber bottom sets up to cylindric, enrichment chamber top lateral wall sets up towards the slope of enrichment chamber axis direction, forms coniformly.
Furthermore, the chip body is located rotary valve department is provided with the mounting, is applicable to and carries out spacing sealing to the rotary valve, compresses tightly and the sealing contact face to the rotary valve on the chip body.
Further, the mounting is including setting up the piece of placing on the chip body, place and seted up the standing groove on the piece, standing groove bottom and chip body intercommunication, the rotary valve is located place the piece and rotatory in placing the piece, the notch department of standing groove is provided with the gland, the inner wall of gland outer wall and standing groove is provided with the screw thread, the outer wall of gland and the inner wall threaded connection of standing groove, gland and rotary valve support tightly.
Further, the mounting includes that first rotor compresses tightly the piece, first rotor compresses tightly and is provided with outer border on the lateral wall of piece, outer border is provided with two at least, the bottom that first rotor compressed tightly the piece is provided with first holding tank, the rotary valve is located first holding tank and contradicts with first holding tank bottom, chip body is located rotary valve department and is provided with and outer border matching's the tight limit of supporting, first rotor compresses tightly the piece and is located a plurality of tight limits of supporting between, and first rotor compresses tightly the piece and rotates on chip body, is located the tight limit below of supporting and supports tight limit and contradicts until outer border.
Furthermore, the abutting edge and the outer edge abut against one side of the frame in an inclined mode, the abutting edge and the outer edge are consistent in inclined direction, and the outer edge and the abutting edge are matched and inclined.
Further, an amplification module is arranged at the position of the amplification carrier and used for applying pressure and heat to the amplification carrier so that the collected microdroplets in the amplification carrier are subjected to thermal cycles for several times to complete nucleic acid amplification.
Further, the amplification module includes annular hot lid, advance kind passageway and go out kind passageway and all set up in annular hot lid, the bottom that annular hot lid was covered and sealed with annular hot lid is established to the carrier cover that amplifies, annular hot is covered and is provided with first heating member, second heating member and the laminating of annular hot lid.
Further, the annular thermal cover is constructed of a metallic material.
Further, the amplification module is protruding including the tube cap that sets up in the bottom of chip body, introduction channel and appearance passageway all set up in the tube cap is protruding, the protruding chucking of amplification carrier and tube cap, chip body amplification carrier department of amplification dorsad is provided with the second heating member, second heating member and chip body laminating.
Further, one end of the generation oil duct, the backflow oil duct and the sample channel back to the rotary valve is set to be a generation oil port, a backflow oil port and a sample port, the generation oil port, the backflow oil port and the sample port are communicated with the outside, and the chip body is located at the generation oil port, the backflow oil port and the sample port and is provided with a generation oil liquid storage tank, a backflow oil liquid storage tank and a sample liquid storage tank
A method of detection using any of the rotary valve based microfluidic chips, comprising the steps of:
droplet generation: the rotary valve is rotated to a generation position, the sample channel is communicated with the sample feeding channel, the sample outlet channel is communicated with the ventilation channel, the generated oil is introduced into the sample channel and the generation oil channel and is pressurized to be positive pressure, the generated oil enters the sample channel to form shear flow, the sample is divided into micro-droplets, and the micro-droplets are introduced into the sample feeding channel under the action of the rotary valve and finally enter the amplification carrier;
and (3) microdroplet amplification: the amplification carrier starts to carry out a plurality of thermal cycles, and microdroplets in the cavity of the amplification carrier are heated to complete microdroplet amplification;
droplet backflow detection: rotating the rotary valve again, moving the rotary valve to the detection position, communicating the sample outlet channel with the sample channel, communicating the sample inlet channel with the return oil channel, applying positive pressure to one side of the return channel, introducing return oil from the return oil channel into the sample inlet channel and pressing the return oil into the amplification carrier, firstly pressing microdrops in the amplification carrier into the sample outlet channel and returning the microdrops into the sample channel, applying positive pressure to the generated oil to the inside, introducing the generated oil into the sample channel from the communication position of the generated oil channel and the sample channel, spacing the microdrops, aligning an external optical detection module with the sample channel, and detecting nucleic acid of the microdrops when the microdrops pass through the return channel.
