WO2020007098A1 - 数字pcr芯片及包含它的液滴生成系统和检测系统 - Google Patents

数字pcr芯片及包含它的液滴生成系统和检测系统 Download PDF

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Publication number
WO2020007098A1
WO2020007098A1 PCT/CN2019/083435 CN2019083435W WO2020007098A1 WO 2020007098 A1 WO2020007098 A1 WO 2020007098A1 CN 2019083435 W CN2019083435 W CN 2019083435W WO 2020007098 A1 WO2020007098 A1 WO 2020007098A1
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Prior art keywords
liquid
droplet
chip
digital pcr
channel
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PCT/CN2019/083435
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English (en)
French (fr)
Inventor
李昂
周阳
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北京致雨生物科技有限公司
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Publication date
Priority claimed from CN201810737039.XA external-priority patent/CN110684650A/zh
Priority claimed from CN201810738786.5A external-priority patent/CN110684828A/zh
Application filed by 北京致雨生物科技有限公司 filed Critical 北京致雨生物科技有限公司
Priority to US17/257,236 priority Critical patent/US20210229101A1/en
Publication of WO2020007098A1 publication Critical patent/WO2020007098A1/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
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • 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
    • B01L3/50273—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 the means or forces applied to move the fluids
    • 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
    • B01L3/56—Labware specially adapted for transferring fluids
    • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
    • 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
    • C12M1/10—Apparatus for enzymology or microbiology rotatably mounted
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings
    • 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
    • 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
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
    • 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]

Definitions

  • the invention relates to a digital PCR chip, a droplet generation system and a detection system containing the same.
  • PCR polymerase chain reaction
  • Droplet Digital PCR (Droplet Digital PCR) technology is a water-in-oil droplet technology based on a microfluidic chip. It encapsulates a single DNA molecule into individual droplets through a water-in-oil structure and uses the inertness of the oil to achieve the DNA molecule They are isolated from each other, and each DNA molecule is restricted to be amplified separately in its own droplet to avoid competition from other sequences. After the DNA molecule is amplified under appropriate temperature conditions, by recording the total number of droplets and the number of droplets where fluorescence signals can be detected, the Poisson distribution algorithm can be used to accurately quantify the number of DNA copies.
  • micro liquid operation is to further divide the microliter liquid into microreaction systems with nanoliter or even picoliter volume.
  • One of the main technical branches of microreaction system generation is the formation of emulsified micro-droplets.
  • micro-droplet generation techniques have been reported in the literature, such as membrane emulsification, spray emulsification, micro-fluidic chip method, micro-pipeline injection / jet method, and the like.
  • the recent Chinese invention patent No. ZL201410655191.5 and the Chinese patent publication No. CN104815709A further optimize the method of generating emulsified micro-droplets through micro-channels. These methods of emulsifying micro-droplets each have certain disadvantages in practical applications.
  • the Chinese invention patent method with the patent number ZL201410655191.5 utilizes the interfacial energy and fluid shear force of the trace liquid at the gas-liquid phase interface to overcome the surface tension and adhesion of the liquid at the outlet of the microchannel, so that it flows out of the mouth of the microchannel
  • the droplets can smoothly escape from the microchannel and form a controllable droplet in a heterogeneous solution.
  • this method requires cutting movement of the micro-pipes on the liquid surface, and requires high-precision positioning of the start and end positions of the micro-pipes relative to the liquid surface, which is very difficult to realize in engineering.
  • CN104815709A uses the shear force generated by the circular or spiral uniform movement of the micro-pipe in the liquid to cut off the injected immiscible liquid to form droplets.
  • This method The size is greatly affected by changes in various system factors (such as the viscosity of the liquid, the temperature of the environment, the speed of the movement, the trajectory of the movement, etc.), and this error will accumulate with the increase in the number of droplets generated, so large quantities It is difficult to control the uniformity of the volume generated by the droplets.
  • the purpose of the present invention is to provide an improved digital PCR chip to solve one or more shortcomings of the existing PCR chip.
  • the invention also provides a digital PCR detection system and detection method based on a digital PCR chip, and a droplet generation system and a digital PCR detection method including the digital PCR chip and used for digital PCR detection.
  • a technical solution adopted by the present invention is: a digital PCR chip, which includes a chip body with a droplet storage cavity and a liquid inlet provided on the chip body.
  • the digital PCR chip is further
  • the chip body includes an accommodation cavity which is vertically arranged on the chip body and communicates with the liquid inlet, and a liquid discharge port provided on the chip body.
  • the chip body further includes separately separating the liquid inlet and the liquid inlet.
  • the accommodating cavity extends upward from the upper surface of the chip body, and the liquid inlet is located at the bottom of the accommodating cavity.
  • the accommodating cavity has a length of 2 to 30 mm, a width of 2 to 30 mm, and a height of 20 to 2000 um.
  • the accommodating cavity is integrally formed with the chip body, or the accommodating cavity is fixedly connected to the chip body.
  • the first channel is connected to the droplet storage cavity at an end portion; and / or, the second channel is connected to the droplet storage cavity at an end portion.
  • the first channel is located on one side of the droplet storage cavity; and / or, the second channel is located on one side of the droplet storage cavity.
  • the first inner passage and the second inner passage are divided on different sides of the droplet storage cavity.
  • the droplet storage chamber has a first communication port communicating with the first channel, and a second communication port communicating with the second channel, and the first communication port is in communication with the second channel.
  • the second communication port is divided on two opposite sides of the droplet storage cavity.
  • first communication port is directly opposite to the second communication port.
  • the droplet storage cavity has at least one arc-shaped chamfer, and the first communication port is provided at the arc-shaped chamfer.
  • the droplet storage cavity is a polygon having a chamfer within an arc; or, the droplet storage cavity is circular or oval.
  • the droplet storage cavity is a square or a rectangle, and the first communication port and the second communication port are located at opposite corners of the droplet storage cavity.
  • the passage is separated from the second inner passage on opposite sides of the droplet storage cavity, and the first inner passage and the second inner passage are respectively connected to the first communication port and the second communication port at the ends. Connected.
  • a part or the whole of the first channel and the second channel are curved.
  • the first channel includes at least one straight extension and at least one arc-shaped extension, and one end of the straight extension is in communication with the liquid inlet, and the at least one straight extension and The inner space of at least one arc-shaped extension constitutes the first inner passage.
  • the first inner passage is composed of an internal space of a straight extension and an arc-shaped extension, and the straight extension is located outside the droplet storage cavity and communicates with the droplet storage cavity.
  • One side of the droplet storage cavity is parallel, one end of the straight extension is bent toward the droplet storage cavity and extended to form the arc-shaped extension, and the arc-shaped extension is far from the straight extension. The end of the is in communication with the droplet storage cavity.
  • the droplet storage cavity, the first channel, and the second channel constitute a center-symmetric structure.
  • the bottom surfaces of the first channel, the second channel, and the droplet storage cavity are located at the same height position.
  • the height of the liquid inlet in the vertical direction is higher than the first channel, and / or the height of the liquid outlet in the vertical direction is higher than the second channel.
  • the inner diameter of the liquid inlet can be, for example, 4mm-8mm.
  • the height of the liquid inlet can be, for example, 5 mm to 15 mm.
  • the height of the droplet storage cavity may be, for example, 50-1000um.
  • the length and width may be, for example, 5-30 mm.
  • the inner diameters of the first channel and the second channel may be, for example, 4 mm to 10 mm.
  • the thickness of the chip body is 1 to 6 mm.
  • a cross section of the droplet storage cavity is a square, and a side length of the square is 2-30 mm; and a height of the droplet storage cavity is 20-2000 um.
  • the chip further includes a sealing cover for sealing the receiving cavity.
  • the digital PCR chip further includes a liquid discharge tube vertically arranged on the chip body, and the liquid discharge tube is in communication with the liquid discharge port.
  • the drain pipe extends upward from the upper surface of the chip body, and is integrally formed with or fixedly connected to the chip body.
  • the liquid discharge port is provided with a negative pressure connector for matching with an outlet of the negative pressure device.
  • the chip body is formed by stacking a chip cover and a chip substrate in a thickness direction, the chip cover is a flat plate, and the chip substrate is provided with a groove. The grooves are superimposed and compacted on each other to form the droplet storage cavity, the first channel, and the second channel.
  • the groove opening faces downward
  • the chip substrate is located above the chip cover
  • the chip cover is a transparent glass plate, a transparent PC board, a transparent acrylic plate, and a COP transparent Plate or black non-reflective plate.
  • the groove opening faces upward
  • the chip substrate is located below the chip cover
  • the chip substrate and the chip cover are made of plastic, respectively.
  • the droplet storage cavity, the first channel and the second channel together constitute a chip unit, and a plurality of the chip units are provided on the chip body.
  • the chip body is elongated, and the plurality of chip units are distributed along a length direction of the chip body.
  • the invention also provides a digital PCR detection system, which includes a digital PCR detection device, and also includes a digital PCR chip as described above, and a negative pressure device used in conjunction with the digital PCR chip.
  • the negative pressure device is used for A negative pressure is generated in the first channel, the droplet storage cavity, and the second channel.
  • the present invention also provides a digital PCR detection method based on the digital PCR chip as described above or the digital PCR detection system as described above, which includes a loading step of delivering a droplet to the droplet storage cavity, and the loading step include:
  • the liquid droplets are transported to the liquid droplet storage cavity through the liquid inlet and the first channel.
  • the liquid droplet storage cavity, the first channel, and the second channel are filled with an oil phase.
  • both the liquid inlet and the liquid outlet are kept in a sealed state, and the PCR chip is allowed to stand horizontally for more than 5 minutes.
  • the negative pressure device is turned on to promote the discharge of the oil phase from the liquid discharge port and the droplets to the droplet storage cavity.
  • the present invention also provides a droplet generation system including the above-mentioned digital PCR chip and used for digital PCR detection.
  • the droplet generation system further includes:
  • a microchannel having a first opening and a second opening for liquid to enter and exit;
  • the end of the first opening of the micropipeline can be inserted into the receiving cavity of the digital PCR chip and can be reciprocated in the receiving cavity under the driving of the rotary driving mechanism.
  • the liquid droplet generating system further includes a liquid discharge pipe vertically arranged on the chip body, and the liquid discharge pipe is in communication with the liquid discharge port.
  • drain pipe extends upward from the upper surface of the chip body, and is integrally formed with or fixedly connected to the chip body. Furthermore, a negative pressure connector is provided on the liquid discharge port for matching with the outlet of the negative pressure device.
  • the droplet storage cavity, the first channel and the second channel together constitute a chip unit, and a plurality of the chip units are provided on the chip body.
  • the reciprocating swing of the micro-pipe is horizontal swing; there is a liquid storage cavity with a volume of 10 ⁇ L to 100 ⁇ L between the first opening and the second opening of the micro-pipe.
  • the fluid driving mechanism includes a syringe and a delivery tube, and the liquid inlet and outlet of the syringe communicate with the second opening of the micropipe through the delivery tube, and the inner diameter of the delivery tube is less than The inner diameter of the micro-channel.
  • the fluid driving mechanism further includes a syringe driving assembly for driving the syringe to work.
  • the syringe driving assembly includes a screw nut driving mechanism or a rack and pinion driving mechanism.
  • the droplet generation system further includes the liquid storage tank having a liquid outlet, the liquid outlet of the liquid storage tank, the liquid inlet and outlet of the syringe, and the delivery tube.
  • the three at one end are connected through a three-way directional valve.
  • the driving mechanism is detachably connected to the micro-channel.
  • the driving mechanism includes a rotating electric machine, a rotating shaft, and a joint, and an output end of the rotating electric machine is connected to the rotating shaft, and the joint is fixedly connected in a direction perpendicular to the axis of the rotating shaft.
  • the micropipe is detachably mounted on the joint.
  • the fluid driving mechanism includes a syringe and a delivery tube
  • the joint is tubular, and has a first liquid inlet and outlet and a second liquid inlet and outlet connected internally, and one end of the delivery tube is connected to the inlet and outlet of the syringe. The other end is connected to the first liquid inlet and outlet of the joint, and the second opening of the micropipe is connected to the second liquid inlet and outlet of the joint; one of the rotating shafts is provided with A plurality of the joints are connected to a plurality of the microfluidic tubes.
  • the micro-droplet generating device further includes a needle withdrawal mechanism, which is used to separate the micro-pipe from the joint. Further, an end where the second opening of the micropipe is located is sleeved on one end of the joint, and the needle withdrawal mechanism includes a needle withdrawal plate slidably disposed on the joint and drives the needle.
  • the needle ejection plate driving component of the needle ejection plate slides, and the needle ejection plate slides against the microchannel to separate the microchannel from the joint.
  • the needle ejection plate driving component is preferably a screw nut driving structure or an air cylinder driving structure.
  • the liquid droplet generating system further includes a base frame, the rotation driving mechanism can be slidably arranged on the base frame, and the liquid droplet generating system further includes: A longitudinal movement driving mechanism that drives the driving mechanism to slide.
  • the droplet generation system further includes a negative pressure device used in conjunction with the PCR chip, and the negative pressure device is configured to generate in the first channel, the droplet storage cavity, and the second channel. Negative pressure.
  • the invention also provides a digital PCR detection method based on the droplet generation system as described above.
  • the droplets are formed by mixing an aqueous phase and an oil phase.
  • the detection method includes a sample loading step, and the sample loading step includes:
  • the first opening of the micropipe is inserted below the oil phase liquid level in the accommodating cavity, and a rotation mechanism is started to drive the micropipe to reciprocate.
  • the fluid phase is injected into the water phase using the fluid drive mechanism and the micropipe Micro-droplets are formed in the oil phase;
  • the liquid droplets are transported to the liquid droplet storage cavity through the liquid inlet and the first channel.
  • the liquid droplet storage cavity, the first channel, and the second channel are filled with an oil phase.
  • the liquid inlet and the liquid outlet are kept in a sealed state, and the digital PCR chip is allowed to stand horizontally for more than 5 minutes.
  • the negative pressure device is turned on to promote the discharge of the oil phase from the liquid discharge port and the liquid droplets to the liquid droplet storage chamber.
  • the swing angle of the micro-pipe is 0.1 ° to 10 °; the frequency of the back-and-forth swing of the micro-pipe is 1 Hz to 1000 Hz.
  • oil phase and water phase have the general meaning in the art and are not particularly limited.
  • the oil phase is usually less dense than the water phase.
  • the structural design of the digital PCR chip of the present invention is based on a completely different design principle from the traditional digital PCR chip.
  • the accommodating cavity constitutes a generating container for droplet generation. After the droplet is generated in the accommodating cavity, it is deposited by its own gravity at the liquid inlet, and then gradually enters the droplet storage cavity through the first channel.
  • the structure design of the first channel, the second channel and the droplet storage cavity on the chip enables the droplet to maintain a good stability during the transportation process, and realizes the uniform tiling of the droplets in the droplet storage cavity.
