WO2019144905A1 - 微液滴容器、微液滴容器制备方法、微液滴平铺方法、微液滴生成试剂盒、温控装置、微液滴生成用油相组合物及其处理方法 - Google Patents

微液滴容器、微液滴容器制备方法、微液滴平铺方法、微液滴生成试剂盒、温控装置、微液滴生成用油相组合物及其处理方法 Download PDF

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Publication number
WO2019144905A1
WO2019144905A1 PCT/CN2019/072969 CN2019072969W WO2019144905A1 WO 2019144905 A1 WO2019144905 A1 WO 2019144905A1 CN 2019072969 W CN2019072969 W CN 2019072969W WO 2019144905 A1 WO2019144905 A1 WO 2019144905A1
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Prior art keywords
microdroplet
container
liquid
micro
outlet end
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Application number
PCT/CN2019/072969
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English (en)
French (fr)
Inventor
盛广济
Original Assignee
北京光阱管理咨询合伙企业（有限合伙）
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Priority claimed from CN201810070377.2A external-priority patent/CN110066857B/zh
Priority claimed from CN201810843257.1A external-priority patent/CN110763667A/zh
Application filed by 北京光阱管理咨询合伙企业（有限合伙） filed Critical 北京光阱管理咨询合伙企业（有限合伙）
Priority to EP19743237.0A priority Critical patent/EP3739323A4/en
Priority to JP2020560538A priority patent/JP7138301B2/ja
Priority to US16/964,607 priority patent/US11946100B2/en
Priority to CA3089402A priority patent/CA3089402A1/en
Publication of WO2019144905A1 publication Critical patent/WO2019144905A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • 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
    • B01L3/52—Containers specially adapted for storing or dispensing a reagent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • 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
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • 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
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • 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
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • 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
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
    • B01L2400/021—Drop detachment mechanisms of single droplets from nozzles or pins non contact spotting by inertia, i.e. abrupt deceleration of the nozzle or pin
    • 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
    • B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
    • B01L2400/028—Pin is moved through a ring which is filled with a fluid
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/34—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements
    • H01L23/345—Arrangements for heating
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/34—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements
    • H01L23/36—Selection of materials, or shaping, to facilitate cooling or heating, e.g. heatsinks
    • H01L23/367—Cooling facilitated by shape of device
    • H01L23/3672—Foil-like cooling fins or heat sinks
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/34—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements
    • H01L23/38—Cooling arrangements using the Peltier effect
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/34—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements
    • H01L23/46—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements involving the transfer of heat by flowing fluids
    • H01L23/467—Arrangements for cooling, heating, ventilating or temperature compensation ; Temperature sensing arrangements involving the transfer of heat by flowing fluids by flowing gases, e.g. air
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L23/00—Details of semiconductor or other solid state devices
    • H01L23/48—Arrangements for conducting electric current to or from the solid state body in operation, e.g. leads, terminal arrangements ; Selection of materials therefor
    • H01L23/488—Arrangements for conducting electric current to or from the solid state body in operation, e.g. leads, terminal arrangements ; Selection of materials therefor consisting of soldered or bonded constructions
    • H01L23/498—Leads, i.e. metallisations or lead-frames on insulating substrates, e.g. chip carriers
    • H01L23/4985—Flexible insulating substrates

Definitions

  • the present application relates to the field of microdroplets, and in particular to a microdroplet container, a microdroplet container preparation method, a microdroplet tiling method, a microdroplet generation kit, a temperature control device, and an oil phase for generating microdroplets Composition and method of treatment thereof.
  • Digital PCR is an absolute quantification technique for nucleic acid molecules. Compared to qPCR, digital PCR allows you to directly count the number of DNA molecules, which is an absolute quantification of the starting sample. In recent years, digital PCR technology has developed rapidly.
  • the traditional microfluidic control chip, micro-hole array chip and droplet micro-flow control chip based on micro-nano manufacturing and micro-flow control technology are disposable consumables. It is discarded to prevent cross-contamination.
  • the overall liquid level of the conventional micro-droplet container is curved, the liquid surface presents a concave liquid surface, and a plurality of micro-droplets are concentrated in the middle part of the micro-droplet container during the downward sedimentation process, and are gathered together. It interferes with the acquisition of batch micro-droplet fluorescence images.
  • the physical properties of the conventional oil phase composition change relatively large during use, and the volume uniformity of the generated micro-droplets is poor.
  • the traditional temperature control device has a slow rate of temperature rise and fall, which leads to a long time required to complete nucleic acid amplification, which affects the detection efficiency of digital PCR.
  • a microdroplet container including a bottom surface, a first annular side surface disposed around the bottom surface, and an annular surface.
  • the first annular side surface is connected to the bottom surface and surrounds to form a receiving space having an opening, the first annular side being perpendicular to the bottom surface.
  • the annular surface is disposed around the opening and is coupled to the first annular side, the annular surface being parallel to the bottom surface.
  • the microdroplet container includes a bottom surface, a first annular side surface disposed around the bottom surface, and an annular surface.
  • the first annular side surface is connected to the bottom surface and surrounds to form a receiving space having an opening, the first annular side being perpendicular to the bottom surface.
  • the annular surface is disposed around the opening and is coupled to the first annular side, the annular surface being parallel to the bottom surface.
  • the storage space is for accommodating a plurality of micro-droplets and an oil phase composition.
  • the oil phase composition is added to the annular face during use, at which point the liquid surface in the microdroplet container can be ensured to be a horizontal plane.
  • the liquid level of the micro-droplet container can be made flat, avoiding the overall liquid level of the micro-droplet container being curved. Therefore, the micro-droplet container does not affect the observation of the micro-droplets near the edge portion of the bottom plate of the container, which facilitates photo imaging and improves the number of detection of the plurality of micro-droplets.
  • a microdroplet container including a first container bottom plate, a polygonal frame, and a container cover.
  • the first container bottom plate is provided with a plurality of polygonal ridges.
  • the polygonal frame surrounds and forms a first storage space, the polygonal frame is connected to the first container bottom plate, and the plurality of polygonal ridges are disposed in the first storage space.
  • the container cover is disposed on a surface of the polygonal frame away from the bottom surface of the first container, and the container cover is detachably connected to the polygonal frame, and the container cover is surrounded by the polygonal frame to form an oil storage groove. .
  • the two sides of the polygonal frame are respectively connected to the first container bottom plate and the container cover, and the container cover is detachably connected to the polygonal frame, and the micro droplet container can be sealed.
  • the container cover is surrounded by the polygonal frame to form an oil receiving groove, and when the microdroplet container is sealed by the container cover, excess oil in the microdroplet container can be squeezed into the In the oil storage tank, the influence of the oil substances in the upper part of the micro-droplet on the detection process can be avoided as much as possible, and the fluorescent background caused by the oil substance is avoided.
  • the liquid in the microdroplet container can be prevented from flowing out, and the microfluid can also be The bubbles in the drip container are discharged, which avoids the influence of the bubbles on the image when the photograph is detected.
  • the present application provides an oil phase composition for forming micro droplets, comprising the following components of mineral oil and a surfactant.
  • the volume percentage of the mineral oil in the oil phase composition is from 88% to 98.5%.
  • the surfactant includes a chain alkyl group-containing silicon oxide chain nonionic surfactant.
  • the above oil phase composition for microdroplet formation comprising mineral oil and a long-chain alkyl group-containing silicon oxide chain nonionic surfactant has a density of less than 1 g/ml, and is capable of allowing most types of first liquid to be detached from the squirt gun. The outlet end of the head forms a droplet and then falls in the second liquid.
  • the chain-type alkyl-containing silicon oxide chain nonionic surfactant prevents the fusion of numerous micro-droplets.
  • the present application provides a microdroplet tiling method, comprising: S311, providing a microdroplet container, the microdroplet container having an opening, and the microdroplet container containing a second liquid; S312, providing a first liquid, the first liquid has a density greater than the second liquid and is immiscible with the second liquid, and stacking the first liquid to generate a plurality of microdroplets stacked on the micro liquid Dropping the bottom plate of the container; S313, performing high and low temperature circulation on the plurality of microdroplets until the plurality of microdroplets are laid on the bottom plate of the microdroplet container.
  • the plurality of microdroplets are subjected to high and low temperature cycles until the plurality of microdroplets are laid flat on the microdroplet container bottom plate.
  • the tiling is carried out by the microdroplet tiling method using the principle of thermal expansion and contraction. As the temperature changes, as the temperature increases, the viscosity of the sample droplets becomes lower and the volume shrinks. At the same time, the higher the temperature, the lower the viscosity, and the sample droplet shape is the softest, so that the plurality of micro-droplets are laid flat on the bottom surface of the micro-droplet container, which is advantageous for photograph detection.
  • the present application provides a temperature control device including a flexible circuit board, a heating substrate spaced apart from the flexible circuit board, and a plurality of semiconductor galvanic pairs.
  • the heating substrate includes a first surface and a second surface disposed opposite each other.
  • the plurality of semiconductor galvanic pairs are disposed between the flexible circuit board and the first surface, and the plurality of semiconductor galvanic pairs are connected in series, in parallel, or in a mixed manner.
  • the flexible circuit board has the characteristics of high wiring density, light weight, thin thickness and good bending property. The flexible circuit board eliminates thermal stress by its own deformation during the temperature rise and fall process, thereby prolonging the service life of the temperature control device.
  • the temperature control device can realize instantaneous temperature rising and cooling, and the process of heating and cooling is shortened, thereby achieving high and low temperature circulation, shortening the detection time of the digital PCR detector, and improving the detection efficiency.
  • FIG. 1 is a schematic diagram of an overall structure of a digital PCR detector provided by the present application
  • FIG. 2 is a micro-droplet generating apparatus of a digital PCR detector provided by the present application
  • FIG. 3 is an outlet of a liquid-spraying gun head according to an embodiment of the present application.
  • FIG. 4 is a schematic diagram showing the change of the velocity of the outlet end of the ejection head according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram of the movement of the outlet end of the ejection head according to an embodiment of the present application
  • FIG. 6 is a schematic diagram of the force of the droplets when the outlet end of the ejection head is provided according to another embodiment of the present application;
  • FIG. 1 is a schematic diagram of an overall structure of a digital PCR detector provided by the present application
  • FIG. 2 is a micro-droplet generating apparatus of a digital PCR detector provided by the present application
  • FIG. 3 is an outlet of a liquid-spraying gun head according to
  • FIG. 7 is a droplet dispensing gun according to an embodiment of the present application
  • FIG. 8 is a schematic view showing a process of generating a micro-droplet in two movement cycles of the outlet end of the squirting lance head according to an embodiment of the present invention
  • FIG. 10 is a schematic diagram of generating two micro-droplets in one movement period of the outlet end of the squirting lance head according to an embodiment of the present application; of FIG.
  • FIG. 11 is a schematic view showing a process of generating microdroplets when a spit gun head is oscillated according to an embodiment of the present invention
  • FIG. 12 is a process of generating microdroplets when a viscosity of a second liquid is changed according to an embodiment of the present application
  • FIG. 13 is a schematic diagram of a process of generating a micro-droplet when a liquid sprinkler head is replaced according to an embodiment of the present invention
  • FIG. 14 is a schematic view of the outlet end of the spit gun head according to an embodiment of the present invention.
  • FIG. 15 is a schematic diagram showing the variation of the velocity of the outlet end of the squirting lance head according to another embodiment of the present invention.
  • FIG. 26 is a schematic view showing the structure of the temperature control device according to the fourth embodiment of the present invention
  • FIG. 29 is a schematic diagram of a semiconductor galvanic pair electrode connection structure of the temperature control device of the present application
  • FIG. 30 is a schematic diagram of transient performance testing of the temperature control device of the present application
  • FIG. 31 is a steady state performance test of the temperature control device of the present application
  • Figure 32 is a schematic view showing the structure of the micro-droplet container of the present application
  • Figure 33 is a schematic view showing the structure of the micro-droplet container of the present application
  • Figure 34 is a schematic structural view of the reaction unit of the micro-droplet container of the present application
  • FIG. 36 is a schematic structural view of a micro-droplet container of the present application
  • FIG. 29 is a schematic diagram of a semiconductor galvanic pair electrode connection structure of the temperature control device of the present application
  • FIG. 30 is a schematic diagram of transient performance testing of the temperature control device of the present application
  • FIG. 31 is
  • FIG. 37 is a schematic cross-sectional view of the micro-droplet container of the present application
  • FIG. 39 is a schematic structural view of a micro-droplet container provided by the present application
  • FIG. 40 is a schematic structural view of a container cover of the micro-droplet container provided by the present application
  • FIG. 42 is a schematic structural view of a container cover of a micro-droplet container provided by the present application
  • FIG. 43 is a schematic structural view of a first container bottom plate of the micro-droplet container provided by the present application;
  • Figure 44 is a schematic structural view of a plurality of support plates of the polygonal frame of the micro-droplet container provided by the present application;
  • Figure 45 is a partially enlarged structural view of a plurality of support plates of the polygonal frame of the micro-droplet container provided by the present application;
  • 46 is a schematic structural view of a plurality of second support rods of a polygonal frame of the microdroplet container provided by the present application;
  • FIG. 47 is a multi-droplet container provided by the present application.
  • FIG. 48 is a schematic view showing the overall structure of the micro-droplet container provided by the present application;
  • FIG. 49 is a flow chart of the micro-droplet tiling method of the present application;
  • the present application provides a digital PCR detector 1 including a microdroplet generating device 10, a temperature controlling device 20, a fluorescent signal detecting device 30, and a quantitative analyzing device. 40 and controller 50.
  • the microdroplet generating device 10 is for microdropping a nucleic acid amplification reaction solution to form a plurality of microdroplets.
  • the temperature control device 20 and the micro-droplet generating device 10 are connected by a track for transferring a plurality of micro-droplets to the temperature control device 20 to perform temperature cycling to realize nucleic acid amplification.
  • the fluorescent signal detecting device 30 is disposed opposite to the temperature controlling device 20 for performing photodetection on a plurality of micro-droplets after nucleic acid amplification.
  • the quantitative analysis device 40 and the fluorescent signal detecting device 30 are connected by a data line for realizing the transmission of a plurality of micro-droplet fluorescence information for quantitative analysis.
  • the controller 50 is connected to the microdroplet generating device 10, the temperature controlling device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, respectively, for controlling the microdroplet generating device 10, the temperature controlling device 20, the fluorescent signal detecting device 30, and Quantitative analysis device 40.
  • the digital PCR detector 1 can integrate the microdroplet generating device 10, the temperature controlling device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, thereby enabling an operator to perform an automated operation.
  • the digital PCR detector 1 has a high working efficiency.
  • the microdroplet generating device 10 can dropletize the nucleic acid amplification reaction solution to be tested to form a plurality of microdroplets.
  • the temperature control device 20 can perform nucleic acid amplification on a plurality of microdroplets.
  • the fluorescent signal detecting device 30 captures a fluorescence change picture of a plurality of microdroplets in real time.
  • a fluorescence change curve of a plurality of microdroplets can be obtained by a fluorescence change picture of a plurality of microdroplets.
  • the Ct values of the plurality of microdroplets can be obtained, and the concentration of the initial DNA is quantitatively analyzed by the relationship between the Ct value and the initial copy number.
  • the Ct value refers to the number of cycles experienced when the fluorescence signal of each microdroplet reaches a set threshold.
  • the temperature control device 20 performs a nucleic acid amplification reaction on the plurality of microdroplets, and collects, by the fluorescence signal detecting device 30, product signals of a plurality of microdroplets after the nucleic acid amplification reaction, such as fluorescence, ultraviolet absorption, turbidity and the like.
  • product signals of a plurality of microdroplets after the nucleic acid amplification reaction such as fluorescence, ultraviolet absorption, turbidity and the like.
  • the difference in composition between the plurality of amplified and non-amplified microdroplets is used to analyze the number of droplets amplified by the target sequence, and finally quantitative analysis of the nucleic acid molecule is realized.
  • the detection results are direct and can solve the problem of false positives and false negatives in multiple microdroplets.
  • the digital PCR detector 1 integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, so that the operator can realize the automatic operation, improve the working efficiency, and have a quick response.
  • the microdroplet generating device 10 includes a ejector head 110, a fluid drive mechanism 120, a motion control mechanism 130, and a first controller 170.
  • the ejector tip 110 has an outlet end and an inlet end and is used to store the first liquid.
  • the microdroplet generating device 10 can be used in conjunction with a microdroplet container.
  • the second liquid is stored in the microdroplet container, and the outlet end of the ejector head 110 is inserted under the liquid surface of the second liquid.
  • the first liquid and the second liquid are mutually incompatible or have an interfacial reaction.
  • the first liquid and the second liquid may be any two liquids that are immiscible.
  • the first liquid is an aqueous solution
  • the second liquid is an oily liquid that is immiscible with water, such as mineral oil (including Tetrapropane or the like, vegetable oil, silicone oil, perfluoroalkane oil, etc.
  • the generated droplets are aqueous droplets.
  • the first liquid is a mineral oil such as an organic phase such as tetradecane or n-hexane
  • the second liquid is a perfluoroalkane oil which is immiscible with mineral oil.
  • the first liquid and the second liquid may be immiscible aqueous two phases.
  • the first liquid is an aqueous solution
  • the second liquid is an aqueous liquid that is immiscible with water, such as the first liquid being dextran.
  • the solution, the second liquid is a polyethylene glycol (PEG) aqueous solution, and the resulting droplets are droplets of a dextran solution.
  • PEG polyethylene glycol
  • the first liquid and the second liquid may also be two liquids having an interfacial reaction.
  • the first liquid is an aqueous solution of sodium alginate
  • the second liquid is an aqueous solution of calcium oxide, such as a mass concentration of 1%.
  • the aqueous calcium oxide solution has an interfacial reaction
  • the resulting droplets are calcium alginate gel microspheres.
  • the application can also form a plurality of droplets of different components and volumes in the open container by replacing the components of the first liquid flowing out of the spit gun head or the spit gun head, which can be used for realizing large quantities. Micro-volume high-throughput screening can also achieve multi-step ultra-micro biochemical reactions and detection, and has broad application prospects.
  • the fluid drive mechanism 120 is coupled to the inlet end of the ejector head 110 for discharging the first liquid stored inside the ejector head 110 from the outlet end of the squirt head 110.
  • the motion control mechanism 130 is configured to control a relative movement between the outlet end of the ejector head 110 and the second liquid to generate a set trajectory or a set speed or set an acceleration so that the first end of the discharge end of the ejector head 110 is discharged.
  • the liquid overcomes the surface tension and the adhesion of the vomiting tip 110 to the microdroplets.
  • the first controller 170 is coupled to the fluid drive mechanism 120 and the motion control mechanism 130, respectively, for controlling the fluid drive mechanism 120 and the motion control mechanism 130 to coordinate operations.
  • micro-droplet generation method with stable micro-droplet generation process is provided.
  • the outlet end 112 of the ejector head 110 can perform a motion including instantaneous acceleration under the second liquid level, and the acceleration is a 1 .
  • the first liquid is discharged from the outlet end 112 of the ejector head 110 to form a droplet 195 attached to the outlet end 112 of the squirt head 110.
  • the droplet 195 exits the outlet end 112 of the ejector tip 110 at the instant of instantaneous acceleration of the outlet end 112 of the ejector tip 110 to form microdroplets.
  • the forces received by the microdroplets before exiting the outlet end 112 of the ejector tip 110 are gravity G, the buoyancy f 1 of the second liquid, the viscous resistance f 2 of the second liquid, and the ejector tip 110
  • Micro-droplets in the mass exit from the extruding liquid tip 110 before end 112 is m, is a magnitude of the acceleration a 2. According to Newton’s second law of motion,
  • the viscous resistance f 2 6 ⁇ rv received by the droplet 195 as it moves in the second liquid, where ⁇ is the viscosity coefficient of the second liquid and r is the radius of the droplet 195 , v is the speed of movement of the droplet 195.
  • the velocity of the droplet 195 is zero before the instantaneous end of the ejection tip 110 is instantaneously accelerated, so that the droplet 195 is viscous in the second liquid at the moment the tip end 112 of the ejection tip 110 is instantaneously accelerated.
  • f 2 is zero or very small.
  • the droplet diameter typically in the range of pi to 195 microliters of magnitude, and opposite to gravity and buoyancy G droplets 195 f 1 of the second liquid direction, the droplets of gravity G 195
  • the vector sum with the buoyancy f 1 of the second liquid is about zero. Since the viscous resistance f 2 is zero or very small, and the vector sum of gravity G and buoyancy f 1 is about zero, It can be known from Newton's second law of motion that the maximum acceleration that the droplet 195 can reach in the second liquid is a 2 ⁇ f 3 /m, where m is the droplet when the outlet end 112 of the ejector tip 110 is subjected to the instantaneous acceleration motion. The quality of 195.
  • the droplet 195 falls from the outlet end 112 of the ejector tip 110 to form a microdroplet.
  • the condition under which the droplet 195 exits the outlet end 112 of the ejector tip 110 i.e., produces a microdroplet
  • a 2 ⁇ (f 3 /m) ⁇ a 1 is approximately: a 2 ⁇ (f 3 /m) ⁇ a 1 .
  • the motion control mechanism 130 is capable of accurately controlling the magnitude of the instantaneous acceleration of the outlet end 112 of the ejector head 110. Therefore, by controlling the outlet end 112 of the ejector head 110 to have a large value of the instantaneous acceleration, the outlet end 112 of the ejector head 110 is instantaneously accelerated to generate the droplet 195 efficiently.
  • micro-droplet generation method comprising the following steps:
  • the outlet end 112 of the ejector head 110 performs a motion including instantaneous acceleration under the second liquid level, while the first liquid is discharged from the outlet end 112 of the ejector head 110, and the outlet end of the ejector head 110 is discharged.
  • the first liquid of 112 forms a droplet 195 attached to the outlet end 112 of the ejector tip 110, and the droplet 195 exits the outlet end of the squirt head 110 during the instantaneous acceleration movement of the outlet end 112 of the ejector tip 110.
  • 112 forms microdroplets under the second liquid level.
  • the droplet 195 attached to the outlet end 112 of the ejector head 110 and the sputum
  • the adhesion between the outlet ends 112 of the tips 110 is insufficient to cause the droplets 195 to accelerate in synchronism with the outlet end 112 of the ejector tip 110 such that the fluid adheres to the outlet end 112 of the ejector tip 110
  • the drop 195 is detached from the outlet end 112 of the ejector tip 110 to form microdroplets under the second liquid level.
  • the outlet end 112 of the ejector head 110 generates micro-droplets during the instantaneous acceleration movement under the liquid surface of the second liquid, and the ejector tip 110 is reduced.
  • the disturbance of the second liquid caused by the movement of the outlet end 112 ensures the stability of the micro-droplet generation process.
  • the manner in which the first liquid is discharged from the outlet end 112 of the ejector head 110 may be continuous discharge or discontinuous discharge.
  • the specific discharge method can be designed according to the actual working conditions.
  • the first liquid is continuously discharged from the outlet end 112 of the ejector head 110 to fully utilize the instantaneous acceleration of the outlet end 112 of the ejector head 110 to generate microdroplets.
  • the first liquid is discharged by the outlet end 112 of the ejector head 110 at a constant flow rate, that is, at an equal time interval, the outlet end 112 of the ejector head 110 is discharged.
  • the first liquid volume is always equal.
  • the first liquid is discharged by the outlet end 112 of the ejector tip 110 at a constant flow rate, facilitating control of microdroplet generation by controlling the movement of the outlet end 112 of the ejector head 110.
  • the outlet end 112 of the ejector head 110 performs a periodic motion including instantaneous acceleration under the second liquid level.
  • the outlet end 112 of the ejector tip 110 is periodically moved under the second liquid level, meaning that the displacement, velocity and acceleration of the outlet end 112 of the ejector tip 110 exhibit periodic changes.
  • the outlet end 112 of the ejector tip 110 performs a periodic motion including a momentary acceleration motion, and the first liquid is discharged from the outlet end 112 of the ejector head 110 at a constant flow rate to effect equal time interval generation of the microdroplets.
  • the flow rate of the outlet end 112 of the first liquid discharge jetting head 110 is varied, but during a period of motion of the outlet end 112 of the jetting head 110, the first liquid exits the outlet end 112 of the jetting head 110.
  • the volume remains the same. This ensures that the volume of the droplets 195 is the same each time the outlet end 112 of the ejection tip 110 is instantaneously accelerated to generate microdroplets of uniform size.
  • the surface free energy of the ejector head 110, the geometry of the ejector head 110, and the surface tension of the droplet 195 act as an outlet affecting the ejector head 110 without replacing the ejector tip 110 and the first liquid. factors maximal adhesive force f between two ends 112 and 195 is determined by the droplet. Thus, without replacing the tip 110 and the liquid discharge of the first liquid, the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 it is fixed. Driven by the fluid drive mechanism 120, the first liquid is capable of continuously discharging the outlet end 112 of the ejector head 110 at a uniform flow rate.
  • the motion control mechanism 130 can precisely control the timing at which the exit end 112 of the ejector tip 110 makes the instantaneous acceleration a 1 and the magnitude of the instantaneous acceleration a 1 .
  • Fluid drive mechanism 120 and the motion control mechanism 130 cooperate with each other can be easily achieved when the volume of the droplet 195 reaches a fixed value of the moment, the outlet tip 110 of the liquid discharge drive end 112 generates acceleration instantaneous acceleration a 1 to generate the size of the volume Consistent microdroplets.
  • the fluid drive mechanism 120 controls the first liquid to uniformly and continuously discharge the outlet end 112 of the ejector tip 110
  • only the motion control mechanism 130 drives the outlet end 112 of the ejector tip 110 to produce an instantaneous acceleration movement at equal intervals, i.e., Microdroplets of uniform size can be generated.
  • the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 act as an outlet end 112 and fluid that affect the ejector tip 110.
  • the two factors of maximum adhesion f 3 between drops 195 are varied.
  • batch processing can control the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 to vary within a certain interval.
  • the surface tension of the droplet 195 as another factor affecting the maximum adhesion force f 3 between the outlet end 112 of the ejection head 110 and the droplet 195 also varies only in a small range.
  • the fluid drive mechanism 120 is capable of driving the first liquid to continuously discharge the outlet end 112 of the ejector head 110 at a uniform flow rate.
  • the motion control mechanism 130 can precisely control the timing at which the exit end 112 of the ejector tip 110 makes the instantaneous acceleration a 1 and the magnitude of the instantaneous acceleration a 1 .
  • Fluid drive mechanism 120 and the motion control mechanism 130 cooperate with each other can be easily achieved when the volume of the droplet 195 reaches a fixed value of the moment, the outlet tip 110 of the liquid discharge drive end 112 generates acceleration instantaneous acceleration a 1 to generate the size of the volume Consistent microdroplets. If the fluid drive mechanism 120 controls the first liquid to uniformly and continuously discharge the outlet end 112 of the ejector tip 110, only the motion control mechanism 130 drives the outlet end 112 of the ejector tip 110 to produce an instantaneous acceleration movement at equal intervals, i.e., Microdroplets of uniform size can be generated.
  • the fluid drive mechanism 120 when the first liquid is uniformly discharged from the outlet end 112 of the ejector head 110, cooperates with the motion control mechanism 130 to perform an instantaneous acceleration motion with a large acceleration value at the moment when the volume of the droplet 195 reaches a set value.
  • the micro-droplet generation method provided by the present application not only ensures that the same ejector tip 110 is used to generate the droplet 195 having a uniform size, and at the same time, the volume of the droplets simultaneously or sequentially generated by the plurality of ejector tips 110 can be ensured. Uniformity.
  • the micro-droplet generation method provided by the embodiment can ensure the generation efficiency of the micro-droplets by simultaneously generating the micro-droplets by the plurality of ejector tips 110 while ensuring the uniformity of the volume of the micro-droplets.
  • the outlet end 112 of the ejector head 110 includes multiple instantaneous acceleration motions in a periodic motion, and the accelerations of the multiple instantaneous acceleration motions are the same, and multiple instantaneous acceleration motions The moments are equally divided into one cycle of motion of the outlet end 112 of the jetting tip 110.
  • the exit end 112 of the ejector tip 110 includes a plurality of transient acceleration motions within a periodic motion to assist in generating a plurality of microdroplets during an exercise cycle at the outlet end 112 of the ejector tip 110.
  • the movement trajectory of the outlet end 112 of the ejector head 110 under the second liquid level includes a combination of one or more of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • the outlet end 112 of the ejector tip 110 When the outlet end 112 of the ejector tip 110 includes more than two instantaneous acceleration movements in a periodic motion, the outlet end 112 of the ejector tip 110 is traversed into a regular polygon in the second liquid, including an equilateral triangle, Square, regular pentagon, regular hexagon, etc.
  • step S203 during the periodic movement of the outlet end 112 of the ejector head 110 under the second liquid level, the velocity of the outlet end 112 of the ejector head 110 is a rectangular wave. Variety.
  • the velocity of the outlet end 112 of the ejector tip 110 changes in a rectangular wave.
  • the velocity is entered into a uniform phase, which facilitates the motion control mechanism 130 to achieve precise control of the motion state of the outlet end 112 of the ejector tip 110.
  • the high-order time and the low-order time of the rectangular wave indicating the change in the moving speed of the outlet end 112 of the ejector tip 110 may be equal or different.
  • step S203 during the periodic movement of the outlet end 112 of the ejector head 110 under the second liquid level, the velocity of the outlet end 112 of the ejector head 110 changes in a square wave.
  • the high-order time and the low-order time of the rectangular wave indicating the change in the moving speed of the outlet end 112 of the ejector head 110 are equal.
  • the velocity of the outlet end 112 of the ejection head 110 is zero or has a velocity in the opposite direction with respect to the high position. As shown in FIG.
  • the trajectory of the outlet end 112 of the ejector head 110 under the second liquid level is a straight line segment, and the outlet end 112 of the ejector head 110 is instantaneously accelerated from one end of the straight section.
  • the other end of the straight line segment performs the instantaneous acceleration motion in the opposite direction.
  • the acceleration of the two instantaneous acceleration motions is a 1 .
  • the trajectory of the outlet end 112 of the ejector tip 110 below the second liquid level is a circular arc segment or a polygon.
  • the frequency at which the outlet end 112 of the ejector head 110 periodically moves under the second liquid level is between 0.1 Hz and 200 Hz, which is easy to implement in engineering.
  • the fluid drive mechanism 120 controls the first liquid to exit the outlet end 112 of the ejector head 110 at a constant flow rate.
  • the motion control mechanism 130 controls the output end of the ejector head 110 to perform a periodic motion in which the motion trajectory is a straight line and the speed is a square wave.
  • the direction of velocity of the outlet end 112 of the ejector tip 110 changes, the instantaneous acceleration of the outlet end 112 of the ejector tip 110 reaches a maximum.