The technical scheme of the invention has the following advantages:
1. when the micro-fluidic chip based on the rotary valve is used, when micro-droplets are generated, the rotary valve is rotated at the moment, the rotary valve is rotated to a generation position, the sample channel is communicated with the sample introduction channel under the action of the rotary valve, the sample outlet channel is communicated with the air channel, so that the sample outlet channel is communicated with the atmosphere, the generated micro-droplets can smoothly enter the amplification carrier, when the micro-droplets are generated, positive pressure is applied to inlets of the generation oil channel and the sample channel, so that the generated oil and the samples are conveyed in the generation oil channel and the sample channel, at the moment, as the generation oil channel is communicated and intersected with the sample channel, shear flow is formed at the communicated position, the samples in the sample channel can be divided into micro-droplets with consistent volume, the micro-droplets are introduced to the rotary valve from the sample channel after being generated, and the mixture of the micro-droplets and the oil can enter the enrichment cavity, because the density of the microdroplets is less than that of the oil, when the mixture of the microdroplets and the oil enters the enrichment wall, the microdroplets generate an enrichment effect in the enrichment cavity, namely the microdroplets entering the enrichment cavity firstly float above the enrichment cavity, when the enrichment cavity is filled with the mixture of the microdroplets and the oil, the microdroplets firstly flow out of the rotary valve and enter the sample feeding channel, and the redundant oil phase remains in the enrichment cavity, so that the higher microdroplet volume fraction is obtained in the amplification carrier, the on-off of the microdroplets and the oil phase mixed fluid is realized by the rotary valve when the microdroplets are generated, and meanwhile, the enrichment of the microdroplets is also realized, so that the microdroplet volume fraction in the amplification carrier is high, the collected microdroplets are prevented from being accompanied by excessive oil phase to influence the amplification effect, meanwhile, the enrichment on the rotary valve is realized, and an additional function is given, the method comprises the steps of rotating the rotary valve, rotating the rotary valve to a detection position, communicating a sample outlet channel with a sample channel, communicating a sample inlet channel with a return oil channel, applying positive pressure to one end of the return oil channel, which is back to the rotary valve, introducing return oil from the return oil channel into the sample inlet channel and from the sample inlet channel into an amplification carrier, wherein droplets are attached to the upper layer of the amplification carrier due to the fact that the density of the droplets is smaller than that of the return oil, and after the continuous return oil is introduced into the amplification carrier, the droplets are firstly pressed into the sample outlet channel and flow back into the sample channel from the rotary valve, and the other end of the generation oil channel, which is back to the rotary valve, is also applied with positive pressure.
2. The invention provides a micro-fluidic chip based on a rotary valve, wherein the bottom of the rotary valve is provided with at least four grooves, two of the grooves are respectively communicated with the top and the bottom of an enrichment cavity, the bottom of the enrichment cavity is cylindrical, the side wall of the top of the enrichment cavity is obliquely arranged towards the axial direction of the enrichment cavity to form a cone shape, and the top of the cone-shaped enrichment cavity can guide microdroplets, reduce the possibility of accumulation of microdroplets on the top of the enrichment cavity, so that the microdroplets entering the enrichment cavity can enter an enrichment channel and are finally transferred into an amplification carrier.
3. According to the micro-fluidic chip based on the rotary valve, the fixing piece is arranged at the position, located on the rotary valve, of the chip body, the chip body is suitable for limiting and sealing the rotary valve, the rotary valve is pressed on the chip body and a contact surface is sealed, after the rotary valve is placed on the upper surface of the chip body, the rotary valve is pressed through the fixing piece, the position, located on the chip body, of the rotary valve is limited while the rotary valve can be ensured to be along the axis of the rotary valve, the rotary valve is pressed and sealed, the abutting surface between the rotary valve and the chip body is sealed, and therefore the situation that fluid does not leak when micro-droplet fluid passes through the rotary valve is guaranteed.
4. The invention provides a micro-fluidic chip based on a rotary valve, wherein a fixing piece comprises a placing block arranged on a chip body, a placing groove is formed in the placing block, the bottom of the placing groove is communicated with the chip body, the rotary valve is positioned in the placing block and rotates in the placing block, a gland is arranged at a notch of the placing groove, threads are arranged on the outer wall of the gland and the inner wall of the placing groove, the outer wall of the gland is in threaded connection with the inner wall of the placing groove, the gland is abutted against the rotary valve, when the rotary valve is placed on the chip body, the rotary valve is placed in the placing groove and can rotate in the placing groove, the bottom of the rotary valve is attached to the upper surface of the chip body, the gland is placed at the notch of the placing groove and extends into the placing groove to be in threaded connection with the inner wall of the rotary valve of the placing groove, the gland is continuously rotated, the gland is moved towards the direction until the upper surface of the gland is abutted against the rotary valve, the rotary valve is tightly pressed and sealed on the chip body, and the abutting surface of the seal with the chip body is ensured that fluid can not leak when the micro-drop fluid passes through.
5. The invention provides a micro-fluidic chip based on a rotary valve, which comprises a first rotor pressing piece, wherein an outer edge is arranged on the side wall of the first rotor pressing piece, at least two outer edges are arranged on the outer edge, a first accommodating groove is arranged at the bottom of the first rotor pressing piece, the rotary valve is positioned in the first accommodating groove and is abutted against the bottom of the first accommodating groove, a butting edge matched with the outer edge is arranged at the position of the chip body, the first rotor pressing piece is positioned among a plurality of butting edges, the first rotor pressing piece rotates on the chip body until the outer edge is positioned below the butting edge and abutted against the butting edge, when the rotary valve is installed, the first rotor pressing piece is placed right above the rotary valve, the outer edge and the butting edge are staggered, then the first rotor pressing piece moves towards the rotary valve until the rotary valve extends into the first accommodating groove, then the first rotor pressing piece rotates on the rotary valve, and the first rotor pressing piece rotates to drive the rotary valve to move until the outer edge moves to the lower part of the outer edge, so that the top wall of the rotary valve and the first rotor pressing piece do not leak in the subsequent rotary valve, and the top wall of the rotary valve can not be pressed.
