WO2023088148A1 - 一体式微液滴芯片 - Google Patents

一体式微液滴芯片 Download PDF

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Publication number
WO2023088148A1
WO2023088148A1 PCT/CN2022/130910 CN2022130910W WO2023088148A1 WO 2023088148 A1 WO2023088148 A1 WO 2023088148A1 CN 2022130910 W CN2022130910 W CN 2022130910W WO 2023088148 A1 WO2023088148 A1 WO 2023088148A1
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Prior art keywords
droplet
micro
oil
reaction chamber
pipeline
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PCT/CN2022/130910
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English (en)
French (fr)
Inventor
苏世圣
王博
夏雷
刘金伟
杨文军
王勇斗
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Targetingone Technology Beijing Corp
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Targetingone Technology Beijing Corp
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Priority to US18/708,388 priority Critical patent/US20250018394A1/en
Priority to JP2024526652A priority patent/JP7756461B2/ja
Priority to EP22894685.1A priority patent/EP4424813A4/en
Publication of WO2023088148A1 publication Critical patent/WO2023088148A1/zh
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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
    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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
    • B01L3/502769—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 multiphase flow arrangements
    • B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/14—Means for pressure control
    • 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
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0469—Buoyancy
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • the invention belongs to the technical field of digital PCR analyzers, and in particular relates to an integrated micro-droplet chip.
  • Droplet-based microfluidics is a technology platform developed on microfluidic chips in recent years to manipulate small-volume liquids. , one of which is the oil phase and the other is the water phase. After the oil phase and the water phase enter the microchannel at the same time, under the action of the microchannel, the water phase is distributed in the oil phase in the form of tiny volume units, forming a A series of discrete micro-droplets. Each droplet acts as a microreactor to complete a set of chemical or biological reactions.
  • Digital PCR technology is known as the third-generation PCR technology, which has the advantages of absolute quantification and single-molecule detection sensitivity, and has important application prospects in the field of molecular diagnosis.
  • One of the mainstream technical routes of digital PCR technology is to use droplet microfluidic chips to divide the reaction system into tens of thousands or even millions of droplets of uniform size, and complete the generation, amplification and fluorescence detection.
  • a mathematical model calculates the exact copy number of the target molecule in the sample.
  • the structure in the droplet generation chip is often used to complete the droplet generation, and then the droplet is transferred to the reaction tube for amplification, and finally the structure in the droplet detection chip is used to form the droplet.
  • This method of using droplet microfluidics to realize digital PCR has the advantages of uniform droplet size, unrestricted number of droplets, and high signal-to-noise ratio of fluorescence detection. Complete, low integration, difficult to automate and other deficiencies.
  • the technical problem to be solved by the present invention is to provide an integrated micro-droplet chip to overcome the shortcomings of the prior art that droplet generation and detection are completed in different droplet chips, and the degree of integration and automation is low.
  • the present invention provides an integrated micro-droplet chip, including a chip body, the chip body has a reaction chamber and a sample loading chamber, and the chip body is constructed with a micro-droplet generating structure, an oil interface, A gas-liquid interface and a fluorescence detection area, the gas-liquid interface communicates with the reaction chamber, the sample loading chamber communicates with the micro-droplet generating structure, and the oil-liquid interface communicates with the micro-droplet generating structure;
  • a first pressure difference is formed between the sample injection chamber and the gas-liquid interface
  • a second pressure difference is formed between the oil-liquid interface and the gas-liquid interface
  • the first pressure difference and The second pressure difference respectively drives the sample in the sample loading chamber and the generated oil of the oil interface to enter the micro-droplet generating structure, and the generated micro-droplets enter and store in the reaction chamber;
  • the external pressure drives the detection to push the oil from the gas-liquid interface into the reaction chamber, so that the micro-droplets in the reaction chamber flow out from the reaction chamber to the micro-droplet generating structure, and the external Pressure-driven detection separation oil enters the micro-droplet generation structure from the oil-liquid interface, and the detection separation oil separates the micro-droplets flowing out of the reaction chamber into the micro-droplet generation structure to form a queue, into the fluorescent detection zone.
  • the micro-droplet generating structure includes an oil pipeline and a communication pipeline
  • the oil pipeline crosses the communication pipeline
  • the communication pipeline includes a first side at the intersection point and connects with the reaction
  • the first pipeline communicated with the chamber and the second pipeline located on the second side of the cross point and communicated with the sample injection chamber, and the oil interface communicated with the oil pipeline.
  • the reaction chamber and the sample loading chamber are on the first side, and the reaction chamber and the chip body are on the first side.
  • the connection interface on the first side extends upwards and forms a bell mouth with a small bottom and a big top.
  • a gas-liquid pipeline extending from bottom to top is also configured in the reaction chamber, the lower opening of the gas-liquid pipeline communicates with the gas-liquid interface, and the upper opening of the gas-liquid pipeline is higher than the connection.
  • the interface is catchy.
  • the sample loading chamber includes an opening and a sealing cover sealingly connected to the opening of the opening.
  • the sample loading chamber is provided with a filter membrane or an exhaust hole.
  • the fluorescence detection zone is on the second pipe.
  • the sample loading chamber is located on the first side
  • the reaction chamber is located on the second side
  • the second side and the first side are opposite sides of the chip body.
  • light oil is preset in the reaction chamber before the micro-droplets enter the reaction chamber.
  • the chip body is integrated with a sample loading chamber, a reaction chamber, a micro-droplet generating structure and a fluorescence detection area, through time-division multiplexing of the micro-droplet generating structure , integrating droplet generation, amplification and detection in one chip, realizing a fully integrated and fully closed digital PCR process.
  • FIG. 1 is a schematic diagram of a three-dimensional structure of an integrated micro-droplet chip according to an embodiment of the present invention
  • FIG. 2 is a schematic structural view of the micro-droplet generation structure in the integrated micro-droplet chip of the embodiment of the present invention
  • FIG. 3 is a schematic diagram of the internal structure of the reaction chamber in the integrated droplet chip of the embodiment of the present invention.