  • the digital PCR chip has the significant advantages of simple structure and low cost. Further, based on the structure and method of the digital PCR chip of the present invention, multi-layer tiling of droplets in a droplet storage cavity can also be realized, greatly improving detection throughput, and meeting clinical automation and high-throughput droplets per unit area. Requirements for analysis.
  • the invention also provides a new liquid droplet generating system and generating idea.
  • the droplet generation system integrates the formation and detection of droplets. Not only can it generate droplets of uniform volume in large quantities, the droplets can be directly used for detection, and the droplets can be uniform in the droplet storage cavity. The tiling helps to obtain significantly more accurate detection results.
  • the droplet generation system has the obvious advantages of simple structure and low cost.
  • the digital PCR detection system and detection method of the invention have many advantages such as high detection throughput and more accurate detection results.
  • FIG. 1 is a schematic structural diagram of a micro-droplet generating device used in the present invention
  • FIG. 2 is a schematic diagram of a reciprocating swing of a micro-droplet generating device used in the present invention
  • FIG. 3 is a schematic diagram of micro-droplet generation by a micro-droplet generating device used in the present invention.
  • FIG. 4 is a schematic diagram of a case where a micro-pipe is not connected to any vibration motor in the prior art
  • FIG. 5 is a schematic diagram of a situation in which a micro-pipe is fixed on a vibration motor capable of generating uniform linear motion in the prior art
  • FIG. 6 is a schematic diagram of a micro-droplet generating device used in the present invention to fix a micro-channel to a rotatable driving mechanism
  • FIG. 7 is a schematic diagram of related factors affecting the formation of micro-droplets
  • FIG. 8 is a front view of the micro-droplet generating device according to the embodiment.
  • FIG. 9 is a front sectional view of a micro-droplet generating device according to an embodiment
  • FIG. 10 is a left side view of the micro-droplet generating device according to the embodiment.
  • FIG. 11 is a rear view of the micro-droplet generating device according to the embodiment.
  • FIG. 12 is a cross-sectional view at A-A in FIG. 11;
  • FIG. 13 is a partially enlarged view at C in FIG. 12; FIG.
  • FIG. 14 is a partially enlarged view at D in FIG. 12; FIG.
  • FIG. 16 is a rear view of another embodiment of the micro-droplet generating device used in the present invention.
  • FIG. 17 is a front cross-sectional view of another embodiment of a micro-droplet generating device used in the present invention.
  • FIG. 18 is a front view of another embodiment of the micro-droplet generating device used in the present invention.
  • FIG. 19 is a left side view of another embodiment of the micro-droplet generating device used in the present invention.
  • 20 is a schematic diagram of a closed-loop motor for controlling a vibration angle or position involved in a micro-droplet generating device used in the present invention
  • FIG. 21 is a schematic structural diagram of Embodiment 1 of a digital PCR chip system according to the present invention.
  • FIG. 22 is a schematic exploded view of the digital PCR chip system according to the first embodiment of the present invention.
  • FIG. 23 is a front view of a chip substrate in the digital PCR chip of Example 1;
  • FIG. 24 is a schematic structural sectional view taken along the M-M direction in FIG. 23; FIG.
  • 25 is a plan view of the chip body of FIG. 23;
  • 26 is a bottom view of the chip body of FIG. 23;
  • FIG. 27 is a schematic structural diagram of a sealing cover in the digital PCR system of Embodiment 1;
  • FIG. 28 is a first structural decomposition diagram 1 of a digital PCR chip system according to the present invention.
  • FIG. 29 is a second structural decomposition diagram 2 of a digital PCR chip system according to the present invention.
  • FIG. 30 is a perspective view of a chip cover in the digital PCR chip of Example 2.
  • Figure 31 is a front view of the chip cover of Figure 30;
  • FIG. 32 is a schematic structural cross-sectional view taken along the N-N direction in FIG. 31; FIG.
  • FIG. 33 is a bottom view of the chip cover of FIG. 30;
  • FIG. 34 is a top view of the chip cover of FIG. 30; FIG.
  • 35 is a perspective view of a chip substrate in the digital PCR chip of Example 2.
  • Figure 36 is a top view of Figure 35;
  • FIG. 37 to FIG. 39 are schematic diagrams of implementing droplet tiling during the process of droplets entering the droplet storage cavity through the first channel;
  • Figure 40 is a schematic plan view of a droplet when it is tiled in the droplet storage cavity
  • 41 and 42 are schematic diagrams when two or three layers of the droplet are tiled in the droplet storage cavity.
  • the invention provides a new liquid droplet generating system and generating ideas.
  • the droplet generation system integrates droplet formation and detection.
  • the basic structure of the droplet generation system provided by the present invention includes a microchannel, a rotation driving mechanism, and a PCR chip. Parts other than the PCR chip are collectively referred to as a droplet generation device in the present invention.
  • the liquid droplet generating system of the present invention can not only generate liquid droplets of uniform volume in large quantities, but also can be directly used for detection.
  • a microchannel 100 and a rotation driving mechanism 200 of a droplet generation system are shown.
  • the microchannel 100 has a first opening 110 for outputting a first liquid 130, and the rotation driving mechanism 200 is used for driving the microchannel.
  • 100 makes horizontal reciprocating swing.
  • the rotation driving mechanism 200 drives the micro-pipe 100 to reciprocate around the rotation center 221, so that the first opening 110 of the micro-pipe 100 also reciprocates to generate micro-droplets 131 under the liquid surface of the second liquid 610 .
  • the micro-droplet generating device further includes a fluid driving mechanism 300.
  • the fluid driving mechanism 300 is connected to the micro-pipe 100 through a transfer pipe 310.
  • the second opening 120 is communicated.
  • the second opening 120 of the micropipe 100 communicates with the first opening 110, and the fluid driving mechanism 300 can apply a stable driving force to the micropipe 100 through the delivery pipe 310, so that the first liquid 130 in the micropipe 100 can stably and continuously follow Micro-droplets 131 flow out from the first opening 110.
  • the droplet generation method provided by the present invention is a very complicated dynamic process, and there are many factors that affect the volume of droplet generation.
  • the main factors are: the surface tension of the droplet (related to the area of the micropipe opening, the difference in surface energy between the first and second liquids), and the adhesion between the micropipe opening and the droplet (subject to the size of the pipe opening and surface properties) Influence); shear force (determined by the viscosity of the second liquid, the velocity of the microchannel and the surface area of the droplet), centrifugal force (related to the mass of the droplet, the radial acceleration of the microchannel's oscillation), and The tangential acceleration of the pipe swing is proportional to the mass of the droplet).
  • Centrifugal force is essentially radial inertial force.
  • the first liquid 130 in the microchannel 100 is formed at the nozzle of the first opening 110 of the microchannel 100.
  • the shear force of the microdroplets 131 on the second liquid 610, the speed of the nozzle movement of the first opening 110 of the microchannel 100 and the surface area of the microdroplets 131, the mass of the microdroplets 131 and the microchannels The centrifugal force related to the radial acceleration of the swing of the nozzle of the first opening 110 of 100, and the tangential acceleration of the swing of the nozzle of the first opening 110 of the micropipe 100 to the tangential inertial force proportional to the mass of the microdroplet 131 Under the common action, the micro-droplets 131 are separated from the nozzle of the first opening 110 of the micro-pipe 100 under the liquid level of the second liquid 610.
  • FIG. 4 is a schematic diagram of a case where a micro-pipe is not connected to any vibration motor in the prior art.
  • the fluid driving device continuously injects the first liquid 130 into the second liquid 610 through the micro-pipe 100 at a constant speed
  • the droplets will gradually increase.
  • the volume of the liquid is incompressible
  • the volume of the droplet will also increase at a constant rate under the condition of uniform injection.
  • Acting on the droplets are surface tension and adhesion that keep the droplets from detaching, and downward gravity.
  • the droplet grows to a critical volume (see the volume shown by the dividing line in Fig. 4)
  • the force of gravity falls off against surface tension and adhesion. Because droplets must grow to microliters of gravity to overcome tension and adhesion, this method cannot produce microdroplets characterized by nanoliters.
  • FIG. 5 is a schematic diagram of the prior art in which a micro-pipe is fixed on a vibration motor that can generate a uniform linear motion.
  • the fluid driving device continuously injects the first liquid 130 into the second liquid 610 through the micro-pipe 100 at a constant speed
  • the liquid droplets will gradually increase.
  • the volume of the liquid is incompressible
  • the volume of the droplet will also increase at a constant rate under the condition of uniform injection.
  • the linear motor is driven at the same time to drive the micro-pipe 100 to perform a linear motion to the left at a uniform speed. Then, the force of the liquid droplet is shown in FIG. 5.
  • FIG. 6 is a schematic diagram of a micro-droplet generating device of the present invention in which a micro-pipe is fixed on a rotatable driving mechanism.
  • the fluid driving device continuously injects the first liquid 130 into the second liquid 610 through the micro-pipe 100 at a constant speed
  • the high-frequency swing causes a high-frequency modulation of the speed change of the micro-pipe 100 so that the liquid
  • the resulting force is a combination of shear, centrifugal, and tangential inertial forces.
  • the rotation driving mechanism 200 in the droplet generating device of the present invention includes a rotation motor 210, a rotation shaft 220, and a joint 230.
  • the output end of the rotation motor 210 The rotary shaft 220 is connected to the rotary shaft 220.
  • the joint 230 is fixedly connected to the rotary shaft 220 in a direction perpendicular to the axis of the rotary shaft 220.
  • the micropipe 100 is mounted on the joint 230.
  • the rotating motor 210 can drive the rotating shaft 220 and the joint 230 to rotate around the axis of the rotating shaft 220 as a center, so as to drive the micro-pipe 100 to reciprocate.
  • the rotation driving mechanism 200 in the droplet generating device of the present invention may also adopt other rotation driving devices, such as a swing cylinder, a rotating electromagnet, and the like.
  • the microchannel 100 is a tubular structure with openings at both ends.
  • the joint 230 is also tubular. As shown in FIG. 13, the tubular joint 230 has a third opening 231 communicating with each other. With the fourth opening 232, the delivery tube 310 is connected to the third opening 231, and the second opening 120 of the microtube 100 is connected to the fourth opening 232.
  • the fluid driving force output by the fluid driving mechanism 300 can be stably applied in the micro-pipe 100 through the transfer pipe 310 and the joint 230, so that the first liquid 130 in the micro-pipe 100 can stably and continuously flow out of the first opening 110 to generate micro-liquid. Drop 131.
  • the micro-droplet generating device of the present invention can be used in the field of biological detection, in order to avoid cross-contamination of biological materials, the micro-pipe 100 is usually used once, so the micro-pipe 100 needs to be removed from the joint 230 after each use.
  • the micro-droplet generating device of the present invention further includes a needle withdrawal mechanism 400. As shown in FIG. 12 and FIG. 13, the needle withdrawal mechanism 400 includes a needle withdrawal plate 410 and a needle withdrawal plate driving assembly 420, and a needle withdrawal plate.
  • the needle withdrawal hole 411 is provided with a needle withdrawal hole 411, the needle withdrawal hole 411 is sleeved outside the joint 230, the second opening 120 of the microtube 100 is sleeved outside the fourth opening 232, and opposite to the needle withdrawal plate 410, the needle withdrawal plate
  • the driving assembly 420 is used to drive the needle ejection plate 410 to move in the direction of the microchannel 100.
  • a squeeze is applied to the microchannel 100 to release it from the joint 230. Applying force, the needle cannula 410 continues to move to push the micro-tube 100 out of the joint 230.
  • the needle cannula drive assembly 420 drives the needle cannula 410 to move closer to the conveying tube 310, so that the next microtube 100 can Suit over joint 230 on.
  • other structures can also be used to separate the microchannel from the connector.
  • the microchannel is held by a claw, and the microchannel is pulled from the connector by driving the claw to move the two. Person separation.
  • the outside of the fourth opening 232 of the joint 230 is shaped like a large circular table, which reduces the resistance of the micro-pipe 100 during installation and removal.
  • the needle ejection plate drive assembly 420 includes a needle ejection plate drive motor 421, a first lead screw 422, and a first screw nut 423.
  • the needle ejection plate drive motor 421 is fixedly installed on the mounting bracket 240, and the needle ejection plate drive motor 421 The output end is connected to the first screw 422, the first screw nut 423 is installed in cooperation with the first screw 422, and the needle withdrawal plate 410 is connected to the first screw nut 423.
  • the first screw nut 423 cooperates with the first screw 422 to transform the rotary motion output of the needle ejection plate driving motor 421 into a linear movement of the first screw nut 423 along the axial direction of the first screw 422, thereby driving the withdrawal
  • the needle plate 410 performs linear motion.
  • other forms of linear drive components can also be used to drive the needle ejection plate 410. For example cylinder drive.
  • the needle ejection plate driving assembly 420 of another solution includes a first cylinder 1421 and a first fixing nut 1423.
  • the first cylinder 1421 is fixedly installed on the mounting bracket 240, and the first fixing nut 1423 and the first cylinder 1421 piston.
  • the front end of the rod 1422 is fitted and installed, and the needle withdrawal plate 410 is connected to the first fixing nut 1423.
  • the front end of the piston rod 1421 of the first cylinder protrudes outward and moves axially along the piston rod.
  • the first fixing nut 1423 cooperates with the front end of the piston rod of the first air cylinder 1421 to transmit the axial movement output from the first air cylinder 1421 to the needle ejection plate 410, so that the needle ejection plate 410 can be driven to perform linear motion and needle ejection.
  • the rotary driving mechanism 200 further includes a mounting bracket 240, the rotary motor 210 and the needle ejection plate driving motor 421 are respectively fixedly mounted on the mounting bracket 240, and both ends of the rotary shaft 220 are rotatably disposed in the mounting bracket 240 through bearings.
  • the structure of the rotary driving mechanism 200 can be made compact and stable.
  • the micro-droplet generating device further includes a longitudinal movement mechanism 500.
  • the longitudinal movement mechanism 500 includes a first mounting plate 510, a longitudinal movement driving component 520, and a longitudinal sliding component 530.
  • the mounting bracket 240 slides vertically.
  • the assembly 530 is mounted on the first mounting plate 510, and the longitudinal movement driving assembly 520 is used to drive the mounting bracket 240 to slide along the longitudinal sliding assembly 530.
  • the mounting bracket 240 can drive the rotation driving mechanism 200 to move longitudinally, that is, the joint 230 on the rotation shaft 220 can move longitudinally.
  • the micro-pipe 100 on the joint 230 can be driven to move synchronously in the longitudinal direction.
  • the longitudinal movement mechanism 500 can be controlled
  • the micropipe 100 is driven to move downward to a predetermined height; when the micropipe 100 needs to be moved out, the micropipe 100 can be driven to move upward by controlling the longitudinal movement mechanism 500.
