WO2020034479A1 - 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
WO2020034479A1
WO2020034479A1 PCT/CN2018/116929 CN2018116929W WO2020034479A1 WO 2020034479 A1 WO2020034479 A1 WO 2020034479A1 CN 2018116929 W CN2018116929 W CN 2018116929W WO 2020034479 A1 WO2020034479 A1 WO 2020034479A1
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Prior art keywords
droplet
digital pcr
pcr system
forming
chamber
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PCT/CN2018/116929
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English (en)
French (fr)
Inventor
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 US16/622,939 priority Critical patent/US11344892B2/en
Publication of WO2020034479A1 publication Critical patent/WO2020034479A1/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
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/1844—Means for temperature control using fluid heat transfer medium using fans

Definitions

  • the invention belongs to the field of biomedicine, in particular to the field of disease detection, and relates to an integrated in-situ digital PCR system and a droplet formation method.
  • 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 an integrated in-situ digital PCR system and a droplet formation method, which are used to solve the problems of slow droplet formation speed, small flux and complicated operation in the prior art The problem of low PCR oil utilization.
  • the present invention provides a digital PCR system, including:
  • the droplet forming component includes a heat conducting plate and a cover plate, and one side of the cover plate is provided with at least one inverted U-shaped step, and the heat conducting plate, the cover plate and the inverted U-shaped step together form a bottom opening.
  • 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 forming chamber, and a vaporization member is provided in the liquid droplet ejection hole. It is used to vaporize the liquid layer of the digital PCR solution in the droplet spray hole and quickly push it into the droplet-forming oil in the droplet-forming chamber to form digital PCR droplets.
  • the droplet ejection hole assembly includes a thermal bubble printing chip.
  • the height of the inverted U-shaped step is less than twice the diameter of the digital PCR droplets to be formed, so that the obtained digital PCR droplets are tiled into a layer structure in the droplet formation chamber.
  • a side surface of the heat conducting plate facing the cover plate has a boss provided along an outer edge of the inverted U-shaped step.
  • the heat conducting plate gradually extends outward near the opening of the droplet forming chamber to form a slope to expand the size of the opening of the droplet forming chamber.
  • the droplet formation assembly further includes at least one droplet formation oil injection hole, the droplet formation oil injection hole penetrates the heat conduction plate and communicates with the droplet formation chamber.
  • the droplet formation assembly further includes at least one droplet formation chamber exhaust hole, and the droplet formation chamber exhaust hole passes through the heat conducting plate and communicates with the droplet formation chamber.
  • 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.
  • 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, and the PCR reagent chamber is opened from the upper surface of the base and extends toward the lower surface of the base, but does not pass through the base of the base.
  • the droplet spray hole assembly is connected to the upper surface of the base and covers the opening of the PCR reagent chamber.
  • At least one digital PCR solution injection hole is provided on the lower surface of the base, and the digital PCR solution injection hole is in communication with the PCR reagent chamber.
  • At least one PCR reagent chamber exhaust hole is provided on the lower surface of the base, and the PCR reagent chamber exhaust hole is in communication with the PCR reagent chamber.
  • 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.
  • the surface of the base is provided with at least one channel for preventing glue from flowing onto the droplet ejection hole assembly, and the channel is distributed on the outer periphery of the droplet ejection 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.
  • a plurality of circuit-connecting conductive pins connected to the controller circuit connection board on the surface of the bearing-portion, and the circuit-connecting conductive pins correspond to the positions of the second connection pads.
  • 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 controller further includes a machine cover, and the machine cover is connected to the controller housing for covering the base.
  • the digital PCR system further includes an external semiconductor refrigerator for heating or cooling the droplet forming chamber.
  • the external semiconductor refrigerator has a fan.
  • the digital PCR system further includes an external temperature sensor for testing the temperature of the droplet formation chamber.
  • the digital PCR system is further configured with an optical detection system for collecting and detecting PCR signals without transferring a sample.
  • the cover plate is made of transparent material.
  • the invention also provides a digital PCR droplet formation method, which includes the following steps:
  • Adding droplet formation oil to the droplet formation chamber wherein the droplet formation chamber is surrounded by a heat conducting plate, a cover plate, and an inverted U-shaped step provided on one side of the cover plate;
  • a vaporizing member is used to vaporize the liquid layer and quickly push it into the droplet-forming oil in the droplet-forming chamber to form the digital PCR droplet.
  • the vaporization member includes a heating member, and the liquid layer is vaporized by heating the liquid layer.
  • 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 thickness of the liquid layer ranges from 0.2 nm to 30,000 nm.
  • the thickness of the droplet formation chamber is less than twice the diameter of the digital PCR droplets to be formed, so that the resulting digital PCR droplets are tiled into a layer structure within the droplet formation chamber.
  • the PCR reagent chamber is filled with droplet formation oil.
  • an external semiconductor refrigerator is used to raise or lower the temperature of the droplet forming chamber.
  • 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.
  • An external semiconductor refrigerator can be used to precisely control the temperature of the droplet formation chamber, thus realizing in-situ temperature-controlled PCR.
  • the integrated optical system enables detection without sample transfer. This not only reduces the operation time, but also improves the detection accuracy by reducing human error.
  • the in situ digital PCR droplets can be tiled into a layer structure.
  • FIG. 1 is a schematic diagram of the three-dimensional structure of the digital PCR system of the present invention.
  • FIG. 2 is a top view of a digital PCR system according to the present invention.
  • FIG. 3 shows a bottom view of the digital PCR system of the present invention.
  • 4-7 are side views of the digital PCR system of the present invention.
  • FIG. 8 is a schematic diagram showing the exploded structure of the digital PCR system of the present invention.
  • FIG. 9 is a schematic diagram of a three-dimensional structure of a cover plate in the digital PCR system of the present invention.
  • FIG. 10 is a schematic diagram of the three-dimensional structure of the heat conducting plate in the digital PCR system of the present invention.
  • FIG. 11 is a schematic diagram of the three-dimensional structure of the droplet formation module in the digital PCR system of the present invention.
  • FIG. 12 is a top view of a droplet formation module in the digital PCR system of the present invention.
  • FIG. 13 is a bottom view of a droplet formation module in the digital PCR system of the present invention.
  • 14 to 17 are side views of a droplet forming module in the digital PCR system of the present invention.
  • FIG. 18 is a schematic diagram of the front three-dimensional structure of the combination of the liquid droplet ejection hole assembly and the flexible circuit board in the digital PCR system of the present invention.