  • Figure 4 is a schematic diagram of the micro-droplet generation process
  • Figure 5 is a schematic diagram of micro-droplets stored in the reaction chamber after they are generated
  • Fig. 6 is a schematic diagram of the state in the reaction chamber after the integrated micro-droplet chip is turned over 180°;
  • Fig. 7 is a schematic diagram of the state after oil is introduced into the reaction chamber in the state of Fig. 6;
  • Fig. 8 is a schematic diagram of the state after the micro-droplet is forced out from the reaction chamber (the arrows in the figure show the flow direction of the micro-droplet and the oil);
  • FIG. 9 is a schematic perspective view of the three-dimensional structure of an integrated micro-droplet chip according to another embodiment of the present invention.
  • Chip body 11. Reaction chamber; 111. Connection interface; 112. Gas-liquid pipeline; 12. Sample injection chamber; 121. Opening mouth; 122. Sealing cover; 21. Oil pipeline; 22. First pipeline; 23 , the second pipeline; 31, the oil interface; 32, the gas-liquid interface; 33, the fluorescence detection area; 34, the micro-droplet observation area; 4, the micro-droplet;
  • an integrated micro-droplet chip including a chip body 1, and the chip body 1 has a reaction chamber 11 and a sample loading chamber 12, and the chip
  • the body 1 is equipped with a droplet generating structure, an oil-liquid interface 31, a gas-liquid interface 32, and a fluorescence detection area 33.
  • the gas-liquid interface 32 communicates with the reaction chamber 11, and the sample loading chamber 12 communicates with the micro
  • the droplet generating structure is in communication, and the oil interface 31 is in communication with the micro-droplet generating structure;
  • a first pressure difference is formed between the sample adding chamber 12 and the gas-liquid interface 32
  • a second pressure difference is formed between the oil-liquid interface 31 and the gas-liquid interface 32
  • the first The first pressure difference and the second pressure difference respectively drive the sample in the sample loading chamber 12 and the generated oil of the oil interface 31 to enter the micro-droplet generating structure, and the generated micro-droplet 4 enters and is stored in the In the reaction chamber 11;
  • the external pressure drives the detection pusher oil 5 to enter the reaction chamber 11 from the gas-liquid interface 32, so that the micro-droplets 4 in the reaction chamber 11 flow out from the reaction chamber 11 to the micro-droplet.
  • the external pressure drives the detection separation oil to enter the micro droplet generation structure from the oil interface 31, and the detection separation oil flows out from the reaction chamber 11 into the micro droplet generation structure The micro-droplets 4 are separated to form a queue and enter the fluorescence detection area 33 .
  • the chip body 1 is integrated with a sample loading chamber 12, a reaction chamber 11, a micro-droplet generating structure, and a fluorescence detection area 33, so that the generation and detection of the micro-droplets are integrated on the same chip.
  • the degree of integration and automation can be improved; more importantly, through the time-division multiplexing of the micro-droplet generation structure (with the flipping of the integrated micro-droplet chip as the time-sharing boundary), the Droplet generation, amplification and detection are all integrated in one chip, realizing a fully integrated and fully closed digital PCR process, which not only inherits the advantages of uniform droplet size, unrestricted droplet quantity, and high signal-to-noise ratio of fluorescence detection, but also It also overcomes the complexity of the original chip structure, the generation and detection are completed in different chips, the integration is low, and it is difficult to automate. It is an important technological breakthrough in the field of digital PCR.
  • the droplet generating structure includes an oil pipeline 21 and a communication pipeline, the oil pipeline 21 crosses the communication pipeline, and the communication pipeline 21 crosses the communication pipeline.
  • the pipeline includes a first pipeline 22 located on the first side of the intersection and communicating with the reaction chamber 11 and a second pipeline 23 located on the second side of the intersection and communicating with the sample chamber 12, so The oil interface 31 communicates with the oil pipeline 21 .
  • connection interface 111 between the reaction chamber 11 and the first side of the chip body 1 extends upwards, and forms a bell mouth with a small bottom and a big top.
  • the entry of the droplets 4 into the reaction chamber 11 from the first pipeline 22 also facilitates the entry of the microdroplets 4 from the reaction chamber 11 into the first pipeline 22 to prevent the retention of the microdroplets 4 .
  • the reaction chamber 11 and the sample loading chamber 12 are both located on the first side (specifically, the top surface) of the chip body 1, and the micro-droplets 4 entering the reaction chamber 11 are all gathered.
  • the connection interface 111 when PCR amplification is performed, the chip body 1 needs to be turned upside down as a whole, that is, flipped 180°, so that the micro-droplet 4 can be placed in the reaction zone of the reaction chamber 11 .
  • a gas-liquid pipeline 112 extending from bottom to top is also configured in the reaction chamber 11 , the lower opening of the gas-liquid pipeline 112 communicates with the gas-liquid interface 32 , and the gas-liquid pipeline 112
  • the upper opening of the connection interface 111 is higher than the upper opening of the connection interface 111, which can prevent the micro-droplets 4 generated by the micro-droplet generating structure from entering the reaction chamber 11 when the reaction chamber 11 is under negative pressure. Further flow out from the gas-liquid pipeline 112.
  • the flow area of the droplet observation area 34 is much larger than that of the first pipeline 22.
  • Area, that is, the micro-droplet observation area 34 is an enlarged area (width becomes larger) on the first pipeline 22, so that the flow velocity of the micro-droplet 4 entering this area is reduced, which can facilitate external camera imaging , record the shape of the droplet, and judge whether the state of the droplet generation process is normal.
  • the sample adding chamber 12 includes an opening 121 and a sealing cover 122 sealingly connected to the opening of the opening 121 , so that operators can add samples into the sample adding chamber 12 .