  • the longitudinal moving mechanism 500 also provides conditions for the micropipe 100 to be automatically loaded by the joint 230.
  • the micropipe 100 When the micropipe 100 needs to be mounted on the joint 230, the micropipe 100 can be placed below the joint 230 to make the second opening 120 of the micropipe 100 Align the joint 230, start the longitudinal movement driving assembly 520, and drive the joint 230 to move downward, so that the fourth opening 232 of the joint 230 is inserted into the second opening 120 of the microtube 100, and then the joint 230 is moved upward to reset.
  • the longitudinal movement driving component 520 can also drive the micro-tube 100 to move downward so that the first opening 110 is inserted below the liquid level of the second liquid 610, and reciprocatingly swings to produce micro-droplets. .
  • the longitudinal movement driving assembly 520 includes a longitudinal movement driving motor 521, a second lead screw 522, and a second lead screw nut 523.
  • the longitudinal movement driving motor 521 is fixedly installed on the first mounting plate 510.
  • the output end of the longitudinally moving driving motor is connected to the second screw 522, the second screw nut 523 is installed in cooperation with the second screw 522, and the mounting bracket 240 is connected to the second screw nut 523.
  • the second lead screw nut 523 cooperates with the second lead screw 522 to convert the rotational movement output by the longitudinal movement driving motor 521 into a linear movement of the second lead screw nut 523 along the axial direction of the second lead screw 522, thereby driving the mounting bracket.
  • 240 performs linear motion.
  • other forms of linear driving components can also be used to drive the mounting bracket 240. For example rack drive.
  • the longitudinal moving driving assembly 520 of another solution includes a longitudinal moving gear driving motor 1521 with a power-off brake, a first gear 1522 and a first rack 1523.
  • the longitudinal moving gear driving motor 1521 is fixedly installed on the mounting bracket 240.
  • the output end of the longitudinally moving driving motor is connected to the first gear 1522, the first rack 1523 is fixed on the first mounting plate 510, and the first rack 1523 is installed in cooperation with the first gear 1522.
  • the first gear 1522 and the first rack 1523 cooperate to convert the rotational movement output by the longitudinal movement gear drive motor 1521 into a linear movement of the longitudinal movement gear drive motor 1521 and the first gear 1522 along the axial direction of the first rack 1523, thereby
  • the mounting bracket 240 can be driven to perform linear motion.
  • other forms of linear driving components can also be used to drive the mounting bracket 240.
  • the fluid driving mechanism 300 in this embodiment includes a syringe 350 and a syringe driving assembly 320.
  • the liquid inlet and outlet of the syringe 350 are connected to the second opening 120 of the micro-tube 100 through the delivery tube 310. through.
  • the pusher 351 of the syringe 350 slides in the cylinder of the syringe 350 under the drive of the syringe driving assembly 320, and pushes the driving fluid therein into the microtube 100 through the delivery tube 310 and the joint 230, and toward the first liquid 130 in the microtube 100 Provides fluid drive.
  • the fluid driving mechanism provided by the present invention is not limited to the above-mentioned embodiments.
  • a peristaltic pump, a pressure-driven pump, a pneumatically-driven pump, or an electroosmotic-driven pump may be used.
  • the fluid driving mechanism 300 further includes a three-way directional valve 330 and a liquid storage tank, the second opening 120 of the micro-pipe 100, the liquid inlet and outlet of the syringe 350, and the liquid outlet of the liquid storage tank and Three interfaces of the three-way switching valve 330 are communicated.
  • the three-way reversing valve 330 can control the fluid driving mechanism 300 at least to realize the following two modes: First, the liquid inlet and the outlet of the syringe 350 are communicated with the second opening 120 of the micro-tube 100.
  • the syringe 350 With the drive of the syringe driving assembly 320, the syringe 350 provides a liquid driving force to the micro-channel 100 for pushing the first liquid in the micro-channel 100 through the first opening 110, or sucking the first liquid from the first opening 110 into the micro-channel 100; The liquid inlet and outlet of the 350 are connected to the liquid storage tank. Driven by the syringe driving assembly 320, the syringe 350 sucks the driving liquid in the liquid storage tank into the tube of the syringe 350, or pushes the driving liquid in the syringe 350. Into the storage tank.
  • micropipes 100 there are multiple micropipes 100, joints 230, transfer tubes 310, and syringes 350, and multiple The joints 230 are arranged at intervals on the rotating shaft 220.
  • a plurality of micro-pipes 100 are respectively mounted on one joint 230, and two ends of each transfer pipe 310 are respectively connected with the second opening of a micro-pipe 100 and the first of the three-way directional valve 330.
  • One interface is in communication
  • the liquid inlet and outlet of each syringe 350 is in communication with the second interface of the three-way switching valve 330
  • the liquid outlet in the liquid storage tank is in communication with the third interface of the three-way switching valve 330.
  • Multiple micro-channels 100 can perform micro-droplet generation at the same time under the driving of the syringe 350 and the rotating motor 210, and a three-way reversing valve 330 can control the micro-droplet generation states of multiple micro-channels 100 simultaneously.
  • a plurality of three-way switching valves 330 may be provided corresponding to a plurality of micro-pipes 100, joints 230, delivery pipes 310, and syringes 350, and the plurality of three-way switching valves 330 and a plurality of conveyances, respectively, may be provided.
  • the tube 310 is in communication with a plurality of syringes 350.
  • the three droplet switching valves can be independently controlled to independently control the micro-droplet generation status of the plurality of micro-channels 100.
  • the fluid driving mechanism 300 further includes a mounting block 340.
  • a plurality of three-way directional valves 330 and syringes 350 are fixedly mounted on the mounting block 340.
  • First flow passages 341, multiple second flow passages 342, a third flow passage 343, and multiple liquid separation flow passages 344, and two ends of each first flow passage 341 are exchanged with a delivery pipe 310 and a tee, respectively
  • the first port of the valve 330 is in communication with each other.
  • the two ends of each second flow path 342 are in communication with the inlet and outlet of a syringe 350 and the second port of a three-way switching valve 330.
  • the third flow path 343 is in communication with
  • the liquid storage tank is in communication with a plurality of liquid separation flow channels 344, and each liquid separation flow channel 344 is in communication with a third interface of a three-way switching valve 330.
  • the syringe driving assembly 320 includes a syringe driving motor 321, a third screw 322, and a third screw nut 323.
  • the output end of the syringe driving motor 321 is connected to the third screw 322, and the third
  • the screw nut 323 is installed in cooperation with the third screw 322, and the push rods 351 of the plurality of syringes 350 are connected to the third screw nut 323 through a connecting member (not shown in the figure).
  • the third screw nut 323 cooperates with the third screw 322 to convert the rotary motion output by the syringe drive motor 321 into a linear movement of the third screw nut 323 along the axis of the third screw 322, so that the syringe 350 can be driven.
  • the push rod 351 performs a linear motion.
  • other forms of linear driving components may also be used to drive the push rod 351. For example rack drive.
  • the syringe drive assembly 320 of another solution includes a syringe gear drive motor 1321 with a power-off brake, a second gear 1322 and a second rack 1323.
  • the output end of the syringe drive motor 1321 is connected to the second gear 1322.
  • the second rack 1323 is connected to the second mounting plate 360, the second rack 1323 is installed in cooperation with the second gear 1322, and the push rods 351 of the multiple syringes 350 are driven by the syringe gear through a connecting member (not shown in the figure).
  • the motors 1321 are connected.
  • the second gear 1322 and the second rack 1323 cooperate to transform the rotary motion output by the syringe drive motor 1321 into a linear movement of the syringe gear drive motor 1321 and the second gear 1322 along the axial direction of the second rack 1323, thereby driving the syringe
  • the 351 of 350 performs linear motion.
  • other forms of linear drive components can also be used to drive the push rod 351.
  • the fluid driving mechanism 300 further includes a second mounting plate 360.
  • the mounting block 340 and the syringe driving motor 321 are fixedly mounted on the second mounting plate 360.
  • the second mounting plate 360 makes the fluid driving mechanism 300 more compact and stable in structure.
  • the first mounting plate and the second mounting plate can also be combined to save space.
  • the syringe driving assembly 320 can be installed on the integrated mounting plate 1360, and the remaining installation and driving methods remain unchanged.
  • the rotating electrical machine 210 may adopt a galvanometer motor.
  • the galvanometer motor can provide stable and high-speed reciprocating rotation and swing motion, and the swing amplitude and frequency can be set according to requirements, which greatly improves the microfluid of the present invention. Scope of application of the drop generating device.
  • the stepping motor can be used for the needle ejection plate drive motor 421, the longitudinal movement drive motor 521, and the syringe drive motor 321.
  • the combination of the stepper motor and the lead screw lead screw structure can accurately control the linear motion stroke and improve the degree of automation.
  • the rotary motor 210 adopts a motor with closed-loop control of the vibration angle or position, and the rotary drive mechanism 200 is driven by the closed-loop control of the vibration angle or position to perform reciprocating swing, thereby precisely controlling the swing trajectory of the micro-pipe 100, thereby further reducing the environment. And system disturbances.
  • Another advantage of this method is that the system parameters can be adjusted so that the critical volume can be reached in one swing period (as indicated by the arrow in Figure 7). This means that only one droplet is produced in each cycle of the rotary motion. In this way, changes in droplet volume due to fluctuations in various environmental factors will not accumulate in the next cycle. Therefore, droplets of uniform size can be generated in large batches. This is also an advantage that other published solutions of generating nanoliter / picoliter emulsion droplets through mechanical movement do not have.
  • Closed-loop motors that control the angle or position of vibration include components such as infrared position sensors, control circuits, and signal processing circuits.
  • an infrared position sensor is installed on the rotating shaft 220 of the rotary driving mechanism 200, and the position signal obtained by the infrared position sensor is fed back to a control circuit, and the control circuit respectively responds to the feedback position signal according to the PID automatic control principle.
  • Proportional, integral, and differential calculation processing has been done, and combined with signal processing circuits such as position feedforward and speed loop, current loop, etc., the absolute position precise control during motor movement is realized.
  • a closed-loop motor that controls the vibration angle or position can prevent other vibration motors from being subject to complex load environment changes that cause the vibration position to change, which is conducive to the precise control of droplet volume and generation speed in engineering.
  • a liquid storage chamber having a volume of 10 ⁇ L to 100 ⁇ L is provided between the first opening 110 and the second opening 120 of the micropipe 100, and the liquid storage chamber can store a certain amount of the first liquid to ensure that the first liquid is sufficient The required number of micro-droplets are generated.
  • the liquid storage chamber can also prevent the first liquid from being sucked into the joint 230 and the conveying pipe 310 through the micro-pipe 100 to ensure that the system will not be contaminated by the sample.
  • the micro-channel 100 can be made of a non-rigid material and has certain flexibility.
  • the certain flexibility means that the micro-channel 100 can make the movement path of the first opening 110 of the micro-channel 100 have a certain standing wave phenomenon under the driving of the rotation driving mechanism 200.
  • the use of microchannels made of a material with a certain flexibility further reduces the disturbance on the liquid surface, makes it easier and more uniform to generate liquid droplets, and also further reduces the phenomenon of liquid fragmentation.
  • the micro-channel 100 is made of a polypropylene material with a low surface energy; the delivery tube 310 is made of a Teflon material.
  • the inner diameter of the mouth of the first opening 110 of the micropipe 100 is 1 ⁇ m to 250 ⁇ m, and more preferably, the inner diameter of the mouth of the first opening 110 of the micropipe 100 is 10 ⁇ m to 100 ⁇ m.
  • a digital PCR chip there is shown a digital PCR chip, a microchannel 100 according to the present invention (also referred to as an output gun needle in the present invention), and a microtube 100 having a negative pressure gun needle 50 for generating negative pressure. Negative pressure device.
  • the digital PCR chip of this example includes a chip body 10 having a droplet storage cavity 1, a liquid inlet 4 and a liquid outlet 5 provided on the chip body 10, and an upright position and a liquid inlet on the chip body 10. 4 communicating accommodation chamber 61, the chip body 10 further includes a first channel 2 that communicates the liquid inlet 4 with the droplet storage chamber 1, and a second channel 3 that communicates the liquid discharge port 5 with the droplet storage chamber 1, Wherein, the first channel 2 has a first internal path inside the chip body 10, and the second channel 3 also has a second internal path inside the chip body 10.
  • the containing cavity 61 extends upward from the upper surface of the chip body 10, and the liquid inlet 4 is located at the bottom of the containing cavity 61.
  • the output gun needle 40 When in use, first fill the droplet storage chamber 1, the first channel 2, the second channel 3, and the receiving chamber 61 with an oil phase, and then use the output gun needle 40 to inject the water phase into the oil phase of the receiving chamber 61.
  • the output gun needle 40 is swung back and forth, so that liquid droplets are formed in the accommodating cavity 61.
  • the density of the water phase is usually greater than that of the oil phase.
  • the formed droplets will deposit to the bottom of the accommodating cavity 61 due to their own gravity, and then enter the first channel 2 through the liquid inlet 4 to enter the droplet storage cavity 1.
  • the length of the accommodating cavity 61 is 20-1000um, the width is 20-1000um, and the height is 20-2000um.
  • the accommodating cavity 61 may be fixedly connected to the chip body 10, and may also be integrally formed with the chip body 10.
  • the upper surface of the chip body 10 has a liquid inlet guide tube 6 extending upright, and a cavity of the liquid inlet guide tube 6 constitutes an upper receiving cavity 61.
  • the second passage 3 is also connected to the droplet storage chamber 1 at the end.
  • the first path 2 is connected to the droplet storage chamber 1 at an end, and the first inner path is provided on one side of the droplet storage chamber 1; the second path 3 It is also connected to the droplet storage chamber 1 at the end, and the second inner path and the first inner path are separated on different sides of the droplet storage chamber 1.
  • the negative pressure gun needle 50 of the negative pressure device can pass through the liquid discharge port. A negative pressure is generated, and the auxiliary liquid droplet slowly enters the liquid droplet storage chamber 1 from the first channel 2 gradually.
  • the bottom surfaces of the first channel 2, the second channel 3, and the droplet storage chamber 1 are preferably located at the same height position.
  • the height of the liquid inlet 4 in the vertical direction is higher than that of the first channel 2, and the liquid is discharged.
  • the height of the port 5 in the vertical direction is higher than that of the second channel 3.
  • the inner diameter of the liquid inlet 4 is preferably set to 4mm-8mm, and the height is preferably 5mm-15mm.
  • the inner diameters of the first channel 2 and the second channel 3 are 4mm-10mm, respectively.
  • the length and width of the droplet storage cavity 1 are 2-30 mm, and the height is 20-2000 um.
  • the droplet storage chamber 1 has a first communication port 1a communicating with the first channel 2 and a second communication port 1b communicating with the second channel 3.