6. According to the micro-fluidic chip based on the rotary valve, the abutting edges and the outer edge abutting sides are obliquely arranged, the inclining directions of the abutting edges are consistent, the outer edge and the abutting edges are inclined in a matched mode, in the installation process of the rotary valve, the first rotor pressing piece is placed right above the rotary valve, meanwhile, the outer edge and the abutting edges are staggered, then the first rotor pressing piece moves towards the rotary valve until the rotary valve extends into the first accommodating groove, the first rotor pressing piece is rotated, the outer edge on the first rotor pressing piece moves towards the abutting edge, the small end of the outer edge is in front, when the outer edge moves to the position of the abutting edges, the small end of the outer edge at the moment firstly enters the position right below the abutting edges, the first rotor pressing piece is moved continuously, the small end of the outer edge moves towards the large end of the abutting edges until the outer edge and the abutting edges, the outer edge cannot move forwards, the first rotor pressing piece and the inner edge of the rotary valve cannot be pressed tightly, and the rotor cannot be tightly pressed in the subsequent rotor pressing process, and the rotor cannot be tightly pressed.
7. The invention provides a micro-fluidic chip based on a rotary valve, wherein an amplification module comprises an annular heat cover, a sample inlet channel and a sample outlet channel are arranged in the annular heat cover, an amplification carrier is sleeved at the bottom of the annular heat cover and is sealed with the annular heat cover, a first heating element is arranged on the annular heat cover, a second heating element is attached to the annular heat cover, when droplets enter the amplification carrier for amplification, the amplification module starts to perform heating and cooling circulation on the amplification carrier, so that the droplets in the amplification carrier undergo several times of thermal circulation to complete nucleic acid amplification, and meanwhile, when the droplets are amplified, the first heating element starts to drive the annular heat cover to heat, and the heated heat cover heats the top of the amplification carrier to prevent the droplets in the amplification carrier from evaporating.
8. According to the micro-fluidic chip based on the rotary valve, the annular hot cover is made of the metal material, so that the temperature rising speed is increased, the evaporation prevention effect is better, and the amplification efficiency is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the overall structure of a rotary valve-based microfluidic chip according to the present invention;
FIG. 2 is a schematic view of the internal structure of the placement block of the present invention;
FIG. 3 is a schematic view of the bottom of the rotary valve of the present invention;
FIG. 4 is a schematic view showing the internal structure of a rotary valve according to the present invention;
FIG. 5 is a schematic diagram of the internal structure of the chip body according to the present invention;
FIG. 6 is a schematic diagram of the rotation valve on/off during droplet generation, droplet amplification and droplet detection according to the present invention;
FIG. 7 is a schematic view of another embodiment of a fastener of the present invention;
FIG. 8 is a schematic view of another embodiment of a reservoir according to the present invention;
FIG. 9 is a schematic view of an abutment edge of another embodiment of the fastener of the present invention;
FIG. 10 is a schematic view of a first rotor hold down of the present invention;
FIG. 11 is a bottom view of the first rotor hold down of the present invention;
FIG. 12 is a schematic diagram of a first embodiment of an amplification module according to the invention;
FIG. 13 is a schematic diagram of a second embodiment of an amplification module according to the invention;
FIG. 14 is a schematic diagram showing the overall structure of an amplification module according to the present invention;
FIG. 15 is a step diagram of the detection method of the present invention.
Description of reference numerals:
1. a chip body; 2. amplifying the vector; 3. rotating the valve; 4. generating an oil passage; 5. a return oil passage; 6. a sample channel; 7. a vent passage; 8. a sample introduction channel; 9. a sample outlet channel; 10. generating an oil port; 11. a return oil port; 12. a sample port; 13. a trench; 14. an enrichment chamber; 15. rotating the wrench groove; 16. a fixing member; 161. placing the blocks; 162. a placement groove; 163. a gland; 164. a first communication hole; 165. a first rotor pressing member; 166. an outer edge; 167. a first accommodating groove; 169. abutting the edge; 1610. a second communication hole; 17. a first protrusion; 18. an amplification module; 181. heating the base; 182. a Peltier; 183. a heat sink; 184. the pipe cover is raised; 185. a second heating member; 186. an annular thermal cover; 187. a first heating member; 19. generating an oil storage tank; 20. a return oil reservoir; 21. a sample reservoir; 22. and (3) an enrichment channel.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope 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., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present 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 explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; 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 specific cases to those skilled in the art.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Examples
Referring to fig. 1 to 14, the invention provides a micro-fluidic chip based on a rotary valve, which includes a chip body 1, an amplification carrier 2 and a rotary valve 3, wherein a generation oil passage 4, a return oil passage 5, a sample passage 6 and a ventilation passage 7 are arranged in the chip body 1, one end of the generation oil passage 4 is communicated with the side wall of the sample passage 6, generation oil in the generation oil passage 4 enters the sample passage 6 to shear a sample to form micro-droplets, the generation oil passage 4 is provided with two generation oil passages, one end of each of the two generation oil passages 4 facing the rotary valve 3 is communicated with the sample passage 6 and is perpendicular to the sample passage 6, a cross flow passage is formed at the joint of the two generation oil passages 4 and the sample passage 6, and one end of the ventilation passage 7 is communicated with the outside to facilitate the micro-droplets transportation into the amplification carrier 2;
the amplification carrier 2 is sealed in the chip body 1, a sample inlet channel 8 and a sample outlet channel 9 are also arranged at the position, located at the amplification carrier 2, of the chip body 1, and the sample inlet channel 8 and the sample outlet channel 9 are both communicated with the amplification carrier 2; the rotary valve 3 is rotatably arranged on the chip body 1 along the axis thereof and is positioned between the amplification carrier 2 and the sample channel 6, one end of the sample channel 6, the oil return channel, the sample feeding channel 8 and the sample discharging channel 9 are positioned under the rotary valve 3 and protrude out of the bottom surface of the chip body 1 to be contacted with the rotary valve 3, when the rotary valve 3 rotates to a generation position, the sample channel 6 is communicated with the sample feeding channel 8, the sample discharging channel 9 is communicated with the ventilation channel 7, when the rotary valve 3 rotates to a detection position, the sample discharging channel 9 is communicated with the sample channel 6, the sample feeding channel 8 is communicated with the oil return channel 5, and droplets in the amplification carrier 2 enter the sample channel 6 through the sample discharging channel 9 for backflow detection; wherein be provided with enrichment chamber 14 in the rotary valve 3, when rotary valve 3 rotated to the generation position, the bottom and the top in enrichment chamber 14 communicate with sample channel 6 and sample channel 8 respectively.