  • the droplet 195 attached to the outlet end 112 of the ejector tip 110 also exits the outlet end 112 of the ejector tip 110 when the instantaneous acceleration of the outlet end 112 of the ejector tip 110 reaches a maximum to form microdroplets 199. Since the first liquid is discharged at the outlet end 112 of the ejector head 110 at a constant flow rate, when the droplet 195 is detached from the outlet end 112 of the ejector head 110, the new droplet 195 enters the generated state. When the outlet end 112 of the ejector tip 110 is again accelerated in the reverse direction, the newly generated droplet 195 also falls from the outlet end 112 of the ejector tip 110 to form a new droplet 199.
  • two micro-droplets 199 can be generated in one motion cycle of the outlet end 112 of the ejector head 110, and the square wave is relatively easy to implement in engineering.
  • a droplet 199 is created during one cycle of the outlet end 112 of the ejector tip 110.
  • the outlet end 112 of the ejector head 110 performs a square wave motion of the trajectory in any direction in the second liquid 699, including: a plane perpendicular to the direction in which the squirting head 110 extends.
  • the square wave motion in which the trajectory is a straight line the square wave motion in which the trajectory is a straight line in a plane at an arbitrary angle with the extending direction of the ejector head 110, and the trajectory in a straight line along the extending direction of the ejector head 110 Wave movements, etc.
  • the trajectory of the outlet end 112 of the ejector head 110 is a circular arc segment or a polygon
  • the outlet end 112 of the ejector tip 110 is traversed in any direction in the second liquid 699.
  • the linear square wave motion includes: performing a square wave motion in which a trajectory is a straight line in a plane perpendicular to the extending direction of the ejector head 110, and making a trajectory in a plane at an arbitrary angle to the extending direction of the ejector head 110
  • the square wave motion of the straight line, the square wave motion in which the trajectory is a straight line along the extending direction of the ejector head 110 is performed by performing a square wave motion in which a trajectory is a straight line in a plane perpendicular to the extending direction of the ejector head 110, and making a trajectory in a plane at an arbitrary angle to the extending direction of the ejector head 110.
  • the outlet end 112 of the ejector head 110 under the driving of the motion control mechanism 130, performs a periodic change of velocity under the second liquid level, in the first half cycle of the change in speed.
  • the velocity of the outlet end 112 of the ejector tip 110 varies monotonically.
  • the monotonous change means that the velocity value at the rear end of the outlet end 112 of the ejector tip 110 is always greater than or equal to or less than the velocity value at the previous moment during the first half cycle or the second half cycle of the change in the speed magnitude.
  • the velocity of the outlet end 112 of the ejector tip 110 continues to increase or the segment continues to increase while the segment remains unchanged.
  • the velocity of the outlet end 112 of the ejector tip 110 continues to decrease or the segment continues to decrease while the segment remains unchanged.
  • the first liquid is discharged from the outlet end 112 of the ejector head 110 to form a droplet 195 attached to the outlet end 112 of the squirt head 110.
  • the droplet 195 exits the outlet end 112 of the ejector tip 110 to form microdroplets 199 as the speed of movement of the outlet end 112 of the ejector tip 110 reaches a certain level.
  • the forces received by the microdroplets 199 before exiting the outlet end 112 of the ejector tip 110 are gravity G, the buoyancy f 1 of the second liquid 699, and the viscous resistance of the second liquid 699, respectively.
  • Mass microdroplets outlet 199 from the liquid discharge end 112 of the tip 110 before is m, speed v, acceleration a 2.
  • the droplet 195 is subjected to the combined action of the viscous force f 2 , the gravity G, the buoyancy f 1 and the adhesion force f 3 during the movement of the second liquid 699, ie
  • the diameter of the droplets 195 generally ranges from aspirated to microliters, while the viscosity of the second liquid 699 is generally relatively large. Therefore, there are generally Therefore, during the shifting cycle of the outlet end 112 of the ejector tip 110 under the liquid level of the second liquid 699, the droplet 195 is detached from the outlet end 112 of the ejector tip 110 (i.e., a microdroplet 199 is formed). Approximate
  • micro-droplet generation method including the following steps:
  • the outlet end 112 of the ejection head 110 is inserted into the liquid surface of the second liquid 699 by the opening of the microdroplet container 60;
  • the outlet end 112 of the ejector head 110 is subjected to a periodic change in velocity under the liquid level of the second liquid 699.
  • the outlet end 112 of the ejector head 110 is in the first half cycle and the second half cycle of the change in velocity.
  • the magnitude of the velocity varies monotonically, while the first liquid is uniformly discharged from the outlet end 112 of the ejector tip 110, and the first liquid exiting the outlet end 112 of the ejector tip 110 is formed to adhere to the outlet end 112 of the ejector tip 110.
  • the droplet 195, the droplet 195 exits the outlet end 112 of the ejector tip 110 during the movement of the outlet end 112 of the ejector tip 110 to form a microdroplet 199 under the surface of the second liquid 699.
  • the outlet end 112 of the ejector head 110 is subjected to a periodic change in velocity under the liquid level of the second liquid 699, and the ejector tip is in the first half cycle and the second half cycle of the change in the speed.
  • the velocity of the exit end 112 of 110 is monotonically varied.
  • the viscous force f 2 of the second liquid 699 to the droplet 195 also exhibits a periodic change with the periodic variation of the velocity of the outlet end 112 of the ejector tip 110.
  • the outlet end 112 of the ejector head 110 performs a shifting cycle under the liquid level of the second liquid 699 to generate the microdroplets 199, reducing the outlet of the ejector head 110.
  • the disturbance caused to the second liquid 699 during the movement of the end 112 ensures the stability of the micro-droplet 199 generation process.
  • step S213 the first liquid is continuously discharged from the outlet end 112 of the ejector head 110. Further, in step S213, the first liquid is discharged by the outlet end 112 of the ejector head 110 at a constant flow rate, that is, the first liquid volume of the outlet end 112 of the ejector head 110 is discharged at equal time intervals. Always equal. The first liquid is expelled from the outlet end 112 of the ejector tip 110 at a constant flow rate, facilitating the generation of micro-droplets 199 of uniform size by controlling the periodic movement of the outlet end 112 of the ejector tip 110.
  • the velocity v of the droplet 195 is relatively easy to control.
  • the droplet 195 maintains a synchronized motion with the outlet end 112 of the ejector tip 110 prior to exiting the outlet end 112 of the ejector tip 110 to form the microdroplets 199. Therefore, the moving speed v of the liquid droplet 195 can be accurately controlled by controlling the moving speed of the outlet end 112 of the ejector head 110.
  • the first liquid is controlled to exit the outlet end 112 of the ejector tip 110 at a uniform flow rate, and the magnitude r of the radius of the droplet 195 also exhibits a periodic change over a fixed time interval.
  • the viscosity coefficient ⁇ of the second liquid 699 may vary within a certain range during use, but the second liquid 699 The range of variation of the viscosity coefficient ⁇ is small.
  • the surface free energy of the ejector head 110, the geometry of the ejector head 110, and the surface tension of the droplet 195 act as an outlet affecting the ejector head 110 without replacing the ejector tip 110 and the first liquid. factors maximal adhesive force f between two ends 112 and 195 is determined by the droplet. Thus, without replacing the tip 110 and the liquid discharge of the first liquid, the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 it is fixed.
  • the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 act as an outlet end 112 that affects the ejector tip 110.
  • the two factors of maximum adhesion f 3 between droplets 195 are varied.
  • batch processing can control the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 to vary within a certain interval.
  • the surface tension of the droplet 195 as another factor affecting the maximum adhesion force f 3 between the outlet end 112 of the ejection head 110 and the droplet 195 also varies only in a small range. Extruding liquid tip outlet end 110 of the fluctuations in the three small interval maximum value f adhesion between the droplet 112 and 195.
  • the viscous resistance f 2 that the droplet 195 is subjected to when moving in the second liquid 699 is greater than the interval value of the maximum value f 3 of the adhesion between the outlet end 112 of the ejector head 110 and the droplet 195.
  • the size r of the radius of the droplet 195 should be fixed during the process of generating the microdroplets 199 in the same batch. Once the experimental parameters are determined, the magnitude r of the radius of the droplet 195 is also determined. The speed of movement of the outlet end 112 of the ejector tip 110 below the level of the second liquid 699 varies.
  • the outlet end 112 of the ejector tip 110 performs a periodic change in velocity at the level of the second liquid 699.
  • the first liquid is controlled to be discharged from the outlet end 112 of the ejector head 110 at a uniform flow rate, and the volume of the droplet 195 attached to the outlet end 112 of the ejector head 110 is also uniformly increased.
  • the radius of the microdroplet 199 is referred to as the critical radius and the velocity of the microdroplet 199 becomes the critical velocity.
  • the droplets 195 attached to the outlet end 112 of the ejector tip 110 simultaneously reach a critical radius and critical velocity, and new droplets 199 are formed. Since the first liquid is discharged at the outlet end 112 of the ejector head 110 at a uniform flow rate, the volume of the generated microdroplets 199 is the same.
  • step S213 the velocity of the outlet end 112 of the ejector head 110 is center-symmetric with the intermediate point in time during a speed change period. Further, in step S213, the acceleration, velocity, and motion trajectory of the outlet end 112 of the ejector tip 110 under the liquid level of the second liquid 699 are periodically changed. Further, in step S213, the velocity of the outlet end 112 of the discharge gun head 110 under the liquid level of the second liquid 699 changes cosine.
  • the movement trajectory of the outlet end 112 of the ejector head 110 under the liquid surface of the second liquid 699 includes one or more of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon. combination.
  • the frequency at which the outlet end 112 of the ejector head 110 periodically moves under the liquid level of the second liquid 699 is between 0.1 Hz and 200 Hz, which is easily accomplished in engineering.
  • the outlet end 112 of the ejector head 110 is actually oscillating.
  • the motion displacement can be represented by a sinusoid, as shown by curve a in FIG.
  • the first liquid is discharged from the outlet end 112 of the ejector head 110 at a uniform flow rate by the fluid control mechanism. It is assumed that the drop 195 does not exit the outlet end 112 of the ejector tip 110.
  • the viscosity resistance f viscosity experienced by the droplet 195 as it moves in the second liquid 699 changes with time as shown by the curve b in FIG.
  • the radius r of the droplet 195 increases significantly.
  • a uniform increase in the volume of the droplet 195 can only cause a slow increase in the radius r of the droplet 195.
  • the maximum value of the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 increases rapidly, and then gradually increases slowly. . 7, the liquid droplets 195 moving in the second viscous resistance by 699 f 2 also presents a similar solution discharge outlet tip 110 of the end 112 of the periodic motion periodically, i.e. droplets 195
  • the viscous resistance f 2 experienced when moving in the second liquid 699 varies with the speed of the outlet end 112 of the ejector head 110.
  • the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 increases and is greater than the maximum adhesion between the outlet end 112 of the ejector tip 110 and the droplet 195.
  • f 3 the discharge liquid droplet 195 from the outlet end 112 of tip 110 is formed microdroplets 199 off.
  • the outlet end 112 of the ejector head 110 is controlled to be a circular arc, and the displacement is sinusoidally varied.
  • the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 is fixed.
  • the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 also increases.
  • the viscous resistance f 2 of the droplet 195 as it moves in the second liquid 699 is greater than the instant of the maximum value f 3 of the adhesion between the outlet end 112 of the squirt head 110 and the droplet 195, and the droplet 195 is spit from The outlet end 112 of the liquid gun head 110 is detached to form microdroplets 199, which are droplets I in FIG. Entering the generation cycle of the next round of microdroplets 199.
  • a first microdroplet 199 is produced at the end of the second cycle of the oscillating motion of the sinusoidal variation of the displacement end 112 of the ejector tip 110, which is the droplet I in FIG.
  • the radius of the droplet 195 attached to the outlet end 112 of the squirt head 110 is The increase is faster, and the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 does not decrease immediately but presents a small increase. Thereafter, the radius r of the droplet 195 is slowly increased, and the viscous resistance f 2 received by the droplet 195 as it moves in the second liquid 699 mainly changes with the speed of movement of the outlet end 112 of the ejector head 110.
  • the outlet end 112 of the ejector tip 110 is again generated with the previous one at the time of the two movement cycles after the generation of the last droplet 199.
  • the microdroplet 199 is an equal volume of new droplet 195, which is droplet II in FIG.
  • the moving speed of the outlet end 112 of the ejection head 110 is also the same as before the two motion periods.
  • a new volume 195 of equal volume with the last microdroplet 199 is detached from the outlet end 112 of the ejector tip 110.
  • the outlet end 112 of the ejector head 110 is controlled to be a circular arc with a sinusoidal variation in displacement.
  • the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 is fixed.
  • the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 also increases.
  • the viscous resistance f 2 of the droplet 195 as it moves in the second liquid 699 is greater than the instant of the maximum value f 3 of the adhesion between the outlet end 112 of the squirt head 110 and the droplet 195, and the droplet 195 is spit from The outlet end 112 of the liquid gun head 110 is detached to form microdroplets 199. Entering the generation cycle of the next round of microdroplets 199.
  • a first microdroplet 199 is produced at the end of the first cycle of the oscillating motion of the sinusoidal shift of the outlet end 112 of the ejector tip 110, which is droplet I in FIG.
  • the radius of the droplet 195 attached to the outlet end 112 of the squirt head 110 is The increase is faster, and the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 does not decrease immediately but presents a small increase. Thereafter, the radius r of the droplet 195 is slowly increased, and the viscous resistance f 2 received by the droplet 195 as it moves in the second liquid 699 mainly changes with the speed of movement of the outlet end 112 of the ejector head 110.
  • the outlet end 112 of the ejector tip 110 is again generated with the last micro at the moment of the movement of the last microdroplet 199.
  • the droplet 199 is equal in volume to the new droplet 195, and at this point the velocity of the exit end 112 of the jetting tip 110 is also the same as before one motion cycle.
  • a new volume 195 of equal volume with the last microdroplet 199 is detached from the outlet end 112 of the ejector tip 110, which is droplet II in FIG. This cycle generates droplet III, droplet IV, and the like.
  • the uniform discharge of the first liquid and the oscillating movement of the outlet end 112 of the ejector tip 110 in a sinusoidal variation ensure the uniformity of the volumetric size of the generated microdroplets 199.
  • the outlet end 112 of the ejector head 110 is controlled to be a circular arc with a sinusoidal variation in displacement.
  • the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 is fixed.
  • the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 also increases.
  • the viscous resistance f 2 of the droplet 195 as it moves in the second liquid 699 is greater than the instant of the maximum value f 3 of the adhesion between the outlet end 112 of the squirt head 110 and the droplet 195, and the droplet 195 is spit from The outlet end 112 of the liquid lance head 110 is detached to form droplets 199, which are droplets I in FIG. Entering the generation cycle of the next round of microdroplets 199.
  • the first micro-droplet 199 is generated in the acceleration phase of the first half cycle of the oscillating motion in which the displacement of the ejection tip 110 is displaced at a sinusoidal variation, and the droplet I is in FIG.
  • the speed of movement of the outlet end 112 of the ejector tip 110 is reduced, but due to the radius of the droplet 195 attached to the outlet end 112 of the ejector tip 110
  • the increase is faster, and the viscous resistance f 2 experienced by the droplet 195 as it moves in the second liquid 699 does not decrease immediately but presents a small increase.
  • the radius r of the droplet 195 is slowly increased, and the viscous resistance f 2 received by the droplet 195 as it moves in the second liquid 699 mainly changes with the speed of movement of the outlet end 112 of the ejector head 110.
  • the first liquid is controlled to exit the outlet end 112 of the ejector tip 110 at a uniform flow rate.
  • the outlet end 112 of the ejector tip 110 produces a second microdroplet 199 during the second half cycle acceleration phase of the sinusoidal oscillating motion, which is droplet II in FIG. Thereafter, the stage of stably generating the microdroplets 199 is entered.
  • a new droplet 195 of equal volume to the second micro-droplet 199 is generated, and at this time the squirt gun
  • the speed of movement of the outlet end 112 of the head 110 is also the same as before the half of the motion cycle.
  • a new droplet 195 of equal volume with the second microdroplet 199 is detached from the outlet end 112 of the ejector tip 110, and thus circulated, thereby generating droplet III, droplet IV, droplet V, etc. shown in FIG. .
  • the uniform discharge of the first liquid and the oscillating movement of the outlet end 112 of the ejector tip 110 in a sinusoidal variation together ensure the uniformity of volumetric size of the generated microdroplets 199.
  • the condition that the droplet 195 attached to the outlet end 112 of the ejector tip 110 is separated from the outlet end 112 of the ejector tip 110 is approximately:
  • the size of the generated micro-droplets 199 is uniform: the droplets 199 are equally spaced from the ejector tip The outlet end 112 of the 110 is detached.
  • Factors affecting the maximum value f 3 of the adhesion between the outlet end 112 of the ejector tip 110 and the droplet 195 include: surface free energy of the ejector tip 110, geometric dimensions, and surface tension of the first liquid. In the case where the tips 110 without replacing the extruding liquid and the first liquid, the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 is fixed.
  • the factors affecting the viscous resistance f 2 that the droplet 195 is subjected to when moving in the second liquid 699 include the viscosity coefficient ⁇ of the second liquid 699, the radius r of the droplet 195, and the moving velocity v of the droplet 195.
  • the radius r of the droplet 195 is determined by the interval between the generation of the droplets 199.
  • the droplet 195 moves synchronously with the outlet end 112 of the ejector head 110 before exiting the outlet end 112 of the ejector head 110, and the speed of movement of the outlet end 112 of the ejector head 110 can be precisely controlled by the motion control mechanism 130.
  • the viscosity coefficient ⁇ of the second liquid 699 varies within a certain range during the formation of the droplet 195, but the variation range of the viscosity coefficient ⁇ of the second liquid 699 is small. As shown in Fig.
  • the curve a indicates the displacement change of the outlet end 112 of the ejector head 110
  • the curve b and the curve c are the droplets 199 when the viscous coefficient ⁇ of the second liquid 699 varies within a small range.
  • the generation process curve When the viscosity coefficient ⁇ of the second liquid 699 varies within a small range, the generation timing of the micro-droplets 199 is changed only in a small range. The generation interval of the microdroplets 199 is not changed. As shown in FIG. 12, the generation time interval of the micro-droplets 199 represented by the curves b and c is half a period t/2, which ensures the uniformity of the volume size of the generated micro-droplets 199.
  • the maximum adhesion force between the outlet end 112 of the ejection head 110 and the droplet 195 when the surface of the first liquid changes due to a temperature change or the like is changed when the ejection head 110 is replaced.
  • f 3 is difficult to control precisely, so if the volume of the generated microdroplets 199 is insensitive to changes in f 3 over a certain range, then it is important to generate microdroplets 199 of uniform size.
  • the curve a indicates the displacement change of the outlet end 112 of the ejector head 110
  • the curve b and the curve c are the generation process curves of the microdroplet 199 when the ejector head 110 is replaced.
  • the maximum value f 3 of the adhesion between the outlet end 112 of the spit gun head 110 and the droplet 195 fluctuates within a certain range, which causes the outlet end of the spit gun head 110 when the droplet 195 falls off.
  • 112 corresponds to different speeds.
  • the generation of the microdroplets 199 reaches a steady state, the velocity of the outlet end 112 of the spit gun head 110 is fixed during each wobble period when the droplets 195 fall off, as shown in FIG. 13, curve b and curve.
  • the generation time interval of the micro-droplets 199 represented by c is half a period t/2. It is therefore possible to ensure that the interval at which the microdroplets 199 are generated is fixed.
  • the volume of the generated microdroplets 199 is uniform. Simultaneously adjusting the flow rate of the outlet end 112 of the first liquid discharge ejector head 110 and the swing frequency of the outlet end 112 of the ejector head 110 in the second liquid 699 can simultaneously control the volume of the uniform volume microdroplet 199. And the generation rate.
  • the maximum value of the adhesion force f 3 and the viscous resistance f 2 are tolerated, that is, the maximum adhesion is obtained.
  • the value f 3 or the viscous resistance f 2 is varied within a certain range, it is still possible to generate the micro droplets 199 having a uniform volume.
  • the range of variation of the maximum value f 3 that can be tolerated is determined as a platform under the premise of generating a micro-droplet 199 having a uniform volume. period.
  • the presence of the plateau period is of great significance for the processing of the ejection tip 110 and the control of the formation temperature of the microdroplets 199.
  • the presence of the plateau period allows the processing precision of the ejector head 110 to be reduced to a certain extent, and even if there is a difference in surface free energy between the squirting tips 110 of the same batch, it is possible to generate a micro-liquid of uniform size. Drop 199.
  • the presence of the plateau period also allows the temperature control requirements of the microdroplet 199 generation process to be reduced to some extent.
  • the presence of the plateau period allows the processing accuracy requirements of the ejector tip 110 or the temperature control requirements of the microdroplet 199 generation process to be reduced to a certain extent, further reducing consumable costs and control costs during the generation of the microdroplets 199.
  • two micro-droplets 199 are generated in each movement cycle of the outlet end 112 of the ejector head 110, and it is easily understood that as long as the outlet end 112 of the ejector tip 110 is displaced, the sinusoidal cyclic movement is performed.
  • the outlet end 112 of the ejector tip 110 can be displaced in a sinusoidal periodic motion in any direction within the second liquid 699.
  • the trajectory of the exit end 112 of the ejector tip 110 is an arc, line or other shaped trajectory.
  • the ejection head 110 is obliquely inserted into the second liquid 699, and the outlet end 112 of the ejection head 110 is swung under the liquid surface of the second liquid 699.
  • a microdroplet 199 is produced.
  • the outlet end 112 of the ejector head 110 performs a periodic motion in which the trajectory is a horizontal straight line and the displacement is sinusoidal in the second liquid 699 to generate a micro Droplet 199.
  • the outlet end 112 of the ejector head 110 is moved in a periodic motion in which the second liquid 699 is in a straight line and the displacement is sinusoidal to generate Microdroplets 199.
  • step S213 the outlet end 112 of the ejector head 110 is in a uniform shifting motion during the first half period and the second half period in one cycle of the change in the speed. Further, in step S213, the outlet end 112 of the ejector head 110 is equal in magnitude to the acceleration of the first half period and the second half period.
  • the first liquid is controlled to exit the outlet end 112 of the ejector tip 110 at a uniform flow rate. As the first liquid is continuously discharged, the viscous resistance f 2 of the droplets 195 adhering to the outlet end 112 of the ejector tip 110 during the movement is also continuously increased.
  • the droplet 195 is detached from the ejector tip 110 to form the microdroplet 199. This is followed by the generation of the next microdroplet 199.
  • the frequency of movement and the speed of movement of the outlet end 112 of the ejector tip 110 are adapted to match the flow rate of the first liquid to ensure volume uniformity of the resulting microdroplets 199.
  • the oil phase composition capable of ensuring the uniformity of the volume of the microdroplets is provided by using the conventional oil phase composition in the jetting head injection/spraying method, and the uniformity of the generated microdroplet volume is poor. And its processing methods.
  • the present application provides an oil phase composition for forming micro droplets, that is, the above second liquid 699, comprising the following components:
  • Mineral oil the volume percentage of mineral oil in the oil phase composition is 88% - 98.5%;
  • surfactants include silica-based nonionic surfactants containing chain alkyl groups.
  • the above oil phase composition for microdroplet formation comprising mineral oil and a chain alkyl group-containing silicon oxide chain nonionic surfactant has a density of less than 1 g/ml, and is capable of allowing most types of first liquid to be detached from the squirt gun.
  • the exit end 112 of the head 110 forms a droplet 199 which then falls in the second liquid 699.
  • the chain-chain alkyl-containing silicon oxide chain nonionic surfactant can prevent a plurality of micro-droplets 199 from intermingling with each other.
  • the volume fraction of the chain alkyl-containing silicon oxide chain nonionic surfactant in the oil phase composition is from 1.5% to 12%.
  • the chain alkyl-containing silicon oxide chain nonionic surfactant includes with One or two of them.
  • the surfactant further comprises a chain alkane ester, and the mass ratio of the chain alkane ester to the oil phase composition in the oil phase composition is from 0.015 g/mL to 0.05 g/mL.
  • the chain alkane ester includes one or more of dihydroxy hydroxystearate (PEG-30), stearic acid glycerin, polyethylene glycol (30) dipolyhydroxystearate (P135), and the like.
  • the chain alkane ester is polyethylene glycol (30) dipolyhydroxystearate (P135).
  • the volume fraction of the chain alkyl-containing silicone chain nonionic surfactant in the oil phase composition is from 1.5% to 5.0%.
  • the chain alkyl group-containing silicon oxide chain nonionic surfactant is
  • the gas In mineral oil, the gas has a certain amount of solubility. And it is related to the temperature of gas and mineral oil. For example, at room temperature, air is dissolved in mineral oil and is not visible. Dissolved gases in mineral oil affect the physical properties of mineral oil such as viscosity, bulk modulus, heat transfer, and boundary lubrication, forming foaming and cavitation. If the gas content in the mineral oil is above saturation, the visible bubbles form and are suspended in the mineral oil, and the mineral oil becomes blurred. This is called entrained gas. The bubbles slowly rise to the surface of the mineral oil. In the oil film, air bubbles cause continuity of the oil film, thereby reducing the ability of the oil film to prevent other phases from coming into contact.
  • the temperature will rise to 95 °C.
  • the solubility of the gas in the mineral oil is lowered, and the gas content in the mineral oil is higher than the saturation value, and bubbles are generated at this time.
  • the bubbles rise to the surface of the mineral oil and eventually rupture.
  • bubbles can affect the acquisition of fluorescent signals during this process.
  • the bubbles interact with the micro-droplets 199, the stability of the micro-droplets 199 is affected, and the fusion reaction between the micro-droplets 199 is promoted.
  • the present application also provides a method of treating an oil phase composition for treating the oil phase composition of any of the above aspects.
  • the method of treating the oil phase composition comprises heating the oil phase composition while placing the oil phase composition in an environment of negative pressure and ultrasonic vibration. In a negative pressure environment, air and other gases dissolved in the oil phase composition are allowed to overflow, minimizing the amount of air and other gases dissolved in the oil phase composition. Ultrasonic waves can promote the escape of gases dissolved in the oil phase composition.
  • the micro-droplet 199 includes an aqueous phase, after the micro-droplet 199 is formed, moisture may be dissolved in the oil phase composition upon subsequent operation of the micro-droplet 199.
  • Varying the size of the microdroplets 199 affects the positional arrangement of the microdroplets 199, which in turn affects the real-time detection of the microdroplets 199.
  • the method of treating the oil phase composition further comprises the step of bringing the oil phase composition to water saturation.
  • the step of bringing the oil phase composition to water saturation comprises adding distilled water to the oil phase composition prior to heating the oil phase composition. After the heating process of the oil phase composition is completed, the oil phase composition is naturally cooled in an environment of 25 ° C to 35 ° C. Distilled water is added to the oil phase composition while heating to bring the oil phase composition to water saturation. The insoluble water is removed at room temperature.
  • the oil phase composition is already in a water saturated state prior to the generation of the microdroplets 199, minimizing the amount of moisture in the aqueous phase droplets 199 entering the oil phase composition. Further, the cooled oil phase composition was protected with nitrogen. Nitrogen has a very low solubility in mineral oil-based oil phase compositions. The use of nitrogen as a shielding gas prevents the air or other gases in the environment from being dissolved in the oil phase composition during storage of the oil phase composition to reduce the quality of the oil phase composition. As an achievable way, when there is space left in the vessel in which the oil phase composition is stored, the remaining space is filled with nitrogen.
  • Protocol 1 The experiment explored the volume size uniformity of the microdroplets 199 produced in the oil phase composition of different components (second liquid 699).
  • second liquid 699 Using the outlet end 112 of the ejector tip 110 described above to perform a periodic instantaneous acceleration motion in the oil phase composition (second liquid 699), the first liquid in the ejector tip 110 is detached from the ejector tip 110. The outlet end 112 forms a microdroplet 199 in the oil phase composition (second liquid 699).
  • the first liquid is an aqueous phase
  • the components of the oil phase composition (second liquid 699) are as follows:
  • the volume uniformity of the micro-droplets 199 in the dotted line frame in FIG. 16 is good, corresponding to the first embodiment, the second embodiment, the third embodiment, and the fourth embodiment.
  • the composition of the oil phase composition (second liquid 699).
  • second liquid 699) when the oil phase composition (second liquid 699) When the volume percentage is increased to 15% and 16%, the volumetric uniformity of the generated microdroplets 199 is poor. Easy to draw, when the oil phase composition (second liquid 699) When the volume percentage is from 1.5% to 12%, the volumetric uniformity of the generated microdroplets 199 is good.
  • the volume uniformity of the microdroplets 199 in the dotted line frame in Fig. 19 is good.
  • the oil phase composition second liquid 699
  • the volume percentage is 1.5% to 12% and the content of P135 is less than 5%, as shown in FIGS. 19 and 20, the volume uniformity of the generated microdroplets 199 is good.
  • the content of P135 is more than 5%, as shown in Figs. 19 and 21, the volume of the generated microdroplets 199 exhibits a different size.
  • the thermal stability of the microdroplets 199 in the solid line in Fig. 19 is good.
  • the thermal stability of the microdroplets 199 is good.
  • the thermal stability of the microdroplet 199 is poor.
  • the oil phase composition (second liquid 699) contains mineral oil as a main component
  • the volume percentage is 1.5%-5.0% and the content of P135 is 1.5%-5.0%
  • the volume uniformity of the micro-droplets 199 formed by the first liquid in the oil phase composition (second liquid 699) is good.
  • the thermal stability of the microdroplets 199 is also good.
  • Option 3 Experimental exploration Potential alternative ingredients. versus The same as the silica-based nonionic surfactant containing a chain alkyl group, using the experimental conditions of the first scheme, All or part of instead.
  • the composition of the oil phase composition (second liquid 699) in Scheme 3 is as follows:
  • the micro-droplet 199 generation experiment was carried out using the oil phase composition (second liquid 699) of the different components shown in Examples 23 to 28 in the above table, and the generated micro-droplets 199 each have Better volume uniformity.
  • Option 4 Exploring potential alternatives to P135.
  • Polyethylene glycol (30) dipolyhydroxystearate (P135) and dipolyhydroxystearate (PEG-30) and stearic acid glycerin are both chain alkane esters.
  • P135 was replaced in whole or in part by dihydroxy hydroxystearate (PEG-30) and stearic acid glycerol.
  • the composition of the oil phase composition (second liquid 699) in Scheme 4 is as follows:
  • the oil phase composition (second liquid 699) of the different components shown in Examples 29 to 32 in the above table was used to carry out the formation of the microdroplet 199 and the thermal stability test.
  • the micro-droplets 199 have good volume uniformity and good thermal stability.
  • the device for generating microdroplets 199 and the method for generating the same are applied in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the microdroplet 199 is a nucleic acid amplification reaction solution to be tested, and the nucleic acid amplification reaction solution is microdropleted by the microdroplet generating device 10 to form a plurality of microdroplets.