  • the first communication port 1a and the second communication port 1b The first communication port 1a is opposite to the second communication port 1b, and the first communication port 1a is arranged opposite to the second communication port 1b.
  • the first communication port 1 1a and the second communication port 1b are preferably disposed on a set of diagonals of the polygon.
  • the droplet storage chamber 1 has at least one arc-shaped chamfer, and the first communication port 1a is provided at the arc-shaped chamfer, so that the first channel 2 is connected to the first communication port 1a and communicates with the first communication port 1a.
  • the liquid droplet storage cavity 1 is switched on, which is more conducive to the tile movement after the liquid droplets enter the liquid droplet storage cavity 1.
  • the droplet storage cavity 1 may be a polygon with a chamfer in an arc, or a circle or an oval.
  • the above-mentioned arc chamfer may be formed at the position where two adjacent edges meet, or a large chamfer treatment may be performed on one of the sides to form an arc chamfer.
  • a large chamfer treatment may be performed on one of the sides to form an arc chamfer.
  • the cross section of the liquid droplet storage chamber 1 is other irregular shapes, it is desirable to also perform a large chamfering process.
  • an angle between at least two adjacent sides in the cross section of the droplet storage cavity 1 is a right angle, and the first communication port 1a is provided at the right angle.
  • the cross-section of the droplet storage cavity 1 is set to be square, and the first communication port 1a and the second communication port 1b are on a set of diagonal corners, as shown in FIG. 26, and the other inner corners are all rounded.
  • the setting of the chamfer in the arc is beneficial for the droplet to maintain good stability in the droplet storage cavity 1.
  • the square side of the cross section of the droplet storage cavity 1 is 5-30 mm, and the height of the droplet storage cavity 1 is 50-1000um. When the diameter of the droplet is reduced, the side length of the droplet storage cavity 1 can be further reduced, and the height setting needs to meet the needs of the droplet tiling on the one hand, and the formula oil must be taken into account on the other. The problem of exploitation.
  • the first channel 2 includes a liquid inlet section 2 a having one end in communication with the liquid inlet 4, and an arc curvedly extending from the other end of the liquid inlet section 2 a toward the droplet storage chamber 1. ⁇ ⁇ ⁇ ⁇ 2b ⁇ The output section 2b.
  • the liquid inlet section 2a is a linearly extending section, which is located outside the droplet storage chamber 1 and is parallel to one side of the droplet storage chamber 1.
  • One end of the liquid inlet section 2a is bent toward the droplet storage chamber 1 and extends to form The liquid discharge section 2b of the arc-shaped extension section, and the end of the liquid discharge section 2b remote from the liquid inlet section 2a is connected to the first communication port 1a and is connected to the liquid droplet storage chamber 1.
  • This design facilitates the liquid droplets to pass through the liquid inlet. After the port 4 enters the first channel 2, it smoothly enters the droplet storage chamber 1 under the action of gravity.
  • the second channel 3 is arranged symmetrically with the center of the first channel 2.
  • the second channel 3 includes a liquid discharge section 3a connected at one end to the liquid discharge port 5 and a liquid inlet section 3b which is curved and extended toward the droplet storage chamber 1 from the other end of the liquid discharge section 3a.
  • the segment 3b has an arc shape that is gradually arched away from the liquid discharge segment 3a.
  • the end of the liquid inlet segment 3b is connected to the second communication port 1b and is connected to the liquid droplet storage chamber 1.
  • the first channel 2, the droplet storage chamber 1, and the second channel 3 constitute a center-symmetric structure.
  • This structure design realizes the stable transportation of liquid droplets and ensures the stability of liquid droplets.
  • the chip body 10 is mainly formed by stacking a chip cover and a chip substrate in a thickness direction.
  • the chip cover is a flat plate, and a groove is formed on the chip substrate.
  • the flat plate and the groove are superimposed and pressed to form a liquid droplet storage cavity 1.
  • the groove opening faces downward, and the chip substrate 101 is located above the chip cover plate 102.
  • the chip cover plate 102 is a transparent glass plate, a transparent PC plate, a transparent acrylic plate, a COP transparent plate, or a non-reflective material such as POM or PP. Made of black non-reflective sheet.
  • the chip substrate 101 and the chip cover plate 102 can be welded to the seal by gluing or ultrasonic or thermocompression bonding, and the edge between the two must maintain absolute tightness.
  • the groove opening faces upward, and the chip substrate 104 is located below the chip cover 103.
  • the liquid inlet 4 and the liquid outlet 5 are both opened on the chip cover.
  • the liquid inlet guide tube 6 and the liquid discharge guide tube 7 are also integrally formed on the chip cover 103; the droplet storage chamber 1, the first channel 2 and the second channel 3 are disposed on the chip substrate 104.
  • the above-mentioned chip cover 103 and chip substrate 104 are made of plastic, and the two are welded to the seal by a thermocompression bonding process.
  • the upper surface of the chip body 10, that is, the upper surface of the chip substrate 101 also has a drainage drainage tube 7 extending upright and communicating with the drainage port 5,
  • the drainage guide tube 7 is mainly used as a negative pressure connector that is matched with the negative pressure gun needle 50.
  • the negative pressure gun needle 50 is connected with the drainage guide tube 7 to form a negative pressure on the liquid droplet storage chamber 1.
  • the liquid inlet diversion tube 6 and the liquid drainage diversion tube 7 are integrally formed on the chip substrate 101. Of course, in some other embodiments, they can also be formed by independent processing. Then, it is connected to the chip substrate 101 by ultrasonic welding or glue bonding.
  • each liquid-conducting diversion tube 6 can be sealed by a detachably connected sealing cover 201, which means that the accommodation cavity 61 is sealed; the mouth of the liquid-diversion diversion tube 7 can be sealed by a sealing film 30. Seal it.
  • the liquid droplet storage chamber 1, the first channel 2 and the second channel 3 together constitute a chip unit.
  • the chip body 10 is provided with a plurality of spacers arranged along the length direction.
  • Each of the above chip units can perform multiple sets of sample loading and analysis detection at the same time.
  • there are multiple groups of the liquid inlet 4, the liquid outlet 5 and the accommodating cavity 61, and the liquid inlet guide tube 6 and the liquid discharge guide tube 7 are also provided with multiple groups.
  • all the sealing caps 201 adapted to the plurality of liquid inlet guide tubes 6 are integrally formed to form an integral sealing cap member 20, and the sealing film 30 can also be used as an integral member to serve as all the drainage guides at the same time.