  • the sample loading chamber 12 is provided with a filter membrane or an air vent, and when the sample loading chamber 12 becomes a waste liquid pool (that is, when the droplet chip is turned upside down), a certain amount of air is removed to prevent the Pressure builds up in the sample loading chamber 12 .
  • the fluorescence detection area 33 is located on the second pipeline 23, and on the second pipeline 23, the micro-droplets 4 flowing out of the reaction chamber 11 can pass through the cross intersection point. Under the action of the detection oil in the oil pipeline 21 , it is separated into a queue of liquid droplets with appropriate spacing, so that the fluorescence detection can be completed under the action of the external system.
  • FIG. 9 another implementation of the integrated micro-droplet chip is provided.
  • the difference between it and the integrated micro-droplet chip shown in FIG. The sample chambers 12 are respectively located on two opposite sides of the chip body 1, specifically, the sample application chamber 12 is located on the first side, the reaction chamber 11 is located on the second side, and the second The side and the first side are the opposite sides of the chip body 1.
  • the working principle and process of the integrated micro-droplet chip are basically the same as the above-mentioned integrated micro-droplet chip.
  • the reaction chamber 11 since the reaction chamber 11 is located on the bottom side of the chip body 1 (the sample loading chamber 12 is on the top side), the micro-droplet 4 will directly drop to the reaction chamber 11 when it enters the reaction chamber.
  • the reaction area at the bottom of chamber 11 is collected in the reaction area. Therefore, the integrated micro-droplet chip does not need to be turned 180° after the droplet generation is completed, and can directly enter the subsequent amplification link.
  • the reaction chamber 11 before the micro-droplets 4 enter the reaction chamber 11, the reaction chamber 11 is preset with light oil (that is, oil with a lower density), so as to ensure that the light oil can always
  • the top of the micro-droplet 4 in the reaction chamber 11 solves the problem of micro-droplet evaporation during amplification and realizes heat-free lid PCR.
  • a digital PCR method for an integrated micro-droplet chip includes a chip body 1 as described above, and the chip body 1 has a reaction chamber 11 and a Cavity 12, the micro-droplet generating structure, oil-liquid interface 31, gas-liquid interface 32 and fluorescence detection area 33 are constructed in the chip body 1, the gas-liquid interface 32 communicates with the reaction chamber 11, and the sample loading
  • the cavity 12 communicates with the micro-droplet generating structure, the oil interface 31 communicates with the micro-droplet generating structure,
  • Described digital PCR method comprises the steps:
  • the micro-droplet segmentation and generation step is to control the formation of a first pressure difference between the sample loading chamber 12 and the gas-liquid interface 32, and the formation of a second pressure difference between the oil-liquid interface 31 and the gas-liquid interface 32, so that the first pressure difference and the second pressure difference respectively drive the sample in the sample loading chamber 12 and the generated oil of the oil interface 31 to enter the micro-droplet generating structure, and the generated micro-droplets 4 enters and stores in the reaction chamber 11, specifically, oil is provided to the oil interface 31, and negative pressure is provided to the gas-liquid interface 32, and under the action of the negative pressure, the sample addition
  • the sample in the chamber 12 and the oil at the oil interface 31 are driven to converge at the intersection of the droplet generation structure along the second pipeline 23 and the oil pipeline 21 respectively.
  • micro-droplets 4 water-in-oil droplets
  • the first pipeline 22 finally enters the reaction chamber 11 for storage. It should be noted that when the micro-droplets enter the micro-droplet observation area 34, the flow rate of the micro-droplets 4 forms a dense droplet community, which is convenient. Camera imaging record;
  • the reaction chamber 11 is placed in a heating module (not shown in the figure) to heat and amplify according to a preset cycle, and the heating module can be an existing heating module;
  • micro-droplet detection step control the external pressure to drive the detection push oil 5 from the gas-liquid interface 32 into the reaction chamber 11, so that the micro-droplets 4 in the reaction chamber 11 flow out from the reaction chamber 11 to the reaction chamber 11.
  • the external pressure drives the detection separation oil to enter the droplet generation structure from the oil interface 31, and the detection separation oil flows out from the reaction chamber 11 to the droplet generation structure.
  • the micro-droplets 4 in the structure are separated to form a queue, enter the fluorescence detection area 33, and complete the fluorescence detection.
  • the micro-droplet 4 after the amplification reaction in the reaction chamber 11 is floated by the buoyancy of the oil, and under the buoyancy of the oil, the micro-droplet 4 can pass through the connection interface 111 flows out of the reaction chamber 11 and enters the first pipeline 22, and flows through the droplet observation area 34 into the intersection, and enters the second pipeline 23, and is carried out in the fluorescence detection area 33. detection, and then finally enters the sample loading chamber 12, at this time, the sample loading chamber 12 is a waste liquid pool.
  • the droplet generation, amplification and detection are all integrated into one Among the chips, the fully integrated and fully closed digital PCR process not only inherits the advantages of uniform droplet size, unrestricted droplet quantity, and high signal-to-noise ratio of fluorescence detection, but also overcomes the complexity of the original chip structure, generation and The detection is completed in different chips, the integration level is low, and it is difficult to automate, which is an important technological breakthrough in the field of digital PCR.
  • the reaction chamber 11 and the sample loading chamber 12 are on the first side, before the amplification reaction step
  • the micro-droplet segmentation and generation step also includes: a chip flipping step, controlling the chip body 1 to turn up and down 180°, at this time, the micro-droplet 4 in the reaction chamber 11 is close to the connection interface 111 Turn over to the side away from the connection interface 111, at this time the position of the reaction chamber 11 corresponding to the micro-droplet 4 is the reaction area of the reaction chamber 11, and the reaction area is in contact with the heating module for temperature adjustment A temperature-adjusted reaction of the sample is achieved.
  • the 30 microliters of the system (that is, the aforementioned sample) is added to the sample chamber 12, and the 30 microliters of the PCR system includes 10 microliters of ddPCR Supermix for Probes from Bio-Rad, GJB2 gene upstream and downstream primer reagent 5 ⁇ l and 5 ⁇ l of template containing 1 ng of genomic DNA.