In use, when droplet generation is performed, the rotary valve 3 is rotated to rotate the rotary valve 3 to the generation position, the sample channel 6 is communicated with the sample inlet channel 8 under the action of the rotary valve 3, the sample outlet channel 9 is communicated with the air vent channel 7, so that the sample outlet channel 9 is communicated with the atmosphere, smooth entry of generated droplets into the amplification carrier 2 is ensured, when droplets are generated, positive pressure is applied at inlets of the generation oil channel 4 and the sample channel 6, so that generated oil and samples are conveyed in the generation oil channel 4 and the sample channel 6, when the generation oil channel 4 and the sample channel 6 intersect crosswise, a shear flow is formed at the communication position, the samples in the sample channel 6 are divided into droplets with consistent volume, the droplets are introduced into the rotary valve 3 after being generated, when the mixture of the droplets and the oil enters the enrichment chamber 14, when the mixture of the droplets and the mixture of the droplets enters the enrichment chamber 14, the droplets generate an enrichment effect in the enrichment chamber 14, that the droplets enter the rotary valve 14, when the mixture of the droplets and the oil phase 2 enter the enrichment chamber, the enrichment chamber 14, the effect of the mixture of droplets is achieved, and when the volume of the mixture of the droplets is increased by the oil phase is increased, the volume of the droplets is further, thereby, the mixture of the droplets is obtained by the oil phase enrichment chamber 14, and the volume of the droplet enrichment chamber 14 is further increased by the enrichment chamber 14, and the volume of the oil phase enrichment chamber 14, and the oil phase enrichment of the oil phase enrichment chamber 14 is further enriched oil phase enriched carrier is achieved, meanwhile, the rotary valve 3 is fully utilized, and the chip is not required to be additionally arranged, so that the size of the microfluidic chip is reduced, and the integration level of the microfluidic chip is improved.
In addition, after the micro-droplets are amplified, the rotary valve 3 is rotated again, the rotary valve 3 is rotated to the detection position, the sample outlet channel 9 is communicated with the sample channel 6, the sample inlet channel 8 is communicated with the return oil channel 5, and a positive pressure is applied to one end of the return oil channel 5 opposite to the rotary valve 3, the return oil is introduced into the sample inlet channel 8 from the return oil channel 5 through the rotary valve 3 and is introduced into the amplification carrier 2 from the sample inlet channel 8, because the density ratio of the micro-droplets is less than that of the return oil, the micro-droplets are attached to the upper layer of the amplification carrier 2, and after the continuous return oil is introduced into the amplification carrier 2, the micro-droplets are firstly pressed into the sample outlet channel 9 and are returned into the sample channel 6 from the rotary valve 3, and a positive pressure is also applied to one end of the generation oil channel 4 opposite to the rotary valve 3, because the side wall of the sample channel 6 facing one side of the rotary valve 3 is crossed with the generating oil channel 4, the generated oil is also input into the sample channel 6 at the moment, the continuously reflowed microdroplets are separated, an external optical detection module starts to work to align to the sample channel 6, when the microdroplets reflow, the digital nucleic acid detection of the microdroplets can be completed, and the detection is performed in a mode of reflowing into the sample channel 6, so that a detection channel and detection equipment do not need to be additionally designed for detection, sample introduction and detection are unified to the same sample channel 6 for transmission, the production cost is reduced, the resources are saved, the size of the microfluidic control equipment is reduced, the integration level of a microfluidic chip is further improved, the microdroplet generation, amplification and detection steps are completed in the same chip, the functions are integrated in the same device, and the whole volume is greatly reduced.
One end of the generation oil duct 4, the return oil duct 5 and the sample passage 6 back to the rotary valve 3 is set to be 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 port 12 are communicated with the outside, two the generation oil duct 4 shares the generation oil port 10, the chip body 1 is located at the generation oil port 10, the return oil port 11 and the sample port 12 and is provided with a generation oil storage tank 19, a return oil storage tank 20 and a sample storage tank 21.