  • the present invention also provides a detection method using the above-mentioned digital PCR chip or digital PCR detection system, which includes a sample loading step of delivering a droplet to the droplet storage cavity 1, and the loading step includes:
  • the micro-pipe 100 (ie, the output gun needle 40) is used to inject the water phase into the oil phase in the accommodating chamber 61, and the micro-pipe 100 is reciprocated to swing while being injected, so that liquid droplets are formed in the accommodating chamber 61;
  • the liquid droplets are transported to the liquid droplet storage chamber 1 through the liquid inlet 4 and the first channel 2.
  • the liquid droplet storage chamber 1, the first channel 2, and the second channel 3 are preferably filled with the oil phase.
  • the inlet 4 and the outlet 5 are kept horizontally for more than 5 minutes; after the formation of droplets, or after the completion of the generation of the droplets, turn on the negative pressure.
  • the device promotes the discharge of the oil phase from the liquid discharge port 5 and the flow of the liquid droplets to the liquid droplet storage chamber 1.
  • the specific detection process is performed according to the following steps: the liquid droplet storage chamber 1, the first channel 2, the second channel 3, and the accommodation chamber 61 of the chip body 10 are filled with an oil phase in advance, and the sealing cover 201 and the sealing film are respectively used 30 seals the nozzles of the liquid inlet guide tube 6 and the liquid outlet guide tube 7.
  • the sealing cover 201 is opened, and the output needle of the output gun needle 40 of the droplet generating device is inserted into the accommodation cavity 61 of the liquid guide tube 6 so that the port of the output needle (i.e., the micropipe
  • the first opening 110 of the 100 is located below the liquid surface of the oil phase to inject the water phase and swing the output needle back and forth while injecting, so that liquid droplets are formed in the accommodating cavity 61.
  • the generated droplets are accumulated at the bottom of the accommodating cavity 61 due to the effect of their own gravity, and some of the droplets naturally fall to the first pipe 2 through the liquid inlet 4.
  • the intervention of the droplets will cause the height of the oil phase liquid level to be There is an increase in the liquid inlet 4, but it will not affect the stability of droplet formation.
  • puncture the sealing film 30 at the mouth of the drainage diversion tube 7 an actionable mechanism can be provided in conjunction with the instrument to perform puncture
  • a negative pressure needle 50 connected to a negative pressure device Negative pressure is slowly generated, and the liquid droplets will slowly pass through the first channel 2 into the liquid droplet storage chamber 1 as the pressure acts, and they will be tiled to the liquid droplet storage region 1 as shown in Figure 37 to Figure As shown at 39, the droplet generation and preliminary tiling process is completed at this time, and the chip body 10 can be compressed by the mechanical structure.
  • the entire upper surface of the chip body 10 can be pressed or several fixed points can be pressed, and the pressure is buffered by a structure such as a spring. If several fixed points are used for pressing, it is necessary to avoid excitation light irradiation or camera detection.
  • the light path area enables real-time fluorescence reading and observes the movement state of the droplet at any time.
  • the thickness and area of the droplet storage cavity 1 by adjusting the thickness and area of the droplet storage cavity 1, the volume of the droplet, and the total volume of the sample, a precise one layer, or two layers as shown in FIG. 41, or three layers as shown in FIG. 42, Even more layers of tiling.
  • a sample volume of 20 microliters a droplet volume of 1 nanoliter, a chip area of 16mm ⁇ 16mm, and a chip thickness of 125-150 microns
  • the resulting 20,000 droplets can only be tiled into one layer.
  • the chip area is adjusted to 11.5mm ⁇ 11.5mm and the thickness is adjusted to 200-275 microns, 20,000 droplets can only be tiled into two layers.
  • This multi-layer droplet tiling method can realize multi-layer observation of droplets with higher throughput in a unit area. This is very important for improving the overall detection throughput of digital PCR equipment for image-type detection of droplets, and solves the bottleneck problem of low detection throughput faced by such equipment.

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Abstract

本发明公开了一种数字PCR芯片及包含它的液滴生成系统和检测系统，其中数字PCR芯片包括具有液滴存储腔的芯片本体和设置在芯片本体上的进液口，数字PCR芯片还包括直立设置在芯片本体上且与进液口连通的容置腔，以及设置在芯片本体上的排液口；芯片本体还包括分别将进液口与液滴存储腔连通的第一通道、所述排液口与液滴存储腔连通的第二通道，第一通道具有位于芯片本体内部的第一内通路，第二通道具有位于芯片本体内部的第二内通路。液滴在容置腔中生成后便经进液口、第一通道进入液滴存储腔中，液滴在输送过程中能够保持很好的稳定性及封闭性以及实现液滴在液滴存储腔中均匀的单层或多层平铺，有利于获得显著更准确的检测结果。

Description

数字PCR芯片及包含它的液滴生成系统和检测系统 技术领域
本发明涉及一种数字PCR芯片及包含它的液滴生成系统和检测系统。
背景技术
聚合酶链式(PCR)反应技术是现代生物学最重要的工具之一，它广泛应用在医学诊断、个体化医疗、食品检验、转基因生物检测、病原体鉴定、免疫分析、法医科学等方面。而作为PCR技术的最新一代，基于微流控技术发展产生的数字PCR(dPCR)，具有比传统qPCR更小的反应体积、更快的反应速度、更低的系统噪声和更高的灵敏度。
液滴数字PCR(Droplet Digital PCR)技术是基于微流控芯片的油包水液滴技术，其通过油包水结构，将单个DNA分子封装到单独的微滴中，利用油的惰性实现DNA分子间的相互隔离，每个DNA分子被限制在自己的微滴中单独的进行扩增，避免来自其他序列竞争。在合适的温度条件下完成DNA分子的扩增后，通过记录微滴的总体个数和可以检测到荧光信号的微滴个数，利用泊松分布算法就可以实现对DNA拷贝数的精确定量。
微量液体操作的核心技术之一是把微升量级的液体进一步分割为纳升甚至皮升体积的微反应体系。微反应体系生成的一个主要技术分支是乳化微液滴生成。近些年来，在文献中报道了多种微液滴生成技术如膜乳化法、喷雾乳化法、微流控芯片法、微管道注射/喷射法等。近期专利号为ZL201410655191.5的中国发明专利和公开号为CN104815709A的中国专利申请进一步优化了通过微管道生成乳化微液滴的方法。乳化微液滴这些方法在实际应用中都各存在一定的缺点。专利号为ZL201410655191.5的中国发明专利的方法利用微量液体在气液相界面变换时的界面能和流体剪切力，克服液体在微通道出口的表面张力和附着力，使流出微管道管口的液滴能顺利地脱离微管道，并在不相溶液体中形成大小可控的液滴。但是这种方法需要微管道在液面上下切割运动，需要对微管道相对于液面的起始和终点位置进行高精度的定位，在工程实现上难度很大。公开号为CN104815709A的中国专利申请的方法通过微管道在液体里的圆周或者螺旋匀速运动产生的剪切力切断注入的不相溶液体而形成液滴，但是这种方法由于微管道产生液滴的大小受到各种系统因素变化的影响较大(比如液体的粘稠度、环境的温度、运动速度、运动轨迹等)，并且这一误差会随着产生液滴的数量增多而积累，因而大批量液滴生成的体积大小均一性的控制难度很大。
现有技术中，也有一些数字PCR芯片集液滴生成与液滴收集存储于一体，使得液滴收集后即可直接地被用作检测，然而，该种结构的芯片不易稳定地生成液滴或液滴的稳定性和均一性较差；芯片检测通量小，不能满足临床上自动化及单位面积内高通量液滴分析的要求；此外，该种芯片结构复杂、加工精度要求高，成本高。
发明内容
本发明的目的在于提供一种改进的数字PCR芯片，以解决现有PCR芯片所存在的一个或多个不足。
本发明同时还提供基于数字PCR芯片的数字PCR检测系统及检测方法，以及包含该数字PCR芯片且用于数字PCR检测的液滴生成系统及数字PCR检测方法。
为达到上述目的，本发明采用的一种技术方案是：一种数字PCR芯片，包括具有液滴存储腔的芯片本体和设置在所述芯片本体上的进液口，所述的数字PCR芯片还包括直立设置在所述芯片本体上且与所述进液口连通的容置腔，以及设置在所述芯片本体上的排液口；所述芯片本体还包括分别将所述进液口与所述液滴存储腔连通的第一通道、将所述排液口与所述液滴存储腔连通的第二通道，所述第一通道具有位于所述芯片本体内部的第一内通路，所述第二通道具有位于芯片本体内部的第二内通路。
根据本发明的一些优选方案，所述容置腔自所述芯片本体的上表面向上延伸，所述进 液口位于所述容置腔的底部。
根据本发明的一些优选方案，所述容置腔的长度为2～30mm、宽度为2～30mm、高度为20～2000um。
根据本发明的一些优选方案，所述容置腔与所述芯片本体一体成型设置，或者，所述容置腔与所述芯片本体相固定连接。
根据本发明的一些优选方案，所述第一通道在端部与所述液滴存储腔接通；和/或，所述第二通道在端部与所述液滴储存腔接通。
根据本发明的一些优选方案，所述第一通道位于所述液滴存储腔的一侧；和/或，所述第二通道位于所述液滴存储腔的一侧。
根据本发明的一些优选方案，所述第一内通路与所述第二内通路分设于所述液滴存储腔相异的两侧。
根据本发明的一些优选方案，所述液滴存储腔具有与所述第一通道连通的第一连通口、与所述第二通道连通的第二连通口，所述第一连通口与所述第二连通口分设于所述液滴存储腔相对的两个侧部上。
进一步地，所述第一连通口正对所述第二连通口设置。
根据本发明的进一步实施方案，所述液滴储存腔具有至少一个弧形倒角，所述第一连通口设于所述弧形倒角处。
根据本发明的进一步实施方案，所述液滴存储腔为具有圆弧内倒角的多边形；或者，所述液滴存储腔为圆形或椭圆形。
根据本发明的进一步实施方案，所述液滴储存腔为正方形或矩形，所述第一连通口与所述第二连通口分设于所述液滴存储腔的对角上，所述第一内通路与所述第二内通路分设于所述液滴存储腔相对的两侧，所述第一内通路与所述第二内通路分别在端部与所述第一连通口、第二连通口连通。
根据本发明的一些优选方案，所述第一通道、第二通道的部分或整体为曲线状。
进一步地，所述第一通道包括至少一个直形延伸段和至少一个弧形延伸段，且由所述直形延伸段的一端与所述进液口连通，所述至少一个直形延伸段和至少一个弧形延伸段的内部空间构成所述第一内通路。
根据本发明的进一步实施方案，所述第一内通路由一个直形延伸段和一个弧形延伸段的内部空间构成，所述直形延伸段位于所述液滴存储腔的外侧且与所述液滴存储腔的一条边平行，所述直形延伸段的一端向着所述液滴存储腔的方向弯折并延伸形成所述弧形延伸段，所述弧形延伸段的远离直形延伸段的端部与所述液滴存储腔连通。
作为一种优选的实施方案，所述液滴存储腔、所述第一通道、所述第二通道构成中心对称的结构。
根据本发明的一些优选方案，所述第一通道、所述第二通道及所述液滴存储腔的底面位于同一高度位置。
根据本发明的一些优选方案，所述进液口在竖直方向的高度高于所述第一通道，和/或，所述排液口在竖直方向的高度高于所述第二通道。
根据本发明，所述进液口的内径可以为例如4mm-8mm。
根据本发明，所述进液口的高度可以为例如5mm-15mm。
根据本发明，液滴储存腔的高度可以为例如50-1000um。长度、宽度可以分别为例如5-30mm。