  • the nucleic acid amplification reaction solution to be tested is passed through the micro-droplet generating device 10 of the integrated digital PCR detector 1 and converted into a plurality of micro-droplets 199 by micro-drip processing, so that the detected fragments in the sample to be tested are from a large amount.
  • the complex background is separated and placed in the microdroplet container 60 for inspection.
  • a plurality of droplets 199 of uniform size can be generated by the microdroplet generating device 10.
  • Each of the microdroplets 199 is on the order of micrometers, and each of the microdroplets 199 can be considered as a separate reactor, corresponding to a test tube commonly used in biochemical reactions.
  • the plurality of microdroplets 199 are placed in the microdroplet container 60 for easy inspection and observation.
  • a plurality of micro-droplets of different volumes can also be generated by the micro-droplet generating device 10 for medical clinical detection.
  • the plurality of microdroplets 199 are small in volume and large in number, and have the advantage that many conventional test tubes do not.
  • a large number of micro-droplets 199 can be generated by the micro-droplet generating device 10, so that the digital PCR detector 1 has the advantages of high flux, low consumable cost and low background noise, and has a good industrialization prospect.
  • the traditional temperature control device has a slow temperature rise and fall rate, and it takes tens of seconds to several minutes for each temperature rise or fall. It takes about 1 to 2 hours to do dozens of PCR cycles, which leads to prolonged time required for nucleic acid amplification.
  • the nucleic acid amplification efficiency is low and the service life is low.
  • a temperature control device with a fast temperature rise and a high service life is provided.
  • the present application further provides a temperature control device 20 including a flexible circuit board 220, a heating substrate 240 spaced apart from the flexible circuit board 220, and a plurality of semiconductor galvanic pairs 230.
  • the heating substrate 240 includes a first surface 241 and a second surface 242 that are disposed opposite each other.
  • the plurality of semiconductor galvanic pairs 230 are disposed between the flexible circuit board 220 and the first surface 241, and the plurality of semiconductor galvanic pairs 230 are connected in series, in parallel, or in a mixed manner.
  • the temperature control device 20 is generally used in a high and low temperature cycle environment, and the temperature needs to be rapidly raised and lowered, so the temperature control device 20 is required to be extremely high.
  • the temperature control device 20 employs the flexible circuit board 220.
  • the flexible circuit board 220 has the characteristics of high wiring density, light weight, thin thickness, and good bending property.
  • the flexible circuit board 220 eliminates thermal stress by its own deformation during the temperature rise and fall process. The thermal stress existing in the temperature rise and fall process can be reduced by the flexible circuit board 220, thereby extending the service life of the temperature control device 20. At the same time, the problem of uneven temperature distribution is solved by the flexible circuit board 220.
  • the temperature control device 20 can realize instantaneous temperature rise and temperature decrease, and the process of temperature rise and temperature decrease is shortened, thereby achieving high and low temperature cycle, shortening the detection time of the digital PCR detector 1 and improving the detection efficiency.
  • the flexible printed circuit 220 can be a highly reliable and excellent flexible printed circuit board made of polyimide or polyester film.
  • the flexible circuit board has the characteristics of high wiring density, light weight, thin thickness, and good bending property.
  • the flexible circuit board is light in weight and thin in thickness, and can effectively save product volume.
  • the semiconductor electric cooler (TEC) is made by utilizing the Peltier effect of a semiconductor material.
  • the so-called Peltier effect refers to the phenomenon that when a direct current passes through a galvanic couple composed of two kinds of semiconductor materials, one end absorbs heat and one end radiates heat. Replacing one of the conventional semiconductor refrigerators by the flexible circuit board 220 makes the semiconductor cooler more thermally conductive.
  • Thermal stress is also called temperature change stress.
  • the thermal stress is balanced with the zero external phase. It is a self-balancing stress caused by the thermal deformation. The compression occurs at a high temperature and the tensile deformation occurs at a low temperature. Under certain conditions, by controlling the stress to make it reasonably distributed, the mechanical properties and service life of the parts can be improved, and the damage can be improved.
  • the heating substrate 240 can be a superconducting aluminum substrate circuit.
  • the aluminum substrate is a metal-based copper clad plate with good heat dissipation function.
  • the single panel is composed of a three-layer structure, which is a circuit layer (copper foil), an insulating layer and a metal base layer.
  • the superconducting aluminum substrate circuit is a material of a circuit board which is an aluminum alloy and can conduct heat quickly.
  • the aluminum substrate minimizes thermal resistance and provides excellent thermal conductivity of the aluminum substrate, and its mechanical properties are excellent compared to thick film ceramic circuits.
  • the semiconductor galvanic pair 230 includes a P-type galvanic coupler 231 and an N-type galvanic coupler 232 spaced from the P-type galvanic coupler 231.
  • the P-type coupler 231 and the N-type couple 232 are soldered between the flexible circuit board 220 and the substrate 240.
  • the semiconductor galvanic pair 230 includes a pair of galvanic couples formed by the P-type 232 and the N-type 232, and a plurality of pairs of the galvanic pairs 230 are connected by electrodes, and Sandwiched between the flexible circuit board 220 and the first surface 241.
  • a "hot" side and a "cold” side are produced. Whether it is cooling or heating, as well as the rate of cooling and heating, is determined by the direction and magnitude of the current passing through it.
  • the thermoelectric effect produced by a pair of said semiconductor couples 230 is small, so in practice, hundreds of pairs of said semiconductor couples 230 are connected in series, and the resulting thermoelectric effect is increased.
  • the first surface 241 includes a plurality of spaced apart first electrode sheets 243, one of the first electrode sheets 243 corresponding to one of the semiconductor couple pairs 230, the semiconductor couple pair 230
  • the P-type coupler 231 and the N-type couple 232 are connected in series through the first electrode piece 243.
  • the flexible circuit board 220 includes a plurality of second electrode sheets 221 spaced apart from each other and connected in series, and two adjacent semiconductor couple pairs 230 are connected in series by one of the second electrode sheets 221.
  • the temperature control device 20 further includes a thermally conductive enhancement layer 250 disposed on the second surface 242.
  • the thermally conductive reinforcing layer 250 has very good strength, flexibility, electrical conductivity, thermal conductivity, and optical properties.
  • the thermally conductive reinforcing layer 250 is directly in contact with the micro-droplet container 60, and the plurality of micro-droplets can be heated uniformly, thereby realizing nucleic acid amplification by controlling temperature.
  • the heat conduction enhancement layer 250 may be a graphite heat conduction layer or a silicone grease heat conduction layer to accelerate heat conduction, increase temperature uniformity of the second surface 242 of the heating substrate 240, thereby ensuring proximity to the microdroplet container 60.
  • the surface temperature is uniform so that the plurality of microdroplets are heated uniformly. Further, the nucleic acid amplification is completed, the detection efficiency is improved, and time is saved.
  • the material of the thermally conductive enhancement layer 250 includes graphene.
  • the graphene is a flat film which has very good heat conduction properties and can achieve uniform heat conduction in the lateral direction.
  • the temperature control device 20 further includes a second controller 210 electrically coupled to the flexible circuit board 220 for controlling the magnitude of the current.
  • the temperature control device 20 further includes a temperature sensor 260 disposed on the second surface 242 and electrically connected to the second controller 210 for detecting the temperature of the second surface 242. And sending the temperature to the second controller 210.
  • the temperature sensor 260 is disposed on the second surface 242 of the heat conduction enhancement layer 250 to detect the real-time temperature of the second surface 242, and then feed back temperature information to the second controller 210, thereby implementing Control of the heating temperature of the plurality of microdroplets.
  • the temperature sensor 260 is configured to measure the temperature of the micro-droplet container 60 by detecting a change in resistance of the metal, to detect the temperature change of the plurality of micro-droplets during the nucleic acid amplification process in real time, thereby Feedback to the second controller 210, thereby controlling the control circuit to perform temperature regulation, achieving temperature control, and better performing nucleic acid amplification.
  • the second controller 210 includes a temperature control unit 212 and a control circuit 214.
  • the temperature control unit 212 is coupled to the temperature sensor 260 for detecting the temperature of the second surface 242 in real time.
  • the control circuit 214 is coupled to the flexible circuit board 220 for regulating temperature changes of the plurality of semiconductor galvanic pairs 230.
  • the temperature control unit 212 and the control circuit 214 are disposed on a circuit board.
  • the relationship between the temperature control unit 212 and the control circuit 214 is a logical operation relationship of the internal algorithm, and a Packet Identifier closed-loop control algorithm, that is, a PID closed-loop control algorithm, may be used.
  • the temperature detected by the temperature control unit 212 is the temperature feedback of the nucleic acid amplification.
  • the control circuit 214 calculates the result as an output of the internal algorithm, thereby forming a closed loop relationship.
  • the temperature feedback of the circuit part is actually a sampling circuit.
  • the electrical signal on the platinum resistor is collected and converted to a temperature value that is passed to the input of the control circuit.
  • the temperature sensor 260 and the temperature control unit 212 are connected by a standard platinum resistance three-wire system.
  • the flexible circuit board 220 is provided with a first electrode 222 and a second electrode 223.
  • the plurality of second electrode sheets 221 are connected in series with the first electrode 222 and the second electrode 223 in series.
  • the first electrode 222 and the second electrode 223 are respectively connected to the control circuit.
  • the connection between the control circuit 214 and the flexible circuit board 220 is two wires, and the first electrode 222 and the second electrode 223 are respectively connected.
  • the temperature control device 20 further includes a heat sink 270 that includes a substrate 271 and a heat sink 272 coupled to the substrate 271.
  • the flexible circuit board 220 is disposed on a surface of the substrate 271. Since the heat sink 272 is disposed on the surface of the substrate 271, the heat exchange area is increased without increasing the area of the substrate 271, and the time during which the cool air acts on the surface of the substrate 271 is extended.
  • the heat dissipation air passage is also beneficial for accelerating heat exchange, and more heat is taken away from the surface of the substrate 271, thereby achieving a more ideal heat dissipation effect.
  • the temperature control device further includes a fan 273 disposed around the heat sink 273.
  • the heat sink 270 can be assisted by the fan 273 for heat dissipation.
  • the fan 273 is disposed around the heat sink 273, and a plurality of fans 273 can be disposed, so that a better heat dissipation effect can be achieved, and the temperature control device 20 can be warmed and cooled more quickly.
  • the temperature control device 20 is circulated with alternating current, and the current controller is adjusted by the second controller 210 to control whether the temperature control device 20 is cooled or heated, and cooled, heated. rate.
  • the temperature sensor 260 is used to detect the real-time heating temperature of the micro-droplet container 60, and then feed back the temperature information to the temperature control unit 212.
  • the temperature control unit 212 feeds back a temperature change condition to the control circuit 214 to control the temperature of the plurality of microdroplets.
  • the plurality of microdroplets can be subjected to nucleic acid amplification by the temperature control device 20. Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle.
  • the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C.
  • the primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA.
  • the primer strand is extended along the template to amplify the nucleic acid in a suitable temperature range.
  • the bottom of the micro-droplet container 60 is closely attached to the temperature control device 20, and there is no gap between the two, which improves the accuracy of the digital PCR detector 1.
  • the temperature control device 20 can realize instantaneous temperature rise and temperature decrease, and the process of temperature rise and temperature decrease is shortened, thereby achieving high and low temperature cycle, shortening the detection time of the digital PCR detector 1 and improving the detection efficiency.
  • the method of performing the accounting amplification by the temperature control device 20 is as follows:
  • the micro-droplet container 60 is placed on the thermally conductive enhancement layer 250 of the temperature control device 20.
  • the plurality of microdroplets were subjected to elevated temperature heating, the temperature was heated to 95 ° C, and heated for 10 min.
  • the plurality of microfluids were heated to 95 ° C and heated for 10 min to thermally initiate the enzymes in the plurality of microdroplets.
  • the plurality of micro-droplets are denatured for 30 s;
  • the temperature is lowered to 55 ° C, and the annealing is extended for 45 s, and the plurality of pico liquids are photographed and subjected to 45 cycles;
  • the temperature control device 20 generally has two indexes for testing the temperature control performance, and observes the temperature rise and fall of the temperature control device 20 in the transient state and the steady state, respectively.
  • the temperature rise and fall rate can reach 13.34448 ° C / s at the maximum.
  • the control accuracy is 0.02722 °C.
  • the rate of measurement of the temperature control device 20 when it is warmed to a steady state can be as fast as 18.953894 ° C / s. Therefore, the transient response of the temperature control device 20 is good, and the temperature control device 20 can realize instantaneous temperature rise and temperature reduction, save time, and improve detection efficiency.
  • the temperature control device 20 when the temperature control device 20 is in a steady state, that is, the temperature floats after reaching stability.
  • the temperature change is relatively stable and the temperature fluctuation is small. Therefore, the temperature control device 20 can achieve a rapid temperature rise and fall cycle, and the temperature after the stabilization is small, which saves the time for the digital PCR to detect the sample of the solution and improves the work efficiency.
  • This rate of temperature rise and fall shortens the time required to complete nucleic acid amplification, improves nucleic acid amplification efficiency, and improves the accuracy of the digital PCR detection system.
  • the microdroplet generating device 10 microdrops the nucleic acid amplification reaction solution to be tested to form a plurality of microdroplets. Then, during the heating of the plurality of micro-droplets by the temperature control device 20, the fluorescence signal detecting device 30 is used to take a real-time measurement of the fluorescence change image of the plurality of micro-droplets. The fluorescence change image of the plurality of microdroplets is analyzed by the quantitative analysis device 40, the Ct value of the plurality of microdroplets is obtained, and the concentration of the initial nucleic acid is determined by the relationship between the Ct value and the initial copy number. Perform quantitative analysis.
  • the microdroplet generating device 10 microdrops the nucleic acid amplification reaction solution to be tested to form a plurality of microdroplets.
  • the plurality of microdroplets are then subjected to nucleic acid amplification by the temperature control device 20.
  • the fluorescent signal detecting device 30 is used to take a picture of the fluorescence changes of the plurality of micro-droplets in real time.
  • a fluorescence change curve of the plurality of microdroplets is obtained by a fluorescence change picture of the plurality of microdroplets.
  • the Ct value of the plurality of microdroplets can be obtained, and the concentration of the initial DNA is quantitatively analyzed by the relationship between the Ct value and the initial copy number.
  • the Ct value refers to the number of cycles experienced when the fluorescence signal of each microdroplet reaches a set threshold.
  • the micro-droplet generating device 10 generates uniform-sized micro-droplets, and performs nucleic acid amplification reaction on the plurality of micro-droplets through the temperature control device 20, and collects product signals such as fluorescence, ultraviolet absorption, turbidity, and the like. signal. Utilizing the difference in composition between the plurality of amplified and non-amplified microdroplets, the number of droplets obtained by obtaining the target sequence is analyzed, and finally quantitative analysis of the nucleic acid molecule is realized. By monitoring the fluorescence change pictures of the plurality of micro-droplets in real time, the sequencing results are direct, and the problems of false positives and false negatives among the plurality of micro-droplets can be solved.
  • the fluorescent signal detecting device 30 includes an excitation light source, a fluorescence detecting component, and a third controller.
  • the excitation light source is disposed above the detection area of the micro-droplet container 60, and is irradiated at an oblique angle to the detection area of the micro-droplet container 60 to form an oblique light path.
  • the fluorescence detecting component is disposed directly above the detection area of the microdroplet container 60 for collecting a fluorescent image of the plurality of microdroplets.
  • the third controller is coupled to the excitation light source and the fluorescence detecting component, respectively, for controlling the excitation light source and the fluorescence detecting component.
  • the fluorescent signal detecting device can perform imaging of a plurality of fluorescent channels on the microdroplets and perform bright field dark field imaging. Among them, multiple fluorescent channel imaging is used for detecting the droplet reaction signal, and bright field dark field imaging is used to detect the size information of the formed microdroplet and monitor the state of the droplet during the reaction.
  • the generation of a fluorescent image of the plurality of microdroplets is accomplished primarily by a camera in the fluorescence detecting assembly.
  • the fluorescent signal detecting device 30 can cause the plurality of micro-droplets to be fluorescently imaged, take a certain number of fluorescent images of the plurality of micro-droplets at a time, and then use image processing technology to perform droplet fluorescence in the image. Automatic identification to obtain fluorescence information of the droplets. Irradiating the micro-droplet container 60 from the micro-droplet container 60, the fluorescence signal detecting device 30 is used to perform periodic two-dimensional scanning on the plurality of micro-droplets, and real-time Take a photo.
  • the oblique light path can effectively reduce the excitation light scattering background and improve the sensitivity of fluorescence detection.
  • the internal fluorescence of the plurality of microdroplets within the microdroplet container 60 is excited into the camera such that the camera captures a fluorescent picture of the plurality of microdroplets.
  • micro-droplet container For the traditional micro-droplet container, the cost of consumables is relatively high, the number of micro-droplets is small, and the liquid level of the micro-droplet container is concave, which affects the problem of photo detection, and provides a low cost and can accommodate a large amount of consumables. Microdroplets, and are advantageous for photodetection of microdroplet containers.
  • an embodiment of the present application provides a micro-droplet container 60 including a bottom surface 611, a first annular side surface 621 disposed around the bottom surface 611, and an annular surface 641.
  • the first annular side surface 621 is connected to the bottom surface 611 and surrounds a receiving space 630 having an opening 631 perpendicular to the bottom surface 611.
  • the annular surface 641 is disposed around the opening 631 and is connected to the first annular side surface 621, and the annular surface 641 is parallel to the bottom surface 611.
  • the annular surface 641 is parallel to the bottom surface 611 to ensure that the surface of the liquid in the microdroplet container 60 is a horizontal plane.
  • the liquid level of the micro-droplet container 60 can be made flat, avoiding the overall liquid level of the micro-droplet container 60 being curved. Therefore, the micro-droplet container 60 does not affect the observation of the micro-droplets near the edge portion of the container bottom plate, facilitating the camera to perform photographing imaging, and improving the detection efficiency of the plurality of micro-droplets.
  • the microdroplet container 60 further includes a container bottom plate 610, a first annular side plate 620 disposed around the container bottom plate 610, and an annular plate 640.
  • the surface of the container bottom plate 610 is the bottom surface 611.
  • the inner surface of the first annular side plate 620 is the first annular side surface 621, and the first annular side plate 620 is fixedly connected to the container bottom plate 610 and is surrounded by the container bottom plate 610 to form the storage.
  • Space 630 The surface of the annular plate 640 is the annular surface 641.
  • the annular plate 640 is fixedly coupled to an end of the first annular side plate 620 away from the container bottom plate 610, and the annular plate 640 is parallel to the container bottom plate 610.
  • the second liquid (oil phase composition) is first placed in the microdroplet container 60.
  • the liquid level of the second liquid is the same as the horizontal surface of the annular surface 641
  • the addition of the second liquid is stopped.
  • the liquid level of the second liquid is on the same level as the surface of the annular surface 641, which can ensure that the oil surface of the second liquid in the micro-droplet container 60 is flat, which is convenient for ensuring the bottom surface of the container.
  • the top surface of the upper oil is a horizontal plane for ease of imaging, increasing the utilization of the microdroplet container 60 to accommodate a larger number of microdroplets.
  • an inner circumference of the annular plate 640 is coupled to an end of the first annular side plate 620 away from the container bottom plate 610.
  • the microdroplet container 60 also includes a second annular side panel 650.
  • the second annular side plate 650 is disposed around the annular plate 640 and is fixedly coupled to the annular plate 640 .
  • the radius of the second annular side plate 650 is greater than the inner diameter of the annular plate 640 .
  • the annular plate 640, the first annular side plate 620, the container bottom plate 610, and the second annular side plate 650 may be respectively associated with the annular surface 641, the first annular side surface 621, and the bottom surface
  • the 611 cooperates to form the storage space 630.
  • the storage space 630 is for accommodating the second liquid (oil phase composition).
  • the liquid level of the second liquid is on the same level as the surface of the annular surface 641, which can ensure that the oil level of the second liquid in the micro-droplet container 60 is flat, and the oil above the bottom surface of the container is conveniently ensured.
  • the top surface of the liquid is a horizontal plane, which avoids the problem that the entire liquid surface of the micro-droplet container is curved and the liquid surface presents a concave liquid surface. Therefore, when the fluorescent signal detecting device 30 performs fluorescence detection on the plurality of micro-droplets, the imaging is more convenient, and the utilization rate of the micro-droplet container 60 is improved to accommodate a larger number of micro-drop
  • the outer circumference of the annular plate 640 is coupled to an end of the first annular side plate 620 that is remote from the container floor 610.
  • the outer circumference of the first annular side plate 620 is fixedly coupled to the first annular side surface 621.
  • the microdroplet container 60 can be formed into a water platform by connecting the annular plate 640 to the first annular side plate 620.
  • the liquid level of the second liquid may be at the same level as the surface of the annular surface 641, so that the micro-droplet container can be secured.
  • the oil level of the second liquid in 60 is a plane, which is convenient for ensuring that the top surface of the oil above the bottom surface of the container is a horizontal surface, and the whole liquid surface of the micro-droplet container is prevented from being curved, and the liquid surface is concave. The problem. Therefore, when the fluorescent signal detecting device 30 performs fluorescence detection on the plurality of micro-droplets, the imaging is more convenient, and the utilization rate of the micro-droplet container 60 is improved to accommodate a larger number of micro-droplets. .
  • the microdroplet container 60 further includes a third annular side panel 660.
  • One end of the third annular side plate 660 is fixedly connected to the bottom surface 611.
  • the other end of the third annular side plate 660 is fixedly coupled to the inner circumference of the first annular side plate 620.
  • the third annular side plate 660 and the container bottom plate 610 together form the storage space 630.
  • the third annular side panel 660 is perpendicular to the container floor 610.
  • the liquid level of the second liquid can be made at the same level as the surface of the annular surface 641, so that the micro-droplets
  • the liquid level in the container 60 is flat, and the surface is formed by the conventional concave liquid surface to facilitate imaging, and the utilization rate of the micro-droplet container 60 is improved.
  • the micro-droplet container 60 further includes a plurality of annular ribs 613 spaced apart from the bottom surface 611, each of the annular ribs 613 and the bottom surface 611 surrounding to form a micro-droplet storage. Slot 614.
  • the micro-droplet receiving groove 614 is configured to receive the generated plurality of micro-droplets, and the plurality of micro-droplets are tiled on the bottom surface 611 by a micro-droplet tiling method to form a single-layer micro-liquid Drop, used for photo observation.
  • the interval between the plurality of the micro-droplet receiving grooves 614 may be set according to the distance between the pin headers of the micro-droplet generating device 10, so that the plurality of the micro-droplets may be accommodated at one time.
  • a plurality of micro-droplets are formed in the groove 614 to increase the capacity of the micro-droplet container 60, and can also be used to detect different kinds of nucleic acids.
  • the plurality of annular ribs 613 have a height of 0.1 mm to 1 mm. By setting the height of the annular rib 613, it is possible to eliminate the shadow caused by the excitation light from the side, so that the camera can acquire the fluorescence information of all the micro droplets, and the fluorescence detecting device is improved. Sensitivity.
  • the inner wall surface of the micro-droplet receiving groove 614 is provided with an oleophobic layer.
  • an oleophobic layer By performing oleophobic treatment on the surface of the bottom plate 610 of the container, the adhesion between the bottom plate 610 and the micro-droplets is reduced, the surface tension is lowered, and the frictional force is reduced, which is easy to slip, and the micro-droplet will be Automatic diffusion prevents the plurality of microdroplets from coming together.
  • the plurality of microdroplets can be made more rapid when tiling, facilitating the plurality of microdroplets to be tiled on the microdroplet container bottom plate 610.
  • the oleophobic film also called oleophobic layer, is a composite coating material, which is a functional material coating and often has an oleophobic function.
  • the oleophobic layer generally uses nano silica as a raw material (SiO 2 ), and adopts a spraying process to form a coating on the surface, which has good light transmittance and hydrophobic oleophobicity.
  • the contact angle thereof can reach 90 degrees, and automatic roll-off can be achieved without leaving traces, so that the plurality of micro-droplets can be tiled on the micro-liquid. Drop the bottom 610 of the container.
  • a microdroplet generation kit includes the microdroplet container 60, the seal cap 670, and the oil phase composition mentioned in the above embodiments, the oil phase composition being placed in the storage In the space 630, the sealing cover 670 is disposed at the opening 631 for sealing the storage space 630.
  • each reaction unit 612 includes each of the annular ribs 613, and each of the annular ribs 613 is surrounded by the bottom surface 611 to form a micro-droplet receiving groove 614.
  • the container bottom plate 610 is provided with a plurality of reaction units 612, each of which can place a plurality of micro-droplets so that the micro-droplet container 60 can accommodate a large number of micro-droplets, so that the truly detected
  • the number of droplets will be far more than 20,000, and there is no limit to the number of droplets. At the same time, it takes more time to perform a large number of microdroplet detection.
  • the plurality of reaction units 612 are rectangular in shape.
  • the microdroplet container 60 is square or rectangular. Since most of the film and digital photosensitive elements CCD/CMOS are square in the past, the shape of the micro-droplet container 60 is square, which can improve the space utilization of the micro-droplet container, and It facilitates the splicing of the formed fluorescent images to achieve real-time tracking.
  • a plurality of the reaction units 612 are equally spaced on the container floor 610.
  • the distance between the plurality of the reaction units 612 is the same as the distance between the pins of the micro-droplet generating device 10, so that a large number of micro-droplets are simultaneously formed in the plurality of the reaction units 612, and the micro-liquid is improved.
  • the speed of droplet generation saves time.
  • a plurality of micro-droplets of different volumes may be generated in the plurality of the reaction units 612 by the micro-droplet generating device 10.
  • the plurality of annular ribs 613 have a height of 0.1 mm to 1 mm. By setting the height of the annular rib 613, it is possible to eliminate the shadow caused by the excitation light from the side, so that the camera can acquire the fluorescence information of all the micro droplets, and the fluorescence detecting device is improved. Sensitivity.
  • Each individual reaction unit of a conventional digital PCR detection system is typically placed with a microdroplet. Moreover, during the actual detection process, the number of droplets actually detected does not reach 20,000, and there is still a limit to the number of the microdroplets. Therefore, the above problem can be solved by using the micro-droplet container 60 without limiting the number of the micro-droplets.
  • a plurality of the reaction units 612 on the container bottom plate 610 can accommodate a large number of micro-droplets, increasing the storage amount of the micro-droplet container 60, and detection of more than 20,000 micro-droplets can be achieved, and Different types of nucleic acids can be detected.
  • the reaction unit bezel it is avoided that the plurality of microdroplets are scattered into the adjacent reaction unit 612.
  • the microdroplet container 60 has a rectangular cross section.
  • the microdroplet container 60 is square or rectangular.
  • the shape of the microdroplet container 60 is consistent with the shape of the camera lens, which improves the space utilization of the microdroplet container, and facilitates the splicing of the formed fluorescent image, thereby realizing real-time tracking.
  • the annular face 641 is a square frame.
  • the adhesion between the bottom plate 610 and the micro-droplets is reduced, the surface tension is lowered, and the frictional force is reduced, which is easy to slip, and the micro-droplet will be Automatic diffusion prevents the plurality of microdroplets from coming together.
  • the plurality of microdroplets can be made more rapid when tiling, facilitating the plurality of microdroplets to be tiled on the microdroplet container bottom plate 610.
  • the oleophobic film also called oleophobic layer, is a composite coating material, which is a functional material coating and often has an oleophobic function.
  • the oleophobic layer generally uses nano silica as a raw material (SiO 2 ), and adopts a spraying process to form a coating on the surface, which has good light transmittance and hydrophobic oleophobicity.
  • the contact angle thereof can reach 90 degrees, and automatic roll-off can be achieved without leaving traces, so that the plurality of micro-droplets can be tiled on the micro-liquid. Drop the bottom 610 of the container.
  • the first annular side panel 620 or the second annular side panel 650 has a height of 5 mm to 15 mm.
  • the microdroplet generating device 10 can be prevented from avoiding the plurality of microdroplets in the process of preparing the plurality of microdroplets A microdroplet is thrown out.
  • it can be advantageous to exclude the shadow caused by the excitation light from the side illumination, so that the camera can acquire the fluorescence information of all the micro droplets, and the sensitivity of the fluorescence detecting device 30 is improved.
  • the container bottom plate 610 is made of glass, quartz or stainless steel or the like.
  • the container bottom plate 610 is made of glass, which is inexpensive, and has low consumable cost.
  • the micro-droplet container 60 is made of glass material, which is inexpensive, and has low cost of consumables. It can be discarded by performing one inspection, preventing cross-contamination, saving detection time, and increasing the number. The detection efficiency of the PCR detector 1.
  • the annular bead 613 is the same material as the microdroplet container bottom plate 610.
  • the microdroplet container bottom plate 610 can be formed into a plurality of the reaction units 612 by process techniques.
  • a plurality of the reaction units 612 are disposed in an array form on the microdroplet container bottom plate 610 to form a plurality of nucleic acid amplification units.
  • the shape and size of the container bottom plate 610 is the same as that of the 24-hole plate and the 96-well plate, so that the micro-droplet container 60 can be conveniently applied to other types of instruments, and is more practical and compatible. .
  • the first annular side plate 620 or the second annular side plate 650 is made of a high-low temperature, oil-resistant, non-fluorescent black silicone rubber.
  • the black silicone rubber has the characteristics of being odorless, non-toxic, not afraid of high temperature and resisting severe cold. Moreover, the black silicone rubber has the advantages of good electrical insulation, oxygen and aging resistance, light and aging resistance, mildew resistance and chemical stability, and has been paid attention to in the modern medical field.
  • the micro-droplet container 60 is made of glass or stainless steel to reduce the cost of inspection.
  • a plurality of the reaction units 612 on the bottom plate 610 of the container can accommodate a large number of micro-droplets, which increases the storage amount of the micro-droplet container, and can detect more than 20,000 micro-droplets, and can It is economical to test different types of nucleic acids.
  • the excitation light source When the excitation light source is obliquely irradiated to the microdroplet container 60, the plurality of microdroplets are irradiated.
  • the oblique light path formed by the excitation light source can effectively reduce the excitation light scattering background.
  • reducing the height of the first annular side plate 620 or the second annular side plate 650 of the micro-droplet container 60 is advantageous for eliminating the shadow caused by the excitation light from the side, so that the fluorescent signal
  • the camera of the detecting device 30 is capable of acquiring fluorescence information of all the microdroplets, improving the sensitivity of the fluorescence detecting device 30.
  • the present application provides a microdroplet container 60 that includes a first container bottom plate 680, a polygonal frame 684, and a container cover 690.
  • the first container bottom plate 680 is provided with a plurality of polygonal ridges 681.
  • the polygonal frame 684 is surrounded by a first storage space 685.
  • the polygonal frame 684 is connected to the first container bottom plate 680, and the plurality of polygonal protrusions 681 are disposed in the first storage space 685.
  • the container cover 690 is disposed on a surface of the polygonal frame 684 away from the first container bottom plate 680, and the container cover 690 is detachably connected to the polygonal frame 684, the container cover 690 and the polygonal frame 684 An oil accommodating groove 698 is formed to surround.