As an alternative embodiment, the production oil reservoir 19 and the return oil reservoir 20 may not be disposed at the production oil port 10 and the return oil port 11, and only the sample reservoir 21 is disposed at the sample port 12, and the production oil and the return oil are injected with external oil by an external injection device, thereby further reducing the volume.
Specifically, the bottom of rotary valve 3 is provided with four grooves 13, two of them the groove 13 communicates with the top and the bottom of enrichment chamber 14 respectively, enrichment chamber 14 bottom sets up to cylindricly, 14 top lateral wall of enrichment chamber sets up towards the slope of enrichment chamber 14 axis direction, forms coniform, the coniform enrichment chamber 14 top at this moment can play the guide effect to the droplet, reduce the droplet at the accumulational possibility in enrichment chamber 14 top, make the droplet that enters into in the enrichment chamber 14 can both enter into enrichment passageway 22, finally transfer to in the amplification carrier 2, when the droplet generates, two grooves 13 that this moment communicate with enrichment chamber 14 communicate respectively with sample passageway 6 and sampling channel 8, and communicate with the groove 13 and the sample passageway 6 that enrichment chamber 14 bottom communicates, guarantee that the droplet can enter into the bottom of enrichment chamber 14 through sample passageway 6, enrich.
Meanwhile, in order to rotate the rotary valve 3 conveniently, a rotary wrench groove 15 is formed in one side, back to the chip body 1, of the rotary valve 3, the rotary wrench groove 15 is a waist-shaped groove, after the rotary valve 3 is compressed and limited by the fixing piece 16, the rotary valve 3 can rotate at the moment, the rotary valve extends into the rotary wrench groove 15 through an external rotary mechanism, and then the rotary valve 3 is driven to rotate to achieve connection and disconnection or reversing.
As a specific embodiment, the fixing member 16 includes a placing block 161 disposed on the chip body 1, a placing groove 162 is disposed on the placing block 161, the bottom of the placing groove 162 is communicated with the chip body 1, the rotary valve 3 is disposed in the placing block 161 and rotates in the placing block 161, a pressing cover 163 is disposed at a notch of the placing groove 162, the outer wall of the pressing cover 163 and the inner wall of the placing groove 162 are provided with threads, the outer wall of the pressing cover 163 is in threaded connection with the inner wall of the placing groove 162, the pressing cover 163 and the rotary valve 3 are tightly abutted, when the rotary valve 3 is placed on the chip body 1, the rotary valve 3 is placed in the placing groove 162, and the rotary valve 3 can rotate in the placing groove 162, the bottom of the rotary valve 3 is abutted against the upper surface of the chip body 1, the pressing cover 163 is placed at the notch of the placing groove 162, and the pressing cover 163 is continuously rotated, the pressing cover is moved in the direction of the rotary valve 3 until the pressing cover 163 and the upper surface of the chip body 3 are tightly abutted, the rotary valve 3 is pressed, the rotary valve 3 and the chip body is tightly pressed, and fluid leakage of the fluid can be prevented, and the fluid leakage of the chip body can occur.
In addition, the top of the pressing cover 163 is opened with a first communicating hole 164, and the first communicating hole 164 is communicated with the rotating wrench groove 15, so that an external rotating mechanism can enter the rotating wrench groove 15 through the first communicating hole 164, and then the rotating valve 3 is rotated.
As an alternative embodiment, the fixing member 16 includes a first rotor pressing member 165, an outer rim 166 is provided on a side wall of the first rotor pressing member 165, at least two outer rims 166 are provided, a first receiving groove 167 is provided at a bottom of the first rotor pressing member 165, the rotary valve 3 is located in the first receiving groove 167 and abuts against a bottom of the first receiving groove 167, the chip body 1 is located at the rotary valve 3 and is provided with abutting rims 169 matched with the outer rims 166, the first rotor pressing member 165 is located between the abutting rims 169, the first rotor pressing member 165 rotates on the chip body 1 until the outer rims 166 are located below the abutting rims 169 and abut against the abutting rims 169, when the rotary valve 3 is in the installation process, the first rotor pressing member 165 is placed directly above the rotary valve 3, the outer rims 166 and the abutting rims 169 are misaligned, then the first rotor pressing member 165 moves toward the rotary valve 3 until the rotary valve 3 extends into the first receiving groove 167, the first rotor pressing member rotates on the receiving groove 3, the outer rims 166 and the abutting rims 169 are misaligned, so that the first rotor pressing member 165 and the rotary valve 3 can move to press against the rotary valve 3, when the rotary valve 3 rotates, the rotary valve 3 and the rotary valve 3 is pressed by the rotary valve, the rotary valve 3, and the rotary valve can move, thereby preventing the top wall of the rotary valve from the rotary valve 3 and the rotary valve.
In addition, the top of the first rotor pressing member 165 is opened with a second communication hole 1610, the first communication hole 164 is communicated with the first receiving groove 167, and the first communication hole 164 is also communicated with the rotation wrench groove 15, so that an external rotation mechanism can enter the rotation wrench groove 15 through the first communication hole 164 and then rotate the rotation valve 3.