  • the chip as a whole is shown in Figure 1.
  • One chip contains 8 parallel and independent droplet chip structures, and each structure includes a sampling chamber 12, an oil-liquid interface 31, a gas-liquid interface 32, a micro-droplet generating structure, Reaction chamber 11.
  • the sealing cover 122 is tightly closed or bonded to seal the sample loading chamber 12, preferably there is a vent hole with a filter membrane or a small diameter in the sample loading chamber 12.
  • the sample loading chamber 12 is connected with the second pipeline 23 in the micro-droplet generating structure
  • the oil-liquid interface 31 is connected with the oil-liquid pipeline 21 in the micro-droplet generating structure
  • the first pipeline 22 and the gas-liquid interface 32 are connected with the reaction chamber. 11, wherein the first pipeline 22 is connected to the connection interface 111, and the gas-liquid interface 32 is connected to the gas-liquid pipeline 112.
  • the oil pipeline 21 has two branches, which are respectively located on both sides of the second pipeline 23 and the first pipeline 22 , and both are connected to the oil interface 31 .
  • the first pipeline 22 may include a micro-droplet observation area 34. The pipe in the observation area becomes wider, and the flow velocity decreases after the droplet enters, which is convenient for an external camera to image, record the shape of the droplet, and judge whether the state of the droplet generation process is normal.
  • the reaction system Driven by the pressure difference, the reaction system enters the second pipeline 23, and the generated oil enters the oil pipeline 21 and meets at the cross structure (that is, the aforementioned cross intersection).
  • water-in-oil micro-droplets 4 with uniform size are formed.
  • the depth of the channel at the cross is about 70 microns, the width is 80 microns, and the droplet size is about 100 microns.
  • the micro-droplet 4 enters the first pipeline 22, and after entering the micro-droplet observation area 34, the flow rate is reduced to form a dense droplet community, which is convenient for camera imaging and recording.
  • the schematic diagram of the droplet generation process is shown in Figure 4.
  • the generated droplets flow through the first pipeline 22 and reach the connection interface 111 of the reaction chamber 11 .
  • the bottom of the connection interface 111 has a slope structure (that is, the aforementioned bell mouth), and the bottom of the slope communicates with the first pipeline 22 .
  • the pressure difference applied to the interface of the chip is removed, and the droplet should still be located under the gas-liquid pipeline 112 at this time.
  • the chip is turned upside down, so that the droplets are transferred from the connection interface 111 to the reaction area (that is, away from the connection interface 111 ), as shown in FIG. 5 .
  • the structure of the reaction zone should adopt a design with high heat transfer efficiency, such as a flat design with high depth and thin thickness, so that the external system (that is, the heating module) can heat and cool the reaction zone from the left and right sides, not only ensuring the temperature conduction The short distance also ensures a large contact area for efficient heat transfer.
  • the temperature cycle flow is a pre-denaturation at 95°C for 10 minutes, followed by 40 temperature cycles, each cycle is 95°C for 5 seconds, 60°C for 15 seconds, and finally kept at 4°C.
  • 30 microliters of low-density anti-volatile reagent can be placed in the reaction chamber 11 in advance.
  • the amplification reaction in the droplet containing the template is correspondingly completed, and it needs to enter the droplet fluorescence detection link.
  • Inject detection oil that is, the above-mentioned detection driving oil 5
  • the liquid level of the liquid droplets continuously rises, and then is guided through the slope of the connection interface 111 and enters the first pipe 22 . This process is shown in Figure 7.
  • the camera can also be used to perform bright-field imaging on the droplet, so as to evaluate the state of the droplet after the amplification reaction.
  • the detection oil is injected into the oil interface 31 , and the detection oil passes through the oil pipeline 21 and merges with the droplet queue at the cross pipe, and separates the closely arranged droplets into a droplet queue with a suitable spacing.
  • the droplet queue passes through the fluorescence detection area 33 located in the second pipeline 23 in sequence, as shown in FIG. 8 .
  • the position corresponding to the fluorescence detection area 33 is the focus of fluorescence detection of the external system.
  • An external system focuses excitation light, such as a laser or LED narrow-band light with wavelengths of 488nm and 532nm, into the detection focus.
  • excitation light such as a laser or LED narrow-band light with wavelengths of 488nm and 532nm
  • the fluorescence excited in the droplets will also be received by the collection optical path of the external system, so as to obtain the fluorescence information of each droplet.
  • delineate the signal threshold distinguish the negative and positive of the droplets, and use the Poisson distribution model to calculate the copy number of the target molecule in the sample.
  • the liquid droplets that have completed the fluorescence detection enter the sample loading chamber 12. Since the sample loading chamber 12 has been sealed by the sealing cover 122, it will not be in contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully enclosed digital chip. PCR process.
  • a method of time-division multiplexing the micro-droplet generation structure is pioneered.
  • the micro-droplet generating structure is used to realize the droplet generation.
  • the micro-droplet generation structure completes the separation of the droplet queue, ensuring the droplet fluorescence signal detection.
  • the camera when the droplet passes through the observation area in the first pipeline 22, the camera is used to perform fluorescence imaging on the droplet, and the imaged photo is analyzed to obtain the fluorescence intensity information of each droplet in the photo, and complete Droplet fluorescence signal detection.