  • FIG. 19 is a top view of a combination of a liquid droplet ejection hole assembly and a flexible circuit board in the digital PCR system of the present invention.
  • FIG. 20 is a bottom view of a combination of a liquid droplet ejection hole assembly and a flexible circuit board in the digital PCR system of the present invention.
  • 21 to 24 are side views of a combination of a liquid droplet ejection hole assembly and a flexible circuit board in the digital PCR system of the present invention.
  • FIG. 25 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. 26 is a partial cross-sectional view of a droplet ejection hole assembly in the digital PCR system of the present invention.
  • FIG. 27 is a schematic diagram of the front three-dimensional structure of the base in the digital PCR system of the present invention.
  • FIG. 28 is a top view of a base in the digital PCR system of the present invention.
  • FIG. 29 is a bottom view of a base in the digital PCR system of the present invention.
  • 30-33 are side views of a base in the digital PCR system of the present invention.
  • FIG. 34 is a schematic diagram of the three-dimensional structure of the controller in the digital PCR system of the present invention.
  • FIG. 35 is a top view of the controller after the cover is removed in the digital PCR system of the present invention.
  • FIG. 36 is a bottom view of the controller shown in the digital PCR system of the present invention after the controller housing bottom plate is removed.
  • FIG. 37 is a schematic diagram showing that an external semiconductor refrigerator is set in a controller housing in the digital PCR system of the present invention.
  • FIG. 38 shows an optical microscope image of a digital PCR droplet formed using the digital PCR system of the present invention.
  • FIG. 39 is a fluorescence diagram of a digital PCR droplet formed using the digital PCR system of the present invention.
  • Oil droplets form an oil injection hole
  • FIG. 1 is a schematic diagram of the three-dimensional structure of the digital PCR system
  • FIG. 2 is a top view of the digital PCR system
  • FIG. A bottom view of the digital PCR system is described
  • FIGS. 4, 5, 6, and 7 are side views of the digital PCR system in four directions.
  • FIG. 8 is a schematic diagram of the exploded structure of the digital PCR system. It can be seen that the digital PCR system includes a droplet forming component 1 and a droplet ejection hole component 5, wherein the droplet ejection hole component 5 is connected to Below the liquid droplet forming component 1.
  • the droplet forming component 1 includes a heat conducting plate 2 and a cover plate 3.
  • FIGS. 9 and 10 wherein FIG. 9 is a schematic diagram of the three-dimensional structure of the cover plate 3, and FIG. 10 is a schematic diagram of the three-dimensional structure of the heat conductive plate 2.
  • FIG. 9 is a schematic diagram of the three-dimensional structure of the cover plate 3
  • FIG. 10 is a schematic diagram of the three-dimensional structure of the heat conductive plate 2.
  • at least one inverted U-shaped step 4 is provided on one side of the cover plate 3.
  • FIG. 11 is a schematic view showing the three-dimensional structure of the droplet forming component 1 by combining the heat conducting plate 2 and the cover plate 3, and FIG. 12 is the droplet forming component 1.
  • FIG. 13 is a bottom view of the droplet formation module 1
  • FIGS. 14, 15, 16, and 17 are side views of the droplet formation module 1 in four directions.
  • the heat conducting plate 2, the cover plate 3, and the inverted U-shaped step 4 together form a liquid droplet forming chamber with a bottom opening.
  • the number of the inverted U-shaped steps 4 is taken as an example, and accordingly, the number of the droplet forming chambers is also one. In other embodiments, the number of the inverted U-shaped steps 4 may be multiple, so as to construct multiple droplet forming chambers.
  • the height of the inverted U-shaped step 4 is less than twice the diameter of the digital PCR droplets to be formed, so that the obtained digital PCR droplets are tiled into a layer in the droplet formation chamber.
  • the inverted U-shaped step 4 is formed by connecting three segments of straight steps.
  • the segments of the inverted U-shaped step 4 may not be limited to straight lines.
  • the inverted U-shaped step 4 may be a combination of a straight step and a curved step, and the protection scope of the present invention should not be excessively limited here.
  • the inverted U-shaped step 4 may be obtained by performing processes such as photolithography and etching on the cover plate 3, and may also be double-sided adhesive with an appropriate thickness. There is a preset distance between the inverted U-shaped step 4 and the outer edge of the cover plate 3. If an adhesive connection is used between the cover plate 3 and the heat conductive plate 2, the inverted U-shaped step 4 The peripheral area can be used for dispensing.
  • the material of the cover plate 3 includes, but is not limited to, any one of transparent or opaque plastic and glass, and the cover plate 3 may also be made of a metal material. In this embodiment, the cover plate 3 is preferably made of transparent material.
  • a side surface of the heat conductive plate 2 facing the cover plate 3 has a boss 8 provided along an outer edge of the inverted U-shaped step 4, and the boss 8 faces the cover plate.
  • the bonding of 3 plays a positioning role.
  • the height of the boss 8 is equivalent to the thickness of the cover plate 3.
  • the absolute height of the boss 8 is not important and can be adjusted as needed.
  • the heat conducting plate 2 gradually extends outward near the opening of the droplet forming chamber to form a slope 9 to enlarge the opening size of the droplet forming chamber.
  • the slope 9 can reserve a space for the ejected droplets to prevent the ejected droplets from colliding with the formed droplets at high speed.
  • the droplet forming assembly further includes at least one droplet forming oil injection hole 10, the droplet forming oil injection hole 10 penetrates the heat conducting plate 2 and forms a cavity with the droplet. Room connected.
  • the droplet-forming oil injection hole 10 is preferably disposed at the slope 9.
  • the droplet forming assembly further includes at least one droplet forming chamber exhaust hole 11, and the droplet forming chamber exhaust hole 11 passes through the heat conducting plate 2 and communicates with the heat conducting plate 2.
  • the droplet formation chamber communicates.
  • the droplet ejection hole assembly 5 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 5 may use an existing thermal bubble printing chip.
  • FIG. 18 is a schematic diagram showing the front three-dimensional structure of the combination of the liquid droplet ejection hole assembly 5 and the flexible circuit board 18, and FIG.
