WO2020034483A1 - 一种数字pcr系统及数字pcr液滴形成方法 - Google Patents

一种数字pcr系统及数字pcr液滴形成方法 Download PDF

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Publication number
WO2020034483A1
WO2020034483A1 PCT/CN2018/117311 CN2018117311W WO2020034483A1 WO 2020034483 A1 WO2020034483 A1 WO 2020034483A1 CN 2018117311 W CN2018117311 W CN 2018117311W WO 2020034483 A1 WO2020034483 A1 WO 2020034483A1
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Prior art keywords
digital pcr
droplet
base
pcr
pcr system
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PCT/CN2018/117311
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English (en)
French (fr)
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吴炫烨
关一民
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上海新微技术研发中心有限公司
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Priority to US17/044,509 priority Critical patent/US20210362158A1/en
Publication of WO2020034483A1 publication Critical patent/WO2020034483A1/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
    • B01L3/502769—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
    • 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
    • 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
    • B01L7/525—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 with physical movement of samples between temperature zones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • B01L2200/10—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
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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
    • B01L2400/0442—Moving fluids with specific forces or mechanical means specific forces thermal energy, e.g. vaporisation, bubble jet
    • 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
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the invention belongs to the field of biomedicine, in particular to the field of disease detection, and relates to a digital PCR system and a method for forming a digital PCR droplet.
  • PCR Polymerase chain reaction
  • dPCR digital PCR
  • Digital PCR also called single-molecule PCR
  • Digital PCR generally includes two parts, namely, PCR amplification and fluorescent signal analysis.
  • digital PCR In the PCR amplification stage, unlike traditional techniques, digital PCR generally requires diluting a sample to the level of a single molecule and distributing it evenly to tens to tens of thousands of units for reaction.
  • digital PCR technology collects the fluorescence signal of each reaction unit after the amplification is completed. Finally, the original concentration or content of the sample was calculated by direct counting or Poisson distribution formula.
  • digital PCR is an absolute quantification technology for nucleic acid molecules, compared to qPCR, it can directly count the number of DNA molecules. It is an absolute quantification of the starting sample. Therefore, it is especially suitable for applications that cannot be distinguished by CT values. For example, copy number variation, mutation detection, relative gene expression studies (such as allelic imbalanced expression), second-generation sequencing results verification, miRNA expression analysis, single-cell gene expression analysis, etc.
  • an object of the present invention is to provide a digital PCR system and a digital PCR droplet formation method, which are used to solve the problems of slow droplet formation speed, small throughput, complicated operation, and PCR in the prior art.
  • the present invention provides a digital PCR system, including:
  • At least one droplet forming assembly including at least one droplet collection tank
  • the liquid droplet ejection hole assembly is connected below the liquid droplet forming assembly, and includes a plurality of liquid droplet ejection holes.
  • the liquid droplet ejection holes are opened from the upper surface of the liquid droplet ejection hole assembly and are directed to the liquid droplet ejection holes.
  • the lower surface of the component extends in the direction, but does not penetrate the lower surface of the liquid droplet ejection hole component.
  • the liquid droplet ejection hole communicates with the liquid droplet collecting tank, and a vaporization member is provided in the liquid droplet ejection hole.
  • the digital PCR solution liquid layer in the droplet spray hole is vaporized and quickly pushed into the droplet forming oil in the droplet collection tank to form digital PCR droplets.
  • the droplet ejection hole assembly includes a thermal bubble printing chip.
  • the vaporization member is disposed on a bottom surface or a side wall of the droplet ejection hole.
  • the opening shape of the droplet ejection hole includes any one of a circle, an oval, and a polygon.
  • the vaporization component includes a heating component, and the digital PCR solution liquid layer is heated to vaporize it.
  • the heating component includes at least one metal layer.
  • a through groove is provided at the bottom of the droplet collection tank, and the through groove exposes a plurality of the droplet spray holes.
  • the PCR system further includes at least one PCR reagent chamber for storing a digital PCR solution.
  • the droplet spray hole assembly is provided with a flow channel, and the droplet spray hole passes through the flow channel and the The PCR reagent chamber is connected.
  • the flow channel includes at least one main flow channel and a plurality of branch flow channels connected to the main flow channel, and each of the droplet ejection holes is respectively connected to one of the branch flow channels.
  • the digital PCR system further includes a base, the PCR reagent chamber is set in the base, and the droplet spray hole assembly is connected above the base.
  • the base includes a first base component and a second base component
  • the PCR reagent chamber includes a PCR reagent upper chamber and a PCR reagent lower chamber
  • the PCR reagent upper chamber is from the
  • the upper surface of the first base component is open and penetrates the lower surface of the first base component
  • the PCR reagent lower chamber is opened from the upper surface of the second base component and faces the second base.
  • the lower surface of the pedestal component extends in a direction but does not penetrate the lower surface of the second base component.
  • the upper chamber of the PCR reagent communicates with the lower chamber of the PCR reagent and partially overlaps.
  • At least one digital PCR solution injection hole is provided on the lower surface of the second base assembly, and the digital PCR solution injection hole is in communication with the lower chamber of the PCR reagent.
  • the PCR reagent lower chamber includes a first end and a second end, and the digital PCR solution injection hole communicates with the PCR reagent lower chamber at the first end, and the PCR reagent lower chamber
  • the chamber is in communication with the upper chamber of the PCR reagent at the second end, and the size of the lower chamber of the PCR reagent gradually increases from the first end to the second end, and then gradually decreases.
  • At least one exhaust hole is provided on the lower surface of the second base assembly, and the exhaust hole communicates with the upper chamber of the PCR reagent through an air passage, and the air passage is from the second base
  • the upper surface of the seat component is open and extends toward the lower surface of the second base component, but does not penetrate the lower surface of the second base component.
  • the first base component is fixed above the second base component by an adhesive method.
  • the digital PCR system further includes a flexible circuit board, which is connected above the base, and the flexible circuit board is provided with a channel for accommodating the droplet ejection hole assembly. Holes, and the surface of the flexible circuit board is provided with a plurality of first connection pads and a plurality of second connection pads, and the droplet spray hole assembly is connected to the first connection pads through a wire.
  • the flexible circuit board is connected to the base by an adhesive method, and a surface of the base is provided with a channel for preventing glue from flowing onto the droplet spray hole assembly.
  • the flexible circuit board is provided with at least two positioning perforations, and the base surface is provided with positioning protrusions corresponding to the positioning perforation positions.
  • the digital PCR system further includes a controller.
  • the controller includes a controller housing and a controller circuit board located in the controller housing.
  • the controller housing has a base for placing the base.
  • one end of the base is provided with at least one limiting slot
  • the controller housing is provided with at least one limiting member corresponding to the limiting slot.
  • the base is provided with a limiting through hole
  • the limiting through hole penetrates the front and back of the base
  • the controller housing is provided with a limiting position corresponding to the limiting through hole. Pieces.
  • the material of the base includes any one of plastic and glass.
  • the invention also provides a method for forming a digital PCR droplet, which includes the following steps: using a vaporizing component to vaporize the digital PCR solution and quickly pushing it into the droplet forming oil to form a digital PCR droplet.
  • the vaporization component includes a heating component, and the digital PCR solution liquid layer is heated to vaporize it.
  • the digital PCR droplet formation speed is controlled by controlling the heating time, the number of heating times, and the heating interval time of the heating member.
  • the vaporization member is used to vaporize the liquid layer and quickly push it into the droplet forming oil in the droplet collection tank to form the digital PCR droplet.
  • the thickness of the liquid layer ranges from 0.2 nm to 30,000 nm.
  • the formation rate of the digital PCR droplets is greater than 1000 per second.