根据本发明，第一通道、第二通道的内径可以分别为例如4mm-10mm。
根据本发明，所述芯片本体的厚度为1～6mm
根据本发明的一些优选方案，所述液滴储存腔的横截面为正方形，该正方形的边长为2-30mm；所述液滴储存腔的高度为20-2000um。
根据本发明的一些优选方案，所述芯片还包括用于密封所述容置腔的密封盖。
进一步地，所述容置腔有多个，对应地，所述密封盖也有多个，所有的所述密封盖一体设置在一个整体部件上。
根据本发明的一些优选方案，所述的数字PCR芯片还包括直立地设于所述芯片本体上的排液管，所述排液管与所述排液口连通。
根据本发明的一些优选方案，所述排液管自所述芯片本体的上表面向上延伸，其与所述芯片本体一体成型设置或固定连接。
根据本发明的一些优选方案，所述排液口上设有用于与负压装置的出口配接的负压接头。
根据本发明的一些优选方案，所述芯片本体由芯片盖板与芯片基板沿厚度方向叠加而成，所述芯片盖板为平板，所述的芯片基板上开设有凹槽，所述平板与所述凹槽相互叠加压紧形成所述液滴存储腔、所述第一通道及所述第二通道。
根据本发明的进一步实施方案，所述凹槽开口朝下，所述的芯片基板位于所述芯片盖板上方，所述的芯片盖板为透明玻璃板、透明PC板、透明亚克力板、COP透明板或黑色不反光板。
根据本发明的另一种实施方案，所述凹槽开口朝上，所述的芯片基板位于所述芯片盖板下方，所述芯片基板与所述芯片盖板分别采用塑料制成。
根据本发明的优选实施方案，所述液滴存储腔、所述第一通道及所述第二通道共同构成一个芯片单元，所述芯片本体上设置有多个所述芯片单元。
根据本发明的进一步实施方案，所述芯片本体为长形，所述多个所述芯片单元沿着所述芯片本体的长度方向分布。
本发明还提供了一种数字PCR检测系统，包括数字PCR检测装置，还包括如上述的数字PCR芯片，以及与所述的数字PCR芯片配合使用的负压装置，所述负压装置用于使所述第一通道、液滴存储腔以及第二通道内产生负压。
本发明还提供了一种基于如上述的数字PCR芯片或如上述的数字PCR检测系统的数字PCR检测方法，其包括向所述液滴存储腔输送液滴的上样步骤，所述上样步骤包括：
使所述数字PCR芯片的液滴存储腔、第一通道、第二通道、以及容置腔中充有油相；
利用微管道向所述容置腔内的油相中注入水相并在注入的同时使所述微管道进行往复摆动，使在容置腔内形成液滴；
使所述液滴经所述进液口、第一通道输送到所述液滴存储腔。
根据本发明的一些优选方案，在注入水相之前，使所述液滴存储腔、第一通道、第二通道中充满油相。
根据本发明的一些优选方案，在充入油相之后、注入水相之前，保持所述进液口与所述排液口均处于密封状态，使所述PCR芯片水平静置5min以上。
根据本发明的一些优选方案，在开始形成液滴之后，或完成液滴生成之后，接通负压装置，促进油相从排液口排出以及促进液滴流向液滴存储腔。
本发明还提供了一种包括如上述数字PCR芯片且用于数字PCR检测的液滴生成系统，该液滴生成系统还包括：
微管道，其具有供液体进出的第一开口和第二开口；
旋转驱动机构，其用于驱动所述微管道进行往复摆动；
流体驱动机构，其用于驱动液体通过所述微管道；
所述微管道的所述第一开口所在端能够插入所述数字PCR芯片的所述容置腔中且在所 述旋转驱动机构的驱动下能够在所述容置腔中往复摆动。
根据本发明的一些优选方案，所述的液滴生成系统还包括直立地设于所述芯片本体上的排液管，所述排液管与所述排液口连通。
进一步地，所述排液管自所述芯片本体的上表面向上延伸，其与所述芯片本体一体成型设置或固定连接。更进一步地，所述排液口上设有用于与负压装置的出口配接的负压接头。
根据本发明的一些优选方案，所述液滴存储腔、所述第一通道及所述第二通道共同构成一个芯片单元，所述芯片本体上设置有多个所述芯片单元。
根据本发明的一些优选方案，所述的微管道的往复摆动为水平摆动；所述微管道的第一开口和第二开口之间具有容积为10μL～100μL的储液腔。
根据本发明的一些优选方案，所述流体驱动机构包括注射器、输送管，所述注射器的进出液口通过所述输送管与所述的微管道的第二开口连通，所述输送管的内径小于所述的微管道的内径。优选地，所述流体驱动机构还包括用于驱动所述注射器工作的注射器驱动组件。作为一种具体的优选实施方式，所述注射器驱动组件包括丝杆螺母驱动机构或齿条齿轮驱动机构。
根据本发明的一些优选方案，所述液滴生成系统还包括所述具有出液口的储液罐，所述储液罐的出液口、所述注射器的进出液口、所述输送管的一端三者通过三通换向阀连接。
根据本发明的一些优选方案，所述驱动机构与所述的微管道相可拆卸地连接。进一步地，所述驱动机构包括旋转电机、旋转轴和接头，所述旋转电机的输出端与所述旋转轴相连接，所述接头沿着与所述旋转轴轴线相垂直的方向固定连接在所述旋转轴上，所述微管道可拆卸地安装在所述接头上。
更进一步地，所述流体驱动机构包括注射器、输送管，所述接头为管状，具有内部连通的第一液体进出口和第二液体进出口，所述输送管的一端与所述注射器的进出液口连通，另一端与所述接头的第一液体进出口接通，所述微管道的第二开口所在端与所述的接头的第二液体进出口接通；一个所述旋转轴上设置有多个所述接头，一个所述接头上连接有多个所述的微液管。
根据本发明的一些优选方案，所述微液滴生成装置还包括退针机构，所述退针机构用于使所述微管道与所述接头分离。进一步地，所述的微管道的第二开口所在端套设在所述的接头的一个端部上，所述退针机构包括能够滑动地设置在所述接头上的退针板和驱动所述退针板滑动的退针板驱动组件，通过所述退针板滑动抵触所述的微管道，使微管道与所述接头分离。其中，所述退针板驱动组件优选为丝杆螺母驱动结构或气缸驱动结构。
根据本发明的一些优选方案，所述的液滴生成系统还包括基架，所述的旋转驱动机构能够上下滑动地设置在所述的基架上，所述的液滴生成系统还包括用于驱动所述驱动机构滑动的纵向移动驱动机构。
根据本发明的一些优选方案，液滴生成系统还包括与所述PCR芯片配合使用的负压装置，所述负压装置用于使所述第一通道、液滴存储腔以及第二通道内产生负压。
本发明还提供了一种基于如上述的液滴生成系统的数字PCR检测方法，所述液滴由水相与油相混合形成，所述检测方法包括上样步骤，所述上样步骤包括：
使所述数字PCR芯片的液滴存储腔、第一通道、第二通道、以及容置腔中充有油相；
将所述微管道的第一开口插入所述容置腔中的油相液面下方，启动旋转机构，驱动所述微管道进行往复摆动，同时利用流体驱动机构和微管道，将水相注入到油相中，形成微液滴；
使所述液滴经所述进液口、第一通道输送到所述液滴存储腔。
根据本发明的一些优选方案，在注入水相之前，使所述液滴存储腔、第一通道、第二通道中充满油相。
根据本发明的一些优选方案，在充入油相后、注入水相前，保持所述进液口与所述排液口均处于密封状态下，使所述数字PCR芯片水平静置5min以上。
根据本发明的一些优选方案，在开始形成液滴之后，接通负压装置，促进油相从排液口排出以及促进液滴流向液滴存储腔。
根据本发明的一些优选方案，所述微管道的摆动角度为0.1°～10°；所述微管道往复摆动的频率为1Hz～1000Hz。
根据本发明，所述的“油相”、“水相”具有本领域的一般含义，没有特别限制。油相的密度通常小于水相。
由于上述技术方案的运用，本发明与现有技术相比具有下列优点：本发明的数字PCR芯片的结构设计基于与传统数字PCR芯片完全不同的设计原理，本发明的数字PCR芯片的芯片本体上的容置腔即构成用于液滴生成的生成容器，液滴在该容置腔中生成后，将由自身重力沉积在进液口，随后经第一通道逐渐进入液滴存储腔中，同时，本发明通过芯片上第一通道、第二通道与液滴存储腔的结构设计使得液滴在输送过程中保持很好的稳定性以及实现液滴在液滴存储腔中均匀的平铺，从而有利于获得显著更准确的检测结果，该数字PCR芯片具有结构简单、成本低的显著优势。进一步地，基于本发明的数字PCR芯片结构及方法，还可以实现液滴在液滴存储腔中的多层平铺，大大提高检测通量，满足临床上自动化及单位面积内高通量液滴分析的要求。
本发明同时还提供了一种新的液滴生成系统与生成思路。该液滴生成系统集液滴形成与检测于一体，不仅可以大批量地生成体积大小均一的液滴，且液滴可以直接地被用作检测，且液滴在液滴存储腔中能够实现均匀的平铺，有利于获得显著更准确的检测结果。该液滴生成系统具有结构简单、成本低的显著优势。
本发明的数字PCR检测系统及检测方法具有检测通量高、检测结果更准确等诸多优势。
附图说明
图1为本发明采用的微液滴生成装置的结构示意图；
图2为本发明采用的微液滴生成装置的往复摆动示意图；
图3为本发明采用的微液滴生成装置的微液滴生成示意图；
图4为现有技术中不把微管道和任何振动电机相联的情形示意图；
图5为现有技术中把微管道固定在一个可以产生匀速直线运动的振动电机上的情形示意图；
图6为本发明采用的微液滴生成装置中把微管道固定在一个可以旋转驱动机构上的情形示意图；
图7为影响微液滴生成的相关因素分析示意图；
图8为实施例微液滴生成装置的主视图；
图9为实施例微液滴生成装置的主视剖视图；
图10为实施例微液滴生成装置的左视图；
图11为实施例微液滴生成装置的后视图；
图12为图11中的A-A处的剖视图；
图13为图12中C处的局部放大图；
图14为图12中D处的局部放大图；
图15为图12中的B-B处的剖视图；
图16为本发明采用的微液滴生成装置另一种实施例的后视图；
图17为本发明采用的微液滴生成装置另一种实施例的主视剖视图；
图18为本发明采用的微液滴生成装置另一种实施例的主视图；
图19为本发明采用的微液滴生成装置另一种实施例的左视图；
图20为本发明采用的微液滴生成装置中涉及的闭环控制振动角度或位置的电机原理图；
附图21为本发明的数字PCR芯片系统实施例1的结构示意图；
附图22为本发明的数字PCR芯片系统实施例1的结构分解示意图；
附图23为实施例1的数字PCR芯片中芯片基板的主视图；
附图24为沿图23中M-M向剖视结构示意图；
附图25为附图23的芯片本体的俯视图；
附图26为附图23的芯片本体的仰视图；
附图27为实施例1的数字PCR系统中密封盖的结构示意图；
附图28为本发明的数字PCR芯片系统实施例2的结构分解示意图一；
附图29为本发明的数字PCR芯片系统实施例2的结构分解示意图二；
附图30为实施例2的数字PCR芯片中芯片盖板的轴测图；
附图31为附图30的芯片盖板的主视图；
附图32为沿图31中N-N向剖视结构示意图；
附图33为附图30的芯片盖板的仰视图；
附图34为附图30的芯片盖板的俯视图；
附图35为实施例2的数字PCR芯片中芯片基板的轴测图；
附图36为附图35的俯视图；
附图37至附图39为液滴经第一通道进入液滴存储腔的过程中实现液滴平铺的示意图；
附图40为液滴在液滴存储腔中实现平铺时俯视示意图；
附图41、42为液滴在液滴存储腔中实现两层、三层平铺时示意图。
具体实施方式
下面结合附图和具体的实施例来对本发明的技术方案作进一步的阐述。
首先，结合实施例和附图，详细说明本发明采用的微液滴生成装置的具体结构及工作原理。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
本发明提供一种新的液滴生成系统与生成思路。该液滴生成系统集液滴形成与检测于一体。本发明提供的液滴生成系统的基本构成包括微管道、旋转驱动机构和PCR芯片，除了PCR芯片以外的部分在本发明中又统称为液滴生成装置。本发明的液滴生成系统不仅可以大批量地生成体积大小均一的液滴，且液滴可以直接地被用作检测。
如图1所示，其中显示了液滴生成系统的微管道100、旋转驱动机构200等，微管道100具有用于输出第一液体130的第一开口110，旋转驱动机构200用于驱动微管道100做水平往复摆动。如图2所示，旋转驱动机构200驱动微管道100绕旋转中心221往复摆动，使得微管道100的第一开口110也产生往复摆动，从而在第二液体610的液面下产生微液滴131。
如图1所示，为了使微管道100的第一开口110能够持续的生成微液滴131，微液滴生成装置还包括流体驱动机构300，该流体驱动机构300通过输送管310与微管道100的第二开口120相连通。微管道100的第二开口120与第一开口110相通，流体驱动机构300可以通过输送管310向微管道100内施加稳定的驱动力，使微管道100内的第一液体130能够稳定连续的从第一开口110中流出并生成微液滴131。
本发明所提供的液滴生成方法是一个非常复杂的动态过程，有很多因素影响液滴生成的体积。主要因素有：液滴的表面张力(与微管道开口面积、第一和第二液体之间表面能的差距相关)、微管道开口和液滴之间的附着力(受管道开口大小和表面属性影响)；剪切力(由第二液体粘度、微管道运动的速度和液滴表面积决定)、离心力(与液滴的质量、微管道的摆动径向加速度相关)以及切向惯性力(与微管道的摆动切向加速度和液滴质量成正比)。离心力本质上其实就是径向惯性力。
如图3所示，由于微管道100的第一开口110在旋转驱动机构200的驱动下产生旋转运动，使得微管道100中的第一液体130在微管道100的第一开口110的管口形成的微液滴131在由第二液体610的粘度、微管道100的第一开口110的管口运动的速度和微液滴 131表面积决定的剪切力、与微液滴131的质量和微管道100的第一开口110的管口的摆动径向加速度相关的离心力、以及与微管道100的第一开口110的管口的摆动切向加速度和微液滴131质量成正比的切向惯性力的共同作用下，使得的微液滴131在第二液体610的液面下脱离微管道100的第一开口110的管口。
下面将本发明采用的微液滴生成装置的往复摆动方式(参照图6-7)与其他方式(参照图4-5)进行比较分析，以说明本发明所获得的特有技术效果。需要特别指出的是，下面所有的分析都是把流出微管道的第一液体130液滴作为独立的物体进行受力分析，在图4-7中以虚线框表示第一液体130液滴为独立体系。
图4为现有技术中不把微管道和任何振动电机相联的情形示意图。现有技术中，当流体驱动装置不断通过微管道100匀速将第一液体130注入第二液体610时，液滴会慢慢不断增大。因为液体体积不可压缩，那么在匀速注入的条件下，液滴体积也是匀速变大。作用在液滴上的有保持液滴不脱离的表面张力和附着力，另外还有向下的重力。当液滴增长到临界体积时(参见图4中分割线所示体积)，所受重力克服表面张力和附着力脱落。因为液滴必须成长到微升量级重力才有可能克服张力和附着力，这一方法不能产生在以纳升为特征的微液滴。
图5为现有技术中把微管道固定在一个可以产生匀速直线运动的振动电机上的情形示意图。如图5所示，在现有技术中，当流体驱动装置不断通过微管道100匀速将第一液体130注入第二液体610时，液滴会慢慢不断增大。因为液体体积不可压缩，那么在匀速注入的条件下，液滴体积也是匀速变大。与图4所示的情形不同的地方是，同时开动直线电机驱动微管道100进行匀速向左直线运动。那么，液滴的受力情况如图5所示，因为液滴与第二液体610之间有相对运动而受到向右的剪切力，剪切力与液滴的速度和表面积正相关。因而在匀速情况下，这个力随着液滴体积增大而增大。重力远远小于剪切力而被忽略。在某一临界体积(参照图5分割线所示)，当剪切力克服表面张力和附着力的时候就是液滴分离微管道口的时刻。因为环境和系统的波动，这一临界体积会有上下扰动，这是造成液滴大小不均匀的主要因素(参照图7中虚线a所示)。在这种匀速运动下，这一扰动所带来的误差是很大的(前述图4-5中的微管道液滴生成技术以及背景技术部分所提及的在先专利等现有技术都存在这个问题)。
图6为本发明微液滴生成装置中把微管道固定在一个可以旋转驱动机构上的情形示意图。如图6所示，在当流体驱动装置不断通过微管道100匀速将第一液体130注入第二液体610时，通过高频率的摆动使得微管道100的速度变化有一个高频调制，从而使得液滴所受到的摆脱合力也有一个高频调制。这个摆脱合力是由剪切力、离心力和切向惯性力合并而成的。当临界点有扰动时，这一高频变化的摆脱力足以在极短的时间内冲破临界点的扰动，从而使得扰动带来的体积误差最小化(因为液滴在流体驱动装置驱动下体积匀速增长，所以突破临界扰动所需时间少意味着体积误差小)。如图7所示，可以清楚看到同样的临界扰动下，把微管道固定在一个可以往复摆动电机上的摆动生成的体积误差1远远小于匀速生成的体积误差2。
如图8、图9和图10所示，作为一种优选的实施方式，本发明液滴生成装置中的旋转驱动机构200包括旋转电机210、旋转轴220和接头230，旋转电机210的输出端与旋转轴220相连接，接头230沿着与旋转轴220轴线相垂直的方向固定连接在旋转轴220上，微管道100安装在接头230上。旋转电机210可以驱动旋转轴220和接头230绕旋转轴220的轴线为中心旋转摆动，从而可以带动微管道100往复摆动。本发明液滴生成装置中的旋转驱动机构200还可以采用其他旋转驱动装置，例如摆动气缸、旋转电磁铁等。
在本实施方式中，微管道100为两端都具有开口的管状结构，为了便于安装微管道100，接头230也为管状，结合图13所示，管状接头230具有内部相连通的第三开口231和第四开口232，输送管310连接在第三开口231上，微管道100的第二开口120连接在第四开口232上。流体驱动机构300输出的流体驱动力可以通过输送管310和接头230稳定地作用在微管道100内，使微管道100内的第一液体130能够稳定连续的从第一开口110中流 出生成微液滴131。