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Abstract

本发明提供一种一体式微液滴芯片，包括芯片本体，芯片本体上具有反应腔、加样腔、微液滴生成结构、油液接口、气液接口和荧光检测区；液滴生成时，加样腔与气液接口之间及油液接口与气液接口之间形成压力差，压力差分别驱动加样腔中的样本和油液接口的生成油进入微液滴生成结构生成微液滴进入反应腔；液滴检测时，外部压力驱动检测推动油进入反应腔，使得微液滴从反应腔中流出至微液滴生成结构，外部压力驱动检测分隔油进入微液滴生成结构，检测分隔油将从反应腔中流出至微液滴生成结构中的微液滴分隔形成队列，进入荧光检测区。本发明通过对微液滴生成结构的分时复用，将液滴生成、扩增和检测都集成在一张芯片，实现全集成、全封闭的数字PCR流程。

Description

一体式微液滴芯片 技术领域
本发明属于数字PCR分析仪技术领域，具体涉及一种一体式微液滴芯片。
背景技术
液滴微流控(droplet-based microfluidics)是近年来在微流控芯片上发展起来的一种操控微小体积液体的技术平台，其原理为：将两种互不相溶的液体，示例性的，其中的一种为油相，另一种为水相，油相和水相同时进入微通道后，在微通道的作用下，水相以微小体积单元的形式分布于油相中，形成一系列离散的微液滴。每个液滴作为一个微反应器，完成一组化学或生物反应。
数字PCR技术被誉为第三代PCR技术，具有绝对定量和单分子检测灵敏度的优点，在分子诊断领域有着重要的应用前景。数字PCR技术一种主流的技术路线的采用液滴微流控芯片，将反应体系分割成数万乃至数百万尺寸均一的液滴，并完成生成、扩增和荧光检测，通过荧光检测结果利用数学模型计算出样本中目标分子的精确拷贝数。
在液滴数字PCR技术中，常常利用液滴生成芯片中的结构，完成液滴生成，然后将液滴转移至反应管中扩增，最后再利用液滴检测芯片中的结构，将液滴形成有一定间隔的液滴队列，每个液滴依次通过荧光检测区域，激发并检测液滴内的荧光信号。这种利用液滴微流控实现数字PCR的方法，具有液滴尺寸均一、液滴数量不易受限制、荧光检测信噪比高等优点，但同时也存在芯片结构复杂，生成和检测在不同芯片中完成，集成度低，难以自动化等不足。
发明内容
因此，本发明要解决的技术问题在于提供一种一体式微液滴芯片，以克服现有技术中液滴生成与检测在不同液滴芯片中完成，集成度及自动化程度低的不足。
为了解决上述问题，本发明提供一种一体式微液滴芯片，包括芯片本体，所述芯片本体上具有反应腔及加样腔，所述芯片本体内构造有微液滴生成结构、油液接口、气液接口和荧光检测区，所述气液接口与所述反应腔连通，所述加样腔与所述微液滴生成结构连通，所述油液接口与所述微液滴生成结构连 通；
液滴生成时，所述加样腔与所述气液接口之间形成第一压力差，所述油液接口与所述气液接口之间形成第二压力差，所述第一压力差和所述第二压力差分别驱动所述加样腔中的样本和所述油液接口的生成油进入所述微液滴生成结构，生成的微液滴进入并存储于所述反应腔中；
液滴检测时，外部压力驱动检测推动油从所述气液接口进入所述反应腔，使得所述反应腔中的微液滴从所述反应腔中流出至所述微液滴生成结构，外部压力驱动检测分隔油从所述油液接口进入所述微液滴生成结构，所述检测分隔油将所述从反应腔中流出至所述微液滴生成结构中的微液滴分隔形成队列，进入所述荧光检测区。
优选地，所述微液滴生成结构包括油液管道以及连通管道，所述油液管道与所述连通管道成十字交叉，所述连通管道包括处于十字交叉点的第一侧且与所述反应腔连通的第一管道以及处于所述十字交叉点的第二侧且与所述加样腔连通的第二管道，所述油液接口与油液管道连通。
优选地，以所述芯片本体的所述第一侧面处于水平方位为参照，所述反应腔与所述加样腔处于所述第一侧面上，所述反应腔与所述芯片本体的所述第一侧面的连接接口向上延伸，且成下小上大的喇叭口。
优选地，所述反应腔内还构造有自下而上延伸的气液管道，所述气液管道的下口与所述气液接口连通，所述气液管道的上口高于所述连接接口的上口。
优选地，所述第一管道与所述连接接口之间具有微液滴观测区。
优选地，所述加样腔包括开口腔以及密封连接于所述开口腔的开口处的密封盖。
优选地，所述加样腔设置有滤膜或者排气孔。
优选地，所述荧光检测区处于所述第二管道上。
优选地，所述加样腔处于第一侧面上，所述反应腔处于第二侧面上，所述第二侧面与所述第一侧面为所述芯片本体的相对两侧。
优选地，在所述微液滴进入所述反应腔内之前，所述反应腔内预置轻油。
本发明提供的一种一体式微液滴芯片，所述芯片本体上集成具有加样腔、反应腔、微液滴生成结构以及荧光检测区，通过对所述微液滴生成结构的分时复用，将液滴生成、扩增和检测都集成在一张芯片当中，实现全集成、全封闭的数字PCR流程。
附图说明
图1为本发明实施例的一体式微液滴芯片的立体结构示意图；
图2为本发明实施例的一体式微液滴芯片中的微液滴生成结构的结构示意图；
图3为本发明实施例的一体式微液滴芯片中的反应腔的内部结构示意图；
图4为微液滴生成过程示意；
图5为微液滴生成后存储于反应腔内的示意；
图6为一体式微液滴芯片翻转180°后反应腔内的状态示意；
图7为图6状态下向反应腔内通入油液后的状态示意；
图8为微液滴从反应腔内迫出后的状态示意(图中箭头示出微液滴及油液流向)；
图9为本发明另一实施例的一体式微液滴芯片的立体结构示意图。
附图标记表示为：
1、芯片本体；11、反应腔；111、连接接口；112、气液管道；12、加样腔；121、开口腔；122、密封盖；21、油液管道；22、第一管道；23、第二管道；31、油液接口；32、气液接口；33、荧光检测区；34、微液滴观测区；4、微液滴；5、检测推动油。