  • the digital PCR system and digital PCR droplet formation method of the present invention have the following beneficial effects:
  • the present invention uses thermal bubble technology to perform high-speed digital PCR droplet formation.
  • the rapid formation of droplets depends on the instantaneous heating and vaporization of the nano-thickness liquid layer by the vaporized parts in the droplet ejection holes, thereby ejecting the droplets into
  • the digital PCR solution is quickly pushed into the droplet formation oil to form digital PCR droplets.
  • the droplet formation technology in the present invention can achieve greater than 1000 droplets per second. Speed of droplet formation.
  • the oil phase in the technical solution of the present invention is static, so the consumption of the oil phase is greatly reduced, and the oil phase is reduced by about 50%. Dosage.
  • FIG. 1a is a schematic diagram of the three-dimensional structure of the digital PCR system of the present invention.
  • FIG. 1b is a top view of a digital PCR system according to the present invention.
  • Figure 1c shows a bottom view of the digital PCR system of the present invention.
  • Figures 1d-g show side views of the digital PCR system of the present invention.
  • FIG. 2 is a schematic diagram showing the exploded structure of the digital PCR system of the present invention.
  • FIG. 3 is a schematic diagram of the three-dimensional structure of the droplet ejection hole assembly in the digital PCR system of the present invention.
  • FIG. 4 is a partial cross-sectional view of a droplet ejection hole assembly in the digital PCR system of the present invention.
  • FIG. 5a is a schematic diagram of the front three-dimensional structure of the droplet forming component in the digital PCR system of the present invention.
  • FIG. 5b is a schematic diagram of the back three-dimensional structure of the droplet forming component in the digital PCR system of the present invention.
  • FIG. 5c is a top view of a droplet formation module in the digital PCR system of the present invention.
  • FIG. 5d is a bottom view of a droplet formation module in the digital PCR system of the present invention.
  • 5e-5h are side views of the droplet formation module in the digital PCR system of the present invention.
  • FIG. 6a is a schematic diagram of the three-dimensional structure of the base in the digital PCR system of the present invention.
  • FIG. 6b is a top view of a base in the digital PCR system of the present invention.
  • Fig. 6c is a bottom view of the base in the digital PCR system of the present invention.
  • 6d-6g are side views of the base in the digital PCR system of the present invention.
  • FIG. 7 is a partial plan view of a base in the digital PCR system of the present invention.
  • FIG. 8 is a schematic diagram of the front three-dimensional structure of the second base assembly in the digital PCR system of the present invention.
  • FIG. 9 is a schematic diagram of the back three-dimensional structure of the second base assembly in the digital PCR system of the present invention.
  • FIG. 10a is a schematic diagram of the front three-dimensional structure of the droplet ejection hole assembly connected to the flexible circuit board in the digital PCR system of the present invention.
  • FIG. 10b is a schematic structural diagram of a back surface of a droplet ejection hole assembly connected to a flexible circuit board in the digital PCR system of the present invention.
  • FIG. 10c is a top view of a liquid droplet ejection hole assembly connected to a flexible circuit board in the digital PCR system of the present invention.
  • FIG. 10d is a bottom view of the liquid droplet ejection hole assembly connected to the flexible circuit board in the digital PCR system of the present invention.
  • FIG. 10e to FIG. 10h are side views of the liquid droplet ejection hole assembly connected to the flexible circuit board in the digital PCR system of the present invention.
  • FIG. 11 is a schematic diagram of the three-dimensional structure of the controller in the digital PCR system of the present invention.
  • FIG. 12 shows an optical microscope image of a digital PCR droplet formed using the digital PCR system of the present invention.
  • FIGS. 1a to 12 Please refer to FIGS. 1a to 12. It should be noted that the illustrations provided in this embodiment only illustrate the basic idea of the present invention in a schematic manner, and then only the components related to the present invention are shown in the drawings instead of the number, shape and For size drawing, the type, quantity, and proportion of each component can be changed at will in actual implementation, and the component layout type may be more complicated.
  • FIG. 1a is a schematic diagram of the three-dimensional structure of the digital PCR system
  • FIG. 1d, FIG. 1e, FIG. 1f, and FIG. 1g are side views of the digital PCR system in four directions, respectively.
  • FIG. 2 is a schematic diagram showing the exploded structure of the digital PCR system.
  • the digital PCR system includes at least one droplet forming component 1 and a droplet ejection hole component 3.
  • the droplet ejection hole component 3 is connected to Below the liquid droplet forming component 1.
  • the droplet formation assembly 1 includes at least one droplet collection tank 2.
  • FIG. 1 a shows a case where the number of the droplet forming components 1 is one, and the droplet forming component 1 includes four droplet collection tanks 2, where each of the droplet collection tanks 2 is arranged in a row, and Two adjacent droplet collection tanks 2 share one side wall.
  • a plurality of droplet collection tanks 2 may also be arranged in other ways, and may be separately arranged, and the number of the droplet forming components 1 may also be multiple, which should not be excessively limited here. The scope of protection of the invention.
  • FIG. 3 is a schematic diagram of the three-dimensional structure of the liquid droplet ejection hole assembly 3.
  • the liquid droplet ejection hole assembly 3 includes a plurality of liquid droplet ejection holes 4.
  • the droplet ejection holes 4 are arranged in two rows, and the droplet ejection holes in each row are evenly distributed.
  • the droplet spray holes 4 may also be arranged in other ways, and the protection scope of the present invention should not be excessively limited here.
  • FIG. 4 is a partial cross-sectional view of the droplet ejection hole assembly 3.
  • the droplet ejection hole 4 is opened from the upper surface of the droplet ejection hole assembly 3 and faces the
  • the lower surface of the liquid droplet ejection hole assembly 3 extends in a direction, but does not penetrate the lower surface of the liquid droplet ejection hole assembly 3.
  • the opening shape of the droplet ejection hole 4 includes, but is not limited to, any one of a circle, an oval, and a polygon.
  • the volume of the droplet ejection holes 4 determines the volume of digital PCR droplets to be formed.
  • the droplet ejection hole assembly 3 may include a thermal bubble printing chip.
  • Thermal bubble printing technology is a major technology in the field of printers, and its basic principle is to eject ink droplets by heating.
  • the liquid droplet ejection hole assembly 3 may use an existing thermal bubble printing chip.
  • FIG. 5a is a schematic diagram of the front three-dimensional structure of the droplet forming assembly 1
  • FIG. 5b is a schematic diagram of the back three-dimensional structure of the droplet forming assembly 1
  • FIG. 5d is a bottom view of the droplet forming component 1
  • FIGS. 5e, 5f, 5g, and 5h are respectively shown in four directions of the droplet forming component 1.
  • a through slot 6 is provided at the bottom of the droplet collection tank 2 to expose a plurality of the droplet spray holes 4 so that the droplet spray holes 4 communicate with the droplet collection tank 2 .
  • the droplet ejection hole 4 is provided with a vaporization member 5 for vaporizing the liquid layer of the digital PCR solution in the droplet ejection hole 4 and quickly pushing it into the droplet collection.
  • the droplets in tank 2 are formed into oil to form digital PCR droplets.
  • the vaporization member 5 is disposed on the bottom surface of the droplet ejection hole 4, and the vaporization member 5 may be a heating member, which is vaporized by heating the liquid layer of the digital PCR solution.
  • the heating component includes a heating sheet, and the heating sheet may be a single-layer metal layer or a composite multilayer metal layer.
  • the shape of the vaporization member 5 includes, but is not limited to, a circle or a square, and the area may be 0.5 to 2 times the area of the bottom of the droplet ejection hole 4.