  • a plan view of the combination of the flexible circuit board 18 is shown in FIG. 20, and a bottom view of the combination of the droplet ejection hole assembly 5 and the flexible circuit board 18 is shown in FIG. 21, FIG. 22, FIG. 23 and FIG. Side views of the liquid droplet ejection hole assembly 5 and 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 5, and the surface of the flexible circuit board 18 is provided with a plurality of first connection pads (not shown). And a plurality of second connection pads 20, the liquid droplet ejection hole assembly 5 is connected to the first connection pads through wires, so as to control the liquid droplet ejection hole assembly 5 to the outside through the flexible circuit board 18 ⁇ ⁇ Connected.
  • the droplet ejection hole assembly 5 may be connected to the first connection pad by a standard wire bonding process.
  • FIG. 25 is a schematic diagram of the three-dimensional structure of the droplet ejection hole assembly 5, and FIG. 26 is a partial cross-sectional view of the droplet ejection hole assembly 5.
  • the liquid droplet ejection hole assembly 5 includes a plurality of liquid droplet ejection holes 6, and the liquid droplet ejection holes 6 communicate with the liquid droplet forming chamber.
  • the droplet ejection holes 6 are arranged in two rows, and the droplet ejection holes in each row are evenly distributed.
  • the droplet spray holes 6 may also be arranged in other ways, and the protection scope of the present invention should not be excessively limited here. As shown in FIG.
  • the droplet ejection hole 6 is opened from the upper surface of the droplet ejection hole assembly 5 and extends toward the lower surface of the droplet ejection hole assembly 5, but does not penetrate the droplet.
  • the opening shape of the droplet ejection hole 5 includes, but is not limited to, any one of a circle, an oval, and a polygon.
  • a vaporization member 7 is provided in the droplet ejection hole 6 to vaporize the liquid layer of the digital PCR solution in the droplet ejection hole 6 and quickly push it into the droplet to form.
  • the droplets in the chamber form oil to form digital PCR droplets.
  • the volume of the droplet ejection holes 6 determines the volume of the digital PCR droplets to be formed.
  • the vaporization member 7 is disposed on the bottom surface of the droplet ejection hole 6, and the vaporization member 7 may be a heating member that vaporizes the liquid layer of the digital PCR solution by heating it.
  • 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 7 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 6.
  • the vaporization member 7 may also be disposed on a side wall of the droplet ejection hole 6, 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 12 for storing a digital PCR solution.
  • a flow channel is provided in the droplet ejection hole assembly 5, and the droplet ejection hole 6 communicates with the PCR reagent chamber 12 through the flow channel.
  • the flow channel includes at least one main flow channel 13 and a plurality of branch flow channels 14 connected to the main flow channel, and each of the droplet ejection holes 6 is respectively connected to one of the branch flow channels 14.
  • 20 and FIG. 25 show a case where the droplet ejection hole assembly 5 includes a main channel 13.
  • the main channel may be correspondingly plural.
  • a material for constructing the flow channel and the droplet discharge hole 6 includes, but is not limited to, silicon, polymer, photoresist, and the like.
  • the digital PCR system further includes a base 15, and the PCR reagent chamber 12 is disposed in the base 15.
  • a material of the base 15 includes, but is not limited to, any one of transparent or opaque plastic and glass, and the base 15 may also be made of a metal material.
  • FIG. 27 is a schematic diagram of the front three-dimensional structure of the base
  • FIG. 28 is a top view of the base
  • FIG. 29 is a bottom view of the base.
  • 31, 32, and 33 are side views of the base in four directions.
  • the PCR reagent chamber 12 is opened from the upper surface of the base 15 and extends toward the lower surface of the base 15 but does not penetrate the lower surface of the base 15 and the droplets
  • the nozzle assembly 5 is connected to the upper surface of the base 15 and covers the opening of the PCR reagent chamber 12.
  • At least one digital PCR solution injection hole 16 is provided on the lower surface of the base 15, and the digital PCR solution injection hole 16 is in communication with the PCR reagent chamber.
  • At least one PCR reagent chamber exhaust hole 17 is provided on the lower surface of the base 15, and the PCR reagent chamber exhaust hole 17 is in communication with the PCR reagent chamber.
  • the flexible circuit board 18 is connected above the base 15.
  • the flexible circuit board 18 is connected to the base 15 by an adhesive method.
  • the surface of the base 15 is further provided with at least one channel 21 for preventing glue from flowing onto the droplet ejection hole assembly 5, and the channel 21 It is distributed on the periphery of the droplet ejection hole assembly 5.
  • the surface of the base 15 has a sunken platform 35 for receiving a flexible circuit board, and four corners of the sunken platform 35 have arc-shaped extension spaces, and the sunken type
  • the protrusions 36 around the platform 35 play a positioning role when the flexible circuit board is adhered to the surface of the sunken platform 35.
  • the flexible circuit board 18 is provided with at least two positioning holes 22, and as shown in FIG. 28, the surface of the base 15 is provided with positioning protrusions corresponding to the positions of the positioning holes 22 twenty three.
  • the digital PCR system further includes a controller 24.
  • FIG. 34 is a schematic diagram showing the three-dimensional structure of the controller 24.
  • the controller 24 includes a controller housing 25 and a controller housing 25. Controller circuit board (not shown).
  • the controller 24 further includes a machine cover 32, which is connected to the controller housing 25 for covering the base 15 to provide a light-shielding environment for the PCR reaction.
  • FIG. 35 is a top view of the controller shown after the cover 32 is removed. It can be seen that the controller housing 25 has a bearing portion 26 for placing the base. There are a plurality of circuit connection conductive pins 27 (also referred to as Pins) connected to the controller circuit connection board, and the circuit connection conductive pins 27 correspond to the positions of the second connection pads 20.
  • circuit connection conductive pins 27 also referred to as Pins
  • FIG. 36 is a bottom view of the controller shown in the digital PCR system of the present invention after the bottom plate of the controller casing is removed.
  • the back of the carrying portion 26 is provided with a plurality of conductive pins 27 connected to the circuit.
  • Corresponding circuit board connection point 37 the controller circuit board can output signals to the circuit connection conductive pin 27 through the circuit board connection point 37.
  • one end of the base 15 is provided with at least one limiting groove 28.
  • the controller housing 25 is provided with at least one corresponding to the limiting groove 28. ⁇ ⁇ ⁇ 29 ⁇ 29 of the limit pieces.
  • the limiting member 29 may be a spring plunger.
  • the base 15 is provided with a limiting through hole 30 that penetrates the front and back of the base 15.
  • the controller is provided with a limiting piece 31 corresponding to the limiting through hole 30.
  • the limiting member 31 may be a spring plunger.