  • the two sides of the polygonal frame 684 are respectively connected to the first container bottom plate 680 and the container cover 690.
  • the container cover 690 is detachably connected to the polygonal frame 684, and the micro-droplet container 60 can be sealed.
  • the plurality of polygonal ridges 681 may have a polygonal shape such as a square, a rectangle, or a pentagon.
  • the container cover 690 is surrounded by the polygonal frame 684 to form an oil receiving groove 698.
  • the fluorescent signal detecting device 30 may take a picture of the fluorescence change of the plurality of microdroplets in real time. Therefore, by sealing the micro-droplet container 60, when the micro-droplet container 60 is tilted at an angle of 3 to 5 degrees, the liquid in the micro-droplet container 60 can be prevented from flowing out, and the The bubble discharge in the micro-droplet container 60 avoids the influence of the bubble on the image when the photograph is detected.
  • the container closure 690 includes a flat plate 691 and a polygonal container closure frame 692.
  • the flat plate 691 is transparent.
  • the polygonal container cover frame 692 is surrounded to form a mounting space 697 for mounting the flat plate 691.
  • the plate 691 is a glass plate.
  • the flat plate 691 and the polygonal container cover frame 692 can be adhered and fixed by glue which is resistant to water, high temperature, no fluorescence, and non-toxic, and has no inhibitory effect on the PCR reaction.
  • the flat plate 691 is inexpensive for the glass plate and has low consumable cost. If a large amount of micro-droplet detection is performed, the micro-droplet container 60 is made of glass material, which is inexpensive, and has low cost of consumables. It can be discarded by performing one inspection, preventing cross-contamination, saving detection time, and increasing the number. The detection efficiency of the PCR detector.
  • the polygonal container closure frame 692 includes a polygonal frame body 693, a plurality of extension panels 695, and a plurality of flat panel mounting frames 696.
  • the polygonal frame body 693 includes a plurality of frames 694 that are fixedly coupled.
  • Each of the extension plates 695 is fixedly coupled to each of the bezels 694 and extends obliquely toward the installation space 697.
  • Each of the flat mounting frames 696 is fixedly coupled to each of the extending plates 695 and extends toward the first receiving space 685 formed by the polygonal bezel 684.
  • the plurality of bezels 694 are fixedly coupled to form the polygonal frame body 693.
  • An angle formed by a surface of each of the bezels 694 and a surface of each of the extension plates 695 is greater than 90 degrees, so that the oil receiving groove can be formed when the container cover 690 is coupled to the polygonal bezel 684. 698.
  • the plurality of extension plates 695 are fixedly connected in sequence to form an annular frame and are fixedly coupled to the polygonal frame body 693.
  • the plurality of flat mounting frames 696 are fixedly connected in sequence to form an annular frame, and are fixedly connected to the annular frame formed by the plurality of extending plates 695.
  • the polygonal container cover frame 692 is integral.
  • each of the extension plates 695 forms an angle with the surface of each of the flat mounting frames 696 that is greater than 90 degrees.
  • the surface of each of the flat mounting frames 696 forms an angle of 90 degrees with the surface of each of the bezels 694, that is, the flat mounting frame 696 is perpendicular to the level of the oily liquid in the microdroplet container 60.
  • a rectangular interface can be formed, which can be made the same as the shape of the camera lens, so that the liquid level of the micro-droplet container 60 can be made flat, avoiding the micro-droplet container 60.
  • the overall liquid surface is curved.
  • the micro-droplet container 60 does not affect the observation of the micro-droplets of the first container bottom plate 680 near the edge portion, facilitating the camera to perform photographing imaging, and improving the detection efficiency of the plurality of micro-droplets.
  • the utilization of the microdroplet container 60 is increased by the microdroplet container 60 to accommodate a larger number of microdroplets.
  • the excess oily substance in the micro-droplet container 60 can be squeezed into the oil-receiving tank 698, and the micro-liquid can be avoided as much as possible. The effect of the oily substance in the upper part of the drop on the detection process.
  • the first container bottom plate 680 further includes a substrate 683 , and a plurality of the polygonal ribs 681 are disposed in an array on the first side of the substrate 683 adjacent to the polygonal frame 684 .
  • the surface, each of the polygonal ribs 681 and the surface of the substrate 683 adjacent to the polygonal frame 684 surrounds to form a first micro-droplet receiving groove 682.
  • the plurality of polygonal ribs 681 are rectangular.
  • the microdroplet container 60 is square or rectangular. Since most of the film and digital photosensitive elements CCD/CMOS are square in the past, the shape of the micro-droplet container 60 is square, which can improve the space utilization of the micro-droplet container, and It facilitates the splicing of the formed fluorescent images to achieve real-time tracking.
  • a plurality of the polygonal ribs 681 are disposed in an array on the surface of the substrate 683 adjacent to the frame 684, and each of the polygonal ridges 681 and the substrate 683 are surrounded to form a first micro-droplet receiving groove 682.
  • the first micro-droplet receiving groove 682 is configured to receive the generated plurality of micro-droplets, and the plurality of micro-droplets are laid flat on the first container bottom plate 680 by a micro-droplet tiling method to form a single Layer microdroplets or multilayer microdroplets for photo observation.
  • the spacing between the plurality of the polygonal ribs 681 may be set according to the distance between the pins of the micro-droplet generating device 9680, so that the plurality of the first micro-droplets may be accommodated at one time.
  • a plurality of micro-droplets are formed in the groove 682 to increase the capacity of the micro-droplet container 60, and can also be used to detect different kinds of nucleic acids.
  • each of the polygonal ribs 681 has a height of 0.2 mm to 0.8 mm.
  • Each of the polygonal ribs 681 has a height of 0.2 mm to 0.8 mm, and preferably may be between 0.3 mm and 0.5 mm.
  • the polygonal ribs 681 are not caused to pass over, and the polygonal ridges 681 each of the polygonal ridges 681 The height cannot be too low. Therefore, the height of the polygonal ridges 681 is at least above one droplet diameter.
  • micro-droplet image When the micro-droplet image is collected by the fluorescence detecting device 30, two or more layers of micro-droplets may be formed in the first micro-droplet receiving groove 682 of the micro-droplet container 60, such that The detection range of the digital PCR detector can be expanded to facilitate processing of large quantities of microdroplets.
  • the nucleic acid amplification reaction solution is microdropped by the microdroplet generating device 10 to form a plurality of microdroplets, and the volume of each microdroplet is generally between 0.1 mm and 0.2 mm in diameter.
  • the height of the polygonal ridges 681 is set to be larger than the diameter of the two micro-droplets, so that the micro-droplet generating device 10 can be made In the process of preparing a plurality of the microdroplets, a plurality of the microdroplets are prevented from scooping out. Moreover, it is possible to eliminate the shadow caused when the excitation light is irradiated from the side, so that the fluorescence signal detecting device 30 can acquire the fluorescence information of all the micro droplets, and the sensitivity of the fluorescence detecting device 30 is improved.
  • the height of each of the polygonal ridges 681 does not affect the image collection, which may be advantageous for eliminating the excitation light from the side.
  • the shading enables the camera to acquire fluorescence information for all of the microdroplets, increasing the sensitivity of the fluorescence detecting device 30.
  • the inner wall surface of the first micro-droplet receiving groove 682 formed by the plurality of polygonal ribs 681 is disposed with an oleophobic layer. That is, an oleophobic layer is disposed on a surface of the plurality of polygonal ridges 681 close to the space of the first micro-droplet receiving groove 682.
  • the adhesion between the substrate 683 and the micro-droplets is reduced, the surface tension is lowered, and the frictional force is reduced, and the sliding is easy, and the micro-droplets are automatically Diffusion prevents multiple microdroplets from coming together.
  • a plurality of microdroplets can be made more rapid when tiling, facilitating the tiling of a plurality of microdroplets on the substrate 683.
  • the surface tension of the substrate 683 is less than the surface tension of the oily substance, the resistance of the micro-droplet and the bottom plate becomes small, and the micro-droplets are automatically diffused toward the bottom of the first micro-droplet receiving groove 682 to achieve flatness. shop.
  • the oleophobic film also called oleophobic layer, is a composite coating material, which is a functional material coating and often has an oleophobic function.
  • the oleophobic layer generally uses nano silica as a raw material (SiO 2 ), and adopts a spraying process to form a coating on the surface, which has good light transmittance and hydrophobic oleophobicity.
  • the contact angle thereof can reach 90 degrees, and automatic roll-off can be achieved without leaving traces, so that the plurality of micro-droplets can be spread on the substrate 683.
  • the shape and size of the substrate 683 is the same as that of the 24-well plate and the 96-well plate, so that the micro-droplet container 60 is conveniently applied to other types of instruments, and is more practical and compatible.
  • the first micro-droplet receiving groove 682 formed by the plurality of the polygonal ribs 681 can be placed to place a plurality of micro-droplets, so that the micro-droplet container 60 can accommodate a large number of micro-droplets, so that The number of droplets detected will be far more than 20,000, and there is no limit to the number of microdroplets. At the same time, it takes more time to perform a large number of microdroplet detection.
  • the distance between the flat plate 691 and the micro-droplets in the first micro-droplet receiving groove 682 cannot be too high, and the general distance is set at 1 mm. Between 2 mm, the influence of the oily substance on the upper layer of the micro-droplet on the image of the micro-droplet can be avoided, the fluorescent background caused by the oil-like substance is avoided, and a plurality of micro-droplets are facilitated to be laid on the substrate 683. Further, the excess oily substance in the microdroplet container 60 is extruded into the oil accommodating groove 698, and the oil substance in contact with the plurality of microdroplets in the microdroplet container 60 is reduced, and the oil is avoided. The absorption of moisture of a plurality of micro-droplets under the condition that the substance is not saturated solves the case of evaporation of water droplets of the micro-droplets.
  • the polygonal bezel 684 includes a plurality of first support bars 686, a plurality of support plates 688, and a plurality of second support bars 689.
  • Each of the first support bars 686 is annularly connected.
  • Each of the support plates 688 is fixedly disposed on an inner wall of each of the first support bars 686 and extends toward the first storage space 685.
  • the plurality of support plates 688 are annularly connected to support the first Container bottom plate 680.
  • Each of the second support bars 689 is fixedly disposed on a first support bar surface 687 of each of the first support bars 686 away from the first container bottom plate 680, and the plurality of second support bars 689 are annularly connected, and The plurality of second support bars 689 and the plurality of first support bars 686 together form the first storage space 685, and the plurality of second support bars 689 and the polygonal frame body 693 are The borders 694 are detachably connected.
  • Each of the support plates 688 is fixedly disposed on an inner wall of each of the first support bars 686 and extends toward the first storage space 685.
  • the plurality of support plates 688 are annularly connected to support the first Container bottom plate 680.
  • the first container bottom plate 680 is mounted on the annular support frame formed by the plurality of support plates 688, and the plurality of the polygonal protrusions 681 are disposed in the first storage space 685.
  • the polygonal frame 684 and the container cover 690 are adhered by the double-sided tape 699, that is, the surface of the polygonal frame formed by the annular connection of the plurality of the second support bars 689 is adhered.
  • the double-sided tape 699 is conveniently connected to the first surface of the frame 694 near the polygonal frame 684, so that the polygonal frame 684 is sealingly connected to the container cover 690.
  • the substrate 683 and the flat plate 691 of the micro-droplet container 60 are all glass plates, which solves the problem of consumables of the sample container when detected by the digital PCR detector, and saves cost.
  • the micro-droplet container 60 has a relatively thin overall structure of the micro-droplet container 60 due to the height setting between each element, increasing the reproducibility of a large number of micro-droplets, so that the micro-droplets The container 60 is rapidly and frequently circulated for easy observation and detection.
  • the width of each of the second support bars 689 is smaller than the width of each of the first support bars 686, and a slit is formed away from the first storage space 685 for convenient disassembly.
  • the polygonal frame 684 is integrally formed.
  • the side surface of the second support rod 689 and the side surface of the first support rod 686 are not in a plane, and the container cover 690 can be prevented from being adhered to the polygonal frame 684. Excess glue is squeezed out to avoid sticking to your hands or other places.
  • the width of each of the second support bars 689 is smaller than the width of each of the first support bars 686, and a gap is formed away from the first storage space 685 for convenient disassembly.
  • a method for preparing a microdroplet container includes:
  • a plurality of polygonal ridges 681 are prepared on the surface of the substrate 683 to form a first container bottom plate 680;
  • a container cover 690 is provided, and the container cover 690 is adhered to the surface of the polygonal frame 684 away from the first container bottom plate 680 for sealing to form a microdroplet container.
  • the substrate 683 can first be connected to the polygonal frame 684 by glue. At this time, the plurality of polygonal ribs 681 are not disposed on the substrate 683. Next, an oily liquid is placed in the container in which the substrate 683 is adhered to the polygonal frame 684. Again, the fluid is dripped and applied to the surface of the substrate 683 by the dispenser to be close to the surface of the polygonal frame 684, which can realize three-dimensional, four-dimensional path dispensing, precise positioning, precise glue control, no drawing, no leakage, no Epoxy, a plurality of the polygonal ribs 681 are formed.
  • the polygonal frame 684 is connected to the container cover 690, and can be adhered by double-sided tape when connected. At this time, the double-sided tape can be set into a polygonal shape, and the polygonal frame 684 and the container cover 690 are Fully stuck between.
  • a plurality of the polygonal ridges 681 may be formed on a surface of the substrate 683 adjacent to the polygonal frame 684.
  • a plurality of the polygonal ribs 681 may be prepared on the surface of the substrate 683 near the polygonal frame 684 by using a dispenser, printing, screen printing, or the like.
  • the first container bottom plate 680 is adhesively coupled to the polygonal frame 684. Again, an oily liquid is placed in the container in which the first container bottom plate 680 is adhered to the polygonal frame 684.
  • the polygonal frame 684 is connected to the container cover 690, and can be adhered by double-sided tape when connected. At this time, the double-sided tape can be set into a polygonal shape, and the polygonal frame 684 and the container cover 690 are Fully stuck between.
  • a plurality of the polygonal ribs 681 are prepared in the step S520, methods such as dispensing, spray printing, screen printing, and hot press forming may be employed.
  • the polygonal rib 681 is made of a high-low temperature resistant, oil-resistant, non-fluorescent polymer, so that the polygon can be avoided when the micro-droplet is subjected to high-temperature cycle and fluorescence photographing. The effect of the ribs 681.
  • the polygonal ribs 681 can be black silicone rubber or silicone sealant.
  • the material of the plurality of polygonal ribs 681 is a high-low temperature, oil-resistant, non-fluorescent black silicone rubber, so that the fluorescence of the polygonal ridges 681 can be avoided when the micro-droplets are fluorescently photographed. influences.
  • the black silicone rubber has the characteristics of being odorless, non-toxic, not afraid of high temperature and resisting severe cold. Moreover, the black silicone rubber has the advantages of good electrical insulation, oxygen and aging resistance, light and aging resistance, mildew resistance and chemical stability, and has been paid attention to in the modern medical field.
  • the fluid is dripped and applied to the surface of the substrate 683 by the dispenser to be close to the surface of the polygonal frame 684, which can realize three-dimensional and four-dimensional path dispensing, precise positioning, precise glue control, no drawing, no leakage, no glue
  • a plurality of the polygonal ridges 681 are formed.
  • the silicone sealant has the characteristics of weather resistance, vibration resistance, moisture resistance, odor resistance, large change in cold and heat, and obvious anti-seepage and leakage prevention effects.
  • the plurality of the polygonal ribs 681 when the plurality of the polygonal ribs 681 are prepared in the step S520, they may also be processed by injection molding or hot press forming.
  • a method for analyzing a digital PCR detector includes the following steps: S10, preparing a nucleic acid amplification reaction solution to be tested; S20, microdropping the nucleic acid amplification reaction solution to form a plurality of a micro-droplet; S30, performing nucleic acid amplification on the plurality of micro-droplets, and acquiring fluorescence information of the plurality of micro-droplets in real time; S40, according to the fluorescence information of the plurality of micro-droplets, Multiple microdroplets were subjected to quantitative analysis.
  • the step S20 microdropping the nucleic acid amplification reaction solution to be used to form a plurality of micro-droplets includes two micro-droplet generation methods: a transiently accelerated micro-droplet generation method and A method of generating a microdroplet in a shifting cycle.
  • the step S30 includes: S310: tiling the plurality of micro-droplets in the micro-droplet container; S320: performing nucleic acid amplification on the plurality of micro-droplets after tiling; S330: When a plurality of microdroplets are subjected to nucleic acid amplification, the plurality of microdroplets are photographed in real time.
  • micro-droplets prepared by the micro-droplet generating device 10 are concentrated in the intermediate portion of the micro-droplet container 60 during the downward sedimentation process, they are gathered together, which is disadvantageous for observation. Therefore, a microdroplet tiling method is provided for the problem of concentrated concentration on the bottom of the microdroplet container.
  • the step S30 includes: S310: tiling the plurality of micro-droplets in the micro-droplet container; S320: the plurality of micro-slices to be tiled The droplets are subjected to nucleic acid amplification; S330: performing photodetection on the plurality of microdroplets in real time when the plurality of microdroplets are subjected to nucleic acid amplification.
  • the step S310 includes a microdroplet tiling method.
  • the microdroplet tiling method comprises: S311, providing a microdroplet container 60 having an opening 631, and the microdroplet container 60 contains a second liquid 699; S312, providing a first liquid 190, the first liquid 190 has a density greater than the second liquid 699 and is immiscible with the second liquid 699, and the first liquid 190 generates a plurality of micro-droplets stacked on the The micro-droplet container bottom plate 610; S313, the high-temperature cycle is performed on the plurality of micro-droplets until the plurality of micro-droplets are laid flat on the container bottom plate 610.
  • a plurality of microdroplets are generated in the microdroplet container 60, and the container bottom plate 610 that falls to the microdroplet container 60 is irregularly stacked.
  • the container bottom plate 610 When a large number of microdroplets are dropped onto the container bottom plate 610, multiple layers of microdroplets are formed in the container bottom plate 610.
  • the plurality of micro-droplets prepared by the micro-droplet generating device are concentrated in the middle portion of the micro-droplet container during the downward sedimentation process, and are gathered together, which is not conducive to observation.
  • the second liquid 699 is an oil phase composition.
  • the components of the oil phase composition include mineral oil and a surfactant.
  • the volume percentage of mineral oil in the oil phase composition is from 88% to 98.5%.
  • the surfactant includes a long-chain alkyl group-containing siloxane chain nonionic surfactant, and the oil phase composition has a long-chain alkyl group-containing siloxane chain nonionic surfactant in a volume percentage of 1.5%. -12%.
  • the first liquid is a nucleic acid amplification reaction solution to be tested.
  • the step S312 includes: S3122, providing a squirting gun head 110 having an outlet end 112,
  • the first liquid 190 is stored in the ejector head 110; S3124, the outlet end 112 of the ejector head 110 is inserted under the liquid surface of the second liquid 699, and the movement of the speed is cyclically changed. During the first half cycle and the second half cycle of the change in the speed, the velocity of the outlet end of the jetting head is monotonously changed; S3126, according to the periodic change of the outlet end 112 of the jetting head 110, The first liquid 190 is discharged from the outlet end 112 of the ejector head 110, and a plurality of micro-droplets are formed under the liquid surface of the second liquid 699 and deposited on the micro-droplet container bottom plate 610.
  • step S3124 the velocity of the outlet end 112 of the ejector head 110 under the liquid surface of the second liquid 699 changes cosine.
  • the trajectory of the periodic change of the outlet end 112 of the ejector head 110 in the step S3124 under the liquid level of the second liquid 699 includes a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon. a combination of one or more of them.
  • step S312 includes:
  • the outlet end 112 of the ejector head 110 in the first half period and the second half of the periodic acceleration of the instantaneous acceleration of the outlet end 112 of the ejector head 110 in the step S3123, the outlet end 112 of the ejector head 110 The speed is the same and the direction is opposite.
  • the motion trajectory of the instantaneously accelerated periodic motion in the step S3123 includes a combination of one or more of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • the step S313 includes: S3131: heating the plurality of micro-droplets; S3133: cooling the plurality of micro-droplets; and S3135: performing the high-low temperature cycle of the plurality of micro-droplets Multiple times until the plurality of microdroplets are laid flat on the bottom plate of the microdroplet container.
  • the second liquid 699 is first placed in the microdroplet container 60 when the plurality of microdroplets are prepared by the microdroplet generating device 10.
  • the addition of the second liquid 699 is stopped.
  • the liquid level of the second liquid 699 is on the same level as the surface of the annular surface 641, and the oil surface of the second liquid 699 in the micro-droplet container 60 can be ensured to be flat. Ensure that the top surface of the oil above the bottom of the container is a horizontal surface for easy imaging.
  • the nucleic acid amplification reaction solution to be tested is microdropped by the microdroplet generating device 10 in the second liquid 699 to form a large number of microdroplets.
  • the plurality of microdroplets are dropped into a plurality of the reaction units 612 of the microdroplet container bottom plate 610.
  • the annular surface 641 of the container bottom plate 610 is parallel to ensure that the second liquid 699 in the microdroplet container is a horizontal plane.
  • a plurality of microdroplets can be placed in each reaction unit 612 such that the microdroplet container 60 can hold more than 20,000 microdroplets.
  • the plurality of microdroplets are laid flat on the microdroplet container bottom plate 610 according to the movement trajectory of the instantaneous acceleration movement of the outlet end 112 of the ejector head 110 in the step S3125. .
  • the trajectory of the instantaneous acceleration movement of the outlet end 112 of the ejector head 110 may cause the plurality of micro-droplets to be staggered from each other when dripping into the micro-droplet container 60, such that the plurality When the microdroplets are dropped into the microdroplet container 60, they do not accumulate with each other. Thereby, the plurality of micro-droplets are tiled in the micro-droplet container 60 to facilitate photographing observation.
  • the microdroplet container bottom plate 610 is coated with an oleophobic layer.
  • the oleophobic layer also called oleophobic coating, is a composite coating material, which is a functional material coating and often has an oleophobic function.
  • the oleophobic layer generally uses nano-silica as a raw material, and adopts a spraying process to form a coating on the surface of the screen, and has good light transmittance and hydrophobic oleophobicity.
  • the temperature control device 20 is employed in the microdroplet tiling method of the above embodiments.
  • the temperature control device 20 includes a flexible circuit board 220, a heating substrate 240 spaced apart from the flexible circuit board 220, and a plurality of semiconductor galvanic pairs 230 disposed between the flexible circuit board 220 and the heating substrate 240. .
  • the principle of thermal expansion and contraction is utilized for tiling.
  • the temperature of the object increases, the kinetic energy of the molecule increases, and the mean free path of the molecule increases, so it appears as thermal expansion.
  • the temperature of the object decreases, the kinetic energy of the molecule decreases, and the mean free path of the molecule decreases, so it appears as cold shrinkage.
  • the viscosity of the sample droplets becomes lower and the volume shrinks.
  • the higher the temperature the lower the viscosity.
  • the shape of the sample droplet is the softest. At this time, the shape is roughly hexagonal. However, at other temperatures, the shape of the droplet is more variability. Poor, it is not easy to achieve tiling in the droplet container.
  • a plurality of microdroplets are dropped into the microdroplet container 60, and the plurality of microdroplets are stacked on the microdroplet container bottom plate 610, that is, the plurality of microdroplets are in the A plurality of microdroplets are formed on the microdroplet container bottom plate 610.
  • the microdroplet container 60 containing the plurality of microdroplets is subjected to high and low temperature circulation.
  • the plurality of microdroplets are subjected to high and low temperature cycles a plurality of times until the plurality of microdroplets are laid flat on the microdroplet container bottom plate 610 such that a large number of the microdroplets are tiled in the reaction In unit 612, large-scale parallel observation of massive droplets is facilitated. Therefore, in order to more accurately acquire information on the nucleic acid amplification reaction of the plurality of microdroplets, it is necessary to tile the plurality of microdroplets in the microdroplet container 60. By layering the plurality of microdroplets in the microdroplet container 60, a layer is formed, thereby avoiding mutual influence between the plurality of microdroplets, so that the fluorescent signal detecting device 30 takes a photo detection, Get more accurate fluorescence information for quantitative analysis.
  • the step S312 generates the micro-droplet by using the micro-droplet generation method of the step S201, the step S202, and the step S203 in the above embodiment, including: S3121, providing an outlet end a spit gun head, the first liquid is stored in the spit gun head; S3123, the outlet end of the spit gun head is inserted under the liquid surface of the second liquid, and the periodic acceleration is performed; S3125 According to the instantaneous acceleration movement of the outlet end of the ejector head, the first liquid is discharged from the outlet end of the ejector head, and a plurality of micro-droplets are formed inside the second liquid, and stacked and stacked The microdroplet container bottom plate.
  • the speed of the outlet end of the ejector head is the same, in the opposite direction.
  • the motion trajectory of the instantaneously accelerated periodic motion in the step S3123 includes a combination of one or more of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • the step S312 generates the micro-droplet by using the micro-droplet generation method of the step S211, the step S212 and the step S213 in the above embodiment, including: S3122, providing an outlet end a spit gun head, the first liquid is stored in the spit gun head; S3124, the outlet end of the spit gun head is inserted under the liquid surface of the second liquid, and the movement of the speed changes periodically During the first half cycle and the second half cycle of the change in the speed, the velocity of the outlet end of the jetting head is monotonously changed; S3126, according to the periodic change of the outlet end of the jetting head, the first The liquid is discharged from the outlet end of the ejector head, and a plurality of microdroplets are formed under the second liquid level and deposited on the bottom plate of the microdroplet container.
  • step S3124 the outlet end of the ejector head changes in a cosine curve at a velocity below the liquid level of the second liquid.
  • the trajectory of the periodic change of the outlet end of the ejector head in the step S3124 under the second liquid level includes one of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon. Combination of species or multiples.
  • the step of performing high and low temperature circulation by the temperature control device 20 is as follows: first, heating the plurality of micro droplets to a temperature of 90 ° C to 95 ° C, and heating for 5 min to 10 min; The plurality of microdroplets are cooled to 40° C. to 60° C. and annealed for 30 s to 60 s. Finally, the cells are sequentially circulated a plurality of times, and the temperature is lowered to 0° C. to 10° C., and the plurality of microdroplets are stored.
  • the step of performing the high and low temperature cycle by the temperature control device 20 further comprises: first, heating the plurality of microdroplets, heating the temperature to 95 ° C, and heating for 10 min; A plurality of microfluidics were heated to 95 ° C and heated for 10 min to thermally initiate the enzymes in the plurality of microdroplets. Then, after the plurality of micro-droplets complete the thermal start of the enzyme, the plurality of micro-droplets are denatured for 30 s; secondly, after the plurality of micro-liquid denaturation, the temperature is lowered to 55 ° C, and the annealing is extended for 45 s. The plurality of picosols were photographed and subjected to 45 cycles; finally, after 45 cycles, the temperature was lowered to 4 ° C, and the plurality of microfluids were stored for a long time.
  • the nucleic acid amplification reaction solution to be tested generates a plurality of microdroplets through the microdroplet generating device 10 for detection.
  • the plurality of micro-droplets prepared by the micro-droplet generating device 10 are concentratedly collected in the intermediate portion of the micro-droplet container 60 during the downward sedimentation process, and are gathered together, which is disadvantageous for observation. Therefore, in order to more accurately acquire information on the nucleic acid amplification reaction of the plurality of microdroplets, it is necessary to tile the plurality of microdroplets in the microdroplet container.
  • the step S320 performs nucleic acid amplification on the plurality of microdroplets as follows: First, the microdroplet container 60 is placed on the heating substrate 240 of the temperature control device 20. And then heating the plurality of micro-droplets to a temperature of 95 ° C and heating for 10 min; heating the plurality of micro-liquids to 95 ° C, and heating for 10 min, to The enzyme in the microdroplets is hot-started. Secondly, after the plurality of micro-droplets complete the thermal initiation of the enzyme, the plurality of micro-droplets are denatured for 30 s; again, after the plurality of micro-liquid denaturation, the temperature is lowered to 55 ° C, and the annealing is extended for 45 s, and is performed. 45 cycles; finally, after 45 cycles, the temperature was lowered to 4 ° C, and the plurality of microliquids were stored for a long time.
  • the temperature control device 20 uses the flexible circuit board 220 and the heat conduction enhancement layer 250 to uniformly distribute the temperature of the microdroplet container 60, thereby accelerating the thermal conductivity of the semiconductor refrigerator.
  • a temperature sensor 260 disposed on a surface of the thermally conductive enhancement layer 250 is coupled to the second controller 210 to detect the microdroplets
  • the real-time temperature of the container 60 feeds back temperature information to the second controller 210 to effect control of the plurality of micro-droplet heating temperatures. It is possible to switch quickly in a matter of seconds.
  • the temperature control device 20 can realize instantaneous temperature rise and temperature decrease, and the process of temperature rise and temperature decrease is shortened, thereby achieving high and low temperature cycle, shortening the detection time of the digital PCR detector 1 and improving the detection efficiency.