  • the vaporization member 5 may also be disposed on a side wall of the droplet spray hole 4, and the protection scope of the present invention should not be excessively limited here.
  • the PCR system further includes at least one PCR reagent chamber for storing a digital PCR solution.
  • the droplet spray hole assembly 3 is provided with a flow channel, and the droplet The spray hole 4 communicates with the PCR reagent chamber through the flow channel.
  • the flow channel includes at least one main flow channel 9 and a plurality of branch flow channels 10 connected to the main flow channel 9, and each of the droplet ejection holes 4 is connected to one of the branch flow channels 10, respectively.
  • FIG. 3 shows a case where the droplet ejection hole assembly 3 includes a main channel 9.
  • the number of the main channels 9 may be matched with the number of the droplet collection tanks 2.
  • FIG. 10b shows a case where the droplet ejection hole assembly 3 includes four main flow channels 9.
  • materials for constructing the flow channel and the droplet ejection hole include, but are not limited to, silicon, polymer, photoresist, and the like.
  • the digital PCR system further includes a base 11, the PCR reagent chamber is disposed in the base 11, and the droplet spray hole assembly 3 is connected to the base 11. Up.
  • the material of the base 11 includes, but is not limited to, any one of transparent or opaque plastic and glass.
  • the base 11 includes a first base component 12 and a second base component 13, and the PCR reagent chamber includes an upper PCR reagent chamber 14 and a lower PCR reagent chamber 15,
  • the PCR reagent upper chamber 14 is opened from the upper surface of the first base assembly 12 and penetrates the lower surface of the first base assembly 12.
  • the PCR reagent lower chamber 15 is opened from the second base.
  • the upper surface of the base assembly 13 is open and extends toward the lower surface of the second base assembly 13, but does not penetrate the lower surface of the second base assembly 13.
  • the PCR reagent upper chamber 14 and the PCR reagent The lower chamber 15 communicates and partially overlaps.
  • FIGS. 6a to 6g wherein FIG. 6a is a schematic view of the three-dimensional structure of the base 11, FIG. 6b is a top view of the base 11, and FIG. 6c is a bottom view of the base 11, 6d, 6e, 6f and 6g are respectively shown as side views of the base 11 in four directions.
  • the first base component 12 is fixed on the second base component 13 by an adhesive method such as double-sided tape or glue.
  • a surface of the second base assembly 13 has a sunken platform for receiving the second base assembly 13, and four corners of the sunken platform have arc-shaped extension spaces. The protrusions around the sunken platform play a positioning role when the first base assembly 12 is adhered to the surface of the sunken platform.
  • FIG. 7 is a partial top view of the base, showing the relative positional relationship between the PCR reagent upper chamber 14 and the PCR reagent lower chamber 15.
  • the PCR The reagent is divided into two parts to hold enough digital PCR solution.
  • FIG. 8 and FIG. 9 are respectively a schematic diagram of the front three-dimensional structure and a schematic diagram of the rear three-dimensional structure of the second base assembly 13.
  • the lower surface of the second base assembly 13 is provided with at least one digital PCR solution injection hole 16, and the digital PCR solution injection hole 16 is under the PCR reagent.
  • the chamber 15 is in communication, and is used to inject a digital PCR solution into the PCR reagent chamber through the digital PCR solution injection hole 16.
  • the PCR reagent lower chamber 15 includes a first end and a second end, and the digital PCR solution injection hole 16 is at the first end and under the PCR reagent.
  • the chamber 15 is in communication, and the PCR reagent lower chamber 15 is in communication with the PCR reagent upper chamber 14 at the second end.
  • the size of the lower chamber 15 of the PCR reagent gradually increases from the first end to the second end, and then gradually decreases. Adopting this design can prevent the formation of air bubbles during liquid addition.
  • At least one exhaust hole 17 is provided on the lower surface of the second base assembly 13, and the exhaust hole 17 passes through the air passage 8 and the PCR reagent upper chamber. 14 ⁇ 14 connected.
  • the air duct 8 is opened from the upper surface of the second base component 13 and extends toward the lower surface of the second base component 13, but does not penetrate the second base. The lower surface of the module 13.
  • the opening area of the digital PCR solution injection hole 16 is larger than the opening area of the exhaust hole 17, and the opening area of the digital PCR solution injection hole 16 is made slightly larger to support displacement. Tip of a liquid gun. Due to the capillary phenomenon, the liquid injected into the PCR reagent chamber will not flow out from the digital PCR solution injection hole 16 or the exhaust hole 17.
  • the digital PCR system further includes a flexible circuit board 18 connected to the base 11, and the droplet ejection hole assembly 3 is connected.
  • the flexible circuit board 18 is connected to the base 11, and the droplet ejection hole assembly 3 is connected.
  • FIG. 10a shows a schematic structural diagram of the front face of the liquid droplet ejection hole assembly 3 connected to the flexible circuit board 18, and FIG. 10b shows the liquid droplet ejection hole assembly 3 connected to FIG. 10c shows a top view of the flexible circuit board 18 connected to the flexible circuit board 18, and FIG. 10d shows the liquid droplet ejection hole module 3 connected to
  • the bottom view of the flexible circuit board 18 is shown in FIG. 10e, FIG. 10f, FIG. 10g, and FIG. 10h as side views of the droplet ejection hole assembly 3 connected to the flexible circuit board 18 in four directions. .
  • the flexible circuit board 18 is provided with through holes 19 for accommodating the droplet ejection hole assembly 3, and the surface of the flexible circuit board 19 is provided with a plurality of first connection pads (not shown). And a plurality of second connection pads 20, the droplet ejection hole assembly 3 is connected to the first connection pads through wires, so as to connect the vaporization component 5 to an external controller through the flexible circuit board 18.
  • the droplet ejection hole assembly 3 may be connected to the first connection pad by a standard wire bonding process.
  • the flexible circuit board 18 is connected to the base 11 by an adhesive method.
  • a surface of the base 11 is provided with a structure for preventing glue from flowing onto the droplet spray hole assembly 3.
  • the number of the channels 21 is multiple, and they are distributed around the droplet ejection hole assembly 3.
  • the arrangement manner of the channels 21 can be adjusted as needed, and is not limited to the manner shown in FIG. 6a.
  • the droplet forming component 1 may also be fixed to the flexible circuit board 18 by an adhesive method. As shown in FIG. 5 b, a protrusion 31 is provided on the back of the droplet forming component 1. This design can ensure a firm adhesion between the droplet forming component 1 and the flexible circuit board 18.
  • the flexible circuit board 18 is provided with at least two positioning holes 22, and as shown in FIG. 6a, the surface of the base 11 is provided with a position corresponding to the positioning holes 22.
  • Corresponding positioning protrusions 23, the positioning perforations 22 and the positioning protrusions 23 cooperate with each other, which facilitates the precise positioning of the flexible circuit board 18 on the base 11.
  • the digital PCR system further includes a controller.
  • FIG. 11 is a schematic diagram showing the three-dimensional structure of the controller 24.
  • the controller 24 includes a controller housing 25 and is located in the controller housing 25.
  • a controller circuit board, the controller housing 25 has a bearing portion 26 for placing the base 11, and a surface of the bearing portion 26 is provided with a plurality of circuit connection conductive pins 27 connected to the controller circuit connection board. (Also referred to as Pin), the circuit connection conductive pin 27 corresponds to the position of the second connection pad 30 on the flexible circuit board 18.
  • the controller 24 is connected to the droplet ejection hole assembly 3 through a flexible circuit board 18, and controls the formation of the digital PCR droplets by controlling the heating time, the number of heating times, and the heating interval time of the heating member. speed.
  • the control circuit of the controller 24 may adopt an existing circuit structure.