  • the digital PCR system further includes an external semiconductor refrigerator for heating or cooling the droplet forming chamber to provide reaction conditions at a specific temperature.
  • Semiconductor cooler (Thermo Electric Cooler, TEC for short) is made by using the Peltier effect of semiconductor materials.
  • the so-called Peltier effect refers to a phenomenon in which one end absorbs heat and the other end emits heat when a direct current passes through an electric couple composed of two semiconductor materials.
  • the heavily doped N-type and P-type bismuth telluride is mainly used as a semiconductor material for TEC.
  • the bismuth telluride element is electrically connected in series and generates heat in parallel.
  • TEC includes some P-type and N-type pairs (groups), which are connected together by electrodes and sandwiched between two ceramic electrodes; when a current flows through the TEC, the heat generated by the current will be transferred from one side of the TEC to On the other side, the "hot” and “cold” sides are generated on the TEC. This is the heating and cooling principle of the TEC.
  • the external semiconductor refrigerator 33 is disposed in the controller housing 25 and is located on a side of the heat conducting plate 2 of the droplet forming module 1.
  • the external semiconductor refrigerator also has a fan 34, wherein the fan 34 is used to cool the "hot" side when the external semiconductor refrigerator is cooling, and the external semiconductor refrigerator The cooler is used to warm up the "cold" side when it is heated.
  • a vent 34 is provided beside the fan 34.
  • the digital PCR system further includes an external temperature sensor for testing the temperature of the droplet formation chamber.
  • the external temperature sensor is disposed on a surface of the external semiconductor refrigerator that is in contact with the heat conducting plate.
  • the digital PCR system is further configured with an optical detection system for collecting and detecting PCR signals without transferring a sample.
  • the main parts of the optical system include: a fluorescent light source, a bright-field light source, a control circuit, an optically amplified lens group, a fluorescent filter, a CCD camera, a slide system for moving the lens, and a housing for avoiding light.
  • the photographic shooting area of the optical system is the entire area of the cover. This kind of shooting can be a single imaging or multiple shots and stitching pictures.
  • 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 vaporized 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 oil phase is reduced by about 50%.
  • An external semiconductor refrigerator can be used to precisely control the temperature of the droplet formation chamber, thus realizing in-situ temperature-controlled PCR.
  • the integrated optical system enables detection without sample transfer. This not only reduces the operation time, but also improves the detection accuracy by reducing human error.
  • In situ digital PCR droplets can be tiled into a single layer structure.
  • 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 spray hole communicating with the PCR reagent chamber to form a liquid layer;
  • the vaporization part is used to vaporize the liquid layer and quickly push it into the droplet-forming oil in the droplet-forming chamber to form the digital PCR droplet.
  • 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.
  • the thickness of the droplet formation chamber is less than twice the diameter of the digital PCR droplets to be formed, so that the obtained digital PCR droplets are tiled into a layer structure in the droplet formation chamber.
  • a seal such as a rubber stopper or a sealing film will be used to set the The droplet formation oil injection hole on the wall of the droplet formation chamber is closed.
  • the PCR reagent chamber is filled with droplet formation oil to make the PCR
  • the reagent chamber is in a filled state to prevent the formed droplets from flowing back to the PCR reagent chamber.
  • a seal can be used to seal the droplet formation chamber exhaust hole provided on the droplet formation chamber wall, the digital PCR solution injection hole provided on the PCR reagent chamber wall, and the PCR reagent chamber exhaust hole.