具体实施方式
结合参见图1至图9所示，根据本发明的实施例，提供一种一体式微液滴芯片，包括芯片本体1，所述芯片本体1上具有反应腔11及加样腔12，所述芯片本体1内构造有微液滴生成结构、油液接口31、气液接口32和荧光检测区33，所述气液接口32与所述反应腔11连通，所述加样腔12与所述微液滴生成结构连通，所述油液接口31与所述微液滴生成结构连通；
液滴生成时，所述加样腔12与所述气液接口32之间形成第一压力差，所述油液接口31与所述气液接口32之间形成第二压力差，所述第一压力差和所述第二压力差分别驱动所述加样腔12中的样本和所述油液接口31的生成油进入所述微液滴生成结构，生成的微液滴4进入并存储于所述反应腔11中；
液滴检测时，外部压力驱动检测推动油5从所述气液接口32进入所述反应腔11，使得所述反应腔11中的微液滴4从所述反应腔11中流出至所述微液滴生成结构，外部压力驱动检测分隔油从所述油液接口31进入所述微液滴生成 结构，所述检测分隔油将所述从反应腔11中流出至所述微液滴生成结构中的微液滴4分隔形成队列，进入所述荧光检测区33。
该技术方案中，所述芯片本体1上集成具有加样腔12、反应腔11、微液滴生成结构以及荧光检测区33，从而使所述微液滴的生成、检测集成于同一个芯片上完成，集成度以及自动化程度都能够得到提高；更为重要的，通过对所述微液滴生成结构的分时复用(以所述一体式微液滴芯片的翻转前后为分时界限)，将液滴生成、扩增和检测都集成在一张芯片当中，实现全集成、全封闭的数字PCR流程，不仅继承了液滴尺寸均一、液滴数量不易受限制、荧光检测信噪比高等优点，还克服了原有的芯片结构复杂，生成和检测在不同芯片中完成，集成度低，难以自动化等困难，是数字PCR领域的一个重要的技术突破。
作为所述微液滴生成结构的一种具体的实现方式，所述微液滴生成结构包括油液管道21以及连通管道，所述油液管道21与所述连通管道成十字交叉，所述连通管道包括处于十字交叉点的第一侧且与所述反应腔11连通的第一管道22以及处于所述十字交叉点的第二侧且与所述加样腔12连通的第二管道23，所述油液接口31与油液管道21连通。
一体式微液滴芯片在一些实施方式中，参见图1所示，以所述芯片本体1的所述第一侧面处于水平方位为参照，所述反应腔11与所述加样腔12处于所述第一侧面上，所述反应腔11与所述芯片本体1的所述第一侧面的连接接口111向上延伸，且成下小上大的喇叭口，成喇叭口的连接接口111能够利于微液滴4由第一管道22的进入所述反应腔11，也利于所述微液滴4从所述反应腔11中进入所述第一管道22中，防止微液滴4的羁留。需要说明的是，此时的所述反应腔11及加样腔12皆处于所述芯片本体1的第一侧面(具体为顶面)，进入所述反应腔11内的微液滴4皆集聚于所述连接接口111处，在进行PCR扩增时，需要将所述芯片本体1整体倒置也即翻转180°，使微液滴4能够处于所述反应腔11的反应区内。
在一些实施方式中，所述反应腔11内还构造有自下而上延伸的气液管道112，所述气液管道112的下口与所述气液接口32连通，所述气液管道112的上口高于所述连接接口111的上口，这样能够防止在所述反应腔11内为负压时，所述微液滴生成结构生成的微液滴4在进入所述反应腔11后进一步从所述气液管道112流出。
在一些实施方式中，所述第一管道22与所述连接接口111之间具有微液滴 观测区34，所述微液滴观测区34的通流面积远大于所述第一管道22的流通面积，也即所述微液滴观测区34为一个在所述第一管道22上扩大的区域(宽度变大)，以使进入该区域的微液滴4的流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
作为一种具体的实现方式，所述加样腔12包括开口腔121以及密封连接于所述开口腔121的开口处的密封盖122，以便于操作人员向所述加样腔12内加入样本。进一步的，所述加样腔12设置有滤膜或者排气孔，当加样腔12变成废液池的时候(也即在液滴芯片翻转倒置时)，排除一定的空气，防止所述加样腔12内积累压力。
在一个实施方式中，所述荧光检测区33处于所述第二管道23上，在所述第二管道23上，所述反应腔11内流出的微液滴4在通过十字交叉点时能够在所述油液管道21内的检测油的作用下被分隔成有合适间距的液滴队列，从而在外部系统的作用下完成荧光检测。
如图9所示，给出了所述一体式微液滴芯片的另一种实现方式，其与图1所示的一体式微液滴芯片的不同之处在于，所述反应腔11与所述加样腔12分别处于了所述芯片本体1的两个相对侧面上，具体的，所述加样腔12处于所述第一侧面上，所述反应腔11处于第二侧面上，所述第二侧面与所述第一侧面为所述芯片本体1的相对两侧，此时，一体式微液滴芯片工作原理和流程与前文的一体式微液滴芯片基本一致，不同的地方是，在液滴生成过程中，由于所述的反应腔11位于所述芯片本体1的底侧(加样腔12处于顶侧)，所述微液滴4进入到所述反应腔11之处时会直接下落至反应腔11底部的反应区，并在反应区中收集，也因此，一体式微液滴芯片在液滴生成结束后，不需要进行180°的翻转，直接进入后续的扩增环节即可。
在一些实施方式中，在所述微液滴4进入所述反应腔11内之前，所述反应腔11内预置轻油(也即密度较小的油液)，保证所述轻油能够始终处于所述反应腔11内的微液滴4的顶部，解决扩增时微液滴蒸发的问题，实现免热盖PCR。
根据本发明的实施例，还提供一种一体式微液滴芯片的数字PCR方法，所述一体式微液滴芯片如上所述，包括芯片本体1，所述芯片本体1上具有反应腔11及加样腔12，所述芯片本体1内构造有微液滴生成结构、油液接口31、气液接口32和荧光检测区33，所述气液接口32与所述反应腔11连通，所述加样腔12与所述微液滴生成结构连通，所述油液接口31与所述微液滴生成结 构连通，