  • one end of the base is provided with at least one limiting groove 13.
  • the controller housing 25 is provided with at least one corresponding to the limiting groove 13. ⁇ ⁇ 29 ⁇ Limiting member 29.
  • the base is further provided with a limiting through-hole 7, and the limiting through-hole 7 penetrates the front and back of the base, as shown in FIG. 11.
  • the controller housing 25 is provided with a limiting piece 30 corresponding to the limiting through hole 7.
  • the digital PCR system of the present invention can be used for the formation of digital PCR liquid droplets.
  • the rapid formation of liquid droplets depends on the instantaneous vaporization of a nano-thickness liquid layer by a vaporization component in the liquid droplet spray holes, thereby digitally PCR in the liquid droplet spray holes.
  • the solution is quickly pushed into the droplet formation oil to form digital PCR droplets.
  • the droplet formation technology in the present invention can achieve more than 1,000 droplets per second. Formation speed.
  • the oil phase in the technical solution of the present invention is static, so the consumption of the oil phase is greatly reduced, and the amount of the oil phase is reduced by about 50%. Efficient digital PCR oil utilization.
  • the invention also provides a method for forming a digital PCR droplet, which includes the following steps: using a vaporizing component to vaporize the digital PCR solution and quickly pushing it into the droplet forming oil to form a digital PCR droplet.
  • high-speed digital PCR droplet formation is performed using a thermal bubble technique, and the vaporization part includes a heating part, which is vaporized by heating the liquid layer of the digital PCR solution.
  • the digital PCR droplet formation speed is controlled by controlling the heating time, the number of heating times, and the heating interval time of the heating member.
  • the digital PCR droplet formation method of the present invention can achieve a digital PCR droplet formation speed of more than 1000 per second.
  • the digital PCR droplet formation method includes the following steps:
  • S1 Inject a digital PCR solution into a PCR reagent chamber, so that the digital PCR solution enters a droplet ejection hole communicating with the PCR reagent chamber to form a digital PCR solution liquid layer;
  • the thickness of the liquid layer is nano-scale, and is greater than 0.2 nm. In this embodiment, the thickness of the liquid layer is preferably in a range of 0.2 nm to 30,000 nm.
  • FIG. 12 shows an optical microscope image of digital PCR droplets formed using the digital PCR system of the present invention. It can be seen that the formed digital PCR droplets are symmetrical and uniform in shape.
  • the digital PCR system and the digital PCR droplet formation method of the present invention can satisfy the use of all digital PCR biochemical reagents. Because the concentration of many biomarker molecules in the blood is very low (for example, circulating tumor DNA has only 3 DNA molecules per 2 ml of blood), the digital PCR system and digital PCR droplet formation method of the present invention have the number of droplet formation It is not limited by the amount of oil used and the high speed makes it possible to apply this type of detection to digital PCR.
  • the digital PCR system and digital PCR droplet formation method of the present invention use thermal bubble technology to perform high-speed digital PCR droplet formation.
  • the rapid formation of droplets depends on the nano-thickness liquid from vaporized parts in the droplet ejection holes.