Abstract

一种微液滴容器(60)、微液滴容器制备方法、微液滴平铺方法、微液滴生成试剂盒、温控装置(20)、微液滴生成用油相组合物(699)及其处理方法。

Description

微液滴容器、微液滴容器制备方法、微液滴平铺方法、微液滴生成试剂盒、温控装置、微液滴生成用油相组合物及其处理方法
相关申请
本申请要求2018年01月24日申请的，申请号为201810070377.2，名称为“数字PCR定量检测方法”和2018年07月27日申请的，申请号为201810843257.1，名称为“微液滴容器及其制备方法”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本申请涉及微液滴领域，特别是涉及一种微液滴容器、微液滴容器制备方法、微液滴平铺方法、微液滴生成试剂盒、温控装置、微液滴生成用油相组合物及其处理方法。
背景技术
数字PCR(Digital PCR，dPCR)是一种核酸分子绝对定量技术。相较于qPCR，数字PCR可让你能够直接数出DNA分子的个数，是对起始样品的绝对定量。近年来，数字PCR技术迅速发展，传统的基于微纳制造和微流控制技术的集成微流控制芯片、微孔阵列芯片和液滴微流控制芯片，属于一次性消耗品，进行一次检测需要将其丢弃，防止交叉污染。但是在实际过程中，对微液滴的个数仍然存在限制，且耗材成本较高。传统的微液滴容器的整体液面为弧形，液面呈现凹形液面，并且多个微液滴在向下沉降过程当中，集中集合在微液滴容器的中间部位，聚集在一起，对获取批量微液滴荧光图像造成干扰。在生成多个微液滴的过程中，传统的油相组合物在使用过程中，粘度等物理性质变化比较大，所生成微液滴的体积大小均一性较差。同时，传统的温控装置升降温速率慢，导致完成核酸扩增所需时间长，影响数字PCR的检测效率。
申请内容
有鉴于此，本申请提供一种微液滴容器包括底面、围绕所述底面设置的第一环形侧面以及环形面。所述第一环形侧面与所述底面相连，并包围形成一个具有开口的收纳空间，所述第一环形侧面垂直于所述底面。所述环形面环绕所述开口设置，并与所述第一环形侧面相连，所述环形面与所述底面平行。所述微液滴容器包括底面、围绕所述底面设置的第一环形侧面以及环形面。所述第一环形侧面与所述底面相连，并包围形成一个具有开口的收纳空间，所述第一环形侧面垂直于所述底面。所述环形面环绕所述开口设置，并与所述第一环形侧面相连，所述环形面与所述底面平行。所述收纳空间用以收纳多个微液滴以及油相组合物。通过所述环形面与所述底面平行，在使用时将所述油相组合物加入到所述环形面，此时可以确保所述微液滴容器中的液体表面为水平面。通过设置于所述环形面，可以使得所述微液滴容器的液面呈现出平面状态，避免了所述微液滴容器的整体液面为弧形。因此，通过所述微液滴容器不会影响所述容器底板靠近边沿部位的微液滴的观测，便于拍照成像，提高了所述多个微液滴的检测个数。
有鉴于此，本申请提供一种微液滴容器包括第一容器底板、多边形边框以及容器盖。所述第一容器底板设置有多个多边形凸条。所述多边形边框包围形成一个第一收纳空间，所述多边形边框与所述第一容器底板连接，且所述多个多边形凸条设置于所述第一收纳空 间。所述容器盖设置于所述多边形边框远离所述第一容器底板的表面，且所述容器盖与所述多边形边框可拆卸连接，所述容器盖与所述多边形边框包围形成一个油类收纳槽。所述多边形边框两侧分别与所述第一容器底板和所述容器盖连接，所述容器盖与所述多边形边框可拆卸连接，可以将所述微液滴容器密封。所述容器盖与所述多边形边框包围形成一个油类收纳槽，当通过所述容器盖密封所述微液滴容器时，可以将所述微液滴容器中的多余油类物质挤到所述油类收纳槽中，可以尽量的避免了微液滴上层部分的油类物质对检测过程的影响，避免了油类物质造成的荧光背景。同时，通过将所述微液滴容器中的多余油类物质挤到所述油类收纳槽，所述微液滴容器中与多个微液滴接触的油类物质减少，避免了油类物质不饱和情况下吸收多个微液滴的水分，解决了微液滴水分蒸发的情况。在使用所述微液滴容器放置油性液体时，可能会有空气混入，会产生气泡，所述荧光信号检测装置实时拍测所述多个微液滴的荧光变化图片会受到影响。因此，通过对所述微液滴容器进行密封，当所述微液滴容器以3～5度角进行倾斜时，可以避免所述微液滴容器中的液体流出，还可以将所述微液滴容器中的气泡排出，避免了拍照检测时，气泡造成的对图像的影响。
有鉴于此，本申请提供一种微液滴生成用油相组合物，包括以下组分矿物油以及表面活性剂。所述油相组合物中所述矿物油的体积百分比为88％-98.5％。所述表面活性剂包括含链状烷基的硅氧链非离子型表面活性剂。上述包括矿物油及含长链烷基的硅氧链非离子型表面活性剂的微液滴生成用油相组合物的密度小于1g/ml，能够允许大部分类型的第一液体脱离吐液枪头的出口端形成微液滴后在第二液体中下降。含链状烷基的硅氧链非离子型表面活性剂能够防止众多微液滴之间相互融合。
有鉴于此，本申请提供一种微液滴平铺方法，包括：S311，提供一微液滴容器，所述微液滴容器具有开口，且所述微液滴容器内盛有第二液体；S312，提供第一液体，所述第一液体的密度大于所述第二液体并与所述第二液体不互溶，并将所述第一液体生成多个微液滴层叠堆积于所述微液滴容器底板；S313，对所述多个微液滴进行高低温循环，直至所述多个微液滴平铺于所述微液滴容器底板。对所述多个微液滴进行高低温循环，直至所述多个微液滴平铺于所述微液滴容器底板。通过所述微液滴平铺方法，利用热胀冷缩的原理，进行平铺。随着温度的变化，当温度升高时，样本液滴的粘稠度变低、体积收缩。同时，温度越高粘度越低，样本液滴形状最软，从而使得所述多个微液滴平铺于所述微液滴容器底板，有利于拍照检测。
有鉴于此，本申请提供一种温控装置包括柔性电路板、与所述柔性电路板间隔设置的加热基板以及多个半导体电偶对。所述加热基板包括相对设置的第一表面和第二表面。所述多个半导体电偶对设置于所述柔性电路板与所述第一表面之间，所述多个半导体电偶对相互串联、并联或者混合连接。所述柔性电路板具有配线密度高、重量轻、厚度薄、弯折性好的特点。所述柔性电路板在升降温过程中以自身的变形消除热应力，从而延长了所述温控装置的使用寿命。当所述多个微液滴在不同温度范围内进行核酸扩增时，通过所述柔性电路板、加热基板以及多个半导体电偶对可以实现迅速在几秒时间内进行切换。所述温控装置可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环， 将所述数字PCR检测仪的检测时间缩短了，提高了检测效率。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本申请提供的数字PCR检测仪的整体结构示意图；图2为本申请提供的数字PCR检测仪的微液滴生成装置；图3为本申请一实施例提供的吐液枪头的出口端运动时液滴的受力示意图；图4为本申请一实施例提供的吐液枪头的出口端的速度变化示意图；图5为本申请一实施例提供的吐液枪头的出口端运动时微液滴生成过程示意图；图6为本申请另一实施例提供的吐液枪头的出口端运动时液滴的受力示意图；图7为本申请一实施例提供的液滴随吐液枪头的出口端运动时理想情况下粘滞阻力变化示意图；图8为本申请一实施例提供的吐液枪头的出口端两个运动周期生成一个微液滴的过程示意图；图9为本申请一实施例提供的吐液枪头的出口端一个运动周期生成一个微液滴的过程示意图；图10为本申请一实施例提供的吐液枪头的出口端一个运动周期生成两个微液滴的过程示意图；图11为本申请一实施例提供的吐液枪头摆动时微液滴的生成过程示意图；图12为本申请一实施例提供的第二液体的粘度变化时微液滴的生成过程示意图；图13为本申请一实施例提供的更换吐液枪头时微液滴的生成过程示意图；图14为本申请一实施例提供的吐液枪头的出口端在不同的运动轨迹下微液滴的生成过程示意图；图15为本申请另一实施例提供的吐液枪头的出口端速度变化示意图；图16为本申请一实施例提供的方案一实验结果分布图；图17为本申请一实施例提供的方案一生成的均一性高的微液滴放大图；图18为本申请一实施例提供的方案一生成的均一性低的微液滴放大图；图19为本申请一实施例提供的方案二实验结果分布示意图；图20为本申请一实施例提供的方案二生成的均一性高的微液滴放大图；图21为本申请一实施例提供的方案二生成的均一性低的微液滴放大图；图22为本申请一实施例提供的方案二生成的热稳定性高的微液滴放大图；图23为本申请一实施例提供的方案二生成的热稳定性低的微液滴放大图；图24为本申请一实施例提供的方案三生成的微液滴放大图；图25为本申请一实施例提供的方案四生成的均一性高的微液滴放大图；图26为本申请一实施例提供的方案四生成的热稳定性高的微液滴放大图；图27为本申请温控装置结构示意图；图28为本申请温控装置结构切面结构示意图；图29为本申请温控装置的半导体电偶对电极连接结构示意图；图30为本申请温控装置的瞬态性能测试示意图；图31为本申请温控装置的稳态性能测试示意图；图32为本申请微液滴容器结构示意图；图33为本申请微液滴容器结构平面结构示意图；图34为本申请微液滴容器的反应单元结构示意图；图35为本申请微液滴容器的一种切面结构示意图；图36为本申请微液滴容器的一种切面结构示意图；图37为本申请微液滴容器的一种切面结构示意图；图38为本申请提供的微液滴容器的整体结构示意图；图39为本申请提供的微液滴容器的切面结构示意图；图40为本申请提供的微液滴容器的容器盖的结构示意图； 图41为本申请提供的微液滴容器的多边形容器盖框架的结构示意图；图42为本申请提供的微液滴容器的容器盖的切面结构示意图；图43为本申请提供的微液滴容器的第一容器底板的结构示意图；图44为本申请提供的微液滴容器的多边形边框的多个支撑板的结构示意图；图45为本申请提供的微液滴容器的多边形边框的多个支撑板的局部放大结构示意图；图46为本申请提供的微液滴容器的多边形边框的多个第二支撑杆的结构示意图；图47为本申请提供的微液滴容器的多边形边框的多个第二支撑杆的局部放大结构示意图；图48为本申请提供的微液滴容器的整体结构安装示意图；图49为本申请微液滴平铺方法流程图；图50为本申请微液滴容器底板微液滴堆积示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
为了使本申请的目的、技术方案及优点更加清楚明白，以下通过实施例，并结合附图，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
请参见图1，在一个实施例中，本申请提供一种数字PCR检测仪1，数字PCR检测仪1包括：微液滴生成装置10、温控装置20、荧光信号检测装置30、定量分析装置40以及控制器50。微液滴生成装置10用以将核酸扩增反应液微滴化，形成多个微液滴。温控装置20与微液滴生成装置10通过轨道连接，用以将多个微液滴转移至温控装置20，进行温度循环，实现核酸扩增。荧光信号检测装置30与温控装置20相对设置，用以对核酸扩增后的多个微液滴进行拍照检测。定量分析装置40与荧光信号检测装置30通过数据线连接，用以实现多个微液滴荧光信息的传输，进行定量分析。控制器50分别与微液滴生成装置10、温控装置20、荧光信号检测装置30以及定量分析装置40连接，用以控制微液滴生成装置10、温控装置20、荧光信号检测装置30以及定量分析装置40。
数字PCR检测仪1可以将微液滴生成装置10、温控装置20、荧光信号检测装置30以及定量分析装置40集成化，从而使得操作人员可以实现自动化操作。数字PCR检测仪1具有较高的工作效率。
数字PCR检测仪1在工作时，微液滴生成装置10可以将待测核酸扩增反应液进行微滴化，从而形成多个微液滴。温控装置20可以对多个微液滴进行核酸扩增。荧光信号检测装置30实时拍测多个微液滴的荧光变化图片。通过多个微液滴的荧光变化图片，可以获取多个微液滴的荧光变化曲线。根据荧光变化曲线，可以获取多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始DNA的浓度进行定量分析。其中，Ct值是指每个微液滴的荧光信号达到设定的阈值时所经历的循环数。
温控装置20对多个微液滴进行核酸扩增反应，并通过荧光信号检测装置30采集核酸扩增反应后的多个微液滴的产物信号，如荧光、紫外吸收、浊度等信号。利用多个扩增与 非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定量分析。通过实时监测多个微液滴的荧光变化图片，检测结果具有直接性，可以解决多个微液滴中的假阳性和假阴性的问题。
数字PCR检测仪1将微液滴生成装置10、温控装置20、荧光信号检测装置30以及定量分析装置40集成化，使得操作人员可以实现自动化操作，不进提高了工作效率，还具有反应快速、重复性好、灵敏度高、特异性强和结果清晰的优点。
请参见图2，在一个实施例中，微液滴生成装置10包括吐液枪头110、流体驱动机构120、运动控制机构130以及第一控制器170。吐液枪头110具有出口端及入口端，并用于储存第一液体。微液滴生成装置10可以与微液滴容器配合使用。微液滴容器中储存有第二液体，吐液枪头110的出口端插入第二液体的液面下。
第一液体与第二液体之间互不相溶或具有界面反应。第一液体和第二液体可以为任意不互溶的两种液体，在本申请的一个实施例中，第一液体为水溶液，第二液体为与水不互溶的油性液体，如矿物油(包括正十四烷等)、植物油、硅油和全氟烷烃油等，生成的液滴为水溶液液滴。或者，第一液体为矿物油，如十四烷和正己烷等有机相，第二液体为与矿物油不互溶的全氟烷烃油。第一液体和第二液体可以为不互溶的双水相，在本申请的另一个实施例中，第一液体为水溶液，第二液体为与水不互溶的水性液体，如第一液体为右旋糖酐溶液，第二液体为聚乙二醇(PEG)水溶液，生成的液滴为右旋糖酐溶液液滴。
第一液体和第二液体也可以为具有界面反应的两种液体，在本申请的一个实施例中，第一液体为海藻酸纳水溶液，第二液体为氧化钙水溶液，如质量浓度为1％的氧化钙水溶液，两者存在界面反应，生成的液滴为海藻酸钙凝胶微球。本申请还可以通过更换吐液枪头或吐液枪头内流出第一液体的组分，顺次在开口容器中形成多个不同组分和体积的液滴，既可以用于实现大批量的微体积高通量筛选，也可以实现多步骤的超微量生化反应和检测，具有广阔的应用前景。
流体驱动机构120与吐液枪头110的入口端连接，用于将储存在吐液枪头110内部的第一液体从吐液枪头110的出口端排出。运动控制机构130用于控制吐液枪头110的出口端与第二液体之间产生设定轨迹或设定速度或设定加速度的相对运动，以使排出吐液枪头110的出口端的第一液体克服表面张力及吐液枪头110对其的附着力形成微液滴。第一控制器170分别与流体驱动机构120以及运动控制机构130连接，用以控制流体驱动机构120以及运动控制机构130协调工作。
针对传统的吐液枪头注射/喷射法存在的微液滴生成过程不稳定的问题，提供一种微液滴生成过程稳定的微液滴生成方法。
如图3所示，在本申请一实施例中，在运动控制机构130的带动下，吐液枪头110的出口端112可以在第二液体液面下做包含瞬时加速的运动，加速度大小为a 1。第一液体从吐液枪头110的出口端112排出后形成附着在吐液枪头110的出口端112的液滴195。液滴195在吐液枪头110的出口端112瞬时加速的瞬间脱离吐液枪头110的出口端112形成微液滴。微液滴在脱离吐液枪头110的出口端112之前的所受到的作用力分别为重力G、 第二液体的浮力f 1、第二液体的粘滞阻力f 2以及吐液枪头110的出口端112与液滴195之间的最大附着力f 3。微液滴在脱离吐液枪头110的出口端112之前的质量为m、加速度大小为a 2。根据牛顿第二运动定律，得出
Figure PCTCN2019072969-appb-000001
吐液枪头110的出口端112与液滴195之间附着力的最大值f 3与吐液枪头110的表面自由能、液滴195的表面张力以及吐液枪头110的几何尺寸有关。吐液枪头110的出口端112做瞬时加速运动时，吐液枪头110的出口端112对液滴195附着力的方向与加速度的方向相同。将附着在吐液枪头110的出口端112的液滴195简化为球状。由斯托克斯(Stokes)公式可知，液滴195在第二液体中运动时所受到的粘滞阻力f 2＝6πηrv，其中η为第二液体的粘滞系数，r为液滴195的半径，v为液滴195的运动速度。在吐液枪头110的出口端112做瞬时加速之前液滴195的速度为零，因此液滴195在吐液枪头110的出口端112瞬时加速的瞬间在第二液体中受到的粘滞阻力f 2为零或极小。在微液滴生成的过程中，一般液滴195的直径范围在皮升至微升的数量级，且液滴195的重力G和第二液体的浮力f 1方向相反，因此液滴195的重力G与第二液体的浮力f 1的矢量和约为零。由于粘滞阻力f 2为零或极小，以及重力G与浮力f 1的矢量和约为零，所以
Figure PCTCN2019072969-appb-000002
由牛顿第二运动定律可知，吐液枪头110的出口端112做瞬时加速运动时，液滴195在第二液体中能达到的最大加速度为a 2≈f 3/m，其中m为液滴195的质量。当液滴195的加速度a 2小于吐液枪头110的出口端112的加速度a 1时，液滴195从吐液枪头110的出口端112掉落形成微液滴。因此，液滴195脱离吐液枪头110的出口端112(即生成一个微液滴)的条件近似为：a 2≈(f 3/m)＜a 1。
运动控制机构130能够精确控制吐液枪头110的出口端112瞬时加速度的大小。因此，通过控制吐液枪头110的出口端112每次瞬时加速度的值均较大，吐液枪头110的出口端112做瞬时加速运动能够有效的生成液滴195。
基于此，本申请还提供一种微液滴生成方法，包括以下步骤：
S201，提供具有出口端112的吐液枪头110，吐液枪头110内储存有第一液体；提供储存有第二液体的微液滴容器，微液滴容器具有开口，其中第一液体与第二液体为任意互不相溶的两种液体或具有界面反应的两种液体；
S202，吐液枪头110的出口端112由微液滴容器的开口插入第二液体的液面下；
S203，吐液枪头110的出口端112在第二液体液面下做包含瞬时加速的运动，同时第一液体由吐液枪头110的出口端112排出，排出吐液枪头110的出口端112的第一液体形成附着在吐液枪头110的出口端112的液滴195，液滴195在吐液枪头110的出口端112的瞬时加速运动过程中脱离吐液枪头110的出口端112在第二液体液面下形成微液滴。
上述微液滴生成方法中，由于所述吐液枪头110的出口端112瞬时加速时加速度数值较大，附着在所述吐液枪头110的出口端112的液滴195与所述吐液枪头110的出口端112之间的附着力不足以带动液滴195与所述吐液枪头110的出口端112同步加速，从而使得附着在所述吐液枪头110的出口端112的液滴195脱离所述吐液枪头110的出口端112在第二液体液面下形成微液滴。本申请所提供的微液滴生成方法，所述吐液枪头110的出口 端112在第二液体的液面下做瞬时加速运动时产生微液滴，减小了所述吐液枪头110的出口端112运动时对第二液体造成的扰动，保证了微液滴生成过程的稳定性。
可选的，在步骤S203中，第一液体由吐液枪头110的出口端112排出的方式可以为连续排出或非连续排出。具体的排出方式，可根据实际工况进行相应的设计。在本实施例中，在步骤S203中，第一液体由吐液枪头110的出口端112连续排出，以充分利用吐液枪头110的出口端112的每一次瞬时加速生成微液滴。在一个实施例中，在步骤S203中，第一液体由吐液枪头110的出口端112以恒定的流速排出，意即在相等的时间间隔内，排出吐液枪头110的出口端112的第一液体体积总是相等的。第一液体由吐液枪头110的出口端112以恒定的流速排出，有利于通过控制吐液枪头110的出口端112的运动实现微液滴生成的控制。
在本申请一实施例中，步骤S203中，吐液枪头110的出口端112在第二液体液面下做包含瞬时加速的周期运动。吐液枪头110的出口端112在第二液体液面下做周期运动，意即吐液枪头110的出口端112的位移、速度及加速度均呈现出周期性的变化。吐液枪头110的出口端112做包含瞬时加速运动的周期运动，配合第一液体由吐液枪头110的出口端112以恒定的流速排出，实现了微液滴的等时间间隔生成。或者第一液体排出吐液枪头110的出口端112的流速是变化的，但在吐液枪头110的出口端112的一个运动周期内，第一液体排出吐液枪头110的出口端112的体积保持相同。以此保证每次吐液枪头110的出口端112瞬时加速前液滴195的体积是相同的，以生成体积大小一致的微液滴。
在不更换吐液枪头110及第一液体的情况下，吐液枪头110的表面自由能、吐液枪头110的几何尺寸及液滴195的表面张力作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是确定的。因此，在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。在流体驱动机构120的带动下，第一液体能够实现以均匀的流速连续排出吐液枪头110的出口端112。运动控制机构130能够精确控制吐液枪头110的出口端112做瞬时加速度a 1运动的时刻及瞬时加速度a 1的大小。流体驱动机构120与运动控制机构130相互配合能够容易的实现当液滴195的体积达到固定值的瞬间，驱动吐液枪头110的出口端112产生加速度为a 1的瞬时加速度，以生成体积大小一致的微液滴。如果流体驱动机构120控制第一液体均匀、连续的排出吐液枪头110的出口端112，只需运动控制机构130驱动吐液枪头110的出口端112产生等时间间隔的瞬时加速运动，即可生成体积大小一致的微液滴。
当使用多个吐液枪头110同时或者顺次生成微液滴时，吐液枪头110的表面自由能及吐液枪头110的几何尺寸作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是变化的。但批量加工能够控制吐液枪头110的表面自由能及吐液枪头110的几何尺寸在一定的区间内变化。液滴195的表面张力作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的另一个因素也只是在很小的范围内变化。因此，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3只在很小的区间内波动。流体驱动机构120能够驱动第一液体以均匀的流速连续排出吐液枪头110的出口端112。运动控制机 构130能够精确控制吐液枪头110的出口端112做瞬时加速度a 1运动的时刻及瞬时加速度a 1的大小。流体驱动机构120与运动控制机构130相互配合能够容易的实现当液滴195的体积达到固定值的瞬间，驱动吐液枪头110的出口端112产生加速度为a 1的瞬时加速度，以生成体积大小一致的微液滴。如果流体驱动机构120控制第一液体均匀、连续的排出吐液枪头110的出口端112，只需运动控制机构130驱动吐液枪头110的出口端112产生等时间间隔的瞬时加速运动，即可生成体积大小一致的微液滴。
流体驱动机构120在将第一液体匀速排出吐液枪头110的出口端112的同时，配合运动控制机构130在液滴195的体积达到设定值的瞬间做加速度值较大的瞬时加速运动。本申请提供的微液滴生成方法不仅保证了使用同一根吐液枪头110生成体积大小均一的液滴195，同时能够保证多根吐液枪头110同时或者顺次生成的微液滴体积大小的均一性。本实施例提供的微液滴生成方法在保证微液滴体积大小均一性的同时，可通过多根吐液枪头110同时生成微液滴提高微液滴的生成效率。
进一步的，在运动控制机构130的控制下，吐液枪头110的出口端112在一个周期性运动内包括多次瞬时加速运动，多次瞬时加速运动的加速度大小相同，且多次瞬时加速运动的时刻均分吐液枪头110的出口端112的一个运动周期。吐液枪头110的出口端112在一个周期性运动内包括多次瞬时加速运动有助于在吐液枪头110的出口端112在一个运动周期内生成多个微液滴。可选的，步骤S203中，吐液枪头110的出口端112在第二液体液面下的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。作为一个可实现的方式，当吐液枪头110的出口端112在一个周期性运动内包括两次瞬时加速运动时，吐液枪头110的运动轨迹为直线或者圆弧。当吐液枪头110的出口端112在一个周期性运动内包括两次以上的瞬时加速运动时，吐液枪头110的出口端112在第二液体内做轨迹为正多边形，包括正三角形、正方形、正五边形、正六边形等。
作为一种可实现的方式，在步骤S203中，吐液枪头110的出口端112在第二液体液面下的周期运动过程中，吐液枪头110的出口端112的速度大小呈矩形波变化。吐液枪头110的出口端112的速度大小呈矩形波变化，加速阶段结束以后即进入匀速阶段，有利于运动控制机构130实现对吐液枪头110的出口端112的运动状态的精确控制。可选的，表示吐液枪头110的出口端112运动速度大小变化的矩形波的高位时间和低位时间可以是相等的也可以是相异的。进一步，在步骤S203中，吐液枪头110的出口端112在第二液体液面下的周期运动过程中，吐液枪头110的出口端112的速度大小呈方波变化。表示吐液枪头110的出口端112运动速度大小变化的矩形波的高位时间和低位时间是相等的。表示吐液枪头110的出口端112运动速度大小变化的矩形波处于低位时，吐液枪头110的出口端112的速度为零或具有相对于高位时反方向的速度。如图4所示，更进一步的，所述吐液枪头110的出口端112周期运动的前半周期与后半周期内，所述吐液枪头110的出口端112的速度大小相同，方向相反。在吐液枪头110的出口端112的一个运动周期内包含两次方向相反的瞬时加速运动。
在本实施例中，吐液枪头110的出口端112在第二液体液面下的运动轨迹是直线段， 吐液枪头110的出口端112从直线段的一个端点做瞬时加速运动，从直线段的另一个端点做反方向的瞬时加速运动。两次瞬时加速运动的的加速度大小均为a 1。在其他的实施例中，吐液枪头110的出口端112在第二液体液面下的运动轨迹是圆弧段或者多边形。进一步，步骤S203中，吐液枪头110的出口端112在第二液体液面下周期运动的频率介于0.1赫兹至200赫兹之间，在工程上容易实现。
如图4及图5所示，在本申请一个具体的实施例中，流体驱动机构120控制第一液体以恒定的流速排出吐液枪头110的出口端112。运动控制机构130控制吐液枪头110的输出端做运动轨迹为直线、速度呈方波变化的周期运动。当吐液枪头110的出口端112的速度方向发生改变时，吐液枪头110的出口端112的瞬时加速度达到最大值。附着在吐液枪头110的出口端112的液滴195也在吐液枪头110的出口端112的瞬时加速度达到最大值时脱离吐液枪头110的出口端112而形成微液滴199。由于第一液体是以恒定流速排出吐液枪头110的出口端112，当液滴195从吐液枪头110的出口端112脱落时，新的液滴195进入生成状态。当吐液枪头110的出口端112再次反向加速时，新生成的液滴195也从吐液枪头110的出口端112掉落形成新的微液滴199。
本实施例中，吐液枪头110的出口端112的一个运动周期内可生成两个微液滴199，且方波在工程上较易实现。在其他的实施例中，吐液枪头110的出口端112的一个运动周期内生成一个微液滴199。可选的，在实施例中吐液枪头110的出口端112在第二液体699中沿任意方向做轨迹为直线的方波运动，包括：在与吐液枪头110的延伸方向垂直的平面内做轨迹为直线的方波运动、在与吐液枪头110的延伸方向成任意角度的平面内做轨迹为直线的方波运动、沿吐液枪头110的延伸方向做轨迹为直线的方波运动等。在本申请的其他实施例中，吐液枪头110的出口端112的运动轨迹为圆弧段或多边形时，吐液枪头110的出口端112在第二液体699中沿任意方向做轨迹为直线的方波运动，包括：在与吐液枪头110的延伸方向垂直的平面内做轨迹为直线的方波运动、在与吐液枪头110的延伸方向成任意角度的平面内做轨迹为直线的方波运动、沿吐液枪头110的延伸方向做轨迹为直线的方波运动等。
在本申请一实施例中，在运动控制机构130的带动下，吐液枪头110的出口端112在第二液体液面下做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，吐液枪头110的出口端112的速度大小均单调变化。单调变化指，在速度大小变化的前半周期或后半周期内，吐液枪头110的出口端112的在后时刻的速度值总是大于等于或者小于等于在前时刻的速度值。例如，在速度大小变化的前半周期内，吐液枪头110的出口端112的速度大小持续增加或部分段持续增加而部分段不变。相应的，在速度大小变化的后半周期内，吐液枪头110的出口端112的速度大小持续减小或部分段持续减小而部分段不变。第一液体从吐液枪头110的出口端112排出后形成附着在吐液枪头110的出口端112的液滴195。液滴195在吐液枪头110的出口端112运动速度达到一定大小时脱离吐液枪头110的出口端112形成微液滴199。如图6所示，微液滴199在脱离吐液枪头110的出口端112之前的所受到的作用力分别为重力G、第二液体699的浮力f 1、第二液体699 的粘滞阻力f 2以及吐液枪头110的出口端112与液滴195之间的最大附着力f 3。微液滴199在脱离吐液枪头110的出口端112之前的质量为m、速度为v、加速度为a 2。液滴195在第二液体699的运动过程中受粘滞力f 2、重力G、浮力f 1及附着力f 3的共同作用，即
Figure PCTCN2019072969-appb-000003
吐液枪头110的出口端112与液滴195之间附着力的最大值f 3与吐液枪头110的表面自由能、液滴195的表面张力以及吐液枪头110的几何尺寸有关。将附着在吐液枪头110的出口端112的液滴195简化为球状。由斯托克斯(Stokes)公式可知，液滴195在第二液体699中运动时所受到的粘滞阻力f 2＝6πηrv，其中η为第二液体699的粘滞系数，r为液滴195的半径，v为液滴195的运动速度。在微液滴199生成的过程中，一般液滴195的直径范围在皮升至微升的数量级，而第二液体699的粘滞系数一般比较大。故，一般有
Figure PCTCN2019072969-appb-000004
因此，吐液枪头110的出口端112在第二液体699液面下做变速周期运动过程中，液滴195脱离吐液枪头110的出口端112(即生成一个微液滴199)的条件近似为
Figure PCTCN2019072969-appb-000005
基于此，本申请提供一种微液滴生成方法，包括以下步骤：
S211，提供具有出口端112的吐液枪头110，吐液枪头110内储存有第一液体；提供储存有第二液体699的微液滴容器60，微液滴容器60具有开口；第一液体与第二液体699为任意互不相溶的两种液体或具有界面反应的两种液体；
S212，吐液枪头110的出口端112由微液滴容器60的开口插入第二液体699的液面下；
S213，吐液枪头110的出口端112在第二液体699液面下做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，吐液枪头110的出口端112的速度大小均单调变化，同时第一液体由吐液枪头110的出口端112匀速排出，排出吐液枪头110的出口端112的第一液体形成附着在吐液枪头110的出口端112的液滴195，液滴195在吐液枪头110的出口端112的运动过程中脱离吐液枪头110的出口端112在第二液体699液面下形成微液滴199。
上述微液滴生成方法，吐液枪头110的出口端112在第二液体699液面下做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，吐液枪头110的出口端112的速度大小均单调变化。运动过程中，第二液体699对液滴195的粘滞力f 2随着吐液枪头110的出口端112速度大小的周期变化也呈现出周期变化。当吐液枪头110的出口端112与液滴195之间的最大附着力f 3小于第二液体699对液滴195的粘滞力f 2时，液滴195不能与吐液枪头110的出口端112同步运动，进而附着在吐液枪头110的出口端112的液滴195脱离吐液枪头110的出口端112在第二液体699液面下形成微液滴199。本申请所 提供的微液滴生成方法，吐液枪头110的出口端112在第二液体699的液面下做变速周期运动以产生微液滴199，减小了吐液枪头110的出口端112运动时对第二液体699造成的扰动，保证了微液滴199生成过程的稳定性。