The abutting edge 169 and the abutting edge 166 are arranged in an inclined mode on the abutting side of the outer rim 166, the inclined directions of the abutting edges 169 are the same, the outer rim 166 and the abutting edge 169 are matched and inclined, the rotary valve 3 is installed, the first rotor pressing member 165 is placed right above the rotary valve 3, the outer rim 166 and the abutting edge 169 are staggered, then the first rotor pressing member 165 is moved towards the rotary valve 3 until the rotary valve 3 extends into the first accommodating groove 167, the first rotor pressing member 165 is rotated at the moment, the outer rim 166 on the first rotor pressing member 165 is moved towards the abutting edge 169, the small end of the outer rim 166 is moved forwards until the outer rim 166 is moved to the abutting edge 169, the small end of the outer rim 166 at the moment firstly enters the position right below the abutting edge 169, the first rotor pressing member 165 is continuously moved, the small end of the outer rim 166 is moved towards the large end of the abutting edge 169 until the outer rim 166 and the abutting edge 167 move, the outer rim 167 and the first rotor pressing member 165 is not moved forwards, so that fluid can not flow through the first rotor pressing member 169 and the rotary valve 3 when the outer rim 166 and the first rotor pressing member is not leaked, and the first rotor pressing wall is performed, and the first rotor pressing member 165 is performed.
Meanwhile, the highest position of the side wall of the rotary valve 3 of the abutting edge 169 is provided with a first protrusion 17, the first protrusion 17 is made of elastic plastic materials, when the first rotor pressing piece 165 presses the rotor, the outer edge 166 moves to the abutting edge 169 along with the first rotor pressing piece 165, the small end of the outer edge 166 firstly enters the lower part of the abutting edge 169, the first protrusion 17 is arranged at the small end of the abutting edge 169 at the moment, when the outer edge 166 continues to move towards the large end direction of the abutting edge 169, the outer edge 166 gradually collides with the abutting edge 169 until the abutting edge 169 abuts against the outer edge 166, the outer edge 166 cannot move forwards any more, and the side wall of the outer edge 166 and the side wall of the first protrusion 17 at the moment prevent the first protrusion 17 from colliding with the rotary valve 3, the first rotor pressing piece 165 moves towards the reverse direction, so that the first rotor pressing piece 165 loses the pressing effect, and the first rotor pressing piece 165 moves towards the positive direction and is pressed towards the positive direction.
The amplification carrier 2 is further provided with an amplification module 18, the amplification module 18 is used for applying heat to the amplification carrier 2 to enable the collected microdrops in the amplification carrier 2 to be subjected to a plurality of thermal cycles to complete nucleic acid amplification, the amplification module 18 comprises a heating base 181 arranged at the bottom of the amplification carrier 2, a groove is formed in the heating base 181, the heating base 181 is suitable for the amplification carrier 2 to be located in the groove and to be in contact with the inner wall of the groove, a peltier 182 and a cooling fin 183 are sequentially arranged below the heating base 181, and amplification temperature rise and fall cycles are achieved through the peltier 182 to complete amplification.
In addition, the amplification module 18 further comprises a pipe cover protrusion 184 arranged at the bottom of the chip body 1, the sample introduction channel 8 and the sample discharge channel 9 are arranged in the pipe cover protrusion 184, the amplification carrier 2 and the pipe cover protrusion 184 are clamped, the chip body 1 is back to the amplification carrier 2 and is provided with a second heating element 185, the second heating element 185 is attached to the chip body 1, the chip body 1 and the pipe cover protrusion 184 are heated by starting the second heating element 185, and therefore the micro-droplets are prevented from evaporating when the amplification carrier 2 is heated and cooled, and amplification is influenced.
As an alternative embodiment, since the second heating sheet 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 1mm, and the chip body 1 and the tube cover protrusion 184 are both made of pc material, the temperature rising speed of the chip body 1 and the tube cover protrusion 184 is slow, at this time, in order to increase the temperature rising speed, the amplification module 18 further includes an annular heat cover 186, the sample inlet channel 8 and the sample outlet channel 9 are both disposed in the annular heat cover 186, the amplification carrier 2 is sleeved at the bottom of the annular heat cover 186 and sealed with the annular heat cover 186, the annular heat cover 186 is provided with a first heating element 187, the first heating element 187 and the annular heat cover 186 are attached, when droplets enter the amplification carrier 2 for amplification, the amplification module 18 starts to start up at this time, so as to perform a temperature rising cycle on the amplification carrier 2, so that the droplets in the amplification carrier 2 undergo several thermal cycles to complete nucleic acid amplification, and at the same time, the first heating element 187 is started, so as to drive the annular heat cover 186 to heat the droplets to heat the heating element 2 to prevent evaporation of the droplets. The annular heat cover 186 is made of a metal material, and the annular heat cover 186 is made of metal, thereby increasing the temperature rising speed, improving the evaporation prevention effect, and increasing the amplification efficiency.