  • the layer is instantaneously heated and vaporized, so that the digital PCR solution in the droplet spray hole is quickly pushed into the droplet forming oil to form a digital PCR droplet.
  • the droplet formation technology can achieve a droplet formation speed of more than 1000 per second.
  • the oil phase in the technical solution of the present invention is static, so the consumption of the oil phase is greatly reduced, and the amount of the oil phase is reduced by about 50%. Efficient digital PCR oil utilization. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

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Abstract

本发明提供一种数字PCR系统及数字PCR液滴形成方法，该数字PCR系统包括至少一个液滴形成组件及液滴喷孔组件，所述液滴形成组件包括至少一个液滴收集槽，所述液滴喷孔组件连接于所述液滴形成组件下方，包括若干液滴喷孔，所述液滴喷孔与所述液滴收集槽连通，且所述液滴喷孔内设有汽化部件，用于使所述液滴喷孔中的数字PCR溶液液体层汽化并快速推入所述液滴收集槽中的液滴形成油中，以形成数字PCR液滴。本发明使用热泡技术进行高速数字PCR液滴形成，可以实现大于1000个每秒的液滴形成速度。

Description

一种数字PCR系统及数字PCR液滴形成方法 技术领域
本发明属于生物医药领域，尤其是疾病检测领域，涉及一种数字PCR系统及数字PCR液滴形成方法。
背景技术
聚合酶链式反应(polymerase chain reaction，PCR)提出至今已有20年时间，期间PCR已发展成为分子生物学领域的一项关键技术和常规技术，极大地推动了生命科学各个领域的发展。特别是90年代后期，美国ABI公司推出的实时荧光定量PCR(real time PCR，qPCR)技术及相关产品更是将PCR由体外合成及定性/半定量检测技术发展成为一种高灵敏、高特异性和精确定量的基因分析技术。
尽管经过十几年时间的迅速发展，qPCR技术已经用于除外伤和营养缺乏症外所有疾病的诊断，但是，在PCR扩增过程中影响其扩增效率的因素有很多，不能保证在反应过程中扩增效率保持不变和实际样品与标准样品以及不同样品之间的扩增效率是相同的，由此导至其定量分析所依赖的基础——循环阈值(CT)不是恒定不变的。因此qPCR的定量只是“相对定量”，其准确度和重现性依然不能够满足分子生物学定量分析的要求。
20世纪末，Vogelstein等提出数字PCR(digital PCR，dPCR)的概念，通过将一个样本分成几十到几万份，分配到不同的反应单元，每个单元包含一个或多个拷贝的目标分子(DNA模板)，在每个反应单元中分别对目标分子进行PCR扩增，扩增结束后对各个反应单元的荧光信号进行统计学分析。与qPCR不同的是，数字PCR不依赖于CT值，因此不受扩增效率影响，扩增结束后通过直接计数或泊松分布公式来计算每个反应单元的平均浓度(含量)，能够将误差控制在5％以内，数字PCR可以不需要对照标准样品和标准曲线来实现绝对定量分析。
数字PCR(也可称单分子PCR)一般包括两部分内容，即PCR扩增和荧光信号分析。在PCR扩增阶段，与传统技术不同，数字PCR一般需要将样品稀释到单分子水平，并平均分配到几十至几万个单元中进行反应。不同于qPCR对每个循环进行实时荧光测定的方法，数字PCR技术是在扩增结束后对每个反应单元的荧光信号进行采集。最后通过直接计数或泊松分布公式计算得到样品的原始浓度或含量。
由于数字PCR是一种核酸分子绝对定量技术，相较于qPCR，能够直接数出DNA分子的个数，是对起始样品的绝对定量，因此特别适用于依靠CT值不能很好分辨的应用领域， 例如拷贝数变异、突变检测、基因相对表达研究(如等位基因不平衡表达)、二代测序结果验证、miRNA表达分析、单细胞基因表达分析等。
目前市面上的数字PCR技术主要有三种。一种是通过在特定仪器中使用流动的油切断水相的PCR溶液形成液滴，然后在另外的两台仪器中完成PCR和检测；一种是通过将PCR溶液分布到挖空的硅片上，然后在特定仪器中进行PCR以及另外一台仪器中进行检测；最后一种是在一种仪器上将液体通过狭窄的沟道注入腔体形成液滴，并完成PCR，然后在另一台仪器中完成检测。然而，当前的三种方法的液滴形成速度或者通量各有限制。此外，上述三种技术无一例外的依赖多台大型仪器。这不但增加了仪器的购置的成本，限制了数字PCR的广泛使用；而且增加了实验操作的复杂度。
因此，如何提供一种大于每秒形成1000个液滴的高速的数字PCR液滴形成技术、液滴形成与PCR温控和检测仪器集成的原位PCR技术、高效的数字PCR油利用率方法，成为本领域技术人员亟待解决的一个重要技术问题。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种数字PCR系统及数字PCR液滴形成方法，用于解决现有技术中液滴形成速度慢、通量小、操作复杂、PCR油利用率低的问题。
为实现上述目的及其他相关目的，本发明提供一种数字PCR系统，包括：
至少一个液滴形成组件，包括至少一个液滴收集槽；
液滴喷孔组件，连接于所述液滴形成组件下方，包括若干液滴喷孔，所述液滴喷孔自所述液滴喷孔组件的上表面开口，并往所述液滴喷孔组件的下表面方向延伸，但未贯穿所述液滴喷孔组件的下表面，所述液滴喷孔与所述液滴收集槽连通，且所述液滴喷孔内设有汽化部件，用于使所述液滴喷孔中的数字PCR溶液液体层汽化并快速推入所述液滴收集槽中的液滴形成油中，以形成数字PCR液滴。
可选地，所述液滴喷孔组件包括热泡打印芯片。
可选地，所述汽化部件设置于所述液滴喷孔的底面或侧壁。
可选地，所述液滴喷孔的开口形状包括圆形、椭圆形、多边形中的任意一种。
可选地，所述汽化部件包括加热部件，通过加热所述数字PCR溶液液体层使其汽化。
可选地，所述加热部件包括至少一层金属层。
可选地，所述液滴收集槽底部设有通槽，所述通槽暴露出多个所述液滴喷孔。
可选地，所述PCR系统还包括至少一个用于储存数字PCR溶液的PCR试剂腔室，所述 液滴喷孔组件中设有流道，所述液滴喷孔通过所述流道与所述PCR试剂腔室连通。
可选地，所述流道包括至少一条主流道及与所述主流道连接的多条支流道，每个所述液滴喷孔分别与一条所述支流道连接。
可选地，所述数字PCR系统还包括基座，所述PCR试剂腔室设置于所述基座中，所述液滴喷孔组件连接于所述基座上方。
可选地，所述基座包括第一基座组件与第二基座组件，所述PCR试剂腔室包括PCR试剂上腔室与PCR试剂下腔室，所述PCR试剂上腔室自所述第一基座组件的上表面开口，并贯穿所述第一基座组件的下表面，所述PCR试剂下腔室自所述第二基座组件的上表面开口，并向所述第二基座组件的下表面方向延伸，但未贯穿所述第二基座组件的下表面，所述PCR试剂上腔室与PCR试剂下腔室连通且部分交迭。
可选地，所述第二基座组件的下表面设有至少一个数字PCR溶液注入孔，所述数字PCR溶液注入孔与所述PCR试剂下腔室连通。
可选地，所述PCR试剂下腔室包括第一端及第二端，所述数字PCR溶液注入孔在所述第一端处与所述PCR试剂下腔室连通，所述PCR试剂下腔室在所述第二端处与所述PCR试剂上腔室连通，所述PCR试剂下腔室的尺寸自所述第一端至所述第二端方向首先逐渐增大，然后逐渐减小。
可选地，所述第二基座组件的下表面设有至少一个排气孔，所述排气孔通过气道与所述PCR试剂上腔室连通，所述气道自所述第二基座组件的上表面开口，并向所述第二基座组件的下表面方向延伸，但未贯穿所述第二基座组件的下表面。
可选地，所述第一基座组件通过胶粘方式固定于所述第二基座组件上方。
可选地，所述数字PCR系统还包括软性线路板，所述软性线路板连接于所述基座上方，所述软性线路板中设有用于收容所述液滴喷孔组件的通孔，且所述软性线路板表面设有若干第一连接焊垫及若干第二连接焊垫，所述液滴喷孔组件通过导线与所述第一连接焊垫连接。