  • the sealing member includes, but is not limited to, a rubber stopper, a sealing film, a rubber ring, a cushion for sealing, and the like.
  • the seal can be made of soft plastic such as rubber or PDMS.
  • the droplet forming chamber is heated or cooled by using an external semiconductor refrigerator, and the droplet forming chamber is controlled to a temperature required for performing PCR to achieve in-situ temperature-controlled PCR. .
  • the integrated optical system can also be used to collect and detect PCR signals without transferring samples.
  • FIG. 38 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.
  • FIG. 39 shows a fluorescence chart of a digital PCR droplet formed using the digital PCR system of the present invention.
  • 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 oil phase is reduced by about 50%.
  • An external semiconductor refrigerator can be used to precisely control the temperature of the droplet formation chamber, thus realizing in-situ temperature-controlled PCR.
  • the integrated optical system enables detection without sample transfer. This not only reduces the operation time, but also improves the detection accuracy by reducing human error.
  • In situ digital PCR droplets can be tiled into a single layer structure. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

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Abstract

一种数字PCR系统及数字PCR液滴形成方法，该数字PCR系统包括液滴形成组件(1)及液滴喷孔组件(5)，所述液滴形成组件(1)包括导热板(2)及盖板(3)，所述盖板(3)的一侧面设有至少一个倒U型台阶(4)，所述导热板(2)、所述盖板(3)及所述倒U型台阶(4)共同围成底部开口的液滴形成腔室；所述液滴喷孔组件(5)连接于所述液滴形成组件(1)下方，包括若干液滴喷孔(6)，所述液滴喷孔(6)与所述液滴形成腔室连通，且所述液滴喷孔内设有汽化部件(7)，用于使所述液滴喷孔(6)中的数字PCR溶液液体层汽化并快速推入所述液滴形成腔室中的液滴形成油中，以形成数字PCR液滴。使用热泡技术进行高速数字PCR液滴形成，具有高效的数字PCR油利用率，并可进行原位PCR温控及原位数字PCR信号收集。

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，涉及一种一体化原位数字PCR系统及液滴形成方法。
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一体化原位数字PCR系统及液滴形成方法，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：
液滴形成组件，包括导热板及盖板，所述盖板的一侧面设有至少一个倒U型台阶，所述导热板、所述盖板及所述倒U型台阶共同围成底部开口的液滴形成腔室；
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形成腔室连通，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形成腔室中的液滴形成油中，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，所述倒U型台阶的高度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
可选地，所述导热板面向所述盖板的一侧面具有沿所述倒U型台阶外缘设置的凸台。
可选地，所述导热板靠近所述液滴形成腔室开口处逐渐向外延伸，形成一斜坡，以扩大所述液滴形成腔室的开口尺寸。
可选地，所述液滴形成组件还包括至少一个液滴形成油注入孔，所述液滴形成油注入孔穿通所述导热板，与所述液滴形成腔室连通。
可选地，所述液滴形成组件还包括至少一个液滴形成腔室排气孔，所述液滴形成腔室排气孔穿通所述导热板，与所述液滴形成腔室连通。
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试剂腔室自所述基座的上表面开口，并往所述基座的下表面方向延伸，但未贯穿所述基座的下表面，所述液滴喷孔组件连接于所述基座上表面，并覆盖所述PCR试剂腔室的开口。
可选地，所述基座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试剂腔室连通。
可选地，所述基座的下表面设有至少一个PCR试剂腔室排气孔，所述PCR试剂腔室排气孔与所述PCR试剂腔室连通。
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。
可选地，所述基座表面设有至少一条用于防止胶水流到所述液滴喷孔组件上的沟道，所述沟道分布于所述液滴喷孔组件的外周。
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，且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，所述控制器还包括一机盖，所述机盖与所述控制器外壳连接，用于遮盖所述基座。
可选地，所述数字PCR系统还包括用于加热或冷却所述液滴形成腔室的外置半导体制冷器。
可选地，所述外置半导体制冷器具有一风扇。
可选地，所述数字PCR系统还包括用于测试所述液滴形成腔室温度的外置温度传感器。
可选地，所述数字PCR系统还配置有光学检测系统，用于在不转移样品的情况下进行PCR信号收集检测。
可选地，所述盖板采用透明材质。
本发明还提供一种数字PCR液滴形成方法，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，形成液体层；
向液滴形成腔室中添加液滴形成油，其中，所述液滴形成腔室由导热板、盖板及设置于所述盖板的一侧面的倒U型台阶共同围成；
采用汽化部件使所述液体层汽化并快速推入所述液滴形成腔室中的所述液滴形成油中，以形成所述数字PCR液滴。
可选地，所述汽化部件包括加热部件，通过加热所述液体层使其汽化。
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，所述液体层的厚度范围是0.2nm～30000nm。
可选地，所述液滴形成腔室的厚度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
可选地，待所述PCR试剂腔室内的数字PCR溶液完全推入所述液滴形成腔室中形成数字PCR液滴后，将所述PCR试剂腔室用液滴形成油填充。
可选地，利用外置半导体制冷器对所述液滴形成腔室进行升温或降温。
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。
(3)可以利用外置半导体制冷器对液滴形成腔室进行精确的温控，因此实现了原位温控PCR。而且整合的光学系统可以在不转移样品的情况下进行检测。这既减少了操作时间，也通过减少人为误差提高了检测的准确性。
(4)原位数字PCR液滴可平铺成一层的结构。
附图说明
图1显示为本发明的数字PCR系统的立体结构示意图。
图2显示为本发明的数字PCR系统的俯视图。
图3显示为本发明的数字PCR系统的仰视图。
图4-图7显示为本发明的数字PCR系统的侧视图。
图8显示为本发明的数字PCR系统的分解结构示意图。
图9显示为本发明的数字PCR系统中盖板的立体结构示意图。
图10显示为本发明的数字PCR系统中导热板的立体结构示意图。
图11显示为本发明的数字PCR系统中液滴形成组件的立体结构示意图。
图12显示为本发明的数字PCR系统中液滴形成组件的俯视图。
图13显示为本发明的数字PCR系统中液滴形成组件的仰视图。
图14-图17显示为本发明的数字PCR系统中液滴形成组件的侧视图。