所述数字PCR方法包括如下步骤：
微液滴分割生成步骤，控制所述加样腔12与所述气液接口32之间形成第一压力差、所述油液接口31与所述气液接口32之间形成第二压力差，以使所述第一压力差和所述第二压力差分别驱动所述加样腔12中的样本和所述油液接口31的生成油进入所述微液滴生成结构，生成的微液滴4进入并存储于所述反应腔11中，具体的，向所述油液接口31提供油液，向所述气液接口32提供负压，在所述负压的作用下，所述加样腔12中的样本以及所述油液接口31处的油液被驱动分别沿着所述第二管道23、油液管道21汇聚于所述微液滴生成结构的十字交叉口处，所述样本在油液流体剪切力与表面张力的作用下，形成尺寸均一的微液滴4(油包水液滴)，并最终在所述负压的作用下，所述微液滴4经由所述第一管道22最终进入所述反应腔11内存储，需要说明的是，微液滴在进入所述微液滴观测区34中时，微液滴4的流速，形成密集的液滴群落，方便相机成像记录；
扩增反应步骤，将所述反应腔11置于加热模块(图中未示出)中按照预设循环加热扩增，所述加热模块采用现有的加热模块即可；
微液滴检测步骤，控制外部压力驱动检测推动油5从所述气液接口32进入所述反应腔11，使得所述反应腔11中的微液滴4从所述反应腔11中流出至所述微液滴生成结构，外部压力驱动检测分隔油从所述油液接口31进入所述微液滴生成结构，所述检测分隔油将所述从反应腔11中流出至所述微液滴生成结构中的微液滴4分隔形成队列，进入所述荧光检测区33，完成荧光检测，具体的，向所述气液接口32提供油液(也即检测推动油5，也称上浮油)，通过所述油液的浮力将所述反应腔11内的扩增反应后的微液滴4浮起，并能够在所述油液的浮力作用下，所述微液滴4通过所述连接接口111流出所述反应腔11而进入所述第一管道22，并流经所述微液滴观测区34进入所述十字交叉口，并进入所述第二管道23处，在荧光检测区33进行检测，之后最终进入所述加样腔12内，此时，所述加样腔12为废液池。
该技术方案中，通过对所述微液滴生成结构的分时复用(以所述一体式微液滴芯片的翻转前后为分时界限)，将液滴生成、扩增和检测都集成在一张芯片当中，实现全集成、全封闭的数字PCR流程，不仅继承了液滴尺寸均一、液滴数量不易受限制、荧光检测信噪比高等优点，还克服了原有的芯片结构复杂，生成和检测在不同芯片中完成，集成度低，难以自动化等困难，是数字PCR 领域的一个重要的技术突破。
在一些实施方式中，以所述芯片本体1的第一侧面处于水平方位为参照，所述反应腔11与所述加样腔12处于所述第一侧面上，在所述扩增反应步骤之前，在微液滴分割生成步骤之后还包括：芯片翻转步骤，控制所述芯片本体1上下翻转180°，此时，所述反应腔11内的微液滴4由接近所述连接接口111一侧翻置到远离所述连接接口111的一侧，此时所述微液滴4所对应的反应腔11的位置即为反应腔11的反应区，所述反应区与所述加热模块接触调温实现样本的温度调节反应。
以下结合图1至图8对本发明的采用所述一体式微液滴芯片的作业流程进一步阐述：
首先，将30微升的体系(也即前述样本)加到加样腔12中，30微升的PCR体系包含10微升的Bio-Rad公司的ddPCR Supermix for Probes，GJB2基因上下游引物试剂5微升和含有1ng基因组DNA的模板5微升。芯片整体如图1所示，一张芯片包含了8个平行独立的液滴芯片结构，每个结构都包含有加样腔12、油液接口31、气液接口32、微液滴生成结构、反应腔11。
然后，将密封盖122盖紧或粘接，使加样腔12密封，加样腔12中最好有带滤膜或者口径小的排气孔。
然后，向油液接口31提供液滴生成所需的生成油Bio-Rad Generation Oil，生成油液中含有能使液滴稳定的表面活性剂；向气液接口32提供负压，压力大小为-200mBar，使得与加样腔12和油液接口31形成压力差。其中，加样腔12与微液滴生成结构中的第二管道23相连，油液接口31与微液滴生成结构中的油液管道21相连，第一管道22和气液接口32均与反应腔11相连，其中第一管道22与连接接口111相连，气液接口32与气液管道112相连。微液滴生成结构的各个部分如图2所示，油液管道21有两条分支，分别位于第二管道23和第一管道22两侧，且均与油液接口31相连。第一管道22中可包含微液滴观测区34，观测区管道变宽，液滴进入后流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
在压力差的驱动下，反应体系进入第二管道23中，生成油进入油液管道21中，并在十字结构处(也即前述十字交叉口)交汇，在流体剪切力与表面张力的作用下，形成尺寸均一的油包水微液滴4。十字处管道深度约为70微米，宽度为80微米，液滴尺寸约为100微米。微液滴4进入第一管道22，并在进入微液滴观测区34后降低流速，形成密集的液滴群落，方便相机成像记录，液 滴生成过程原理图如图4所示。
生成的液滴流经第一管道22后到达反应腔11的连接接口111。连接接口111的底部有斜坡结构(也即前述的喇叭口)，斜坡底部与第一管道22相连通。液滴生成流程结束时，撤掉施加在芯片接口处的压力差，此时液滴应仍位于气液管道112下方。
之后，将芯片上下翻转，使液滴从连接接口111转移至反应区(也即远离所述连接接口111)，如图5所示。反应区的结构应采取热传递效率高的设计，比如采用深度高、厚度薄的扁平设计，让外部系统(也即加热模块)从左右两侧给反应区进行加热和制冷，不仅保证温度传导的距离短，还保证较大的接触面积，从而实现高效的热传递。在本实施例中，温度循环流程为先进行一个95℃10分钟的预变性，然后是40个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。为了减少蒸发，可在反应腔11内提前放置30微升低密度的防挥发试剂。