可选地，所述软性线路板通过胶粘方式与所述基座连接，所述基座表面设有用于防止胶水流到所述液滴喷孔组件上的沟道。
可选地，所述软性线路板中设有至少两个定位穿孔，所述基座表面设有与所述定位穿孔位置相对应的定位凸起。
可选地，所述数字PCR系统还包括一控制器，所述控制器包括控制器外壳及位于所述控制器外壳内的控制器电路板，所述控制器外壳具有用于放置所述基座的承载部，所述承载部表面设有若干与所述控制器电路连接板连接的电路连接导电针，所述电路连接导电针与所述第二连接焊垫的位置相对应。
可选地，所述基座的一端设置有至少一个限位槽，所述控制器外壳设置有至少一个与所述限位槽相对应的限位件。
可选地，所述基座设置有一限位通孔，所述限位通孔贯穿所述基座的正面及背面，所述控制器外壳设置有与所述限位通孔相对应的限位件。
可选地，所述基座的材质包括塑料、玻璃中的任意一种。
本发明还提供一种数字PCR液滴形成方法，包括以下步骤：采用汽化部件使数字PCR溶液汽化并快速推入液滴形成油中，以形成数字PCR液滴。
可选地，所述汽化部件包括加热部件，通过加热所述数字PCR溶液液体层使其汽化。
可选地，通过控制所述加热部件的发热时间、发热次数及发热间隔时间来控制所述数字PCR液滴的形成速度。
可选地，包括以下步骤：
向PCR试剂腔室内注入数字PCR溶液，使数字PCR溶液进入与所述PCR试剂腔室连通的液滴喷孔，形成数字PCR溶液液体层；
向液滴收集槽中添加液滴形成油；
采用所述汽化部件使所述液体层汽化并快速推入所述液滴收集槽中的所述液滴形成油中，以形成所述数字PCR液滴。
可选地，所述液体层的厚度范围是0.2nm～30000nm。
可选地，所述数字PCR液滴的形成速度大于1000个/秒。
如上所述，本发明的数字PCR系统及数字PCR液滴形成方法，具有以下有益效果：
(1)本发明使用热泡技术进行高速数字PCR液滴形成，液滴的快速形成依赖于液滴喷孔内的汽化部件对纳米级厚度液体层的瞬间加热汽化，从而将液滴喷孔中的数字PCR溶液快速推入液滴形成油中以形成数字PCR液滴，相比于市面上每秒钟100个液滴的形成速度，本发明中的液滴形成技术可以实现大于1000个每秒的液滴形成速度。
(2)相比于油相与水相共同运动产生液滴的方法，本发明的技术方案中的油相是静态的，因此油相的消耗量被大大减少，减少了50％左右的油相用量。
附图说明
图1a显示为本发明的数字PCR系统的立体结构示意图。
图1b显示为本发明的数字PCR系统的俯视图。
图1c显示为本发明的数字PCR系统的仰视图。
图1d-图1g显示为本发明的数字PCR系统的侧视图。
图2显示为本发明的数字PCR系统的分解结构示意图。
图3显示为本发明的数字PCR系统中液滴喷孔组件的立体结构示意图。
图4显示为本发明的数字PCR系统中液滴喷孔组件的局部剖面图。
图5a显示为本发明的数字PCR系统中液滴形成组件的正面立体结构示意图。
图5b显示为本发明的数字PCR系统中液滴形成组件的背面立体结构示意图。
图5c显示为本发明的数字PCR系统中液滴形成组件的俯视图。
图5d显示为本发明的数字PCR系统中液滴形成组件的仰视图。
图5e-图5h显示为本发明的数字PCR系统中液滴形成组件的侧视图。
图6a显示为本发明的数字PCR系统中基座的立体结构示意图。
图6b显示为本发明的数字PCR系统中基座的俯视图。
图6c显示为本发明的数字PCR系统中基座的仰视图。
图6d-图6g显示为本发明的数字PCR系统中基座的侧视图。
图7显示为本发明的数字PCR系统中基座的局部俯视图。
图8显示为本发明的数字PCR系统中第二基座组件的正面立体结构示意图。
图9显示为本发明的数字PCR系统中第二基座组件的背面立体结构示意图。
图10a显示为本发明的数字PCR系统中液滴喷孔组件连接于软性线路板的正面立体结构示意图。
图10b显示为本发明的数字PCR系统中液滴喷孔组件连接于软性线路板的背面结构示意图。
图10c显示为本发明的数字PCR系统中液滴喷孔组件连接于软性线路板的俯视图。
图10d显示为本发明的数字PCR系统中液滴喷孔组件连接于软性线路板的仰视图。
图10e-图10h显示为本发明的数字PCR系统中液滴喷孔组件连接于软性线路板的侧视图。
图11显示为本发明的数字PCR系统中控制器的立体结构示意图。
图12显示为利用本发明的数字PCR系统形成的数字PCR液滴的光学显微镜图。
元件标号说明
1                     液滴形成组件
2                     液滴收集槽
3                     液滴喷孔组件
4                     液滴喷孔
5                     汽化部件
6                     通槽
7                     限位通孔
8                     气道
9                     主流道
10                    支流道
11                    基座
12                    第一基座组件
13                    第二基座组件
14                    PCR试剂上腔室
15                    PCR试剂下腔室
16                    数字PCR溶液注入孔
17                    排气孔
18                    软性线路板
19                    通孔
20                    第二连接焊垫
21                    沟道
22                    定位穿孔
23                    定位凸起
24                    控制器
25                    控制器外壳
26                    承载部
27                    电路连接导电针
28                    限位槽
29、30                限位件
31                    凸台
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精 神下进行各种修饰或改变。
请参阅图1a至图12。需要说明的是，本实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。
实施例一
本发明提供一种数字PCR系统，请参阅图1a至图1g，其中图1a显示为所述数字PCR系统的立体结构示意图，图1b显示为所述数字PCR系统的俯视图，图1c显示为所述数字PCR系统的仰视图，图1d、图1e、图1f及图1g分别显示为所述数字PCR系统在四个方向上的侧视图。图2显示为所述数字PCR系统的分解结构示意图，本实施例中，所述数字PCR系统包括至少一个液滴形成组件1及液滴喷孔组件3，所述液滴喷孔组件3连接于所述液滴形成组件1下方。
具体的，所述液滴形成组件1包括至少一个液滴收集槽2。图1a中显示的为所述液滴形成组件1的数量为一个，且所述液滴形成组件1包括4个液滴收集槽2的情形，其中，各个液滴收集槽2排成一行，且相邻两个液滴收集槽2共用一面侧壁。当然，在其它实施例中，多个液滴收集槽2也可以采用其它排列方式，并可以分立设置，且所述液滴形成组件1的数量也可以为多个，此处不应过分限制本发明的保护范围。
请参阅图3，显示为所述液滴喷孔组件3的立体结构示意图，所述液滴喷孔组件3包括若干液滴喷孔4。本实施例中，所述液滴喷孔4排列成两行，且每行液滴喷孔均匀分布。在其它实施例中，所述液滴喷孔4也可以采用其它排列方式，此处不应过分限制本发明的保护范围。
请参阅图4，显示为所述液滴喷孔组件3的局部剖面图，本实施例中，所述液滴喷孔4自所述液滴喷孔组件3的上表面开口，并往所述液滴喷孔组件3的下表面方向延伸，但未贯穿所述液滴喷孔组件3的下表面。所述液滴喷孔4的开口形状包括但不限于圆形、椭圆形、多边形中的任意一种。所述液滴喷孔4的容积决定了要形成的数字PCR液滴的体积。
作为示例，所述液滴喷孔组件3可包括热泡打印芯片。热泡打印技术是打印机领域的一项主要技术，其基本原理是通过加热将墨滴喷出。本发明中，所述液滴喷孔组件3可采用现有的热泡打印芯片。