在本实施例中，在步骤S213中，第一液体由吐液枪头110的出口端112连续排出。进一步，在步骤S213中，第一液体由吐液枪头110的出口端112以恒定的流速排出，意即在相等的时间间隔内，排出吐液枪头110的出口端112的第一液体体积总是相等的。第一液体由吐液枪头110的出口端112以恒定的流速排出，有利于通过控制吐液枪头110的出口端112的周期性运动实现生成体积大小一致的微液滴199。
影响液滴195在第二液体699中运动时所受到的粘滞阻力f 2的因素中，液滴195的运动速度v比较容易控制。在脱离吐液枪头110的出口端112而形成微液滴199之前，液滴195与吐液枪头110的出口端112保持同步运动。因此，液滴195的运动速度v可以通过控制吐液枪头110的出口端112的运动速度实现精确控制。控制第一液体以均匀的流速排出吐液枪头110的出口端112，液滴195半径的大小r在固定的时间间隔内也呈现出周期性的变化。影响液滴195在第二液体699中运动时所受到的粘滞阻力f 2的因素中，第二液体699的粘滞系数η会在使用过程中在一定范围内变化，但第二液体699的粘滞系数η的变化范围很小。
在不更换吐液枪头110及第一液体的情况下，吐液枪头110的表面自由能、吐液枪头110的几何尺寸及液滴195的表面张力作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是确定的。因此，在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。当使用多个吐液枪头110同时或者顺次生成微液滴199时，吐液枪头110的表面自由能及吐液枪头110的几何尺寸作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是变化的。但批量加工能够控制吐液枪头110的表面自由能及吐液枪头110的几何尺寸在一定的区间内变化。液滴195的表面张力作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的另一个因素也只是在很小的范围内变化。吐液枪头110的出口端112与液滴195之间附着力的最大值f 3只在很小的区间内波动。
因此，只需控制液滴195在第二液体699中运动时所受到的粘滞阻力f 2大于吐液枪头110的出口端112与液滴195之间附着力的最大值f 3的区间值即可。由于在同一批次生成微液滴199的过程中，液滴195半径的大小r应是固定的。一旦实验参数确定，液滴195半径的大小r也就随之确定。吐液枪头110的出口端112在第二液体699液面下的运动速度是变化的。当吐液枪头110的出口端112在第二液体699液面下的运动速度满足v＞f 3/6πηr时，液滴195从吐液枪头110的出口端112脱离形成微液滴199。
吐液枪头110的出口端112在第二液体699液面下做速度大小周期变化的运动。控制第一液体以均匀的流速从吐液枪头110的出口端112排出，附着在吐液枪头110的出口端112的液滴195体积也是均匀增大的。将第一个微液滴199从吐液枪头110的出口端112掉落时，微液滴199的半径称为临界半径，微液滴199的速度成为临界速度。调整吐液枪 头110的出口端112的运动周期及第一液体排出吐液枪头110的出口端112的流速，以使经过相同的时间间隔(吐液枪头110的出口端112运动周期的倍数)后，附着在吐液枪头110的出口端112的液滴195同时达到临界半径及临界速度，新的微液滴199形成。由于第一液体是以均匀的流速排出吐液枪头110的出口端112，所生成的微液滴199的体积大小相同。
作为一种可实现的形式，在步骤S213中，在一个速度大小变化周期内，吐液枪头110的出口端112的速度大小以中间时刻点为中点呈中心对称。进一步，在步骤S213中，吐液枪头110的出口端112在第二液体699液面下的加速度、速度及运动轨迹均呈周期性变化。更进一步，在步骤S213中，吐液枪头110的出口端112在第二液体699液面下的速度大小呈余弦曲线变化。
可选的，在步骤S213中，吐液枪头110的出口端112在第二液体699液面下的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。在步骤S213中，吐液枪头110的出口端112在第二液体699液面下周期运动的频率介于0.1赫兹与200赫兹之间，在工程上容易实现。
以吐液枪头110的出口端112在第二液体699液面下做轨迹为圆弧、速度呈余弦变化的周期运动为例，此时吐液枪头110的出口端112实际上做摆动运动，运动位移可以用正弦曲线表示，如图7中曲线a所示。在流体控制机构的驱动下，第一液体以均匀的流速从吐液枪头110的出口端112排出。假设液滴195不脱离吐液枪头110的出口端112。通过计算，液滴195在第二液体699中运动时所受到的粘滞阻力f 粘随时间变化如图7中曲线b所示。第一液体以均匀的流速从吐液枪头110的出口端112排出的初始阶段，随着液滴195体积的增大，液滴195的半径r也明显增大。随着液滴195半径r的不断增大，液滴195体积的匀速增大只能引起液滴195半径r的缓慢增大。因此，吐液枪头110的出口端112的前几个摆动周期内，液滴195在第二液体699中运动时所受到的粘滞阻力f 2的最大值迅速增加，而后逐渐趋于缓慢增加。如图7所示，液滴195在第二液体699中运动时所受到的粘滞阻力f 2也呈现出与吐液枪头110的出口端112的周期运动相似的周期性，即液滴195在第二液体699中运动时所受到的粘滞阻力f 2随吐液枪头110的出口端112的速度变化而变化。在实际工况中，当液滴195在第二液体699中运动时所受到的粘滞阻力f 2增大并大于吐液枪头110的出口端112与液滴195之间附着力的最大值f 3时，液滴195从吐液枪头110的出口端112脱落形成微液滴199。
在本申请一实施例中，如图8所示，控制吐液枪头110的出口端112做轨迹为圆弧、位移呈正弦变化的摆动。在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。随着附着在吐液枪头110的出口端112的液滴195半径r不断增大，液滴195在第二液体699中运动时所受到的粘滞阻力f 2也不断增大。液滴195在第二液体699中运动时所受到的粘滞阻力f 2大于吐液枪头110的出口端112与液滴195之间附着力的最大值f 3的瞬间，液滴195从吐液枪头110的出口 端112脱落形成微液滴199，图8中为液滴Ⅰ。进入下一轮微液滴199的生成循环中。
在本实施例中，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3＝1.8×10 -4N，吐液枪头110的出口端112的摆动频率是50赫兹。在吐液枪头110的出口端112做位移呈正弦变化的摆动运动的第二个周期末尾生成第一个微液滴199，图8中为液滴I。在生成第二个微液滴199的初始阶段，虽然吐液枪头110的出口端112的运动速度有所减小，但由于附着在吐液枪头110的出口端112的液滴195半径r增加较快，液滴195在第二液体699中运动时所受到的粘滞阻力f 2并没有立刻下降反而呈现出小范围的增加。此后，液滴195半径r缓慢增加，液滴195在第二液体699中运动时所受到的粘滞阻力f 2主要随吐液枪头110的出口端112的运动速度变化而变化。
当控制第一液体以均匀流速排出吐液枪头110的出口端112时，吐液枪头110的出口端112在生成上一个微液滴199后的两个运动周期的时刻又生成与上一个微液滴199等体积的新的液滴195，图8中为液滴II。且此时吐液枪头110的出口端112的运动速度也与两个运动周期之前相同。与上一个微液滴199等体积的新的液滴195从吐液枪头110的出口端112脱落。第一液体的匀速排出及吐液枪头110的出口端112做位移呈正弦变化的摆动运动共同保证了生成微液滴199的体积大小均一性。
在本申请一实施例中，如图9所示，控制吐液枪头110的出口端112做轨迹为圆弧、位移呈正弦变化的摆动。在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。随着附着在吐液枪头110的出口端112的液滴195半径r不断增大，液滴195在第二液体699中运动时所受到的粘滞阻力f 2也不断增大。液滴195在第二液体699中运动时所受到的粘滞阻力f 2大于吐液枪头110的出口端112与液滴195之间附着力的最大值f 3的瞬间，液滴195从吐液枪头110的出口端112脱落形成微液滴199。进入下一轮微液滴199的生成循环中。
在本实施例中，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3＝1.5×10 -4N，吐液枪头110的出口端112的摆动频率是50赫兹。在吐液枪头110的出口端112做位移呈正弦变化的摆动运动的第一个周期末尾生成第一个微液滴199，图9中为液滴I。在生成第二个微液滴199的初始阶段，虽然吐液枪头110的出口端112的运动速度有所减小，但由于附着在吐液枪头110的出口端112的液滴195半径r增加较快，液滴195在第二液体699中运动时所受到的粘滞阻力f 2并没有立刻下降反而呈现出小范围的增加。此后，液滴195半径r缓慢增加，液滴195在第二液体699中运动时所受到的粘滞阻力f 2主要随吐液枪头110的出口端112的运动速度变化而变化。
当控制第一液体以均匀流速排出吐液枪头110的出口端112时，吐液枪头110的出口端112在生成上一个微液滴199后的一个运动周期的时刻又生成与上一个微液滴199等体积的新的液滴195，且此时吐液枪头110的出口端112的运动速度也与一个运动周期之前相同。与上一个微液滴199等体积的新的液滴195从吐液枪头110的出口端112脱落，图9中为液滴II。如此循环，生成液滴III、液滴IV等。第一液体的匀速排出及吐液枪头110 的出口端112做位移呈正弦变化的摆动运动共同保证了生成微液滴199的体积大小均一性。
在本申请一实施例中，如图10及图11所示，控制吐液枪头110的出口端112做轨迹为圆弧、位移呈正弦变化的摆动。在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。随着附着在吐液枪头110的出口端112的液滴195半径r不断增大，液滴195在第二液体699中运动时所受到的粘滞阻力f 2也不断增大。液滴195在第二液体699中运动时所受到的粘滞阻力f 2大于吐液枪头110的出口端112与液滴195之间附着力的最大值f 3的瞬间，液滴195从吐液枪头110的出口端112脱落形成微液滴199，图10中为液滴I。进入下一轮微液滴199的生成循环中。
在本实施例中，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3＝1.0×10 -4N，吐液枪头110的出口端112的摆动频率是50赫兹。在吐液枪头110的出口端112做位移呈正弦变化的摆动运动的前半周期的加速阶段生成第一个微液滴199，图10中为液滴I。在生成第二个微液滴199的初始阶段，当吐液枪头110的出口端112的运动速度有所减小，但由于附着在吐液枪头110的出口端112的液滴195半径r增加较快，液滴195在第二液体699中运动时所受到的粘滞阻力f 2并没有立刻下降反而呈现出小范围的增加。此后，液滴195半径r缓慢增加，液滴195在第二液体699中运动时所受到的粘滞阻力f 2主要随吐液枪头110的出口端112的运动速度变化而变化。
控制第一液体以均匀流速排出吐液枪头110的出口端112。吐液枪头110的出口端112在做位移呈正弦变化的摆动运动的后半周期加速阶段生成第二个微液滴199，图10中为液滴II。此后进入稳定生成微液滴199的阶段。吐液枪头110的出口端112生成第二个微液滴199后的半个运动周期的时刻又生成与第二个微液滴199等体积的新的液滴195，且此时吐液枪头110的出口端112的运动速度也与半个运动周期之前相同。与第二个微液滴199等体积的新的液滴195从吐液枪头110的出口端112脱落，如此循环，生成图10中所示的液滴III、液滴IV、液滴V等。第一液体的匀速排出及吐液枪头110的出口端112做位移呈正弦变化的摆动运动共同保证了生成微液滴199的体积大小均一性。
由上述可知，附着在吐液枪头110的出口端112的液滴195脱离吐液枪头110的出口端112(即生成一个微液滴199)的条件近似为：
Figure PCTCN2019072969-appb-000006
在控制第一液体以均匀流速排出吐液枪头110的出口端112的情况下，所生成的微液滴199的体积大小均一的条件是：微液滴199等时间间隔的从吐液枪头110的出口端112脱落。
影响吐液枪头110的出口端112与液滴195之间附着力的最大值f 3的因素包括：吐液枪头110的表面自由能、几何尺寸及第一液体的表面张力。在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。影响液滴195在第二液体699中运动时所受到的粘滞阻力f 2的因素包括：第二液体699的粘滞系数η、液滴195的半径r及液滴195的运动速度v。第一液体匀速排出吐液枪头 110的出口端112时，液滴195的半径r由微液滴199生成的间隔时间决定。液滴195在脱离吐液枪头110的出口端112之前与吐液枪头110的出口端112同步运动，可通过运动控制机构130实现精确控制吐液枪头110的出口端112的运动速度。第二液体699的粘滞系数η在液滴195的生成过程中会在一定范围内变化，但第二液体699的粘滞系数η的变化范围很小。如图12所示，曲线a表示吐液枪头110的出口端112的位移变化，曲线b和曲线c为当第二液体699的粘滞系数η在很小的范围内变化时微液滴199的生成过程曲线。当第二液体699的粘滞系数η在很小的范围内变化时，只会在很小范围内改变微液滴199的生成时刻。而不会改变微液滴199的生成时间间隔。如图12所示，曲线b和曲线c所表示的微液滴199的生成时间间隔均为半个周期t/2，保证了所生成微液滴199的体积大小均一性。
如图13所示，在更换吐液枪头110时，或温度变化等引起第一液体的表面张力发生变化时，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3难以精确控制，因此如果生成的微液滴199体积对f 3在一定范围内变化不敏感，那么对生成均一尺寸的微液滴199具有重要意义。图13中，曲线a表示吐液枪头110的出口端112的位移变化，曲线b和曲线c为当更换吐液枪头110的情况下微液滴199的生成过程曲线。更换吐液枪头110后，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3在一定范围内波动会导致液滴195脱落时吐液枪头110的出口端112对应不同的速度。但是当微液滴199的生成达到稳定状态后，液滴195脱落时吐液枪头110的出口端112的速度在每个摆动周期内都是固定的，如图13所示，曲线b和曲线c所表示的微液滴199的生成时间间隔均为半个周期t/2。因此能够保证微液滴199生成的间隔时间是固定的。当第一液体排出吐液枪头110的出口端112的流速固定时，生成的微液滴199的体积是均一的。同时调整第一液体排出吐液枪头110的出口端112的流速及吐液枪头110的出口端112在第二液体699内的摆动频率，即可同时控制均一体积微液滴199的体积大小及生成速率。
上述实施例中吐液枪头110的出口端112做位移呈正弦变化的周期运动时，对附着力的最大值f 3及粘滞阻力f 2的变化具有一定的容忍性，即附着力的最大值f 3或粘滞阻力f 2在一定范围内变化时，仍然能够生成体积大小均一的微液滴199。当吐液枪头110的出口端112做位移呈正弦变化的周期运动时，保证生成体积大小均一的微液滴199的前提下，能够容忍的附着力的最大值f 3的变化范围称为平台期。平台期的存在对于吐液枪头110的加工及微液滴199生成温度的控制具有重要的意义。平台期的存在允许在一定程度内降低吐液枪头110的加工精度要求，即使同批加工的吐液枪头110之间的表面自由能之间存在差异，也能够生成体积大小均一的微液滴199。同理，平台期的存在也允许在一定程度内降低微液滴199生成过程的温度控制要求。
平台期的存在允许在一定程度内降低吐液枪头110的加工精度要求或微液滴199生成过程的温度控制要求，进一步降低了微液滴199生成过程中的耗材成本及控制成本。上述实施例中吐液枪头110的出口端112的每个运动周期内生成两个微液滴199，容易理解的是，只要吐液枪头110的出口端112做位移呈正弦变化的周期运动，当吐液枪头110的出 口端112的每一个运动周期内生成一个微液滴199或者每两个运动周期内生成一个微液滴199时，仍然对附着力的最大值f 3及粘滞阻力f 2的变化具有一定的容忍性，也都存在平台期。
由于微液滴199的生成几乎不受微液滴199的重力及惯性力的影响。因此生成微液滴199时，吐液枪头110的出口端112在第二液体699内可沿任意方向做位移呈正弦变化的周期运动。吐液枪头110的出口端112的运动轨迹是弧线、直线或者其他形状的轨迹。
如图14中的(1)所示，在本申请一实施例中，吐液枪头110倾斜插入第二液体699内，吐液枪头110的出口端112在第二液体699液面下摆动生成微液滴199。作为一种可实现的方式，如图14中的(2)所示，吐液枪头110的出口端112在第二液体699内做轨迹为水平直线、位移呈正弦变化的周期运动以生成微液滴199。作为另一种可实现的方式，如图14中的(3)所示，吐液枪头110的出口端112在第二液体699做轨迹为竖直直线、位移呈正弦变化的周期运动以生成微液滴199。
如图15所示，在本申请另一实施例中，在步骤S213中，速度大小变化的一个周期内，吐液枪头110的出口端112在前半周期与后半周期均是匀变速运动。进一步，在步骤S213中，吐液枪头110的出口端112在前半周期与后半周期的加速度大小相等。控制第一液体以均匀流速排出吐液枪头110的出口端112。随着第一液体的连续排出，附着在吐液枪头110的出口端112的液滴195在运动过程中受到的粘滞阻力f 2也不断增大。当粘滞阻力f 2大于液滴195与吐液枪头110之间附着力的最大值f 3时，液滴195从吐液枪头110脱离形成微液滴199。随后进入下一个微液滴199的生成过程中。控制吐液枪头110的出口端112的运动频率及运动速度与第一液体的流速相适配，以保证生成微液滴199的体积均一性。
传统的油相组合物在使用过程中，粘度等物理性质变化比较大，所生成微液滴的体积大小均一性较差。针对吐液枪头注射/喷射法中使用传统的油相组合物，所生成的微液滴体积大小均一性较差的问题，提供一种能够保证微液滴体积大小均一性的油相组合物及其处理方法。本申请提供一种微液滴生成用油相组合物，即上述的第二液体699，包括以下组分：
矿物油，油相组合物中矿物油的体积百分比为88％-98.5％；
表面活性剂，表面活性剂包括含链状烷基的硅氧链非离子型表面活性剂。
上述包括矿物油及含链状烷基的硅氧链非离子型表面活性剂的微液滴生成用油相组合物的密度小于1g/ml，能够允许大部分类型的第一液体脱离吐液枪头110的出口端112形成微液滴199后在第二液体699中下降。含链状烷基的硅氧链非离子型表面活性剂能够防止众多微液滴199之间相互融合。
在本申请一实施例中，油相组合物中含链状烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-12％。进一步，含链状烷基的硅氧链非离子型表面活性剂包括
Figure PCTCN2019072969-appb-000007
和
Figure PCTCN2019072969-appb-000008
中的一种或两种。在一实施例中，表面活性剂还包括链状烷烃酯，油相组合物中链状烷烃酯与油相组合物的质量体积比为0.015g/mL-0.05g/mL。进一步的，链状 烷烃酯包括二聚羟基硬脂酸酯(PEG-30)、硬脂酸甘油、聚乙二醇(30)二聚羟基硬脂酸酯(P135)等中的一种或多种。具体的，链状烷烃酯为聚乙二醇(30)二聚羟基硬脂酸酯(P135)。在本实施例中，油相组合物中含链状烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-5.0％。在上述方案中，含链状烷基的硅氧链非离子型表面活性剂为
Figure PCTCN2019072969-appb-000009
在矿物油中，气体有一定量的溶解度。而且是和气体及矿物油的温度有关。例如，在室温下，空气会溶解在矿物油中，并且是不可见的。矿物油中溶解气体会影响矿物油的粘度、体积模量、热传导、边界润滑等物理性能，形成发泡和空泡现象。如果矿物油中的气体含量超过饱和，可见的气泡就会形成并悬浮在矿物油中，矿物油就会变得很模糊。这被称为夹带气体。气泡慢慢地上升到矿物油表面。在油膜中，气体的气泡会导致油膜的连续性，从而降低油膜的防止其他相接触的能力。例如在PCR反应过程中，温度会升高到95℃。矿物油中的气体溶解度降低，矿物油中的气体含量高于饱和值，这时就会有气泡产生。气泡会上升到矿物油表面，最终破裂。但在这个过程中气泡会影响荧光信号的采集。此外，若气泡产生时与微液滴199相互作用，会影响微液滴199的稳定性，促进微液滴199之间的融合反应。
本申请还提供一种油相组合物的处理方法，用于处理上述方案任一项所述的油相组合物。油相组合物的处理方法包括加热油相组合物，同时将油相组合物置于负压及超声波振动的环境中。在负压环境中，使溶解在油相组合物中的空气及其他气体溢出，最大程度的降低空气及其他气体在油相组合物中的溶解量。超声波能够促进溶解在油相组合物中的气体的溢出。当微液滴199内包括水相时，生成微液滴199后，在后续对微液滴199进行操作时，可能会有水分溶解在油相组合物中。使微液滴199大小发生改变，会影响微液滴199的位置排列，进而影响到对微液滴199的实时检测。进一步，在所述油相组合物的处理方法还包括使所述油相组合物达到水饱和的步骤。具体的，所述使油相组合物达到水饱和的步骤包括：在加热油相组合物前，在油相组合物中加入蒸馏水。油相组合物的加热过程结束后，油相组合物在25℃-35℃环境下自然冷却。将蒸馏水加入油相组合物中同时加热，使油相组合物达到水饱和。在室温下去掉不能溶解的水。在生成微液滴199之前油相组合物已经处于水饱和状态，最大程度降低了水相的微液滴199中的水分进入油相组合物的量。更进一步，使用氮气保护冷却后的油相组合物。氮气在以矿物油为主的油相组合物中溶解度很低。使用氮气做保护气体，能够防止在储存油相组合物的过程中环境中的空气或其他气体因溶解在油相组合物中而降低油相组合物的品质。作为一种可实现的方式，当储存油相组合物的器皿内有剩余空间时，将剩余空间内填充满氮气。
方案一：实验探索在不同组分的油相组合物(第二液体699)中所生成微液滴199的体积大小均一性。使用上述吐液枪头110的出口端112在油相组合物(第二液体699)内做周期性瞬时加速运动的方法，使吐液枪头110内的第一液体脱离吐液枪头110的出口端112在油相组合物(第二液体699)内形成微液滴199。方案一中，第一液体为水相，油相组合物(第二液体699)的组分如下表：
Figure PCTCN2019072969-appb-000010
Figure PCTCN2019072969-appb-000011
如图16及图17所示的实验结果，图16中虚线框内的微液滴199的体积大小均一性较好，分别对应实施例1、实施例2、实施例3及实施例4所示的油相组合物(第二液体699)的组分。如图18所示，当油相组合物(第二液体699)中
Figure PCTCN2019072969-appb-000012
的体积百分数增大至15％及16％时，所生成微液滴199的体积大小均一性较差。易得出，当油相组合物(第二液体699)中
Figure PCTCN2019072969-appb-000013
的体积百分比为1.5％-12％时，所生成微液滴199的体积大小均一性较好。
方案二：实验探索在不同组分的油相组合物(第二液体699)中所生成微液滴199的热稳定性。微液滴199的生成方法与实施例1-6相同。使含有微液滴199的多组油相组合物(第二液体699)分别经历50次高低温循环。高低温循环过程包括：以6℃/s的温升速率升至95℃并维持10s，然后以6℃/s的温降速率降至65℃并维持10s。方案二中所使用的油相组合物(第二液体699)的组分如下表：
Figure PCTCN2019072969-appb-000014
如图19及图20所示的实验数据，图19中虚线框内的微液滴199的体积大小均一性较好。当油相组合物(第二液体699)中
Figure PCTCN2019072969-appb-000015
的体积百分比为1.5％-12％、P135的含量小于5％时，如图19及图20所示，所生成微液滴199的体积大小均一性均较好。但当P135的含量大于5％时，如图19及图21所示，所生成微液滴199的体积呈现出大小不一的特点。图19中实线内的微液滴199的热稳定性较好。当油相组合物(第二液体699)中P135的含量介于1.5％-5.0％、
Figure PCTCN2019072969-appb-000016
的体积百分比为1.5％-5.0％时，如图22所示，微液滴199的热稳定性较好。当油相组合物(第二液体699)中
Figure PCTCN2019072969-appb-000017
的体积百分比为超过5.0％时，如图23所示，微液滴199的热稳定性较差。
由上述两个方案中的22个实施例的实验数据可以得出，油相组合物(第二液体699)中以矿物油为主要成分、
Figure PCTCN2019072969-appb-000018
的体积百分比为1.5％-5.0％、P135的含量为1.5％-5.0％时，第一液体在油相组合物(第二液体699)中所生成的微液滴199的体积大小均一性较好且微液滴199的热稳定性也较好。
方案三：实验探索
Figure PCTCN2019072969-appb-000019
的潜在替代成分。
Figure PCTCN2019072969-appb-000020
与
Figure PCTCN2019072969-appb-000021
同属于含链状烷基的硅氧链非离子型表面活性剂，使用方案一的实验条件，
Figure PCTCN2019072969-appb-000022
全部或者部分由
Figure PCTCN2019072969-appb-000023
代替。方案三中油相组合物(第二液体699)的组分如下表：
Figure PCTCN2019072969-appb-000024
如图24所示，使用上表中实施例23至实施例28所示不同组分的油相组合物(第二液体699)进行微液滴199生成实验，所生成的微液滴199均具有较好的体积均一性。
方案四：实验探索P135的潜在替代成分。聚乙二醇(30)二聚羟基硬脂酸酯(P135)与二聚羟基硬脂酸酯(PEG-30)及硬脂酸甘油均属于链状烷烃酯。使用方案二的实验条件，P135全部或者部分由二聚羟基硬脂酸酯(PEG-30)及硬脂酸甘油代替。方案四中油相组合物(第二液体699)的组分如下表：
Figure PCTCN2019072969-appb-000025
如图25及图26所示，使用上表中实施例29至实施例32所示不同组分的油相组合物 (第二液体699)进行微液滴199生成及热稳定性实验，所生成的微液滴199均具有较好的体积均一性及较好的热稳定性。
在一个具体的应用环境中，本申请提供的微液滴199的生成装置及生成方法应用在聚合酶链式反应(Polymerase Chain Reaction，PCR)中。
在一个实施例中，所述微液滴199为待测核酸扩增反应液，通过所述微液滴生成装置10将所述待测核酸扩增反应液微滴化，形成多个微液滴，用以通过所述数字PCR检测仪1进行检测。将所述待测核酸扩增反应液通过一体式数字PCR检测仪1的微液滴生成装置10，通过微滴化处理转化为多个微液滴199，使得待测样本中的检测片段从大量的复杂背景中分离出来，并放置于微液滴容器60中，等待检测。通过微液滴生成装置10可以生成多个大小均一的微液滴199。每个所述微液滴199大小在微米级，并且每个所述微液滴199可以视作一个独立的反应器，相当于生化反应中常用的试管。所述多个微液滴199放置于微液滴容器60中，便于检测观察。同时，通过所述微液滴生成装置10也可以生成多个体积不同的微液滴，用以进行医学临床检测。所述多个微液滴199体积小、数量多，具有许多常规试管没有的优势。通过所述微液滴生成装置10可以生成大量微液滴199，使得所述数字PCR检测仪1具有通量高、耗材成本低和背景噪声低的优点，具有很好的工业化前景。
传统的温控装置，升降温速率慢，每一次升温或降温都需要几十秒至数分钟，做几十个PCR循环需要1～2个小时左右，导致完成核酸扩增所需的时间延长，核酸扩增效率低，并且使用寿命低。针对传统的温控装置升降温速率慢的问题，提供一种升降温速率快、使用寿命高的温控装置。
如图27-28所示，本申请申请还提供一种温控装置20，其包括柔性电路板220、与所述柔性电路板220间隔设置的加热基板240以及多个半导体电偶对230。所述加热基板240包括相对设置的第一表面241和第二表面242。所述多个半导体电偶对230设置于所述柔性电路板220与所述第一表面241之间，所述多个半导体电偶对230相互串联、并联或者混合连接。
所述温控装置20通常应用于高低温循环环境中，温度需要快速升降，所以对所述温控装置20要求极高。为了满足所述温控装置20的应用需求，所述温控装置20采用所述柔性电路板220。所述柔性电路板220具有配线密度高、重量轻、厚度薄、弯折性好的特点。所述柔性电路板220在升降温过程中以自身的变形消除热应力。通过所述柔性电路板220可以降低升降温过程中存在的热应力，从而延长了所述温控装置20的使用寿命。同时，通过所述柔性电路板220，解决了温度分布不均匀的问题。当所述多个微液滴在不同温度范围内进行核酸扩增时，通过所述柔性电路板220、加热基板240以及多个半导体电偶对230可以实现迅速在几秒时间内进行切换。所述温控装置20可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环，将所述数字PCR检测仪1的检测时间缩短了，提高了检测效率。
所述柔性电路板220(Flexible Printed Circuit，FPC)可以是以聚酰亚胺或聚酯薄膜为基材制成的一种具有高度可靠性，绝佳的可挠性印刷电路板。所述柔性电路板具有配线密 度高、重量轻、厚度薄、弯折性好的特点。所述柔性电路板重量轻、厚度薄，可以有效节省产品体积。其中，所述半导体致冷器(Thermo Electric Cooler，TEC)是利用半导体材料的珀尔帖效应制成的。所谓珀尔帖效应，是指当直流电流通过两种半导体材料组成的电偶时，其一端吸热，一端放热的现象。通过所述柔性电路板220替换传统半导体致冷器中的一个基板，使得所述半导体致冷器导热性能更好。当温度改变时，物体由于外在约束以及内部各部分之间的相互约束，使其不能完全自由胀缩而产生的应力。热应力又称变温应力。热应力与零外载相平衡，是由热变形受约束引起的自平衡应力，在温度高处发生压缩，温度低处发生拉伸形变。在一定条件下控制应力使之合理分布，就可以提高零件的机械性能和使用寿命，变害为利。
在一个实施例中，所述加热基板240可以为超导铝基板电路。
铝基板是一种具有良好散热功能的金属基覆铜板，一般单面板由三层结构所组成，分别是电路层(铜箔)、绝缘层和金属基层。所述超导铝基板电路是线路板的材料是铝合金，能够导热快。铝基板能够将热阻降至最低，使铝基板具有极好的热传导性能，与厚膜陶瓷电路相比，它的机械性能又极为优良。
如图29所述，在一个实施例中，所述半导体电偶对230包括一个P型电偶231和一个与所述P型电偶231间隔设置的N型电偶232。
所述P型电偶231与所述N型电偶232焊接在所述柔性电路板220和所述基板240之间。所述半导体电偶对230包括一些由所述P型电偶231和所述N型电偶232形成的一对电偶，多对所述半导体电偶对230之间通过电极连在一起，并且夹在所述柔性电路板220与所述第一表面241之间。当有电流流过时，会产生″热″侧和″冷″侧。是致冷还是加热，以及致冷、加热的速率，由通过它的电流方向和大小来决定。一对所述半导体电偶对230产生的热电效应很小，所以在实际中都将上百对所述半导体电偶对230串联在一起，这样产生的热电效应就会增大。
在一个实施例中，所述第一表面241包括多个间隔设置的第一电极片243，一个所述第一电极片243与一个所述半导体电偶对230对应，所述半导体电偶对230中的所述P型电偶231与所述N型电偶232通过所所述第一电极片243串联。