图18显示为本发明的数字PCR系统中液滴喷孔组件与软性线路板组合的正面立体结构示意图。
图19显示为本发明的数字PCR系统中液滴喷孔组件与软性线路板组合的俯视图。
图20显示为本发明的数字PCR系统中液滴喷孔组件与软性线路板组合的仰视图。
图21-图24显示为本发明的数字PCR系统中液滴喷孔组件与软性线路板组合的侧视图。
图25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26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27显示为本发明的数字PCR系统中基座的正面立体结构示意图。
图28显示为本发明的数字PCR系统中基座的俯视图。
图29显示为本发明的数字PCR系统中基座的仰视图。
图30-图33显示为本发明的数字PCR系统中基座的侧视图。
图34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35显示为本发明的数字PCR系统中机盖被拿掉后所呈现的控制器俯视图。
图36显示为本发明的数字PCR系统中控制器外壳底板被拿掉后所呈现的控制器仰视图。
图37显示为本发明的数字PCR系统中外置半导体制冷器设置于控制器外壳中的示意图。
图38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。
图39显示为利用本发明的数字PCR系统形成的数字PCR液滴的荧光图。
元件标号说明
1                     液滴形成组件
2                     导热板
3                     盖板
4                     倒U型台阶
5                     液滴喷孔组件
6                     液滴喷孔
7                     汽化部件
8                     凸台
9                     斜坡
10                    液滴形成油注入孔
11                    液滴形成腔室排气孔
12                    PCR试剂腔室
13                    主流道
14                    支流道
15                    基座
16                    数字PCR溶液注入孔
17                    PCR试剂腔室排气孔
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、31                 限位件
30                     限位通孔
32                     机盖
33                     外置半导体制冷器
34                     风扇
35                     下沉式平台
36                     凸起
37                     电路板连接点
38                     外壳支撑结构
39                     通风口
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1至图39。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1至图7，其中，图1显示为所述数字PCR系 统的立体结构示意图，图2显示为所述数字PCR系统的俯视图，图3显示为所述数字PCR系统的仰视图，图4、图5、图6及图7显示为所述数字PCR系统在四个方向上的侧视图。
请参阅图8，显示为所述数字PCR系统的分解结构示意图，可见，所述数字PCR系统包括液滴形成组件1及液滴喷孔组件5，其中，所述液滴喷孔组件5连接于所述液滴形成组件1下方。
具体的，液滴形成组件1包括导热板2及盖板3。请参阅图9及图10，其中，图9显示为所述盖板3的立体结构示意图，图10显示为所述导热板2的立体结构示意图。如图9所示，所述盖板3的一侧面设有至少一个倒U型台阶4。
请参阅图11至图17，其中，图11显示为所述导热板2及所述盖板3组合成所述液滴形成组件1的立体结构示意图，图12显示为所述液滴形成组件1的俯视图，图13显示为所述液滴形成组件1的仰视图，图14、图15、图16及图17显示为所述液滴形成组件1在四个方向上的侧视图。可见，所述导热板2、所述盖板3及所述倒U型台阶4共同围成底部开口的液滴形成腔室。本实施例中，所述倒U型台阶4的数量以一个为例，相应的，所述液滴形成腔室的数量也为一个。在其它实施例中，所述倒U型台阶4的数量也可以为多个，以构建多个液滴形成腔室。
具体的，所述倒U型台阶4的高度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
作为示例，如图9所示，所述倒U型台阶4由三段直线台阶连接而成，在其它实施例中，所述倒U型台阶4的各段也可以不局限于直线，例如所述倒U型台阶4可以由直线台阶和曲线台阶组合而成，此处不应过分限制本发明的保护范围。
作为示例，所述倒U型台阶4可以是对所述盖板3进行光刻、刻蚀等工艺得到，也可以是厚度合适的双面胶。所述倒U型台阶4与所述盖板3的外缘之间具有预设距离，若所述盖板3与所述导热板2之间采用胶粘连接方式，所述倒U型台阶4的外围区域可以用于点胶水。
具体的，所述盖板3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，所述盖板3还可以采用金属材质。本实施例中，所述盖板3优选采用透明材质。
作为示例，如图10所示，所述导热板2面向所述盖板3的一侧面具有沿所述倒U型台阶4外缘设置的凸台8，所述凸台8对所述盖板3的粘接起了定位作用。本实施例中，所述凸台8的高度与所述盖板3的厚度相当，当然，所述凸台8的绝对高度并不重要，可以根据需要进行调整。
作为示例，如图10所示，所述导热板2靠近所述液滴形成腔室开口处逐渐向外延伸，形成一斜坡9，以扩大所述液滴形成腔室的开口尺寸。所述斜坡9可以为喷射出的液滴预留空 间，防止高速喷出的液滴与已形成的液滴碰撞融合。
作为示例，如图10所示，所述液滴形成组件还包括至少一个液滴形成油注入孔10，所述液滴形成油注入孔10穿通所述导热板2，与所述液滴形成腔室连通。本实施例中，所述液滴形成油注入孔10优选设置于所述斜坡9处。
作为示例，如图10所示，所述液滴形成组件还包括至少一个液滴形成腔室排气孔11，所述液滴形成腔室排气孔11穿通所述导热板2，与所述液滴形成腔室连通。
作为示例，所述液滴喷孔组件5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5可采用现有的热泡打印芯片。
本实施例中，所述液滴喷孔组件5连接于一软性线路板18。请参阅图18至图24，其中，图18显示为所述液滴喷孔组件5与所述软性线路板18组合的正面立体结构示意图，图19显示为所述液滴喷孔组件5与所述软性线路板18组合的俯视图，图20显示为所述液滴喷孔组件5与所述软性线路板18组合的仰视图，图21、图22、图23及图24显示为所述液滴喷孔组件5与所述软性线路板18组合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5的通孔19，且所述软性线路板18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5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将所述液滴喷孔组件5与外部控制器连接。所述液滴喷孔组件5可以通过标准的打线(Wire Bond)工艺与所述第一连接焊垫连接。
请参阅图25及图26，其中，图25显示为所述液滴喷孔组件5的立体结构示意图，图26显示为所述液滴喷孔组件5的局部剖面图。如图25所示，所述液滴喷孔组件5包括若干液滴喷孔6，所述液滴喷孔6与所述液滴形成腔室连通。本实施例中，所述液滴喷孔6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6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。如图26所示，所述液滴喷孔6自所述液滴喷孔组件5的上表面开口，并往所述液滴喷孔组件5的下表面方向延伸，但未贯穿所述液滴喷孔组件5的下表面。作为示例，所述液滴喷孔5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。
具体的，如图26所示，所述液滴喷孔6内设有汽化部件7，用于使所述液滴喷孔6中的数字PCR溶液液体层汽化并快速推入所述液滴形成腔室中的液滴形成油中，以形成数字PCR液滴。其中，所述液滴喷孔6的容积决定了要形成的数字PCR液滴的体积。
作为示例，所述汽化部件7设置于所述液滴喷孔6的底面，所述汽化部件7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7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6底面积的0.5～2倍。在其它实施例中，所述汽化部件7也可以设置于所述液滴喷孔6的侧壁，此处不应过分限制本发明的保护范围。
如图8所示，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12。如图25所示，所述液滴喷孔组件5中设有流道，所述液滴喷孔6通过所述流道与所述PCR试剂腔室12连通。