Example 2
Referring to fig. 15, a detection method using the rotary valve 3-based microfluidic chip described in the examples includes the following steps:
s1: the droplet is generated, at this time, the rotary valve 3 is rotated to a generating position, at this time, the sample channel 6 is communicated with the sample injection channel 8, the sample outlet channel 9 is communicated with the air vent channel 7, and the enrichment cavity 14 is communicated in the groove 13 at the communication position of the sample channel 6 and the sample injection channel 8, a generated oil is introduced into the generating oil channel 4 and applies positive pressure, the generated oil enters the sample channel 6 through the cross flow channel, a shear flow is formed at the cross flow channel, the sample is divided into droplets and is introduced into the rotary valve 3, at this time, when the mixture of the droplets and the oil enters the enrichment cavity 14, the droplets can generate an enrichment effect in the enrichment cavity 14, namely, the droplets entering the enrichment cavity 14 firstly float above the enrichment cavity 14, after the enrichment cavity 14 is filled with the mixture of the droplets and the oil, the droplets can flow out of the rotary valve 3 at this time and enter the sample injection channel 8, and the redundant oil phase can remain in the enrichment cavity 14, thereby obtaining a higher volume fraction in the amplification carrier 2, when the mixture of the droplets and the rotary valve 3 and the oil phase are filled with the oil, the droplet is fully collected, and the droplet collecting effect is achieved, and the chip is also achieved, and the chip is not only the chip is increased.
S2: and (3) carrying out microdroplet amplification, wherein after the microdroplets are sent into the amplification carrier 2, the rotary valve 3 is rotated at the moment to separate the sample channel 6 from the sample feeding channel 8, the sample discharging channel 9 from the ventilation channel 7, the amplification carrier 2 is kept in a sealed state, the Peltier 182 is started, the amplification carrier 2 in the heating base 181 and the heating base 181 is subjected to heating and cooling circulation for a plurality of times under the action of the Peltier 182, and microdroplets in the cavity of the amplification carrier 2 are heated, so that microdroplet amplification is completed.
S3: droplet backflow detection: after the micro-droplets are amplified, rotating the rotary valve 3 again, rotating the rotary valve 3 to a detection position, communicating the sample outlet channel 9 with the sample channel 6, communicating the sample inlet channel 8 with the return oil channel 5, applying positive pressure to one end of the return oil channel 5 opposite to the rotary valve 3, introducing the return oil from the return oil channel 5 to the sample inlet channel 8 through the rotary valve 3, and introducing the return oil into the amplification carrier 2 from the sample inlet channel 8, wherein the micro-droplets are attached to the upper layer of the amplification carrier 2 because the density of the micro-droplets is less than that of the return oil, and after the continuous return oil is introduced into the amplification carrier 2, the micro-droplets are firstly pressed into the sample outlet channel 9 and returned into the sample channel 6 from the rotary valve 3, and simultaneously applying positive pressure to one end of the generation oil channel 4 opposite to the rotary valve 3, because the side wall of the sample channel 6 facing one side of the rotary valve 3 is communicated and tangent with the generating oil channel 4, the generated oil is also input into the sample channel 6 at the moment, the continuously reflowed microdroplets are separated, an external optical detection module starts to work at the same time and is aligned with the sample channel 6, when the microdroplets reflow, the digital nucleic acid detection of the microdroplets can be completed, and the detection is performed by a mode of reflowing into the sample channel 6, so that the detection channel and the detection equipment do not need to be additionally designed for detection, the sample introduction and the detection are unified to the same sample channel 6 for transmission, the production cost is reduced, the resources are saved, the size of the microfluidic equipment is reduced, and the integration level of the microfluidic chip is further improved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (12)
1. A micro-fluidic chip based on a rotary valve comprises a chip body (1), and is characterized in that a generation oil channel (4), a return oil channel (5), a sample channel (6) and a ventilation channel (7) are arranged in the chip body (1), wherein one end of the generation oil channel (4) is communicated with the side wall of the sample channel (6), generation oil in the generation oil channel (4) enters the sample channel (6) to shear a sample to form micro-droplets, and one end of the ventilation channel (7) is communicated with the outside;
further comprising:
the amplification carrier (2) is sealed in the chip body (1), a sample inlet channel (8) and a sample outlet channel (9) are further arranged at the position, located in the amplification carrier (2), of the chip body (1), and the sample inlet channel (8) and the sample outlet channel (9) are both communicated with the amplification carrier (2);
the rotary valve (3) is rotatably arranged on the chip body (1) along the axis of the rotary valve, when the rotary valve (3) rotates to a generation position, the sample channel (6) is communicated with the sample injection channel (8), the sample outlet channel (9) is communicated with the ventilation channel (7), when the rotary valve (3) rotates to a detection position, the sample outlet channel (9) is communicated with the sample channel (6), the sample injection channel (8) is communicated with the backflow oil channel (5), and droplets in the amplification carrier (2) enter the sample channel (6) through the sample outlet channel (9) for backflow detection;
wherein be provided with enrichment chamber (14) in rotary valve (3), when rotary valve (3) rotate to the position of generating, the bottom and the top in enrichment chamber (14) communicate with sample channel (6) and sampling channel (8) respectively.
2. The rotary valve-based microfluidic chip according to claim 1, wherein the bottom of the rotary valve (3) is provided with at least four grooves (13), two of the grooves (13) are respectively communicated with the top and the bottom of the enrichment chamber (14), the bottom of the enrichment chamber (14) is cylindrical, and the top side wall of the enrichment chamber (14) is inclined towards the axis of the enrichment chamber (14) to form a cone shape.