在一个实施例中，所述柔性电路板220包括多个间隔设置并相互串联的第二电极片221，相邻的两个所述半导体电偶对230通过一个所述第二电极片221串联。
当所述P型电偶231和所述N型电偶232联结成的所述半导体电偶对230中有电流通过时，两端之间就会产生热量转移，热量就会从一端转移到另一端，从而产生温差形成冷热端。但是所述P型电偶231和所述N型电偶232自身存在电阻当电流经过所述P型电偶231和所述N型电偶232时就会产生热量，从而会影响热传递。而且所述柔性电路板220与所述加热基板240之间的热量也会通过空气和所述P型电偶231和所述N型电偶232材料自身进行逆向热传递。当冷热端达到一定温差，这两种热传递的量相等时，就会达到一个平衡点，正逆向热传递相互抵消。此时冷热端的温度就不会继续发生变化。为了达到更低的温度，可以采取散热等方式降低热端的温度来实现。
在一个实施例中，所述温控装置20还包括导热增强层250，设置于所述第二表面242。
所述导热增强层250具有十分良好的强度、柔韧、导电、导热、光学特性。所述导热增强层250直接与所述微液滴容器60接触，可以使得所述多个微液滴受热均匀，从而通过对温度的控制实现核酸扩增。所述导热增强层250可以为石墨导热层或者硅脂导热层，加快导热，增加了所述加热基板240的所述第二表面242的温度均匀性，进而保证靠近所述微液滴容器60的表面温度均匀，从而使得所述多个微液滴受热均匀。进而完成核酸扩增，提高了检测效率，节省了时间。
在一个实施例中，所述导热增强层250的材料中包括石墨烯。所述石墨烯是一种平面薄膜具有非常好的热传导性能，横向可以实现均匀导热。在一个实施例中，所述温控装置20还包括第二控制器210，与所述柔性电路板220电连接，用于控制电流大小。
在一个实施例中，所述温控装置20进一步包括温度传感器260，设置于所述第二表面242，并与所述第二控制器210电连接，用于检测所述第二表面242的温度并将该温度发送给所述第二控制器210。所述温度传感器260设置于所述导热增强层250的所述第二表面242，用以检测所述第二表面242的实时温度，进而将温度信息反馈给所述第二控制器210，从而实现对所述多个微液滴加热温度的控制。所述温度传感器260用于通过检测金属的电阻变化来测量所述微液滴容器60的温度，用以实时检测所述多个微液滴进行核酸扩增过程中的温度变化，从而将温度信息反馈给所述第二控制器210，进而控制所述控制电路进行温度的调控，实现温度控制，更好进行核酸扩增。
在一个实施例中，所述第二控制器210包括温度控制单元212以及控制电路214。所述温度控制单元212与所述温度传感器260连接，用以实时检测所述第二表面242的温度。所述控制电路214与所述柔性电路板220连接，用以调控所述多个半导体电偶对230的温度变化。所述温度控制单元212与所述控制电路214设置于一块电路板上。所述温度控制单元212与所述控制电路214的关系为内部算法的逻辑运算关系，可以采用Packet Identifier闭环控制算法，亦即PID闭环控制算法。所述温度控制单元212检测到的温度是核酸扩增的温度反馈，作为内部算法的输入，所述控制电路214计算后的结果作为内部算法的输出，从而形成闭环关系。电路部分的温度反馈实际上是一个采样电路。采集的就是铂电阻上的电信号，转换为温度值传递给控制电路的输入端。所述温度传感器260与所述温度控制单元212通过标准的铂电阻三线制连接。
在一个实施例中，所述柔性电路板220设置有第一电极222以及第二电极223。所述多个第二电极片221串联后与所述第一电极222和所述第二电极223串联。所述第一电极222与所述第二电极223分别与所述控制电路连接。所述控制电路214与所述柔性电路板220之间的连接为两根线，分别连接所述第一电极222以及所述第二电极223。
在一个实施例中，所述温控装置20还包括散热装置270，所述散热装置270包括基板271以及与所述基板271连接的散热片272。所述柔性电路板220设置于所述基板271的表面。由于所述散热片272设置于所述基板271表面，在没有减少所述基板271面积的情况下，更增加了热交换面积，延长凉风作用于所述基板271表面的时间，形成的多股散热 风道，也有利于加快热交换，把更多的热量从所述基板271表面上带走，从而达到更加理想的散热效果。
在一个实施例中，所述温控装置还包括风扇273设置于所述散热片273周围。通过所述风扇273可以协助所述散热装置270进行散热。其中，所述风扇273设置于所述散热片273的周围，可以设置多个，从而可以达到更好的散热效果，进而使得所述温控装置20升温降温更加快速。
在一个实施例中，对所述温控装置20通以交流电，并通过所述第二控制器210对电流大小调节来控制所述温控装置20是致冷还是加热，以及致冷、加热的速率。同时，通过所述温度传感器260用以检测所述微液滴容器60的实时加热温度，进而将温度信息反馈给所述温度控制单元212。所述温度控制单元212将温度变化情况反馈给所述控制电路214，从而控制所述多个微液滴的温度。通过所述温控装置20可以使得所述多个微液滴进行核酸扩增。基于PCR原理三步骤而设置变性-退火-延伸的三个温度点。在标准反应中采用三温度点法，双链DNA在90～95℃变性，再迅速冷却至40～60℃，引物退火并结合到靶序列上，然后快速升温至70～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸，在适合的温度范围内对核酸进行扩增。同时，在核酸扩增过程中，所述微液滴容器60底部与所述温控装置20紧密帖合，两者之间不会有间隙，提高了所述数字PCR检测仪1的准确性。
当所述多个微液滴在不同温度范围内进行核酸扩增时，可以实现迅速在几秒时间内进行切换。所述温控装置20可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环，将所述数字PCR检测仪1的检测时间缩短了，提高了检测效率。
在一个实施例中，通过所述温控装置20进行核算扩增的的操作方法如下：
首先，将所述微液滴容器60放置于所述温控装置20的所述导热增强层250。
然后，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min。将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动。
其次，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；
再次，所述多个微液变性之后，降温至55℃，并退火延伸45s，对所述多个微微液进行拍照，并进行45次循环；
最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
请参见图30，所述温控装置20一般情况下测试温控性能主要有两个指标，分别在瞬时状态和稳定状态下观测所述温控装置20的升降温度变化情况。通过对所述多个微液滴加热过程的监控，所述温控装置20对所述多个微液滴进行温度升降时，升降温速率最大可以达到13.34448℃/s。，控制精度为0.02722℃。并且，有时所述温控装置20升温到稳态的时候测量的速率最快可以到18.953894℃/s。因此，所述温控装置20的瞬态响应好，通过所述温控装置20可以实现瞬时升温降温，节省了时间，提高了检测效率。
请参见图31，当所述温控装置20处于稳定状态时，也就是在达到稳定后温度浮动的情况。当所述温控装置20处于稳定状态时，温度变化比较平稳，温度浮动较小。因此， 所述温控装置20可以达到快速的升温降温循环，且稳定后温服浮动较小，节省了数字PCR检测溶液样本的时间，提高了工作效率。通过这种升降温速率会缩短完成核酸扩增所需的时间，提高了核酸扩增效率，并且提高了数字PCR检测系统的准确性。
所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化，形成多个微液滴。然后，通过所述温控装置20对所述多个微液滴进行加热的过程中，采用所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图像。通过所述定量分析装置40对所述多个微液滴的荧光变化图像进行分析，获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始核酸的浓度进行定量分析。
所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化，形成多个微液滴。然后，通过所述温控装置20对所述多个微液滴进行核酸扩增。同时，采用所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图片。通过所述多个微液滴的荧光变化图片，获取所述多个微液滴的荧光变化曲线。根据所述荧光变化曲线，可以获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始DNA的浓度进行定量分析。其中，Ct值是指每个微液滴的荧光信号达到设定的阈值时所经历的循环数。
所述微液滴生成装置10生成的为均一大小微液滴，通过温控装置20对所述多个微液滴进行核酸扩增反应，并采集产物信号，如荧光、紫外吸收、浊度等信号。利用所述多个扩增与非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定量分析。通过实时监测所述多个微液滴的荧光变化图片，测序结果具有直接性，可以解决所述多个微液滴中的假阳性和假阴性的问题。
在一个实施例中，所述荧光信号检测装置30包括激发光源、荧光探测组件以及第三控制器。所述激发光源设置于所述微液滴容器60检测区域上方，并与所述微液滴容器60检测区域呈倾斜角度进行照射，形成斜射光路。所述荧光探测组件设置于所述微液滴容器60检测区域正上方，用以采集所述多个微液滴的荧光图像。所述第三控制器分别与所述激发光源和所述荧光探测组件连接，用以控制所述激发光源与所述荧光探测组件。所述荧光信号检测装置可以对微液滴进行多个荧光通道成像以及进行明场暗场成像。其中多个荧光通道成像用于微液滴反应信号的探测，明场暗场成像用于检测形成微液滴的尺寸信息以及在反应过程中监测液滴的状态。
所述多个微液滴的荧光图像的生成主要是通过所述荧光探测组件中的相机完成。通过所述荧光信号检测装置30可以使得所述多个微液滴荧光成像，一次拍摄一定数量的所述多个微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。从所述微液滴容器60上方采取倾斜角度照射在所述微液滴容器60上，采用所述荧光信号检测装置30实现对所述多个微液滴进行周期性的二维扫描，并实时进行拍照。斜射光路可有效降低激发光散射背景，提高荧光检测的灵敏度。所述微液滴容器60内的所述多个微液滴的内部荧光被激发，进入所述相机，从而所述相机采集所述多个微液滴的荧光图片。
针对传统的微液滴容器耗材成本较高、容纳微液滴个数偏少，且微液滴容器液面呈现 凹形液面，影响拍照检测的问题，提供一种耗材成本低、可容纳大量微液滴，且有利于拍照检测的微液滴容器。
请参见图32-37，本申请实施例提供一种微液滴容器60，其包括底面611、围绕所述底面611设置的第一环形侧面621以及环形面641。所述第一环形侧面621与所述底面611相连，并包围形成一个具有开口631的收纳空间630，所述第一环形侧面621垂直于所述底面611。所述环形面641环绕所述开口631设置，并与所述第一环形侧面621相连，所述环形面641与所述底面611平行。所述环形面641与所述底面611平行，用以确保所述微液滴容器60中的液体表面为水平面。通过设置于所述环形面641，可以使得所述微液滴容器60的液面呈现出平面状态，避免了所述微液滴容器60的整体液面为弧形。因此，通过所述微液滴容器60不会影响所述容器底板靠近边沿部位的微液滴的观测，便于相机进行拍照成像，提高了所述多个微液滴的检测效率。
在一个实施例中，所述微液滴容器60进一步包括容器底板610、围绕所述容器底板610设置的第一环形侧板620以及环形板640。所述容器底板610的表面为所述底面611。所述第一环形侧板620的内表面为所述第一环形侧面621，所述第一环形侧板620与所述容器底板610固定连接，并与所述容器底板610共同包围形成所述收纳空间630。所述环形板640的表面为所述环形面641。所述环形板640与所述第一环形侧板620远离所述容器底板610的一端固定连接，且所述环形板640与所述容器底板610平行。
在通过所述微液滴生成装置10制备所述多个微液滴时，先将所述第二液体(油相组合物)放置于所述微液滴容器60中。当所述第二液体的液面与所述环形面641水平面相同时，停止加入所述第二液体。此时，所述第二液体的液面与所述环形面641的表面在同一水平面上，可以保证所述微液滴容器60中的所述第二液体的油面为平面，方便保证容器底面上方的油液的顶面是水平面，便于成像，提高了所述微液滴容器60的利用率，用以容纳更多大量的微液滴。
在一个实施例中，所述环形板640的内周与所述第一环形侧板620远离所述容器底板610的一端连接。所述微液滴容器60还包括第二环形侧板650。所述第二环形侧板650环绕所述环形板640设置并与所述环形板640固定连接，所述第二环形侧板650的半径大于所述环形板640的内径。
通过所述环形板640、所述第一环形侧板620、所述容器底板610以及所述第二环形侧板650可以分别与所述环形面641、所述第一环形侧面621与所述底面611相互配合，形成所述收纳空间630。在所述收纳空间630内，用以容纳所述第二液体(油相组合物)。所述第二液体的液面与所述环形面641的表面在同一水平面上，可以保证所述微液滴容器60中的所述第二液体的油面为平面，方便保证容器底面上方的油液的顶面是水平面，避免了所述微液滴容器的整体液面为弧形，液面呈现凹形液面的问题。从而通过所述荧光信号检测装置30对所述多个微液滴进行荧光检测时，更加的便于成像，提高了所述微液滴容器60的利用率，用以容纳更多大量的微液滴。
在一个实施例中，所述环形板640的外周与所述第一环形侧板620远离所述容器底板 610的一端连接。
在一个实施例中，所述第一环形侧板620的外周固定连接于所述第一环形侧面621。通过将所述环形板640与所述第一环形侧板620的连接，可以使得所述微液滴容器60形成一个水平台。当在所述微液滴容器60中加入所述第二液体时，可以使得所述第二液体的液面与所述环形面641的表面在同一水平面上，从而可以保证所述微液滴容器60中的所述第二液体的油面为平面，方便保证容器底面上方的油液的顶面是水平面，避免了所述微液滴容器的整体液面为弧形，液面呈现凹形液面的问题。从而通过所述荧光信号检测装置30对所述多个微液滴进行荧光检测时，更加的便于成像，提高了所述微液滴容器60的利用率，用以容纳更多大量的微液滴。
在一个实施例中，所述微液滴容器60还包括第三环形侧板660。所述第三环形侧板660的一端固定连接于所述底面611。所述第三环形侧板660的另一端固定连接于所述第一环形侧板620的内周。所述第三环形侧板660与所述容器底板610共同包围形成所述收纳空间630。
在一个实施例中，所述第三环形侧板660与所述容器底板610垂直。通过所述微液滴容器60中所述第三环形侧板660的设置，可以使得所述第二液体的液面与所述环形面641的表面在同一水平面上，从而使得所述微液滴容器60中的液面为平面，表面了传统情况下的凹液面的产生，便于成像，提高了所述微液滴容器60的利用率。
在一个实施例中，所述微液滴容器60还包括多个环形凸条613，间隔设置于所述底面611，每个所述环形凸条613与所述底面611包围形成一个微液滴收纳槽614。所述微液滴收纳槽614用来收纳生成的所述多个微液滴，并且所述多个微液滴并通过微液滴平铺方法平铺于所述底面611，形成单层微液滴，用于拍照观察。同时，多个所述微液滴收纳槽614之间的间距可以根据所述微液滴生成装置10的排针之间的距离进行设置，从而可以使得一次性在多个所述微液滴收纳槽614内形成多个微液滴，提高了所述微液滴容器60的容纳量，也可以用来检测不同种类的核酸。
在一个实施例中，多个所述环形凸条613的高度为0.1mm-1mm。通过对所述环形凸条613高度的设置，可以有利于排除激发光从侧面照射时造成的的阴影，使得所述相机能够获取所有微液滴的荧光信息，提高了所述荧光检测装置的的灵敏度。
在一个实施例中，所述微液滴收纳槽614内壁表面设置有疏油层。通过在所述容器底板610表面做疏油处理，使得所述底板610与所述微液滴之间的粘黏性降低，表面张力降低，进而摩擦力降低，容易滑落，所述微液滴会自动扩散，防止了所述多个微液滴聚集在一起。同时，可以使得所述多个微液滴在进行平铺时更加快速，有利于所述多个微液滴平铺于所述微液滴容器底板610。当所述容器底板的表面张力小于所述第二液体(油类)699的表面张力时，所述微液滴与所述底板的阻力变小，所述微液滴会自动向所述微液滴反应单元底部扩散，实现平铺。
所述疏油膜也叫疏油层，是一种复合涂层材料，是一种功能性材料涂层，往往具有疏油功能。所述疏油层一般以纳米二氧化硅为原材料(SiO 2)，采用喷涂工艺，在表面形成涂 层，具备良好的透光性和疏水疏油性。所述多个微液滴与所述反应单元接触时，其接触角可以达到90度，可实现自动滚落而不留痕迹，从而可以实现所述多个微液滴平铺于所述微液滴容器底部610。
在一个实施例中，一种微液滴生成试剂盒包括以上实施例中提到的所述微液滴容器60、密封盖670以及油相组合物，所述油相组合物放置于所述收纳空间630中，所述密封盖670设置于所述开口631，用以将所述收纳空间630密封。
在一个实施例中，每个反应单元612包括每个所述环形凸条613，以及每个所述环形凸条613与所述底面611包围形成一个微液滴收纳槽614。所述容器底板610设置有多个反应单元612，每个反应单元612可以放置多个微液滴，从而可以使得所述微液滴容器60可以容纳大批量的微液滴，使得真正检测到的液滴数目会远远超过20000个，不存在对所述微液滴的个数的限制。同时，如果进行大量的微液滴检测，会需要耗费更多的时间。
在一个实施例中，多个所述反应单元612形状为矩形。所述微液滴容器60为方形或长方形。由于，目前为止绝大多数的胶片和数码感光元件CCD/CMOS都是方形的，所以将所述微液滴容器60形状设计为方形，可以提高所述微液滴容器的空间利用率，并且有利于方便形成的荧光图像的拼接，从而实现实时追踪。
在一个实施例中，多个所述反应单元612等间距排列于所述容器底板610。多个所述反应单元612的间距与所述微液滴生成装置10的排针之间的距离相同，以便于同时在多个所述反应单元612内形成大量的微液滴，提高了微液滴生成的速度，节省了时间。同时，也可以通过所述微液滴生成装置10在所述多个所述反应单元612内生成多个体积不同的微液滴。
在一个实施例中，多个所述环形凸条613的高度为0.1mm-1mm。通过对所述环形凸条613高度的设置，可以有利于排除激发光从侧面照射时造成的的阴影，使得所述相机能够获取所有微液滴的荧光信息，提高了所述荧光检测装置的的灵敏度。
传统的数字PCR检测系统的每个独立反应单元一般放置有一个微液滴。并且，在实际检测过程中，真正检测到的液滴数目不会达到20000个，对所述微液滴的个数仍然存在限制。所以采用所述微液滴容器60可以解决以上问题，不会对所述微液滴个数产生限制。
因此，通过所述容器底板610上的多个所述反应单元612可以容纳大量的微液滴，增大了所述微液滴容器60的收纳量，可以实现大于20000微液滴的检测，并且可以对不同类型的核酸进行检测。通过所述反应单元边框，可以避免使得所述多个微液滴散落至相邻的反应单元612内。
在一个实施例中，所述微液滴容器60的横截面为矩形。所述微液滴容器60为方形或长方形。所述微液滴容器60形状与相机镜头的形状一致，提高了所述微液滴容器的空间利用率，并且有利于方便形成的荧光图像的拼接，从而实现实时追踪。
在一个实施例中，所述环形面641为一个方形框。
通过在所述容器底板610表面做疏油处理，使得所述底板610与所述微液滴之间的粘黏性降低，表面张力降低，进而摩擦力降低，容易滑落，所述微液滴会自动扩散，防止了 所述多个微液滴聚集在一起。同时，可以使得所述多个微液滴在进行平铺时更加快速，有利于所述多个微液滴平铺于所述微液滴容器底板610。当所述容器底板的表面张力小于所述第二液体(油类)的表面张力时，所述微液滴与所述底板的阻力变小，所述微液滴会自动向所述微液滴反应单元底部扩散，实现平铺。
所述疏油膜也叫疏油层，是一种复合涂层材料，是一种功能性材料涂层，往往具有疏油功能。所述疏油层一般以纳米二氧化硅为原材料(SiO 2)，采用喷涂工艺，在表面形成涂层，具备良好的透光性和疏水疏油性。所述多个微液滴与所述反应单元接触时，其接触角可以达到90度，可实现自动滚落而不留痕迹，从而可以实现所述多个微液滴平铺于所述微液滴容器底部610。
在一个实施例中，所述第一环形侧板620或所述第二环形侧板650的高度为5mm-15mm。通过所述第一环形侧板620或所述第二环形侧板650高度的的设置，可以使得所述微液滴生成装置10在制备所述多个微液滴的过程中，避免所述多个微液滴甩出。并且可以有利于排除激发光从侧面照射时造成的的阴影，使得所述相机能够获取所有微液滴的荧光信息，提高了所述荧光检测装置30的的灵敏度。
在一个实施例中，所述容器底板610的材质为玻璃、石英或不锈钢等。
在一个实施例中，所述容器底板610的材质为玻璃，价格便宜，耗材成本低。
如果进行大量的微液滴检测，所述微液滴容器60采用玻璃材质，价格便宜，耗材成本低，进行一次检测可以将其丢弃，防止了交叉污染，节省了检测时间，提高了所述数字PCR检测仪1的检测效率。
在一个实施例中，所述环形凸条613与所述微液滴容器底板610的材料相同。利用工艺技术可以使得所述微液滴容器底板610形成多个所述反应单元612。多个所述反应单元612以阵列形式设置于所述微液滴容器底板610，形成多个核酸扩增单元。
在一个实施例中，所述容器底板610的形状尺寸大小与24孔板和96孔板外形尺寸一致，使得所述微液滴容器60方便应用于其他型号的仪器，更具有实用性与兼容性。
在一个实施例中，所述第一环形侧板620或所述第二环形侧板650的材质为耐高低温、耐油、无荧光的黑色硅橡胶。所述黑色硅橡胶具有无味无毒、不怕高温以及抵御严寒的特点。并且，所述黑色硅橡胶有良好的电绝缘性、耐氧抗老化性、耐光抗老化性、防霉性以及化学稳定性等优点，受到了现代医学领域的重视。
通过所述微液滴容器60，采用玻璃或不锈钢材质，可以降低检测成本。同时，通过所述容器底板610上的多个所述反应单元612可以容纳大量的微液滴，增大了所述微液滴容器的收纳量，可以实现大于20000微液滴的检测，并且可以对不同类型的核酸进行检测，经济实惠。
当激发光源倾斜照射至所述微液滴容器60，用以照射所述多个微液滴。通过所述激发光源形成的斜射光路可以有效降低激发光散射背景。同时，降低所述微液滴容器60的所述第一环形侧板620或所述第二环形侧板650的高度，有利于排除激发光从侧面照射时造成的的阴影，使得所述荧光信号检测装置30的相机能够获取所有微液滴的荧光信息，提 高了所述荧光检测装置30的的灵敏度。
请参见图38-39，在一个实施例中，本申请提供一种微液滴容器60包括第一容器底板680、多边形边框684以及容器盖690。所述第一容器底板680设置有多个多边形凸条681。所述多边形边框684包围形成一个第一收纳空间685，所述多边形边框684与所述第一容器底板680连接，且所述多个多边形凸条681设置于所述第一收纳空间685。所述容器盖690设置于所述多边形边框684远离所述第一容器底板680的表面，且所述容器盖690与所述多边形边框684可拆卸连接，所述容器盖690与所述多边形边框684包围形成一个油类收纳槽698。
所述多边形边框684两侧分别与所述第一容器底板680和所述容器盖690连接，所述容器盖690与所述多边形边框684可拆卸连接，可以将所述微液滴容器60密封。所述多个多边形凸条681形状可以为正方形、长方形、五边形等多边形形状。所述容器盖690与所述多边形边框684包围形成一个油类收纳槽698，当通过所述容器盖690密封所述微液滴容器60时，可以将所述微液滴容器60中的多余油类物质挤到所述油类收纳槽698中，可以尽量的避免了微液滴上层部分的油类物质对检测过程的影响，避免了油类物质造成的荧光背景。同时，通过将所述微液滴容器60中的多余油类物质挤到所述油类收纳槽698，所述微液滴容器60中与多个微液滴接触的油类物质减少，避免了油类物质不饱和情况下吸收多个微液滴的水分，解决了微液滴水分蒸发的情况。
在使用所述微液滴容器60放置油性液体时，可能会有空气混入，会产生气泡，所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图片会受到影响。因此，通过对所述微液滴容器60进行密封，当所述微液滴容器60以3～5度角进行倾斜时，可以避免所述微液滴容器60中的液体流出，还可以将所述微液滴容器60中的气泡排出，避免了拍照检测时，气泡造成的对图像的影响。
请参见图40-42，在一个实施例中，所述容器盖690包括平板691以及多边形容器盖框架692。所述平板691为透明状。所述多边形容器盖框架692包围形成一个安装空间697，用以安装所述平板691。
在一个实施例中，所述平板691为玻璃板。所述平板691与所述多边形容器盖框架692可以通过耐水，耐高温，无荧光，无毒性，对PCR反应无抑制作用的胶水粘贴固定。所述平板691为玻璃板价格便宜，耗材成本低。如果进行大量的微液滴检测，所述微液滴容器60采用玻璃材质，价格便宜，耗材成本低，进行一次检测可以将其丢弃，防止了交叉污染，节省了检测时间，提高了所述数字PCR检测仪的检测效率。
在一个实施例中，所述多边形容器盖框架692包括多边形框架主体693、多个延伸板695以及多个平板安装框696。所述多边形框架主体693包括多个边框694，所述多个边框694固定连接。每个所述延伸板695与每个所述边框694固定连接，并向所述安装空间697倾斜延伸。每个所述平板安装框696与每个所述延伸板695固定连接，并向所述多边形边框684包围形成的所述第一收纳空间685延伸。
在一个实施例中，所述多个边框694固定连接，形成所述多边形框架主体693。每个 所述边框694的表面与每个所述延伸板695的表面形成的夹角大于90度，从而可以使得所述容器盖690与所述多边形边框684连接时，形成所述油类收纳槽698。通过将所述微液滴容器60中的多余油类物质挤到所述油类收纳槽698中，可以尽量的避免了微液滴上层部分的油类物质对检测过程的影响，避免了油类物质造成的荧光背景。
在一个实施例中，所述多个延伸板695依次固定连接，形成一个环形框，并与所述多边形框架主体693固定连接。所述多个平板安装框696依次固定连接形成一个环形框，并与所述多个延伸板695形成的环形框固定连接。所述多边形容器盖框架692为一体。
在一个实施例中，每个所述延伸板695表面与每个所述平板安装框696的表面形成的夹角大于90度。每个所述平板安装框696表面与每个所述边框694的表面形成角度为90度，也就是说所述平板安装框696与所述微液滴容器60中的油性液体液面垂直，这样可以使得安装所述平板691时，形成一个矩形界面，可以使得与相机镜头的形状相同，可以使得所述微液滴容器60的液面呈现出平面状态，避免了所述微液滴容器60的整体液面为弧形。因此，通过所述微液滴容器60不会影响所述第一容器底板680靠近边沿部位的微液滴的观测，便于相机进行拍照成像，提高了所述多个微液滴的检测效率。同时，通过所述微液滴容器60提高了所述微液滴容器60的利用率，用以容纳更多大量的微液滴。
当通过所述容器盖690密封所述微液滴容器60时，可以将所述微液滴容器60中的多余油类物质挤到所述油类收纳槽698中，可以尽量的避免了微液滴上层部分的油类物质对检测过程的影响。
请参见图43，在一个实施例中，所述第一容器底板680还包括基板683，多个所述多边形凸条681以阵列的形式设置于所述基板683靠近所述多边形边框684的第一表面，每个所述多边形凸条681与所述基板683靠近所述多边形边框684的表面包围形成一个第一微液滴收纳槽682。
在一个实施例中，多个所述多边形凸条681为矩形。所述微液滴容器60为方形或长方形。由于，目前为止绝大多数的胶片和数码感光元件CCD/CMOS都是方形的，所以将所述微液滴容器60形状设计为方形，可以提高所述微液滴容器的空间利用率，并且有利于方便形成的荧光图像的拼接，从而实现实时追踪。
多个所述多边形凸条681以阵列的形式设置于所述基板683靠近所述边框684的表面，每个所述多边形凸条681与所述基板683包围形成一个第一微液滴收纳槽682。所述第一微液滴收纳槽682用来收纳生成的所述多个微液滴，并且所述多个微液滴并通过微液滴平铺方法平铺于第一容器底板680，形成单层微液滴或多层微液滴，用于拍照观察。同时，多个所述多边形凸条681之间的间距可以根据所述微液滴生成装置9680的排针之间的距离进行设置，从而可以使得一次性在多个所述第一微液滴收纳槽682内形成多个微液滴，提高了所述微液滴容器60的容纳量，也可以用来检测不同种类的核酸。
在一个实施例中，每个所述多边形凸条681的高度为0.2毫米～0.8毫米。
每个所述多边形凸条681的高度为0.2毫米～0.8毫米，最优可为0.3毫米～0.5毫米之间。在检测过程中，考虑到需要把多个微液滴放置于包围形成的所述第一微液滴收纳槽682 中，不会发生越过，所述多边形凸条681每个所述多边形凸条681的高度不能太低。因此，所述多边形凸条681的高度要至少在一个液滴直径以上。当通过所述荧光检测装置30采集微液滴图像时，也可以在所述微液滴容器60的所述第一微液滴收纳槽682中形成两层或两层以上的微液滴，这样可以扩大数字PCR检测仪的检测范围，有利于处理大批量的微液滴。通过所述微液滴生成装置10将核酸扩增反应液微滴化，形成多个微液滴，每个微液滴的体积一般情况为直径在0.1毫米～0.2毫米之间。由于在检测时，存在多层微液滴或微液滴出现堆叠的情况，通常将所述多边形凸条681的高度设置在2个微液滴直径以上，可以使得所述微液滴生成装置10在制备多个所述微液滴的过程中，避免多个所述微液滴甩出。并且可以有利于排除激发光从侧面照射时造成的阴影，使得所述荧光信号检测装置30能够获取所有微液滴的荧光信息，提高了所述荧光检测装置30的的灵敏度。
同时，在采用所述荧光检测装置30采集多个微液滴的图像时，每个所述多边形凸条681的高度不会影响图像的采集，可以有利于排除激发光从侧面照射时造成的的阴影，使得所述相机能够获取所有微液滴的荧光信息，提高了所述荧光检测装置30的灵敏度。
在一个实施例中，多个所述多边形凸条681包围形成的所述第一微液滴收纳槽682内壁表面设置有疏油层。也就是说在多个所述多边形凸条681靠近所述第一微液滴收纳槽682空间的表面设置有疏油层。
通过在所述基板683表面做疏油处理，使得所述基板683与所述微液滴之间的粘黏性降低，表面张力降低，进而摩擦力降低，容易滑落，所述微液滴会自动扩散，防止了多个微液滴聚集在一起。同时，可以使得多个微液滴在进行平铺时更加快速，有利于多个微液滴平铺于所述基板683。当所述基板683的表面张力小于油类物质的表面张力时，微液滴与所述底板的阻力变小，微液滴会自动向所述第一微液滴收纳槽682底部扩散，实现平铺。
所述疏油膜也叫疏油层，是一种复合涂层材料，是一种功能性材料涂层，往往具有疏油功能。所述疏油层一般以纳米二氧化硅为原材料(SiO 2)，采用喷涂工艺，在表面形成涂层，具备良好的透光性和疏水疏油性。所述多个微液滴与所述反应单元接触时，其接触角可以达到90度，可实现自动滚落而不留痕迹，从而可以实现所述多个微液滴平铺于所述基板683。
在一个实施例中，所述基板683的形状尺寸大小与24孔板和96孔板外形尺寸一致，使得所述微液滴容器60方便应用于其他型号的仪器，更具有实用性与兼容性。
多个所述多边形凸条681包围形成的所述第一微液滴收纳槽682可以放置多个微液滴，从而可以使得所述微液滴容器60可以容纳大批量的微液滴，使得真正检测到的液滴数目会远远超过20000个，不存在对微液滴的个数的限制。同时，如果进行大量的微液滴检测，会需要耗费更多的时间。
当所述容器盖690对所述微液滴容器60进行密封时，所述平板691与所述第一微液滴收纳槽682中微液滴的距离不能太高，一般距离设置在1毫米～2毫米之间，从而可以避免微液滴上层的油类物质对微液滴图像的影响，避免了油类物质造成的荧光背景，并且有利于多个微液滴平铺于所述基板683。并且，通过所述微液滴容器60中的多余油类物质挤到 所述油类收纳槽698，所述微液滴容器60中与多个微液滴接触的油类物质减少，避免了油类物质不饱和情况下吸收多个微液滴的水分，解决了微液滴水分蒸发的情况。