作为示例，所述流道包括至少一条主流道13及与所述主流道连接的多条支流道14，每个所述液滴喷孔6分别与一条所述支流道14连接。图20及图25中显示的为所述液滴喷孔组件5包括一条主流道13的情形，在其它实施例中，当所述液滴形成腔室的数量为多个时，所述主流道13的数量也可以相应地为多个。
作为示例，构建所述流道和所述液滴喷孔6的材料包括但不限于硅、聚合物、光刻胶等。
具体的，如图8所示，所述数字PCR系统还包括基座15，所述PCR试剂腔室12设置于所述基座15中。作为示例，所述基座15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，所述基座15还可以采用金属材质。
请参阅图27至图33，其中，图27显示为所述基座的正面立体结构示意图，图28显示为所述基座的俯视图，图29显示为所述基座的仰视图，图30、图31、图32及图33显示为所述基座在四个方向上的侧视图。
具体的，所述PCR试剂腔室12自所述基座15的上表面开口，并往所述基座15的下表面方向延伸，但未贯穿所述基座15的下表面，所述液滴喷孔组件5连接于所述基座15上表面，并覆盖所述PCR试剂腔室12的开口。
具体的，所述基座15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试剂腔室连通。所述基座15的下表面设有至少一个PCR试剂腔室排气孔17，所述PCR试剂腔室排气孔17与所述PCR试剂腔室连通。
具体的，所述软性线路板18连接于所述基座15上方。作为示例，所述软性线路板18通过胶粘方式与所述基座15连接。如图27及图28所示，本实施例中，所述基座15表面还设有至少一条用于防止胶水流到所述液滴喷孔组件5上的沟道21，所述沟道21分布于所述液滴喷孔组件5的外周。
本实施例中，所述基座15表面具有一用于收容软性线路板的下沉式平台35，且所述下沉式平台35的四角具有圆弧形的延伸空间，所述下沉式平台35四周的凸起36在所述软性线路板黏贴到所述下沉式平台35表面时起到定位作用。
如图19所示，所述软性线路板18中设有至少两个定位穿孔22，如图28所示，所述基座15表面设有与所述定位穿孔22位置相对应的定位凸起23。
具体的，所述数字PCR系统还包括一控制器24，请参阅图34，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(未图示)。本实施例中，所述控制器24还包括一机盖32，所述机盖32与所述控制器外壳25连接，用于遮盖所述基座15，为PCR反应提供一遮光环境。
请参阅图35，显示为所述机盖32被拿掉后所呈现的控制器俯视图，可见所述控制器外壳25具有用于放置所述基座的承载部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)，且所述电路连接导电针27与所述第二连接焊垫20的位置相对应。
请参阅图36，显示为本发明的数字PCR系统中控制器外壳底板被拿掉后所呈现的控制器仰视图，其中，所述承载部26背面设有若干与所述电路连接导电针27相对应的电路板连接点37，控制器电路板可通过所述电路板连接点37输出信号至所述电路连接导电针27。
具体的，如图27所示，所述基座15的一端设置有至少一个限位槽28，如图35所示，所述控制器外壳25设置有至少一个与所述限位槽28相对应的限位件29。所述限位件29可采用弹簧柱塞。
具体的，如图27所示，所述基座15设置有一限位通孔30，所述限位通孔30贯穿所述基座15的正面及背面，如图35所示，所述控制器外壳25设置有与所述限位通孔30相对应的限位件31。所述限位件31可采用弹簧柱塞。
具体的，所述数字PCR系统还包括用于加热或冷却所述液滴形成腔室的外置半导体制冷器，以提供特定温度的反应条件。半导体制冷器(Thermo Electric Cooler，简称TEC)是利用半导体材料的珀尔帖效应制成的。所谓珀尔帖效应，是指当直流电流通过两种半导体材料组成的电偶时，其一端吸热，一端放热的现象。重掺杂的N型和P型的碲化铋主要用作TEC的半导体材料，碲化铋元件采用电串联，并且是并行发热。TEC包括一些P型和N型对(组)，它们通过电极连在一起，并且夹在两个陶瓷电极之间；当有电流从TEC流过时，电流产生的热量会从TEC的一侧传到另一侧，在TEC上产生“热”侧和“冷”侧，这就是TEC的加热与制冷原理。
作为示例，请参阅图37，所述外置半导体制冷器33设置于控制器外壳25中，并位于所述液滴形成组件1的导热板2一侧。本实施例中，所述外置半导体制冷器还具有一风扇34，其中，所述风扇34在所述外置半导体制冷器制冷的时候用来给“热”侧降温，在所述外置半导体制冷器加热时用来给“冷”侧升温。如图37所示，所述风扇34旁还设置有通风口34。 图37中还显示了外壳支撑结构16。
具体的，所述数字PCR系统还包括用于测试所述液滴形成腔室温度的外置温度传感器。作为示例，所述外置温度传感器设置于所述外置半导体制冷器与所述导热板接触的表面。
进一步的，所述数字PCR系统还配置有光学检测系统，用于在不转移样品的情况下进行PCR信号收集检测。光学系统的主要部分包括：荧光光源、明场光源、控制电路、光学放大的镜片组、荧光滤光片、CCD相机、用来移动镜头的滑台系统，以及用来避光的外壳。光学系统的照相拍摄区域是盖板的整个面积。这种拍摄可以是一次成像或者多次拍摄并拼接图片。
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。可以利用外置半导体制冷器对液滴形成腔室进行精确的温控，因此实现了原位温控PCR。而且整合的光学系统可以在不转移样品的情况下进行检测。这既减少了操作时间，也通过减少人为误差提高了检测的准确性。原位数字PCR液滴可平铺成一层的结构。
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，形成液体层；
S2：向液滴形成腔室中添加液滴形成油，其中，所述液滴形成腔室由导热板、盖板及设置于所述盖板的一侧面的倒U型台阶共同围成；
S3：采用汽化部件使所述液体层汽化并快速推入所述液滴形成腔室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。
具体的，所述液滴形成腔室的厚度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
具体的，向液滴形成腔室中添加液滴形成油之后，并且在将液滴喷孔组件(例如热泡打印芯片)放入控制器前，采用胶塞或封口膜等密封件将设置于液滴形成腔室壁上的液滴形成油注入孔封闭。
具体的，待所述PCR试剂腔室内的数字PCR溶液完全推入所述液滴形成腔室中形成数字PCR液滴后，将所述PCR试剂腔室用液滴形成油填充，使所述PCR试剂腔室处于填充状态，以避免形成的液滴回流至所述PCR试剂腔室。随后可以采用密封件将设置于液滴形成腔室壁上的液滴形成腔室排气孔、设置于PCR试剂腔室壁上的数字PCR溶液注入孔及PCR试剂腔室排气孔密封。所述密封件包括但不限于胶塞、封口膜、胶圈环、密封用软垫等。所述密封件可以采用橡胶、PDMS等软塑料材质。
具体的，进行上述密封之后，利用外置半导体制冷器对所述液滴形成腔室进行升温或降温，将所述液滴形成腔室控制在进行PCR所需的温度，实现原位温控PCR。
具体的，还可以利用整合的光学系统可以在不转移样品的情况下进行PCR信号收集检测。
请参阅图38，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。
通过使用标准的数字PCR形成液滴后，通过原位的常规PCR温控反应，经过40个循环后，可见有荧光信号的阳性液滴。请参阅图39，显示为利用本发明的数字PCR系统形成的数字PCR液滴的荧光图。
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。可以利用外置半导体制冷器对液滴形成腔室进行精确的温控，因此实现了原位温控PCR。而且整合的光学系统可以在不转移样品的情况下进行检测。这既减少了操作时间，也通过减少人为误差提高了检测的准确性。原位数字PCR液滴可平铺成一层的结构。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 (37)

  1. 一种数字PCR系统，其特征在于，包括：
    液滴形成组件，包括导热板及盖板，所述盖板的一侧面设有至少一个倒U型台阶，所述导热板、所述盖板及所述倒U型台阶共同围成底部开口的液滴形成腔室；
    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形成腔室连通，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形成腔室中的液滴形成油中，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：所述倒U型台阶的高度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
  4. 根据权利要求1所述的数字PCR系统，其特征在于：所述导热板面向所述盖板的一侧面具有沿所述倒U型台阶外缘设置的凸台。
  5. 根据权利要求1所述的数字PCR系统，其特征在于：所述导热板靠近所述液滴形成腔室开口处逐渐向外延伸，形成一斜坡，以扩大所述液滴形成腔室的开口尺寸。
  6. 根据权利要求1所述的数字PCR系统，其特征在于：所述液滴形成组件还包括至少一个液滴形成油注入孔，所述液滴形成油注入孔穿通所述导热板，与所述液滴形成腔室连通。
  7. 根据权利要求1所述的数字PCR系统，其特征在于：所述液滴形成组件还包括至少一个液滴形成腔室排气孔，所述液滴形成腔室排气孔穿通所述导热板，与所述液滴形成腔室连通。
  8. 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。