在温度循环结束后，包含有模板的液滴内的扩增反应也相应完成了，需要进入液滴荧光检测环节。向气液接口32中注入检测油(也即上述的检测推动油5)，不断充满反应腔11。在这个过程中，液滴液面不断上升，然后经过连接接口111的斜坡引导，进入第一管道22中。这个过程如图7所示。
液滴经过第一管道22中的观测区时，也可利用相机对液滴进行明场成像，从而对扩增反应后的液滴状态进行评估。同时，向油液接口31中注入检测油，检测油经由油液管道21，在十字管道处与液滴队列汇合，并将紧密排列的液滴分隔成有合适间距的液滴队列。液滴队列依次通过位于第二管道23的荧光检测区33，如图8所示。荧光检测区33对应的位置，是外部系统的荧光检测焦点。外部系统将激发光，如波长为488nm和532nm的激光或LED窄带光，聚焦到检测焦点。液滴在依次通过检测焦点的过程中，液滴内激发出的荧光，同样会被外部系统的采集光路接收，从而获取每个液滴的荧光信息。利用液滴的荧光信息，划定信号阈值，对液滴的阴阳性进行区分，并利用泊松分布模型，计算出样本中目标分子的拷贝数。
最后完成荧光检测的液滴进入加样腔12中，由于加样腔12已经被密封盖122密封，因此不会与芯片外的环境接触，杜绝了气溶胶污染的可能，实现了全封闭的数字PCR流程。
在这个实施例中，开创性的采用了对微液滴生成结构进行分时复用的方法。在液滴生成时，利用微液滴生成结构实现液滴生成。而在液滴荧光检测时，微 液滴生成结构完成了对液滴队列的分隔，保证了液滴荧光信号检测。采用这种分时复用的方法，在类流式的数字PCR技术路线上首次实现了一张芯片完成液滴生成，扩增，检测全集成和全封闭的数字PCR流程，是数字PCR领域的一个重要的技术突破。
在另一种实施例中，在液滴经过第一管道22中的观测区时，利用相机对液滴进行荧光成像，并对成像照片进行分析，获取照片内各个液滴的荧光强度信息，完成液滴荧光信号检测。
本领域的技术人员容易理解的是，在不冲突的前提下，上述各有利方式可以自由地组合、叠加。
以上仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种一体式微液滴芯片，其特征在于，包括芯片本体(1)，所述芯片本体(1)上具有反应腔(11)及加样腔(12)，所述芯片本体(1)内构造有微液滴生成结构、油液接口(31)、气液接口(32)和荧光检测区(33)，所述气液接口(32)与所述反应腔(11)连通，所述加样腔(12)与所述微液滴生成结构连通，所述油液接口(31)与所述微液滴生成结构连通；
    液滴生成时，所述加样腔(12)与所述气液接口(32)之间形成第一压力差，所述油液接口(31)与所述气液接口(32)之间形成第二压力差，所述第一压力差和所述第二压力差分别驱动所述加样腔(12)中的样本和所述油液接口(31)的生成油进入所述微液滴生成结构，生成的微液滴(4)进入并存储于所述反应腔(11)中；
    液滴检测时，外部压力驱动检测推动油(5)从所述气液接口(32)进入所述反应腔(11)，使得所述反应腔(11)中的微液滴(4)从所述反应腔(11)中流出至所述微液滴生成结构，外部压力驱动检测分隔油从所述油液接口(31)进入所述微液滴生成结构，所述检测分隔油将从所述反应腔(11)中流出至所述微液滴生成结构中的微液滴(4)分隔形成队列，进入所述荧光检测区(33)。
  2. 根据权利要求1所述的一体式微液滴芯片，其特征在于，所述微液滴生成结构包括油液管道(21)以及连通管道，所述油液管道(21)与所述连通管道成十字交叉，所述连通管道包括处于十字交叉点的第一侧且与所述反应腔(11)连通的第一管道(22)以及处于所述十字交叉点的第二侧且与所述加样腔(12)连通的第二管道(23)，所述油液接口(31)与油液管道(21)连通。
  3. 根据权利要求2所述的一体式微液滴芯片，其特征在于，以所述芯片本体(1)的第一侧面处于水平方位为参照，所述反应腔(11)与所述加样腔(12)处于所述第一侧面上，所述反应腔(11)与所述芯片本体(1)的所述第一侧面的连接接口(111)向上延伸，且成下小上大的喇叭口。
  4. 根据权利要求3所述的一体式微液滴芯片，其特征在于，所述反应腔(11)内还构造有自下而上延伸的气液管道(112)，所述气液管道(112)的下口与所述气液接口(32)连通，所述气液管道(112)的上口高于所述连接接口(111)的上口。
  5. 根据权利要求4所述的一体式微液滴芯片，其特征在于，所述第一管道(22)与所述连接接口(111)之间具有微液滴观测区(34)。
  6. 根据权利要求1所述的一体式微液滴芯片，其特征在于，所述加样腔(12) 包括开口腔(121)以及密封连接于所述开口腔(121)的开口处的密封盖(122)。
  7. 根据权利要求1所述的一体式微液滴芯片，其特征在于，所述加样腔(12)设置有滤膜或者排气孔。
  8. 根据权利要求2所述的一体式微液滴芯片，其特征在于，所述荧光检测区(33)处于所述第二管道(23)上。
  9. 根据权利要求2所述的一体式微液滴芯片，其特征在于，所述加样腔(12)处于第一侧面上，所述反应腔(11)处于第二侧面上，所述第二侧面与所述第一侧面为所述芯片本体(1)的相对两侧。
  10. 根据权利要求1所述的一体式微液滴芯片，其特征在于，在所述微液滴(4)进入所述反应腔(11)内之前，所述反应腔(11)内预置轻油。
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