3. Microfluidic chip based on rotary valve according to claim 1, characterized in that the chip body (1) is provided with a fixture (16) at the rotary valve (3) adapted to perform a positive sealing of the rotary valve (3), to compress the rotary valve (3) and to seal the contact surface on the chip body (1).
4. A rotary valve based microfluidic chip according to claim 3, wherein the fixing member (16) comprises a placing block (161) disposed on the chip body (1), a placing groove (162) is opened on the placing block (161), the bottom of the placing groove (162) is communicated with the chip body (1), the rotary valve (3) is located in the placing block (161) and rotates in the placing block (161), a pressing cover (163) is disposed at the notch of the placing groove (162), the outer wall of the pressing cover (163) and the inner wall of the placing groove (162) are provided with screw threads, the outer wall of the pressing cover (163) and the inner wall of the placing groove (162) are in threaded connection, and the pressing cover (163) and the rotary valve (3) are tightly abutted.
5. A rotary valve based microfluidic chip according to claim 3, wherein the fixing member (16) comprises a first rotor pressing member (165), the first rotor pressing member (165) is provided with at least two outer rims (166) on the side walls, the first rotor pressing member (165) is provided with a first receiving groove (167) at the bottom, the rotary valve (3) is located in the first receiving groove (167) and abuts against the bottom of the first receiving groove (167), the chip body (1) is provided with an abutting edge (169) matching with the outer rim (166) at the position of the rotary valve (3), the first rotor pressing member (165) is located between the abutting edges (169), and the first rotor pressing member (165) rotates on the chip body (1) until the outer rim (166) is located under the abutting edge (169) and abuts against the abutting edge (169).
6. The rotary valve based microfluidic chip according to claim 5, wherein the abutting edge (169) and the outer abutting edge (166) abut against one side in an inclined manner, the inclined direction of the abutting edges (169) is consistent, and the outer abutting edge (166) and the abutting edge (169) are adapted to be inclined.
7. The rotary valve based microfluidic chip according to claim 1, wherein an amplification module (18) is further disposed at the amplification carrier (2), and the amplification module (18) is configured to apply heat to the amplification carrier (2) so that the collected droplets in the amplification carrier (2) undergo several thermal cycles to complete nucleic acid amplification.
8. A rotary valve based microfluidic chip according to claim 7, wherein the amplification module (18) comprises an annular thermal cover (186), the sample inlet channel (8) and the sample outlet channel (9) are both disposed in the annular thermal cover (186), the amplification carrier (2) is sleeved on the bottom of the annular thermal cover (186) and sealed with the annular thermal cover (186), the annular thermal cover (186) is provided with a first heating element (187), and the second heating element (185) is attached to the annular thermal cover (186).
9. The rotary valve-based microfluidic chip of claim 8, the annular thermal cover (186) being comprised of a metallic material.
10. The rotary valve based microfluidic chip according to claim 7, wherein the amplification module (18) comprises a tube cover protrusion (184) disposed at the bottom of the chip body (1), the sample inlet channel (8) and the sample outlet channel (9) are both disposed in the tube cover protrusion (184), the amplification carrier (2) and the tube cover protrusion (184) are clamped, a second heating element (185) is disposed at a position of the chip body (1) opposite to the amplification carrier (2), and the second heating element (185) is attached to the chip body (1).
11. The rotary valve-based microfluidic chip according to any one of claims 1 to 10, wherein one end of the generation oil passage (4), the return oil passage (5), and the 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 port (12) are communicated with the outside, and a generation oil reservoir (19), a return oil reservoir (20), and a sample reservoir (21) are disposed at the generation oil port (10), the return oil port (11), and the sample port (12) of the chip body (1).
12. A method of detection using a rotary valve based microfluidic chip according to any one of claims 1 to 11, comprising the steps of:
droplet generation: rotating the rotary valve (3), rotating the rotary valve (3) to a generation position, communicating the sample channel (6) with the sample introduction channel (8), communicating the sample outlet channel (9) with the ventilation channel (7), introducing generated oil into the sample channel (6) and the generation oil channel (4) and applying positive pressure, wherein the generated oil enters the sample channel (6) to form shear flow, dividing the sample into micro-droplets, and introducing the micro-droplets into the sample introduction channel (8) under the action of the rotary valve (3) and finally entering the amplification carrier (2);
and (3) microdroplet amplification: the amplification carrier (2) starts to carry out thermal cycle for a plurality of times, and microdroplets in the cavity of the amplification carrier (2) are heated to complete microdroplet amplification;
droplet backflow detection: rotating the rotary valve (3) again, moving the rotary valve (3) to the detection position, communicating the sample outlet channel (9) with the sample channel (6), communicating the sample inlet channel (8) with the return oil channel (5), applying positive pressure to one side of the return channel, leading return oil into the sample inlet channel (8) from the return oil channel and pressing the return oil into the amplification carrier (2), firstly pressing microdrops in the amplification carrier (2) into the sample outlet channel (9) and refluxing into the sample channel (6), simultaneously applying positive pressure to the generated oil to the inside, leading the generated oil into the sample channel (6) from the communication position of the generated oil channel (4) and the sample channel (6), spacing the microdrops, aligning an external optical detection module with the sample channel (6), and carrying out nucleic acid detection on the microdrops when the return oil passes through.
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