  9. 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。
  10. 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。
  11. 根据权利要求10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。
  12. 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。
  13. 根据权利要求12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。
  14. 根据权利要求12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试剂腔室自所述基座的上表面开口，并往所述基座的下表面方向延伸，但未贯穿所述基座的下表面，所述液滴喷孔组件连接于所述基座上表面，并覆盖所述PCR试剂腔室的开口。
  15. 根据权利要求14所述的数字PCR系统，其特征在于：所述基座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试剂腔室连通。
  16. 根据权利要求14所述的数字PCR系统，其特征在于：所述基座的下表面设有至少一个PCR试剂腔室排气孔，所述PCR试剂腔室排气孔与所述PCR试剂腔室连通。
  17. 根据权利要求14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。
  18. 根据权利要求17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。
  19. 根据权利要求18所述的数字PCR系统，其特征在于：所述基座表面设有至少一条用于防止胶水流到所述液滴喷孔组件上的沟道，所述沟道分布于所述液滴喷孔组件的外周。
  20. 根据权利要求17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。
  21. 根据权利要求14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，且所述电路连接导电针与所述第二连接焊垫的位置相对应。
  22. 根据权利要求21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。
  23. 根据权利要求21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。
  24. 根据权利要求21所述的数字PCR系统，其特征在于：所述控制器还包括一机盖，所述机盖与所述控制器外壳连接，用于遮盖所述基座。
  25. 根据权利要求1所述的数字PCR系统，其特征在于：所述数字PCR系统还包括用于加热或冷却所述液滴形成腔室的外置半导体制冷器。
  26. 根据权利要求25所述的数字PCR系统，其特征在于：所述外置半导体制冷器具有一风扇。
  27. 根据权利要求25所述的数字PCR系统，其特征在于：所述数字PCR系统还包括用于测试所述液滴形成腔室温度的外置温度传感器。
  28. 根据权利要求1所述的数字PCR系统，其特征在于：所述数字PCR系统还配置有光学检测系统，用于在不转移样品的情况下进行PCR信号收集检测。
  29. 根据权利要求1所述的数字PCR系统，其特征在于：所述盖板采用透明材质。
  30. 一种数字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，形成液体层；
    向液滴形成腔室中添加液滴形成油，其中，所述液滴形成腔室由导热板、盖板及设置于所述盖板的一侧面的倒U型台阶共同围成；
    采用汽化部件使所述液体层汽化并快速推入所述液滴形成腔室中的所述液滴形成油中，以形成所述数字PCR液滴。
  31. 根据权利要求30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，通过加热所述液体层使其汽化。
  32. 根据权利要求31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。
  33. 根据权利要求30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。
  34. 根据权利要求30所述的数字PCR液滴形成方法，其特征在于：所述液滴形成腔室的厚度小于欲形成的数字PCR液滴的直径的2倍，以使得到的数字PCR液滴在所述液滴形成腔室内平铺成一层的结构。
  35. 根据权利要求30所述的数字PCR液滴形成方法，其特征在于：待所述PCR试剂腔室内的数字PCR溶液完全推入所述液滴形成腔室中形成数字PCR液滴后，将所述PCR试剂腔室用液滴形成油填充。
  36. 根据权利要求30所述的数字PCR液滴形成方法，其特征在于：利用外置半导体制冷器 对所述液滴形成腔室进行升温或降温。
  37. 根据权利要求30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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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090023189A1 (en) * 2007-05-18 2009-01-22 Applera Corporation Apparatus and methods for preparation of subtantially uniform emulsions containing a particle
CN104004652A (zh) * 2014-06-16 2014-08-27 东南大学 基于单喷头喷墨打印的数字pcr液滴阵列芯片的制作方法
CN106824313A (zh) * 2017-02-23 2017-06-13 中国科学院上海微系统与信息技术研究所 一种数字pcr芯片及其制备方法
CN107405633A (zh) * 2015-05-22 2017-11-28 香港科技大学 基于高深宽比诱导生成液滴的液滴发生器
CN108248219A (zh) * 2016-12-29 2018-07-06 上海新微技术研发中心有限公司 热泡喷墨打印头芯片及其制造方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4592060B2 (ja) * 2004-04-26 2010-12-01 キヤノン株式会社 Ｐｃｒ増幅反応装置、ならびに、該装置を利用するｐｃｒ増幅反応方法
EP2047910B1 (en) * 2006-05-11 2012-01-11 Raindance Technologies, Inc. Microfluidic device and method
US7909428B2 (en) * 2006-07-28 2011-03-22 Hewlett-Packard Development Company, L.P. Fluid ejection devices and methods of fabrication
CN105849275B (zh) * 2013-06-25 2020-03-17 普罗格诺西斯生物科学公司 检测样品中生物靶标的空间分布的方法和系统
US10486156B2 (en) * 2014-05-09 2019-11-26 Dh Technologies Development Pte. Ltd. Fluid transfer from digital microfluidic device
CN109072292A (zh) * 2016-02-22 2018-12-21 拜奥法尔防护有限责任公司 用于快速pcr的装置和方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090023189A1 (en) * 2007-05-18 2009-01-22 Applera Corporation Apparatus and methods for preparation of subtantially uniform emulsions containing a particle
CN104004652A (zh) * 2014-06-16 2014-08-27 东南大学 基于单喷头喷墨打印的数字pcr液滴阵列芯片的制作方法
CN107405633A (zh) * 2015-05-22 2017-11-28 香港科技大学 基于高深宽比诱导生成液滴的液滴发生器
CN108248219A (zh) * 2016-12-29 2018-07-06 上海新微技术研发中心有限公司 热泡喷墨打印头芯片及其制造方法
CN106824313A (zh) * 2017-02-23 2017-06-13 中国科学院上海微系统与信息技术研究所 一种数字pcr芯片及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, W.F.: "Inkjet Printing Based Droplet Generation for Integrated Online Digital Polymerase Chain Reaction", ANAL. CHEM., vol. 90, 29 March 2018 (2018-03-29), pages 5239 - 5334, XP055688683 *

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