由于本发明微液滴生成装置可以用在生物检测领域中，为了避免生物材料的交叉污染，微管道100通常是一次性使用的，从而需要将每次使用过后的微管道100从接头230上拆卸下来，为了提高拆卸效率，本发明微液滴生成装置还包括退针机构400，结合图12和图13所示，退针机构400包括退针板410和退针板驱动组件420，退针板410上开设有退针孔411，退针孔411套设在接头230的外部，微管道100的第二开口120套设在第四开口232的外部，且与退针板410相对，退针板驱动组件420用于驱动退针板410向微管道100的方向运动，当退针板410抵靠在微管道100的第二开口120时，向微管道100施加了使其脱离接头230的挤压作用力，退针板410继续运动就会将微管道100从接头230上推出，之后，退针板驱动组件420驱动退针板410向靠近输送管310的方向运动，便于下一个微管道100能够套装在接头230上。除了本实施例所提供的退针机构，还可以采用其他的结构实现微管道与接头的分离，例如采用卡爪夹持微管道，通过驱动卡爪运动将微管道从接头上拉拔下来使两者分离。
作为一种优选的实施方式，为了便于安装和拆卸微管道100，接头230的第四开口232的外部呈上大下小的圆台状，减小微管道100安装和拆卸的阻力。
具体的，退针板驱动组件420包括退针板驱动电机421、第一丝杠422和第一丝杠螺母423，退针板驱动电机421固定安装在安装支架240上，退针板驱动电机421的输出端与第一丝杠422相连接，第一丝杠螺母423与第一丝杠422配合安装，退针板410与第一丝杠螺母423相连接。第一丝杠螺母423与第一丝杠422配合将退针板驱动电机421输出的旋转运动转变为第一丝杠螺母423沿着第一丝杠422轴向上的直线运动，从而可以带动退针板410进行直线运动，当然，也可以采用其他形式的直线驱动组件以驱动退针板410。例如气缸驱动。
如图18，另一种方案的退针板驱动组件420包括第一气缸1421和第一固定螺母1423，第一气缸1421固定安装在安装支架240上，第一固定螺母1423与第一气缸1421活塞杆1422前端配合安装，退针板410与第一固定螺母1423相连接。当气体注入第一气缸1421，第一气缸活塞杆1421前端向外伸出，沿活塞杆轴向运动。第一固定螺母1423与第一气缸1421活塞杆前端配合将第一气缸1421输出的轴向运动传递至退针板410，从而可以带动退针板410进行直线运动，进行退针。
进一步的，旋转驱动机构200还包括安装支架240，旋转电机210和退针板驱动电机421分别固定安装在安装支架240上，旋转轴220的两端通过轴承可转动的设置在安装支架240中，使旋转驱动机构200的结构能加紧凑、稳定。
更进一步的，结合图9所示，微液滴生成装置还包括纵向移动机构500，纵向移动机构500包括第一安装板510、纵向移动驱动组件520和纵向滑动组件530，安装支架240通过纵向滑动组件530安装在第一安装板510上，纵向移动驱动组件520用于驱动安装支架240沿着纵向滑动组件530滑动。在纵向移动驱动组件520的作用下，安装支架240可以带动旋转驱动机构200在纵向上移动，即旋转轴220上的接头230可以在纵向上移动。通过控制接头230在纵向上移动，可以带动接头230上的微管道100在纵向上同步移动，当需要将微管道100的第一开口插入第二液体液面以下时，可以通过控制纵向移动机构500带动微管道100向下运动到预定高度；当需要将微管道100移出时，可以通过控制纵向移动机构500带动微管道100向上运动。纵向移动机构500还为接头230自动装载微管道100提供了条件，当需要将微管道100安装在接头230上时，可以将微管道100放置在接头230下方，使微管道100的第二开口120对准接头230，启动纵向移动驱动组件520，带动接头230向下移动，使接头230的第四开口232插入到微管道100的第二开口120中，然后再带动接头230向上移动复位。并且，在接头230上装载微管道100之后，纵向移动驱动组件520还可以驱动微管道100向下移动使第一开口110插入第二液体610的液面之下，进行往复摆动以制造微液滴。
具体的，结合图8和图9所示，纵向移动驱动组件520包括纵向移动驱动电机521、 第二丝杠522和第二丝杠螺母523，纵向移动驱动电机521固定安装在第一安装板510上，纵向移动驱动电机的输出端与第二丝杠522相连接，第二丝杠螺母523与第二丝杠522配合安装，安装支架240与第二丝杠螺母523相连接。第二丝杠螺母523与第二丝杠522配合将纵向移动驱动电机521输出的旋转运动转变为第二丝杠螺母523沿着第二丝杠522轴向上的直线运动，从而可以带动安装支架240进行直线运动，当然，也可以采用其他形式的直线驱动组件以驱动安装支架240。例如齿条驱动。
如图17，另一种方案的纵向移动驱动组件520包括带断电刹车的纵向移动齿轮驱动电机1521、第一齿轮1522和第一齿条1523，纵向移动齿轮驱动电机1521固定安装在安装支架240上，纵向移动驱动电机的输出端与第一齿轮1522相连接，第一齿条1523固定在第一安装板510上，第一齿条1523与第一齿轮1522配合安装。第一齿轮1522和第一齿条1523配合将纵向移动齿轮驱动电机1521输出的旋转运动转变为纵向移动齿轮驱动电机1521与第一齿轮1522沿着第一齿条1523轴向上的直线运动，从而可以带动安装支架240进行直线运动，当然，也可以采用其他形式的直线驱动组件以驱动安装支架240。
如图10、图11和图12所示，本实施方式中的流体驱动机构300包括注射器350和注射器驱动组件320，注射器350的进出液口通过输送管310与微管道100的第二开口120相连通。注射器350的推杆351在注射器驱动组件320的带动下在注射器350的筒体内滑动，推动其中的驱动液通过输送管310和接头230进入微管道100中，向微管道100中的第一液体130提供流体驱动力。本发明所提供的流体驱动机构并不限于上述实施方式，比如，还可以采用蠕动泵、压力驱动泵、气压驱动泵或电渗驱动泵等。
进一步的，如图12所示，流体驱动机构300还包括三通换向阀330和储液罐，微管道100的第二开口120、注射器350的进出液口和储液罐的出液口与三通换向阀330的三个接口相连通。三通换向阀330至少可以控制流体驱动机构300实现以下两种模式：一、使注射器350的进出液口与微管道100的第二开口120相连通，在注射器驱动组件320的带动下，注射器350向微管道100提供液体驱动力，用于将微管道100内的第一液体从第一开口110推出，或者将第一液体从第一开口110抽吸进入微管道100内；二、使注射器350的进出液口与储液罐相连通，在注射器驱动组件320的带动下，注射器350将储液罐中的驱动液抽吸进入注射器350的管体内，或者是将注射器350内的驱动液推入储液罐内。
结合图8、图9和图11所示，为了能够提高微液滴的生成效率，作为一种优选的实施方式，微管道100、接头230、输送管310和注射器350分别为多个，多个接头230在旋转轴220上间隔排列，多个微管道100分别安装在一个接头230上，每根输送管310的两端分别与一个微管道100的第二开口和三通换向阀330的第一接口相连通，每个注射器350的进出液口与三通换向阀330的第二接口相连通，储液罐的出液口与三通换向阀330的第三接口相连通。多个微管道100可以在注射器350和旋转电机210的驱动下同时进行微液滴生成的工作，一个三通换向阀330可以实现同时控制多个微管道100的微液滴生成状态。
作为一种优选的实施方式，也可以对应多个微管道100、接头230、输送管310和注射器350设置多个三通换向阀330，将多个三通换向阀330分别与多个输送管310和多个注射器350相连通，这样可以通过对多个三通换向阀独立控制以实现对多个微管道100的微液滴生成状态进行独立控制。
进一步的，结合图12、图14和15所示，流体驱动机构300还包括安装块340，多个三通换向阀330和注射器350固定安装在安装块340上，安装块340内开设有多个第一流道341、多个第二流道342、一个第三流道343和多个分液流道344，每个第一流道341的两端分别与一根输送管310和一个三通换向阀330的第一接口相连通，每个第二流道342的两端分别与一个注射器350的进出液口和一个三通换向阀330的第二接口相连通，第三流道343与储液罐和多个分液流道344相连通，每个分液流道344与一个三通换向阀330的第三接口相连通。
具体的，结合图10所示，注射器驱动组件320包括注射器驱动电机321、第三丝杠322和第三丝杠螺母323，注射器驱动电机321的输出端与第三丝杠322相连接，第三丝杠螺 母323与第三丝杠322配合安装，多个注射器350的推杆351通过连接件(图中未示出)与第三丝杠螺母323相连接。第三丝杠螺母323与第三丝杠322配合将注射器驱动电机321输出的旋转运动转变为第三丝杠螺母323沿着第三丝杠322轴向上的直线运动，从而可以带动注射器350的推杆351进行直线运动，当然，也可以采用其他形式的直线驱动组件以驱动推杆351。例如齿条驱动。
如图16，另一种方案的注射器驱动组件320包括带断电刹车的注射器齿轮驱动电机1321、第二齿轮1322和第二齿条1323，注射器驱动电机1321的输出端与第二齿轮1322相连接，第二齿条1323与第二安装板360相连接，第二齿条1323与第二齿轮1322配合安装，多个注射器350的推杆351通过连接件(图中未示出)与注射器齿轮驱动电机1321相连接。第二齿轮1322和第二齿条1323配合将注射器驱动电机1321输出的旋转运动转变为注射器齿轮驱动电机1321与第二齿轮1322沿着第二齿条1323轴向上的直线运动，从而可以带动注射器350的351进行直线运动，当然，也可以采用其他形式的直线驱动组件以驱动推杆351。
更进一步的，流体驱动机构300还包括第二安装板360，安装块340和注射器驱动电机321固定安装在第二安装板360上，第二安装板360使流体驱动机构300结构更加紧凑和稳定。同时也可将第一安装板与第二安装板合并设置，以节约空间，例如图19中所示，可将注射器驱动组件320安装在整合安装板1360上，其余安装与驱动方式均不改变。
作为一种优选的实施方式，旋转电机210可以采用振镜电机，振镜电机可以提供稳定且高速的往复旋转摆动动作，且摆幅和频率可以按照需求设定，极大提高了本发明微液滴生成装置的适用范围。同时，退针板驱动电机421、纵向移动驱动电机521、注射器驱动电机321可以采用步进电机，步进电机和丝杠丝母结构配合可以精确地控制直线运动的行程，提高自动化程度。
优选地，旋转电机210采用具有闭环控制振动角度或位置的电机，由闭环控制振动角度或位置的电机驱动旋转驱动机构200进行往复摆动，从而精密的控制微管道100的摆动轨迹，从而进一步减少环境和系统带来的扰动。这一方法的另一个优势在于可以调整系统参数使得临界体积在一个摆动周期内就可以达到(如图7中箭头所指)。这意味着每一个旋转运动的周期内只产生一个液滴。这样使得由于各种环境因素波动所带来的液滴体积的变化不会累积到下一个周期。因而可以大批量的生成大小均匀的液滴。这一点也是其他已经公布的通过机械运动生成纳升/皮升量级乳化液滴方案所不具备的优势。
以下结合图16阐述闭环控制振动角度或位置的电机在本发明中的应用。闭环控制振动角度或位置的电机包括红外位置传感器、控制电路和信号处理电路等部件。在本发明中，在旋转驱动机构200的旋转轴220上安装红外位置传感器，通过红外位置传感器把其所获得的位置信号反馈到控制电路中，控制电路依据PID自动化控制原理分别对反馈的位置信号做了比例、积分、微分运算处理，并且结合位置前馈和速度环、电流环等的信号处理电路，实现了电机运动时的绝对位置精确控制。采用闭环控制振动角度或位置的电机可以避免其它振动电机受到复杂的负载环境变化而引起振动位置的改变，其有利于工程上精确控制液滴体积和生成速度。
在本实施方式中，微管道100的第一开口110和第二开口120之间具有容积为10μL～100μL的储液腔，该储液腔可以存储一定量的第一液体，保证第一液体足够生成所需数量的微液滴，同时，储液腔还可以防止第一液体通过微管道100被吸入到接头230、输送管310中，保障系统不会被样本污染。
优选的，微管道100可以为非刚性材料制成，具有一定柔性。一定柔性指的是微管道100在旋转驱动机构200的驱动下能够使微管道100的第一开口110的运动路径具有一定的驻波现象。采用由具有一定柔性的材料制成的微管道，从而进一步地减少了对液面产生的扰动，使得生成液滴更加容易、均匀，同时也进一步地减少了所生成的液体破碎现象。
在本实施方式中，微管道100由低表面能的聚丙烯材料制成；输送管310由特氟龙(Teflon)材料制成。
在实施方式中，微管道100的第一开口110的管口内径为1μm-250μm，更优选地， 微管道100的第一开口110的管口内径为10μm-100μm。
接下来，结合附图和实施例来详细说明本发明中数字PCR芯片的结构及其工作原理。
参见图21至图22，其中显示了一种数字PCR芯片、本发明所述的微管道100(本发明中也称为输出枪针)以及具有用于产生负压且具有负压枪针50的负压装置。
本例的数字PCR芯片，其包括具有液滴存储腔1的芯片本体10，设置在芯片本体10上的进液口4与排液口5，以及直立设置在芯片本体10上且与进液口4连通的容置腔61，芯片本体10还包括分别将进液口4与液滴存储腔1连通的第一通道2、将排液口5与液滴存储腔1连通的第二通道3，其中，第一通道2具有位于芯片本体10内部的第一内通路，第二通道3也具有位于芯片本体10内部的第二内通路。
该数字PCR芯片中，容置腔61自芯片本体10的上表面向上延伸，进液口4位于容置腔61的底部。使用时，先使液滴存储腔1、第一通道2、第二通道3及容置腔61中充有油相，然后利用输出枪针40向容置腔61的油相中注入水相，并在注入的同时使输出枪针40进行往复摆动，使得在容置腔61内形成液滴。水相的密度通常大于油相，所形成的液滴将因自身重力沉积至容置腔61的底部，随后经进液口4进入第一通道2，从而进入液滴存储腔1中。
具体地，该容置腔61的长度为20～1000um、宽度为20～1000um、高度为20～2000um。该容置腔61可设置为与芯片本体10固定连接，还可以与芯片本体10一体成型设置。本实施例中，芯片本体10的上表面具有向上竖立延伸的进液导流管6，该进液导流管6的管腔即构成上的容置腔61。
该数字PCR芯片中，第二通路3也在端部与液滴存储腔1接通。作为优选地，该数字PCR芯片中，作为优选地，第一通路2在端部与液滴存储腔1接通，第一内通路设置于液滴存储腔1的一侧；该第二通路3也在端部与液滴存储腔1接通，第二内通路与第一内通路分设于液滴存储腔1的相异两侧，可通过负压装置的负压枪针50经由排液口产生负压，辅助液滴缓慢地从第一通道2逐渐地进入液滴存储腔1中。
该芯片本体10上，第一通道2、第二通道3及液滴存储腔1的底面优选设置为位于同一高度位置，进液口4在竖直方向的高度高于第一通道2，排液口5在竖直方向的高度高于第二通道3。进液口4的内径优选设置为4mm-8mm，高度优选为5mm-15mm。第一通道2、第二通道3的内径分别为4mm-10mm。液滴储存腔1的长、宽分别为2-30mm，高度为20-2000um。
参见图26所示，液滴存储腔1具有与第一通道2连通的第一连通口1a、与第二通道3连通的第二连通口1b，该第一连通口1a与第二连通口1b分设于液滴存储腔1相异的两侧侧部上，优选采用第一连通口1a正对第二连通口1b设置；当液滴存储腔1的横截面采用多边形结构时，第一连通口1a与第二连通口1b优选设置于多边形的一组对角上。
在某些实施例中，液滴存储腔1具有至少一个弧形倒角，第一连通口1a设于上述弧形倒角处，使得第一通道2连接在该第一连通口1a上而与液滴存储腔1接通，这更有利于液滴进入液滴存储腔1后实现平铺移动。该液滴存储腔1可以为具有圆弧内倒角的多边形，或者为圆形、椭圆形。
当液滴存储腔1的截面为多边形时，可采用其中两条相邻边交接的位置形成上述的弧形倒角，也可以在其中一条边上进行大倒角处理而形成弧形倒角，当液滴存储腔1的截面为其他不规则形状时，最好也需要进行大倒角处理。作为优选的方案，液滴存储腔1的横截面中至少有两条相邻边之间的夹角呈直角，将第一连通口1a设于该直角处。
本实施例中，液滴存储腔1的横截面设置为正方形，第一连通口1a与第二连通口1b其一组对角上，如图26所示，而其他的各内角则均采用圆弧内倒角设置，有利于液滴在液滴存储腔1中保持良好的稳定性。该液滴存储腔1横截面的正方形边长为5-30mm，液滴存储腔1的高度则为50-1000um。当液滴的直径降低时，也可将该液滴存储腔1的边长尺 寸降低的更小，而高度的设置一方面需要满足液滴平铺的需要，另一方面则需兼顾配方油充分利用的问题。
作为优选地，第一通道2与第二通道3的部分或整体为曲线状。本实施例中，参见图26所示，第一通道2包括一端与进液口4相连通的进液段2a，以及自进液段2a的另一端朝向液滴存储腔1呈弧线弯曲延伸的出液段2b。进液段2a呈直线延伸段，其位于液滴存储腔1的外侧且与液滴存储1腔的一条边平行，进液段2a的一端向着液滴存储腔1的方向弯折并延伸形成呈弧形延伸段的出液段2b，出液段2b的远离进液段2a的端部连接在第一连通口1a上而与液滴存储腔1接通，该设计有利于液滴经由进液口4进入第一通道2后在重力的作用下平稳地进入液滴存储腔1中。
第二通道3与第一通道2中心对称设置。具体地，第二通道3包括一端与排液口5相连通的排液段3a及自排液段3a的另一端朝向液滴存储腔1呈弧线弯曲延伸的进液段3b，该进液段3b呈远离排液段3a逐渐拱起的弧形，进液段3b的端部连接在第二连通口1b上而与液滴存储腔1接通。
整体上，第一通道2、液滴存储腔1、第二通道3构成了中心对称的结构。该结构设计实现了液滴的平稳输送，确保液滴的稳定性。
芯片本体10主要由芯片盖板与芯片基板沿厚度方向叠加而成，芯片盖板为平板，芯片基板上开设有凹槽，所述平板与所述凹槽相互叠加压紧形成液滴存储腔1、第一通道2及第二通道3。本实施例中，凹槽开口朝下，芯片基板101位于芯片盖板102上方，芯片盖板102为透明玻璃板、透明PC板、透明亚克力板、COP透明板或如POM、PP等不反光材料制成的黑色不反光板。芯片基板101与芯片盖板102之间可采用胶合或超声波或者热压键合工艺焊接至密封，两者之间的边缘需保持绝对的密封性。