请参阅图5a至图5h，其中，图5a显示为所述液滴形成组件1的正面立体结构示意图，图5b显示为所述液滴形成组件1的背面立体结构示意图，图5c显示为所述液滴形成组件1 的俯视图，图5d显示为所述液滴形成组件1的仰视图，图5e、图5f、图5g及图5h分别显示为所述液滴形成组件1在四个方向上的侧视图。本实施例中，所述液滴收集槽2底部设有通槽6，用以暴露出多个所述液滴喷孔4，使得所述液滴喷孔4与所述液滴收集槽2连通。
具体的，如图4所示，所述液滴喷孔4内设有汽化部件5，用于使所述液滴喷孔4中的数字PCR溶液液体层汽化并快速推入所述液滴收集槽2中的液滴形成油中，以形成数字PCR液滴。
作为示例，所述汽化部件5设置于所述液滴喷孔4的底面，所述汽化部件5可以采用加热部件，通过加热所述数字PCR溶液液体层使其汽化。本实施例中，所述加热部件包括加热片，所述加热片可以是单层金属层，也可以是复合多层金属层。所述汽化部件5的形状包括但不限于圆形或方形，面积可以是所述液滴喷孔4底面积的0.5～2倍。在其它实施例中，所述汽化部件5也可以设置于所述液滴喷孔4的侧壁，此处不应过分限制本发明的保护范围。
具体的，所述PCR系统还包括至少一个用于储存数字PCR溶液的PCR试剂腔室，如图3及图4所示，所述液滴喷孔组件3中设有流道，所述液滴喷孔4通过所述流道与所述PCR试剂腔室连通。
作为示例，所述流道包括至少一条主流道9及与所述主流道9连接的多条支流道10，每个所述液滴喷孔4分别与一条所述支流道10连接。图3中显示的为所述液滴喷孔组件3包括一条主流道9的情形，在其它实施例中，所述主流道9的数量也可以与所述液滴收集槽2的数量相匹配，例如图10b中显示的为所述液滴喷孔组件3包括四条主流道9的情形。
作为示例，构建所述流道和所述液滴喷孔的材料包括但不限于硅、聚合物、光刻胶等。
具体的，如图1a所示，所述数字PCR系统还包括基座11，所述PCR试剂腔室设置于所述基座11中，所述液滴喷孔组件3连接于所述基座11上方。
作为示例，所述基座11的材质包括但不限于透明或不透明的塑料、玻璃中的任意一种。
作为示例，如图2所示，所述基座11包括第一基座组件12与第二基座组件13，所述PCR试剂腔室包括PCR试剂上腔室14与PCR试剂下腔室15，所述PCR试剂上腔室14自所述第一基座组件12的上表面开口，并贯穿所述第一基座组件12的下表面，所述PCR试剂下腔室15自所述第二基座组件13的上表面开口，并向所述第二基座组件13的下表面方向延伸，但未贯穿所述第二基座组件13的下表面，所述PCR试剂上腔室14与PCR试剂下腔室15连通且部分交迭。
请参阅图6a至图6g，其中，图6a显示为所述基座11的立体结构示意图，图6b显示为所述基座11的俯视图，图6c显示为所述基座11的仰视图，图6d、图6e、图6f及图6g分别显示为所述基座11在四个方向上的侧视图。
作为示例，所述第一基座组件12通过胶粘方式例如双面胶或胶水等固定于所述第二基座组件13上方。本实施例中，所述第二基座组件13表面具有一用于收容所述第二基座组件13的下沉式平台，且所述下沉式平台的四角具有圆弧形的延伸空间，所述下沉式平台四周的凸起在所述第一基座组件12黏贴到所述下沉式平台表面时起到定位作用。
请参阅图7，显示为所述基座的局部俯视图，其中显示了所述PCR试剂上腔室14与所述PCR试剂下腔室15之间的相对位置关系，本实施例中，所述PCR试剂分为两部分是为了装足够多的数字PCR溶液。
请参阅图8及图9，分别显示为所述第二基座组件13的正面立体结构示意图及背面立体结构示意图。如图7及图9所示，本实施例中，所述第二基座组件13的下表面设有至少一个数字PCR溶液注入孔16，所述数字PCR溶液注入孔16与所述PCR试剂下腔室15连通，用于通过所述数字PCR溶液注入孔16往所述PCR试剂腔室内注入数字PCR溶液。
具体的，如图7及图8所示，所述PCR试剂下腔室15包括第一端及第二端，所述数字PCR溶液注入孔16在所述第一端处与所述PCR试剂下腔室15连通，所述PCR试剂下腔室15在所述第二端处与所述PCR试剂上腔室14连通。本实施例中，所述PCR试剂下腔室15的尺寸自所述第一端至所述第二端方向首先逐渐增大，然后逐渐减小。采用这种设计的可以防止加液时形成气泡。
具体的，如图7及图9所示，所述第二基座组件13的下表面设有至少一个排气孔17，所述排气孔17通过气道8与所述PCR试剂上腔室14连通。如图8所示，所述气道8自所述第二基座组件13的上表面开口，并向所述第二基座组件13的下表面方向延伸，但未贯穿所述第二基座组件13的下表面。
具体的，本实施例中，所述数字PCR溶液注入孔16的开口面积大于所述排气孔17的开口面积，所述数字PCR溶液注入孔16的开口面积做的稍微大一点是为了支撑移液枪的枪头。由于毛细现象，注入所述PCR试剂腔室内的液体不会从所述数字PCR溶液注入孔16或者所述排气孔17流出。
具体的，如图1a及图2所示，所述数字PCR系统还包括软性线路板18，所述软性线路板18连接于所述基座11上方，所述液滴喷孔组件3连接于所述软性线路板18中。
请参阅图10a至图10h，其中，图10a显示为所述液滴喷孔组件3连接于所述软性线路板18的正面结构示意图，图10b显示为所述液滴喷孔组件3连接于所述软性线路板18的背面结构示意图，图10c显示为所述液滴喷孔组件3连接于所述软性线路板18的俯视图，图10d显示为所述液滴喷孔组件3连接于所述软性线路板18的仰视图，图10e、图10f、图10g及图10h分别显示为所述液滴喷孔组件3连接于所述软性线路板18在四个方向上的侧视图。
具体的，所述软性线路板18中设有用于收容所述液滴喷孔组件3的通孔19，且所述软性线路板19表面设有若干第一连接焊垫(未图示)及若干第二连接焊垫20，所述液滴喷孔组件3通过导线与所述第一连接焊垫连接，以通过所述软性线路板18将所述汽化部件5与外部控制器连接。所述液滴喷孔组件3可以通过标准的打线(Wire Bond)工艺与所述第一连接焊垫连接。
作为示例，所述软性线路板18通过胶粘方式与所述基座11连接，如图6a所示，所述基座11表面设有用于防止胶水流到所述液滴喷孔组件3上的沟道21。所述沟道21的数量为多个，分布于所述液滴喷孔组件3的四周。所述沟道21的排布方式可以根据需要进行调整，不限于图6a所呈现的方式。
具体的，所述液滴形成组件1也可以通过胶粘方式固定于所述软性线路板18上。如图5b所示，所述液滴形成组件1背面设有一凸台31，这种设计可以保证所述液滴形成组件1与所述软性线路板18之间牢固粘接。
具体的，如图10a及图10b所示，所述软性线路板18中设有至少两个定位穿孔22，如图6a所示，所述基座11表面设有与所述定位穿孔22位置相对应的定位凸起23，所述定位穿孔22与所述定位凸起23相互配合，有助于所述软性线路板18在所述基座11上的精确定位。
具体的，所述数字PCR系统还包括一控制器，请参阅图11，显示为所述控制器24的立体结构示意图，所述控制器24包括控制器外壳25及位于所述控制器外壳25内的控制器电路板，所述控制器外壳25具有用于放置所述基座11的承载部26，所述承载部26表面设有若干与所述控制器电路连接板连接的电路连接导电针27(也称为Pin)，所述电路连接导电针27与所述软性线路板18上的所述第二连接焊垫30的位置相对应。
具体的，所述控制器24通过软性线路板18与所述液滴喷孔组件3相连通过控制所述加热部件的发热时间、发热次数及发热间隔时间来控制所述数字PCR液滴的形成速度。其中，所述控制器24的控制电路可以采用现有的电路结构。
具体的，如图9所示，所述基座的一端设置有至少一个限位槽13，如图11所示，所述控制器外壳25设置有至少一个与所述限位槽13相对应的限位件29。
进一步的，如图8及图9所示，所述基座还设置有一限位通孔7，所述限位通孔7贯穿所述基座的正面及背面，如图11所示，所述控制器外壳25设置有与所述限位通孔7相对应的限位件30。
本发明的数字PCR系统可用于数字PCR液滴的形成，液滴的快速形成依赖于液滴喷孔内的汽化部件对纳米级厚度液体层的瞬间汽化，从而将液滴喷孔中的数字PCR溶液快速推入 液滴形成油中以形成数字PCR液滴，相比于市面上每秒钟100个液滴的形成速度，本发明中的液滴形成技术可以实现大于1000个每秒的液滴形成速度。相比于油相与水相共同运动产生液滴的方法，本发明的技术方案中的油相是静态的，因此油相的消耗量被大大减少，减少了50％左右的油相用量，具有高效的数字PCR油利用率。
实施例二
本发明还提供一种数字PCR液滴形成方法，包括以下步骤：采用汽化部件使数字PCR溶液汽化并快速推入液滴形成油中，以形成数字PCR液滴。