在如图28至图36所示的另一实施例中，凹槽开口朝上，芯片基板104位于芯片盖板103下方，具体地，进液口4与排液口5均开设在芯片盖板103上，进液导流管6与排液导流管7也一体成型于芯片盖板103上；液滴存储腔1、第一通道2及第二通道3则设置在芯片基板104上。上述的芯片盖板103与芯片基板104均采用塑料制成，两者之间采用热压键合工艺焊接至密封。
参见图21、图22、图23及图24所示，芯片本体10的上表面，亦即芯片基板101的上表面还具有向上竖立延伸且与排液口5连通的排液导流管7，该排液导流管7则主要用作与负压枪针50配接的负压接头，该负压枪针50与排液导流管7予以配接以对液滴存储腔1形成负压。本实施例中，进液导流管6与排液导流管7采用的为一体成型地设于芯片基板101上，当然，在其他的一些实施方式中，还可以采用独立加工的方式先成型再通过超声波焊接或胶水粘接等方式连接至芯片基板101上。每个进液导流管6的管口均可通过可拆卸连接的密封盖201予以密封，这亦即对容置腔61实现密封；排液导流管7的管口则可通过密封膜30予以密封。
参见图21至图26所示，芯片本体10的内部，液滴存储腔1、第一通道2及第二通道3共同构成芯片单元，该芯片本体10上设置有沿长度方向间隔排布的多个上述芯片单元，可同时进行多组上样与分析检测。相应地，进液口4、排液口5及容置腔61也有多组，进液导流管6与排液导流管7也设置有多组。为制造方便及操作方便，与多个进液导流管6适配的所有密封盖201一体设置形成了一个整体密封盖部件20，密封膜30也作为一个整体部件而能够同时作为所有排液导流管7管口的密封。
本发明同时还提供了采用上述数字PCR芯片或数字PCR检测系统的检测方法，其包括向液滴存储腔1输送液滴的上样步骤，该上样步骤包括：
使数字PCR芯片的液滴存储腔1、第一通道2、第二通道3、以及容置腔61中充入油相；
利用微管道100(即输出枪针40)向容置腔61内的油相中注入水相并在注入的同时使 微管道100进行往复摆动，使在容置腔61内形成液滴；
使所述液滴经进液口4、第一通道2输送到液滴存储腔1。
其中，在注入水相之前，优选使液滴存储腔1、第一通道2、第二通道3中充满油相。在注入水相之前最好保持进液口4与排液口5均处于密封状态，并使PCR芯片水平静置5min以上；在开始形成液滴之后，或完成液滴生成之后，接通负压装置，促进油相从排液口5排出以及促进液滴流向液滴存储腔1。
具体检测过程按照如下步骤进行：预先在芯片本体10的液滴存储腔1、第一通道2、第二通道3、以及容置腔61中充有油相，并分别采用密封盖201与密封膜30密封住进液导流管6与排液导流管7的管口。待芯片本体10静置5min以上，打开密封盖201，将液滴生成装置的输出枪针40的输出针头插入进液导流管6的容置腔61中，使得输出针头的端口(即微管道100的第一开口110)位于油相的液面之下注入水相并在注入的同时使输出针头进行往复摆动，使在容置腔61内形成液滴。产生的液滴由于自身重力的作用堆积在容置腔61的底部，且有部分液滴经进液口4自然落下至第一管道2，此时液滴的介入，油相液面高度会在进液口4内有所增加，但不会影响到液滴生成的稳定性。完成液滴生成后，扎破排液导流管7管口处的密封膜30(可另外设置配合仪器的可作动机构来执行扎破)，并使连接负压装置的负压枪针50缓慢地产生负压，液滴会随着压力作用而由进液口4缓慢经过第一通道2进入液滴存储腔1，并呈扇形区域平铺至液滴存储区域1，如图37至图39所示，此时液滴生成以及初步平铺过程完成，可通过机械结构压紧芯片本体10。
压紧的过程中可以在整个芯片本体10上表面压紧或几个固定点压紧，并通过弹簧等结构来缓冲压力，若采用几个固定点压紧则需要避开激发光照射或相机检测光路区域，从而可进行实时荧光读取，观察到液滴随时的运动状态。
此外，通过调整液滴存储腔1的厚度和面积、液滴的体积和样品总体积，可以实现精准的一层，或者如图41所示的两层，或者如图42所示的三层，甚至更多层的平铺。假设样品体积为20微升，液滴体积为1纳升，芯片面积为16mm×16mm，芯片厚度为125～150微米，则生成的2万液滴只能平铺为一层。而同样20微升样品体积，1纳升液滴体积，如果芯片面积调整为11.5mm×11.5mm，厚度调整为200～275微米，则2万液滴只能平铺为2层。
这种多层液滴的平铺方式，可以实现在单位面积内更高通量的液滴多层观察。这对提高图像式检测液滴的数字PCR设备的整体检测通量至关重要，解决了这类设备所面临的检测通量低的瓶颈问题。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (60)

  1. 一种数字PCR芯片，包括具有液滴存储腔的芯片本体和设置在所述芯片本体上的进液口，其特征在于：所述的数字PCR芯片还包括直立设置在所述芯片本体上且与所述进液口连通的容置腔，以及设置在所述芯片本体上的排液口；所述芯片本体还包括分别将所述进液口与所述液滴存储腔连通的第一通道、将所述排液口与所述液滴存储腔连通的第二通道，所述第一通道具有位于所述芯片本体内部的第一内通路，所述第二通道具有位于芯片本体内部的第二内通路。
  2. 根据权利要求1所述的数字PCR芯片，其特征在于：所述容置腔自所述芯片本体的上表面向上延伸，所述进液口位于所述容置腔的底部。
  3. 根据权利要求1所述的数字PCR芯片，其特征在于：所述容置腔的长度为2～30mm、宽度为2～30mm、高度为20～2000um。
  4. 根据权利要求1所述的数字PCR芯片，其特征在于：所述容置腔与所述芯片本体一体成型设置，或者，所述容置腔与所述芯片本体相固定连接。
  5. 根据权利要求1所述的数字PCR芯片，其特征在于：所述第一通道在端部与所述液滴存储腔接通；和/或，所述第二通道在端部与所述液滴储存腔接通。
  6. 根据权利要求1所述的数字PCR芯片，其特征在于：所述第一通道位于所述液滴存储腔的一侧；和/或，所述第二通道位于所述液滴存储腔的一侧。
  7. 根据权利要求1所述的数字PCR芯片，其特征在于：所述第一内通路与所述第二内通路分设于所述液滴存储腔相异的两侧。
  8. 根据权利要求1所述的数字PCR芯片，其特征在于：所述液滴存储腔具有与所述第一通道连通的第一连通口、与所述第二通道连通的第二连通口，所述第一连通口与所述第二连通口分设于所述液滴存储腔相对的两个侧部上。
  9. 根据权利要求8所述的数字PCR芯片，其特征在于：所述第一连通口正对所述第二连通口设置。
  10. 根据权利要求8所述的数字PCR芯片，其特征在于：所述液滴储存腔具有至少一个弧形倒角，所述第一连通口设于所述弧形倒角处。
  11. 根据权利要求8所述的数字PCR芯片，其特征在于：所述液滴存储腔为具有圆弧内倒角的多边形；或者，所述液滴存储腔为圆形或椭圆形。
  12. 根据权利要求8所述的数字PCR芯片，其特征在于：所述液滴储存腔为正方形或矩形，所述第一连通口与所述第二连通口分设于所述液滴存储腔的对角上，所述第一内通路与所述第二内通路分设于所述液滴存储腔相对的两侧，所述第一内通路与所述第二内通路分别在端部与所述第一连通口、第二连通口连通。
  13. 根据权利要求1所述的数字PCR芯片，其特征在于：所述第一通道、第二通道的部分或整体为曲线状。
  14. 根据权利要求13所述的数字PCR芯片，其特征在于：所述第一通道包括至少一个直形延伸段和至少一个弧形延伸段，且由所述直形延伸段的一端与所述进液口连通，所述至少一个直形延伸段和至少一个弧形延伸段的内部空间构成所述第一内通路。
  15. 根据权利要求14所述的数字PCR芯片，其特征在于：所述第一内通路由一个直形延伸段和一个弧形延伸段的内部空间构成，所述直形延伸段位于所述液滴存储腔的外侧且与所述液滴存储腔的一条边平行，所述直形延伸段的一端向着所述液滴存储腔的方向弯折并延伸形成所述弧形延伸段，所述弧形延伸段的远离直形延伸段的端部与所述液滴存储腔连通。
  16. 根据权利要求1、13、14或15所述的数字PCR芯片，其特征在于：所述液滴存储腔、所述第一通道、所述第二通道构成中心对称的结构。
  17. 根据权利要求1所述的数字PCR芯片，其特征在于：所述第一通道、所述第二通道及所述液滴存储腔的底面位于同一高度位置。
  18. 根据权利要求1所述的数字PCR芯片，其特征在于：所述进液口在竖直方向的高度高于所述第一通 道，和/或，所述排液口在竖直方向的高度高于所述第二通道。
  19. 根据权利要求1所述的数字PCR芯片，其特征在于：所述进液口的内径为4mm-10mm，高度为5mm-15mm；和/或，所述液滴储存腔的长、宽分别为2-30mm，高度为20-1000um；和/或，所述芯片本体的厚度为1～6mm。
  20. 根据权利要求1所述的数字PCR芯片，其特征在于：所述芯片还包括用于密封所述容置腔的密封盖。
  21. 根据权利要求20所述的数字PCR芯片，其特征在于：所述容置腔有多个，对应地，所述密封盖也有多个，所有的所述密封盖一体设置在一个整体部件上。
  22. 根据权利要求1所述的数字PCR芯片，其特征在于：所述的数字PCR芯片还包括直立地设于所述芯片本体上的排液管，所述排液管与所述排液口连通。
  23. 根据权利要求22所述的数字PCR芯片，其特征在于：所述排液管自所述芯片本体的上表面向上延伸，其与所述芯片本体一体成型设置或固定连接。
  24. 根据权利要求1所述的数字PCR芯片，其特征在于：所述排液口上设有用于与负压装置的出口配接的负压接头。
  25. 根据权利要求1所述的数字PCR芯片，其特征在于：所述芯片本体由芯片盖板与芯片基板沿厚度方向叠加而成，所述芯片盖板为平板，所述的芯片基板上开设有凹槽，所述平板与所述凹槽相互叠加压紧形成所述液滴存储腔、所述第一通道及所述第二通道。
  26. 根据权利要求25所述的数字PCR芯片，其特征在于：所述凹槽开口朝下，所述的芯片基板位于所述芯片盖板上方，所述的芯片盖板为透明玻璃板、透明PC板、透明亚克力板、COP透明板或黑色不反光板。
  27. 根据权利要求25所述的数字PCR芯片，其特征在于：所述凹槽开口朝上，所述的芯片基板位于所述芯片盖板下方，所述芯片基板与所述芯片盖板分别采用塑料制成。
  28. 根据权利要求1所述的数字PCR芯片，其特征在于：所述液滴存储腔、所述第一通道及所述第二通道共同构成一个芯片单元，所述芯片本体上设置有多个所述芯片单元。
  29. 根据权利要求28所述的数字PCR芯片，其特征在于：所述芯片本体为长形，所述多个所述芯片单元沿着所述芯片本体的长度方向分布。
  30. 一种数字PCR检测系统，包括数字PCR检测装置，其特征在于，还包括如权利要求1至29中任一项所述的数字PCR芯片，以及与所述的数字PCR芯片配合使用的负压装置，所述负压装置用于使所述第一通道、液滴存储腔以及第二通道内产生负压。
  31. 一种基于如权利要求1至29中任一项权利要求所述的数字PCR芯片或如权利要求30所述的数字PCR检测系统的数字PCR检测方法，其特征在于，所述检测方法包括向所述液滴存储腔输送液滴的上样步骤，所述上样步骤包括：
    使所述数字PCR芯片的液滴存储腔、第一通道、第二通道、以及容置腔中充有油相；
    利用微管道向所述容置腔内的油相中注入水相并在注入的同时使所述微管道进行往复摆动，使在容置腔内形成液滴；
    使所述液滴经所述进液口、第一通道输送到所述液滴存储腔。
  32. 根据权利要求31所述的数字PCR检测方法，其特征在于：在注入水相之前，使所述液滴存储腔、第一通道、第二通道中充满油相。
  33. 根据权利要求31所述的数字PCR检测方法，其特征在于：在充入油相之后、注入水相之前，保持所述进液口与所述排液口均处于密封状态，使所述PCR芯片水平静置5min以上。
  34. 根据权利要求31所述的数字PCR检测方法，其特征在于：在开始形成液滴之后，或完成液滴生成之后，接通负压装置，促进油相从排液口排出以及促进液滴流向液滴存储腔。
  35. 一种用于数字PCR检测的液滴生成系统，其特征在于：包括如权利要求1至29中任一项权利要求所述的数字PCR芯片，所述液滴生成系统还包括：
    微管道，其具有供液体进出的第一开口和第二开口；
    旋转驱动机构，其用于驱动所述微管道进行往复摆动；
    流体驱动机构，其用于驱动液体通过所述微管道；
    所述微管道的所述第一开口所在端能够插入所述数字PCR芯片的所述容置腔中且在所述旋转驱动机构的驱动下能够在所述容置腔中往复摆动。
  36. 根据权利要求35所述的液滴生成系统，其特征在于：所述的液滴生成系统还包括直立地设于所述芯片本体上的排液管，所述排液管与所述排液口连通。
  37. 根据权利要求36所述的液滴生成系统，其特征在于：所述排液管自所述芯片本体的上表面向上延伸，其与所述芯片本体一体成型设置或固定连接。
  38. 根据权利要求35或36所述的液滴生成系统，其特征在于：所述排液口上设有用于与负压装置的出口配接的负压接头。
  39. 根据权利要求35所述的液滴生成系统，其特征在于：所述液滴存储腔、所述第一通道及所述第二通道共同构成一个芯片单元，所述芯片本体上设置有多个所述芯片单元。
  40. 根据权利要求35所述的液滴生成系统，其特征在于，所述的微管道的往复摆动为水平摆动。
  41. 根据权利要求35所述的液滴生成系统，其特征在于，所述微管道的第一开口和第二开口之间具有容积为10μL～100μL的储液腔。
  42. 根据权利要求35所述的液滴生成系统，其特征在于：所述流体驱动机构包括注射器、输送管，所述注射器的进出液口通过所述输送管与所述的微管道的第二开口连通，所述输送管的内径小于所述的微管道的内径。
  43. 根据权利要求43所述的液滴生成系统，其特征在于：所述流体驱动机构还包括用于驱动所述注射器工作的注射器驱动组件。
  44. 根据权利要求44所述的液滴生成系统，其特征在于：所述注射器驱动组件包括丝杆螺母驱动机构或齿条齿轮驱动机构。
  45. 根据权利要求43所述的液滴生成系统，其特征在于：所述液滴生成系统还包括所述具有出液口的储液罐，所述储液罐的出液口、所述注射器的进出液口、所述输送管的一端三者通过三通换向阀连接。
  46. 根据权利要求35所述的液滴生成系统，其特征在于：所述驱动机构与所述的微管道相可拆卸地连接。
  47. 根据权利要求47所述的液滴生成系统，其特征在于：所述驱动机构包括旋转电机、旋转轴和接头，所述旋转电机的输出端与所述旋转轴相连接，所述接头沿着与所述旋转轴轴线相垂直的方向固定连接在所述旋转轴上，所述微管道可拆卸地安装在所述接头上。
  48. 根据权利要求48所述的液滴生成系统，其特征在于，所述流体驱动机构包括注射器、输送管，所述接头为管状，具有内部连通的第一液体进出口和第二液体进出口，所述输送管的一端与所述注射器的进出液口连通，另一端与所述接头的第一液体进出口接通，所述微管道的第二开口所在端与所述的接头的第二液体进出口接通。
  49. 根据权利要求48或49所述的液滴生成系统，其特征在于，一个所述旋转轴上设置有多个所述接头，一个所述接头上连接有多个所述的微液管。
  50. 根据权利要求47或48或49所述的液滴生成系统，其特征在于，所述微液滴生成装置还包括退针机构，所述退针机构用于使所述微管道与所述接头分离。
  51. 根据权利要求51所述的液滴生成系统，其特征在于，所述的微管道的第二开口所在端套设在所述的接头的一个端部上，所述退针机构包括能够滑动地设置在所述接头上的退针板和驱动所述退针板滑动的退针板驱动组件，通过所述退针板滑动抵触所述的微管道，使微管道与所述接头分离。
  52. 根据权利要求52所述的液滴生成系统，其特征在于：所述退针板驱动组件为丝杆螺母驱动结构或气缸驱动结构。
  53. 根据权利要求35所述的液滴生成系统，其特征在于，所述的液滴生成系统还包括基架，所述的旋转驱动机构能够上下滑动地设置在所述的基架上，所述的液滴生成系统还包括用于驱动所述驱动机构滑动的纵向移动驱动机构。
  54. 根据权利要求35所述的液滴生成系统，其特征在于，液滴生成系统还包括与所述PCR芯片配合使用的负压装置，所述负压装置用于使所述第一通道、液滴存储腔以及第二通道内产生负压。
  55. 一种基于如权利要求35至55中任一项权利要求所述的液滴生成系统的数字PCR检测方法，所述液 滴由水相与油相混合形成，其特征在于，所述检测方法包括上样步骤，所述上样步骤包括：
    使所述数字PCR芯片的液滴存储腔、第一通道、第二通道、以及容置腔中充有油相；
    将所述微管道的第一开口插入所述容置腔中的油相液面下方，启动旋转机构，驱动所述微管道进行往复摆动，同时利用流体驱动机构和微管道，将水相注入到油相中，形成微液滴；
    使所述液滴经所述进液口、第一通道输送到所述液滴存储腔。
  56. 根据权利要求56所述数字PCR检测方法，其特征在于：在注入水相之前，使所述液滴存储腔、第一通道、第二通道中充满油相。
  57. 根据权利要求56所述数字PCR检测方法，其特征在于：在充入油相后、注入水相前，保持所述进液口与所述排液口均处于密封状态下，使所述数字PCR芯片水平静置5min以上。
  58. 根据权利要求56所述数字PCR检测方法，其特征在于：在开始形成液滴之后，接通负压装置，促进油相从排液口排出以及促进液滴流向液滴存储腔。
  59. 根据权利要求56所述数字PCR检测方法，其特征在于：所述微管道的摆动角度为0.1°～10°。
  60. 根据权利要求56或60所述的数字PCR检测方法，其特征在于，所述微管道往复摆动的频率为1Hz～1000Hz。
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