作为示例，使用热泡技术进行高速数字PCR液滴形成，所述汽化部件包括加热部件，通过加热所述数字PCR溶液液体层使其汽化。
具体的，通过控制所述加热部件的发热时间、发热次数及发热间隔时间来控制所述数字PCR液滴的形成速度。利用本发明的数字PCR液滴形成方法可以达到大于1000个/秒的数字PCR液滴形成速度。
作为示例，所述数字PCR液滴形成方法包括以下步骤：
S1：向PCR试剂腔室内注入数字PCR溶液，使数字PCR溶液进入与所述PCR试剂腔室连通的液滴喷孔，形成数字PCR溶液液体层；
S2：向液滴收集槽中添加液滴形成油；
S3：采用汽化部件使所述液体层汽化并快速推入所述液滴收集槽中的所述液滴形成油中，以形成所述数字PCR液滴。
具体的，所述液体层的厚度为纳米级，且大于0.2nm，本实施例中，所述液体层的厚度范围优选为0.2nm～30000nm。
请参阅图12，显示为利用本发明的数字PCR系统形成的数字PCR液滴的光学显微镜图，可见，形成的数字PCR液滴形态对称，均匀。
本发明的数字PCR系统及数字PCR液滴形成方法可以满足所有数字PCR生化试剂的使用。由于许多的生物标志性分子在血液中的浓度非常低(如循环肿瘤DNA在每2ml血液中只有3个DNA分子)，而本发明的数字PCR系统及数字PCR液滴形成方法具有液滴形成数量不受使用油量的限制的特点和高速的特点，使得这类检测在数字PCR的应用成为了可能。
综上所述，本发明的数字PCR系统及数字PCR液滴形成方法使用热泡技术进行高速数字PCR液滴形成，液滴的快速形成依赖于液滴喷孔内的汽化部件对纳米级厚度液体层的瞬间加热汽化，从而将液滴喷孔中的数字PCR溶液快速推入液滴形成油中以形成数字PCR液滴， 相比于市面上每秒钟100个液滴的形成速度，本发明中的液滴形成技术可以实现大于1000个每秒的液滴形成速度。相比于油相与水相共同运动产生液滴的方法，本发明的技术方案中的油相是静态的，因此油相的消耗量被大大减少，减少了50％左右的油相用量，具有高效的数字PCR油利用率。所以，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (28)

  1. 一种数字PCR系统，其特征在于，包括：
    至少一个液滴形成组件，包括至少一个液滴收集槽；
    液滴喷孔组件，连接于所述液滴形成组件下方，包括若干液滴喷孔，所述液滴喷孔自所述液滴喷孔组件的上表面开口，并往所述液滴喷孔组件的下表面方向延伸，但未贯穿所述液滴喷孔组件的下表面，所述液滴喷孔与所述液滴收集槽连通，且所述液滴喷孔内设有汽化部件，用于使所述液滴喷孔中的数字PCR溶液液体层汽化并快速推入所述液滴收集槽中的液滴形成油中，以形成数字PCR液滴。
  2. 根据权利要求1所述的数字PCR系统，其特征在于：所述液滴喷孔组件包括热泡打印芯片。
  3. 根据权利要求1所述的数字PCR系统，其特征在于：所述汽化部件设置于所述液滴喷孔的底面或侧壁。
  4. 根据权利要求1所述的数字PCR系统，其特征在于：所述液滴喷孔的开口形状包括圆形、椭圆形、多边形中的任意一种。
  5. 根据权利要求1所述的数字PCR系统，其特征在于：所述汽化部件包括加热部件，通过加热所述数字PCR溶液液体层使其汽化。
  6. 根据权利要求5所述的数字PCR系统，其特征在于：所述加热部件包括至少一层金属层。
  7. 根据权利要求1所述的数字PCR系统，其特征在于：所述液滴收集槽底部设有通槽，所述通槽暴露出多个所述液滴喷孔。
  8. 根据权利要求1所述的数字PCR系统，其特征在于：所述PCR系统还包括至少一个用于储存数字PCR溶液的PCR试剂腔室，所述液滴喷孔组件中设有流道，所述液滴喷孔通过所述流道与所述PCR试剂腔室连通。
  9. 根据权利要求8所述的数字PCR系统，其特征在于：所述流道包括至少一条主流道及与所述主流道连接的多条支流道，每个所述液滴喷孔分别与一条所述支流道连接。
  10. 根据权利要求8所述的数字PCR系统，其特征在于：所述数字PCR系统还包括基座，所述PCR试剂腔室设置于所述基座中，所述液滴喷孔组件连接于所述基座上方。
  11. 根据权利要求10所述的数字PCR系统，其特征在于：所述基座包括第一基座组件与第二基座组件，所述PCR试剂腔室包括PCR试剂上腔室与PCR试剂下腔室，所述PCR试剂上腔室自所述第一基座组件的上表面开口，并贯穿所述第一基座组件的下表面，所述PCR试剂下腔室自所述第二基座组件的上表面开口，并向所述第二基座组件的下表面方向延伸，但未贯穿所述第二基座组件的下表面，所述PCR试剂上腔室与PCR试剂下腔室连通且部分交迭。
  12. 根据权利要求11所述的数字PCR系统，其特征在于：所述第二基座组件的下表面设有至少一个数字PCR溶液注入孔，所述数字PCR溶液注入孔与所述PCR试剂下腔室连通。
  13. 根据权利要求11所述的数字PCR系统，其特征在于：所述PCR试剂下腔室包括第一端及第二端，所述数字PCR溶液注入孔在所述第一端处与所述PCR试剂下腔室连通，所述PCR试剂下腔室在所述第二端处与所述PCR试剂上腔室连通，所述PCR试剂下腔室的尺寸自所述第一端至所述第二端方向首先逐渐增大，然后逐渐减小。
  14. 根据权利要求11所述的数字PCR系统，其特征在于：所述第二基座组件的下表面设有至少一个排气孔，所述排气孔通过气道与所述PCR试剂上腔室连通，所述气道自所述第二基座组件的上表面开口，并向所述第二基座组件的下表面方向延伸，但未贯穿所述第二基座组件的下表面。
  15. 根据权利要求11所述的数字PCR系统，其特征在于：所述第一基座组件通过胶粘方式固定于所述第二基座组件上方。
  16. 根据权利要求10所述的数字PCR系统，其特征在于：所述数字PCR系统还包括软性线路板，所述软性线路板连接于所述基座上方，所述软性线路板中设有用于收容所述液滴喷孔组件的通孔，且所述软性线路板表面设有若干第一连接焊垫及若干第二连接焊垫，所述液滴喷孔组件通过导线与所述第一连接焊垫连接。
  17. 根据权利要求16所述的数字PCR系统，其特征在于：所述软性线路板通过胶粘方式与 所述基座连接，所述基座表面设有用于防止胶水流到所述液滴喷孔组件上的沟道。
  18. 根据权利要求16所述的数字PCR系统，其特征在于：所述软性线路板中设有至少两个定位穿孔，所述基座表面设有与所述定位穿孔位置相对应的定位凸起。
  19. 根据权利要求16所述的数字PCR系统，其特征在于：所述数字PCR系统还包括一控制器，所述控制器包括控制器外壳及位于所述控制器外壳内的控制器电路板，所述控制器外壳具有用于放置所述基座的承载部，所述承载部表面设有若干与所述控制器电路连接板连接的电路连接导电针，所述电路连接导电针与所述第二连接焊垫的位置相对应。
  20. 根据权利要求19所述的数字PCR系统，其特征在于：所述基座的一端设置有至少一个限位槽，所述控制器外壳设置有至少一个与所述限位槽相对应的限位件。
  21. 根据权利要求19所述的数字PCR系统，其特征在于：所述基座设置有一限位通孔，所述限位通孔贯穿所述基座的正面及背面，所述控制器外壳设置有与所述限位通孔相对应的限位件。
  22. 根据权利要求10所述的数字PCR系统，其特征在于：所述基座的材质包括塑料、玻璃中的任意一种。
  23. 一种数字PCR液滴形成方法，其特征在于，包括以下步骤：采用汽化部件使数字PCR溶液汽化并快速推入液滴形成油中，以形成数字PCR液滴。
  24. 根据权利要求23所述的数字PCR液滴形成方法，其特征在于：所述汽化部件包括加热部件，通过加热所述数字PCR溶液液体层使其汽化。
  25. 根据权利要求24所述的数字PCR液滴形成方法，其特征在于：通过控制所述加热部件的发热时间、发热次数及发热间隔时间来控制所述数字PCR液滴的形成速度。
  26. 根据权利要求23所述的数字PCR液滴形成方法，其特征在于，包括以下步骤：
    向PCR试剂腔室内注入数字PCR溶液，使数字PCR溶液进入与所述PCR试剂腔室连通的液滴喷孔，形成数字PCR溶液液体层；
    向液滴收集槽中添加液滴形成油；
    采用所述汽化部件使所述液体层汽化并快速推入所述液滴收集槽中的所述液滴形成油中，以形成所述数字PCR液滴。
  27. 根据权利要求23所述的数字PCR液滴形成方法，其特征在于：所述液体层的厚度范围是0.2nm～30000nm。
  28. 根据权利要求23所述的数字PCR液滴形成方法，其特征在于：所述数字PCR液滴的形成速度大于1000个/秒。
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