请参见图44-47，在一个实施例中，所述多边形边框684包括多个第一支撑杆686、多个支撑板688以及多个第二支撑杆689。每个所述第一支撑杆686环形连接。每个所述支撑板688固定设置于每个所述第一支撑杆686的内壁，并向所述第一收纳空间685延伸，所述多个支撑板688环形连接，用以支撑所述第一容器底板680。每个所述第二支撑杆689固定设置于每个所述第一支撑杆686远离所述第一容器底板680的第一支撑杆表面687，所述多个第二支撑杆689环形连接，且所述多个第二支撑杆689与所述多个第一支撑杆686共同包围形成所述第一收纳空间685，所述多个第二支撑杆689与所述多边形框架主体693的所述多个边框694可拆卸连接。
每个所述支撑板688固定设置于每个所述第一支撑杆686的内壁，并向所述第一收纳空间685延伸，所述多个支撑板688环形连接，用以支撑所述第一容器底板680。所述第一容器底板680安装于所述多个支撑板688形成的环形支撑架上，并且使得所述多个所述多边形凸条681设置于所述第一收纳空间685内。
请参见图48，所述多边形边框684与所述容器盖690之间通过双面胶条699进行粘连，也就是说在多个所述第二支撑杆689环形连接形成的多边形框架表面粘有所述双面胶条699，方便与所述边框694靠近所述多边形边框684的第一表面连接，进而使得所述多边形边框684与所述容器盖690密封连接。
在一个实施例中，所述微液滴容器60的所述基板683以及所述平板691均为玻璃板，解决了在采用数字PCR检测仪检测时样本容器的耗材问题，节约了成本。同时，所述微液滴容器60由于对每个元件之间的高度设置，使得所述微液滴容器60整体结构比较薄，增加了大量微液滴的可现性，使得所述微液滴容器60快速高低温循环，方便观察检测。
在一个实施例中，每个所述第二支撑杆689的宽度小于每个所述第一支撑杆686的宽度，且远离所述第一收纳空间685形成一个缝隙，用以方便拆卸。
所述多边形边框684为一体成型的，所述第二支撑杆689的侧面与所述第一支撑杆686的侧面不在一个平面上，可以避免所述容器盖690与所述多边形边框684粘连时有多余的胶挤出，避免沾黏到手上或其他地方。每个所述第二支撑杆689的宽度小于每个所述第一支撑杆686的宽度，且远离所述第一收纳空间685形成一个缝隙，用以方便拆卸。
在一个实施例中，一种微液滴容器制备方法，包括：
S510，提供一个基板683；
S520，在所述基板683表面制备多个多边形凸条681，形成第一容器底板680；
S530，提供一个多边形边框684，将所述第一容器底板680与所述多边形边框684连接；
S540，提供一个容器盖690，将所述容器盖690粘贴于所述多边形边框684远离所述第一容器底板680的表面进行密封，形成微液滴容器。
在一个实施例中，首先可以将所述基板683与所述多边形边框684通过胶水连接，此 时，所述基板683上没有设置多个所述多边形凸条681。其次，将油性液体放入所述基板683与所述多边形边框684粘连好的容器中。再次，通过点胶机将流体点滴、涂覆于所述基板683靠近所述多边形边框684的表面，可实现三维、四维路径点胶，精确定位，精准控胶，不拉丝，不漏胶，不滴胶，形成多个所述多边形凸条681。再次，通过数字PCR检测仪的微液滴生成装置10，在多个所述多边形凸条681内准备大量的微液滴。最后，将所述多边形边框684与所述容器盖690连接，连接时可以通过双面胶进行粘连，此时双面胶可以设置成为多边形的形状，使所述多边形边框684与所述容器盖690之间充分粘连。
在一个实施例中，首先，可以在所述基板683靠近所述多边形边框684的表面形成多个所述多边形凸条681。制备多个所述多边形凸条681时，可以采用点胶机、打印、丝网印刷等方式，在所述基板683靠近所述多边形边框684的表面制备多个所述多边形凸条681，以阵列形式排列，形成所述第一容器底板680。其次，将所述第一容器底板680与所述多边形边框684粘连连接。再次，将油性液体放入所述第一容器底板680与所述多边形边框684粘连好的容器中。再次，通过数字PCR检测仪的微液滴生成装置10，在多个所述多边形凸条681内准备大量的微液滴。最后，将所述多边形边框684与所述容器盖690连接，连接时可以通过双面胶进行粘连，此时双面胶可以设置成为多边形的形状，使所述多边形边框684与所述容器盖690之间充分粘连。
在一个实施例中，在所述步骤S520中制备多个所述多边形凸条681时，可以采用点胶、喷涂打印、丝网印刷以及热压成型等方法。
在一个实施例中，所述多边形凸条681的材质为耐高低温、耐油、无荧光的聚合物，从而在对所述微液滴进行高低温循环、荧光拍照时，可以避免了所述多边形凸条681带来的影响。
在一个实施例中，所述多边形凸条681可以为黑色硅橡胶或硅酮密封胶。
多个所述多边形凸条681的材质为耐高低温、耐油、无荧光的黑色硅橡胶，从而在对所述微液滴进行荧光拍照时，可以避免了所述多边形凸条681带来的荧光影响。所述黑色硅橡胶具有无味无毒、不怕高温以及抵御严寒的特点。并且，所述黑色硅橡胶有良好的电绝缘性、耐氧抗老化性、耐光抗老化性、防霉性以及化学稳定性等优点，受到了现代医学领域的重视。通过点胶机将流体点滴、涂覆于所述基板683靠近所述多边形边框684的表面，可实现三维、四维路径点胶，精确定位，精准控胶，不拉丝，不漏胶，不滴胶，形成多个所述多边形凸条681。
所述硅酮密封胶具有耐候性、抗振性，和防潮、抗臭气和适应冷热变化大、防渗防漏效果显著的特点。
在一个实施例中，所述步骤S520中制备多个所述多边形凸条681时也可以采用注塑，热压成型的方式加工。
在一个实施例中，一种数字PCR检测仪的分析方法，包括以下步骤：S10，制备待测核酸扩增反应液；S20，将所述待测核酸扩增反应液微滴化，形成多个微液滴；S30，将所述多个微液滴进行核酸扩增，并实时获取所述多个微液滴的荧光信息；S40，根据所述多 个微液滴的荧光信息，对所述多个微液滴进行定量分析。
在一个实施例中，所述步骤S20将所述待测核酸扩增反应液微滴化，用以形成多个微液滴包括两种微液滴生成方法：瞬时加速的微液滴生成方法以及变速周期的微液滴生成方法。所述步骤S30包括：S310：将所述多个微液滴平铺于所述微液滴容器中；S320：将平铺后的所述多个微液滴进行核酸扩增；S330：在所述多个微液滴进行核酸扩增时，实时对所述多个微液滴进行拍照检测。
由于微液滴生成装置10制备的多个微液滴在向下沉降过程当中，集中集合在微液滴容器60的中间部位，聚集在一起，不利于观察。所以，针对集中聚集在微液滴容器底部的问题，提供一种微液滴平铺方法。
请参见图49，在一个实施例中，所述步骤S30包括：S310：将所述多个微液滴平铺于所述微液滴容器中；S320：将平铺后的所述多个微液滴进行核酸扩增；S330：在所述多个微液滴进行核酸扩增时，实时对所述多个微液滴进行拍照检测。
在一个实施例中，所述步骤S310包括一种微液滴平铺方法。所述微液滴平铺方法包括：S311，提供一微液滴容器60，所述微液滴容器60具有开口631，且所述微液滴容器60内盛有第二液体699；S312，提供第一液体190，所述第一液体190的密度大于所述第二液体699并与所述第二液体699不互溶，并将所述第一液体190生成多个微液滴层叠堆积于所述微液滴容器底板610；S313，对所述多个微液滴进行高低温循环，直至所述多个微液滴平铺于所述容器底板610。
在所述微液滴容器60中生成多个微液滴，并将落至所述微液滴容器60的所述容器底板610，不规则的堆积在一起。当大量的微液滴降落至所述容器底板610时，会在所述容器底板610形成多层的微液滴。并且通过微液滴生成装置制备的多个微液滴在向下沉降过程当中，集中集合在微液滴容器的中间部位，聚集在一起，不利于观察。
在一个实施例中，所述第二液体699为油相组合物。
在一个实施例中，所述油相组合物的组分包括矿物油以及表面活性剂。所述油相组合物中矿物油的体积百分比为88％-98.5％。所述表面活性剂包括含长链烷基的硅氧链非离子型表面活性剂，所述油相组合物中含长链烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-12％。
在一个实施例中，所述第一液体为待测核酸扩增反应液。
在一个实施例中，所述步骤S312包括：S3122，提供具有出口端112的吐液枪头110，
所述吐液枪头110内储存有第一液体190；S3124，将所述吐液枪头110的出口端112插入所述第二液体699的液面下，并做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，所述吐液枪头的出口端的速度大小均单调变化；S3126，根据所述吐液枪头110的出口端112的周期变化的运动，所述第一液体190由所述吐液枪头110的出口端112排出，在所述第二液体699液面下形成多个微液滴，并堆积于所述微液滴容器底板610。
在一个实施例中，所述步骤S3124中，所述吐液枪头110的出口端112在所述第二液 体699的液面下的速度大小呈余弦曲线变化。
在一个实施例中，所述步骤S3124中的所述吐液枪头的110出口端112在第二液体699液面下的周期变化的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
在一个实施例中，所述步骤S312包括：
S3121，提供具有出口端112的吐液枪头110，所述吐液枪头110内储存有第一液体190；S3123，将所述吐液枪头110的出口端112插入所述第二液体699的液面下，瞬时加速运动；S3125，根据所述吐液枪头110的出口端112的瞬时加速运动，所述第一液体190由所述吐液枪头110的出口112端排出，在所述第二液体699液面下形成多个微液滴，并层叠堆积于所述微液滴容器底板610。
在一个实施例中，所述步骤S3123中，所述吐液枪头110的出口端112的瞬时加速的周期运动的前半周期与后半周期内，所述吐液枪头110的出口端112的速度大小相同，方向相反。在一个实施例中，所述步骤S3123中的瞬时加速的周期运动的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
在一个实施例中，所述步骤S313包括：S3131：将所述多个微液滴升温；S3133：将所述多个微液滴降温；S3135：将所述多个微液滴进行高低温循环多次，直至所述多个微液滴平铺于所述微液滴容器底板。
在一个实施例中，在通过所述微液滴生成装置10制备所述多个微液滴时，先将所述第二液体699放置于所述微液滴容器60中。当所述第二液体699的液面与所述环形面641相同时，停止加入所述第二液体699。此时，所述第二液体699的液面与所述环形面641的表面在同一水平面上，可以保证所述微液滴容器60中的所述第二液699体的油面为平面，方便保证容器底面上方的油液的顶面是水平面，便于成像。
在所述第二液体699中通过所述微液滴生成装置10将待测核酸扩增反应液微滴化，形成大量微液滴。所述多个微液滴降落至所述微液滴容器底板610的多个所述反应单元612内。所述环形面641所述容器底板610平行，用以确保所述微液滴容器中的第二液体699为水平面。在每个反应单元612可以放置多个微液滴，从而可以使得所述微液滴容器60可以容纳超过20000个的微液滴。
在一个实施例中，在所述步骤S3125中根据所述吐液枪头110的出口端112的瞬时加速运动的运动轨迹，所述多个微液滴平铺于所述微液滴容器底板610。通过所述吐液枪头110的出口端112的瞬时加速运动的运动轨迹可以使得所述多个微液滴在滴落至所述微液滴容器60中时，可以相互错开，使得所述多个微液滴在滴落至所述微液滴容器60中时，彼此之间没有相互堆积。从而使得所述多个微液滴平铺于所述微液滴容器60中，方便进行拍照观测。
在一个实施例中，所述微液滴容器底板610涂覆有疏油层。所述疏油层也叫疏油涂层，是一种复合涂层材料，是一种功能性材料涂层，往往具有疏油功能。所述疏油层一般以纳米二氧化硅为原材料，采用喷涂工艺，在屏幕表面形成涂层，具备良好的透光性和疏水疏 油性。
在一个实施例中，采用所述温控装置20用于以上实施例中的微液滴平铺方法。所述温控装置20包括柔性电路板220、与所述柔性电路板220间隔设置的加热基板240以及设置于所述柔性电路板220与所述加热基板240之间的多个半导体电偶对230。
通过高低温循环，利用热胀冷缩的原理，进行平铺。当物体温度升高时，分子的动能增加，分子的平均自由程增加，所以表现为热胀。同理，当物体温降低时，分子的动能减小，分子的平均自由程减少，所以表现为冷缩。随着温度的变化，当温度升高时，样本液滴的粘稠度变低、体积收缩。同时，温度越高粘度越低，当温度在60℃左右时，样本液滴形状最软，此时形状大概呈现为六边形，然而在其他的温度情况下，样本液滴形状的可变性较差，不容易实现在液滴容器中平铺。
请参见图50，将多个微液滴滴落至微液滴容器60中，所述多个微液滴堆积在所述微液滴容器底板610，亦即所述多个微液滴在所述微液滴容器底板610上形成多层微液滴。在荧光信号检测过程中，对所述多个微液滴进行拍照时，造成多层之间的相互影响，影响所述多个微液滴的拍照检测。因此，将容纳有所述多个微液滴的所述微液滴容器60进行高低温循环。将所述多个微液滴进行高低温循环多次，直至所述多个微液滴平铺于所述微液滴容器底板610，使得大批量的所述微液滴平铺于所述反应单元612内，便于海量液滴大规模平行观测。所以，为了更加精确的获取所述多个微液滴的核酸扩增反应的相关信息，需要将所述多个微液滴平铺在所述微液滴容器60中。通过将所述多个微液滴平铺在所述微液滴容器60中，形成一层，从而避免了多层微液滴之间的相互影响，使得所述荧光信号检测装置30拍照检测，获取更加精确地荧光信息，以便于定量分析。
在一个实施例中，所述步骤S312采用上述实施例中所述步骤S201、所述步骤S202以及所述步骤S203的所述微液滴生成方法生成微液滴，包括：S3121，提供具有出口端的吐液枪头，所述吐液枪头内储存有第一液体；S3123，将所述吐液枪头的出口端插入所述第二液体的液面下，并做瞬时加速的周期运动；S3125，根据所述吐液枪头的出口端的瞬时加速运动，所述第一液体由所述吐液枪头的出口端排出，在所述第二液体内部形成多个微液滴，并层叠堆积于所述微液滴容器底板。
在一个实施例中，所述步骤S3123中，所述吐液枪头的出口端的瞬时加速的周期运动的前半周期与后半周期内，所述吐液枪头的出口端的速度大小相同，方向相反。在一个实施例中，所述步骤S3123中的瞬时加速的周期运动的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
在一个实施例中，所述步骤S312采用上述实施例中所述步骤S211、所述步骤S212以及所述步骤S213的所述微液滴生成方法生成微液滴，包括：S3122，提供具有出口端的吐液枪头，所述吐液枪头内储存有第一液体；S3124，将所述吐液枪头的出口端插入所述第二液体的液面下，并做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，所述吐液枪头的出口端的速度大小均单调变化；S3126，根据所述吐液枪头的出口端的周期变化的运动，所述第一液体由所述吐液枪头的出口端排出，在所述第二液体液面 下形成多个微液滴，并堆积于所述微液滴容器底板。
在一个实施例中，所述步骤S3124中，所述吐液枪头的出口端在所述第二液体的液面下的速度大小呈余弦曲线变化。
在一个实施例中，所述步骤S3124中的所述吐液枪头的出口端在第二液体液面下的周期变化的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
在一个实施例中，通过所述温控装置20进行高低温循环的步骤如下：首先，将所述多个微液滴进行升温加热至90℃～95℃，并加热5min～10min；然后，将所述多个微液滴降温至40℃～60℃，并退火延伸30s～60s；最后，依次循环多次，降温至0℃～10℃，对所述多个微液滴保存。
在一个实施例中，通过所述温控装置20进行高低温循环的步骤还包括：首先，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min；将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动。然后，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；其次，所述多个微液变性之后，降温至55℃，并退火延伸45s，对所述多个微微液进行拍照，并进行45次循环；最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
所述待测核酸扩增反应液通过所述微液滴生成装置10生成多个微液滴，用以进行检测。通过所述微液滴生成装置10制备的所述多个微液滴在向下沉降过程当中，集中集合在所述微液滴容器60的中间部位，聚集在一起，不利于观察。所以，为了更加精确的获取所述多个微液滴的核酸扩增反应的相关信息，需要将所述多个微液滴平铺在所述微液滴容器中。通过将所述多个微液滴平铺在所述微液滴容器中，形成一层，从而避免了多层微液滴之间的相互影响，使得所述荧光信号检测装置30拍照检测，获取更加精确地荧光信息，以便于定量分析。
在一个实施例中，所述步骤S320将所述多个微液滴进行核酸扩增的步骤如下：首先，将所述微液滴容器60放置于所述温控装置20的所述加热基板240上；然后，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min；将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动。其次，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；再次，所述多个微液变性之后，降温至55℃，并退火延伸45s，并进行45次循环；最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
所述温控装置20采用所述柔性电路板220和所述导热增强层250，使所述微液滴容器60温度分布均匀，加快了所述半导体致冷器的导热性能。当所述多个微液滴在不同温度范围内进行核酸扩增时，设置于所述导热增强层250表面的的温度传感器260与所述第二控制器210连接，可以检测所述微液滴容器60的实时温度，进而将温度信息反馈给所述第二控制器210，从而实现对所述多个微液滴加热温度的控制。可以实现迅速在几秒时间内进行切换。所述温控装置20可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环，将所述数字PCR检测仪1的检测时间缩短了，提高了检测效率。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。
最后，还需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所提供的实施例的上述说明，使本领域专业技术人员能够实现或使用本申请。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本申请的精神或范围的情况下，在其它实施例中实现。因此，本申请将不会被限制于本文所示的这些实施例，而是要符合与本文所提供的原理和新颖特点相一致的最宽的范围。

Claims (56)

  1. 一种微液滴容器，包括：
    底面(611)；
    围绕所述底面(611)设置的第一环形侧面(621)，所述第一环形侧面(621)与所述底面(611)相连，并包围形成一个具有开口(631)的收纳空间(630)，所述第一环形侧面(621)垂直于所述底面(611)；
    环形面(641)，环绕所述开口(631)设置，并与所述第一环形侧面(621)相连，所述环形面(641)与所述底面(611)平行。
  2. 如权利要求1所述的微液滴容器，其特征在于，所述微液滴容器(60)进一步包括：
    容器底板(610)，所述容器底板(610)的表面为所述底面(611)；
    围绕所述容器底板(610)设置的第一环形侧板(620)，所述第一环形侧板(620)的内表面为所述第一环形侧面(621)，所述第一环形侧板(620)与所述容器底板(610)固定连接，并与所述容器底板(610)共同包围形成所述收纳空间(630)；
    环形板(640)，所述环形板(640)的表面为所述环形面(641)，所述环形板(640)与所述第一环形侧板(620)远离所述容器底板(610)的一端固定连接，且所述环形板(640)与所述容器底板(610)平行。
  3. 如权利要求2所述的微液滴容器，其特征在于，所述环形板(640)的内周与所述第一环形侧板(620)远离所述容器底板(610)的一端连接所述微液滴容器(60)还包括：
    第二环形侧板(650)，所述第二环形侧板(650)环绕所述环形板(640)设置并与所述环形板(640)固定连接，所述第二环形侧板(650)的半径大于所述环形板(640)的内径。
  4. 如权利要求2所述的微液滴容器，其特征在于，所述环形板(640)的外周与所述第一环形侧板(620)远离所述容器底板(610)的一端连接。
  5. 如权利要求4所述的微液滴容器，其特征在于，所述第一环形侧板(620)的外周固定连接于所述第一环形侧面(621)。
  6. 如权利要求4所述的微液滴容器，其特征在于，所述微液滴容器(60)还包括第三环形侧板(660)，所述第三环形侧板(660)的一端固定连接于所述底面(611)，所述第三环形侧板(660)的另一端固定连接于所述第一环形侧板(620)的内周，所述第三环形侧板(660)与所述容器底板(610)共同包围形成所述收纳空间(630)。
  7. 如权利要求6所述的微液滴容器，其特征在于，所述第三环形侧板(660)与所述容器底板(610)垂直。
  8. 如权利要求1所述的微液滴容器，其特征在于，所述微液滴容器(60)还包括多个环形凸条(613)，间隔设置于所述底面(611)，每个所述环形凸条(613)与所述底面(611)包围形成一个微液滴收纳槽(614)。
  9. 如权利要求8所述的微液滴容器，其特征在于，所述微液滴收纳槽(614)内壁表 面设置有疏油层。
  10. 一种微液滴生成试剂盒，其特征在于，包括如权利要求1-9中任一项所述微液滴容器(60)、密封盖(670)以及油相组合物，所述油相组合物放置于所述收纳空间(630)中，所述密封盖(670)设置于所述开口(631)，用以将所述收纳空间(630)密封。
  11. 一种微液滴容器，包括：
    第一容器底板(680)，设置有多个多边形凸条(681)；
    多边形边框(684)，包围形成一个第一收纳空间(685)，所述多边形边框(684)与所述第一容器底板(680)连接，且所述多个多边形凸条(681)设置于所述第一收纳空间(685)；
    容器盖(690)，设置于所述多边形边框(684)远离所述第一容器底板(680)的表面，且所述容器盖(690)与所述多边形边框(684)可拆卸连接，所述容器盖(690)与所述多边形边框(684)包围形成一个油类收纳槽(698)。
  12. 如权利要求11所述的微液滴容器，其特征在于，所述容器盖(690)包括：
    平板(691)，所述平板(691)为透明状；以及
    多边形容器盖框架(692)，包围形成一个安装空间(697)，用以安装所述平板(691)。
  13. 如权利要求12所述的微液滴容器，其特征在于，所述多边形容器盖框架(692)包括：
    多边形框架主体(693)，包括多个边框(694)，所述多个边框(694)固定连接；
    多个延伸板(695)，每个所述延伸板(695)与每个所述边框(694)固定连接，并向所述安装空间(697)倾斜延伸；以及
    多个平板安装框(696)，每个所述平板安装框(696)与每个所述延伸板(695)固定连接，并向所述多边形边框(684)包围形成的所述第一收纳空间(685)延伸。
  14. 如权利要求11所述的微液滴容器，其特征在于，所述第一容器底板(680)还包括基板(683)，多个所述多边形凸条(681)以阵列的形式设置于所述基板(683)靠近所述多边形边框(684)的表面，每个所述多边形凸条(681)与所述基板(683)靠近所述多边形边框(684)的表面包围形成一个第一微液滴收纳槽(682)。
  15. 如权利要求14所述的微液滴容器，其特征在于，每个所述多边形凸条(681)的高度为0.2毫米～0.8毫米。
  16. 如权利要求11所述的微液滴容器，其特征在于，所述多边形边框(684)包括：
    多个第一支撑杆(686)，每个所述第一支撑杆(686)环形连接；
    多个支撑板(688)，每个所述支撑板(688)固定设置于每个所述第一支撑杆(686)的内壁，并向所述第一收纳空间(685)延伸，所述多个支撑板(688)环形连接，用以支撑所述第一容器底板(680)；以及
    多个第二支撑杆(689)，每个所述第二支撑杆(689)固定设置于每个所述第一支撑杆(686)远离所述第一容器底板(680)的第一支撑杆表面(687)，所述多个第二支撑杆(689)环形连接，且所述多个第二支撑杆(689)与所述多个第一支撑杆(686)共同包 围形成所述第一收纳空间(685)，所述多个第二支撑杆(689)与所述多边形框架主体(693)的所述多个边框(694)可拆卸连接。
  17. 如权利要求16所述的微液滴容器，其特征在于，每个所述第二支撑杆(689)的宽度小于每个所述第一支撑杆(686)的宽度，且远离所述第一收纳空间(685)形成一个缝隙，用以方便拆卸。
  18. 一种微液滴容器制备方法，包括：
    S510，提供一个基板(683)；
    S520，在所述基板(683)表面制备多个多边形凸条(681)，形成第一容器底板(680)；
    S530，提供一个多边形边框(684)，将所述第一容器底板(680)与所述多边形边框(684)连接；
    S540，提供一个容器盖(690)，将所述容器盖(690)粘贴于所述多边形边框(684)远离所述第一容器底板(680)的表面进行密封，形成微液滴容器。
  19. 如权利要求18所述的微液滴容器制备方法，其特征在于，在所述步骤S520中制备多个所述多边形凸条(681)时，可以采用点胶、喷涂打印、丝网印刷以及热压成型等方法。
  20. 如权利要求19所述的微液滴容器制备方法，其特征在于，所述多边形凸条(681)为黑色硅橡胶或硅酮密封胶。
  21. 一种微液滴生成用油相组合物，包括以下组分：
    矿物油，所述油相组合物中所述矿物油的体积百分比为88％-98.5％；
    表面活性剂，所述表面活性剂包括含链状烷基的硅氧链非离子型表面活性剂。
  22. 如权利要求21所述的微液滴生成用油相组合物，其特征在于，所述油相组合物中所述含链状烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-12％。
  23. 如权利要求21所述的微液滴生成用油相组合物，其特征在于，所述含链状烷基的硅氧链非离子型表面活性剂包括
    Figure PCTCN2019072969-appb-100001
    和
    Figure PCTCN2019072969-appb-100002
    中的一种或两种。
  24. 如权利要求23所述的微液滴生成用油相组合物，其特征在于，所述表面活性剂还包括链状烷烃酯，所述油相组合物中所述链状烷烃酯与所述油相组合物的质量体积比为0.015g/mL-0.05g/mL。
  25. 如权利要求24所述的微液滴生成用油相组合物，其特征在于，所述链状烷烃酯包括二聚羟基硬脂酸酯(PEG-30)、硬脂酸甘油和聚乙二醇(30)二聚羟基硬脂酸酯(P135)中的一种或多种。
  26. 如权利要求25所述的微液滴生成用油相组合物，其特征在于，所述链状烷烃酯为聚乙二醇(30)二聚羟基硬脂酸酯(P135)。
  27. 如权利要求25所述的微液滴生成用油相组合物，其特征在于，所述油相组合物中含链状烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-5.0％。
  28. 如权利要求21-27任一项所述的微液滴生成用油相组合物，其特征在于，所述含链状烷基的硅氧链非离子型表面活性剂为
    Figure PCTCN2019072969-appb-100003
  29. 一种油相组合物的处理方法，其特征在于，用于处理权利要求21-27任一项所述的油相组合物；所述油相组合物的处理方法包括加热所述油相组合物，同时将所述油相组合物置于负压及超声波振动的环境中。
  30. 如权利要求29所述的油相组合物的处理方法，其特征在于，还包括使所述油相组合物达到水饱和的步骤。
  31. 如权利要求30所述的油相组合物的处理方法，其特征在于，所述使所述油相组合物达到水饱和的步骤包括：在加热所述油相组合物前，在所述油相组合物中加入蒸馏水；以及在所述油相组合物的加热过程结束后，所述油相组合物在25℃-35℃环境下自然冷却。
  32. 如权利要求31所述的油相组合物的处理方法，其特征在于，使用氮气保护冷却后的所述油相组合物。
  33. 一种微液滴平铺方法，包括：
    S311，提供一微液滴容器，所述微液滴容器具有开口，且所述微液滴容器内盛有第二液体；
    S312，提供第一液体，所述第一液体的密度大于所述第二液体并与所述第二液体不互溶，并将所述第一液体生成多个微液滴层叠堆积于所述微液滴容器底板；
    S313，对所述多个微液滴进行高低温循环，直至所述多个微液滴平铺于所述微液滴容器底板。
  34. 如权利要求33所述的微液滴平铺方法，其特征在于，所述第二液体为油相组合物。
  35. 如权利要求34所述的微液滴平铺方法，其特征在于，所述油相组合物的组分包括：
    矿物油，所述油相组合物中矿物油的体积百分比为88％-98.5％；
    表面活性剂，所述表面活性剂包括含长链烷基的硅氧链非离子型表面活性剂，所述油相组合物中含长链烷基的硅氧链非离子型表面活性剂的体积百分比为1.5％-12％。
  36. 如权利要求33所述的微液滴平铺方法，其特征在于，所述第一液体为待测核酸扩增反应液。
  37. 如权利要求33所述的微液滴平铺方法，其特征在于，所述步骤S312包括：
    S3122，提供具有出口端的吐液枪头，所述吐液枪头内储存有第一液体；
    S3124，将所述吐液枪头的出口端插入所述第二液体的液面下，并做速度大小呈周期变化的运动，在速度大小变化的前半周期与后半周期内，所述吐液枪头的出口端的速度大小均单调变化；
    S3126，根据所述吐液枪头的出口端的周期变化的运动，所述第一液体由所述吐液枪头的出口端排出，在所述第二液体液面下形成多个微液滴，并堆积于所述微液滴容器底板。
  38. 如权利要求37所述的微液滴平铺方法，其特征在于，所述步骤S3124中，所述吐液枪头的出口端在所述第二液体的液面下的速度大小呈余弦曲线变化。
  39. 如权利要求38所述的微液滴平铺方法，其特征在于，所述步骤S3124中的所述 吐液枪头的出口端在第二液体液面下的周期变化的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
  40. 如权利要求33所述的微液滴平铺方法，其特征在于，所述步骤S312包括：
    S3121，提供具有出口端的吐液枪头，所述吐液枪头内储存有第一液体；
    S3123，将所述吐液枪头的出口端插入所述第二液体的液面下，并做瞬时加速的周期运动；
    S3125，根据所述吐液枪头的出口端的瞬时加速运动，所述第一液体由所述吐液枪头的出口端排出，在所述第二液体内部形成多个微液滴，并层叠堆积于所述微液滴容器底板。
  41. 如权利要求40所述的微液滴平铺方法，其特征在于，所述步骤S3123中，所述吐液枪头的出口端的瞬时加速的周期运动的前半周期与后半周期内，所述吐液枪头的出口端的速度大小相同，方向相反。
  42. 如权利要求41所述的微液滴平铺方法，其特征在于，所述步骤S3123中的瞬时加速的周期运动的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。
  43. 如权利要求33所述的微液滴平铺方法，其特征在于，所述步骤S313包括：
    S3131：将所述多个微液滴升温；
    S3133：将所述多个微液滴降温；
    S3135：将所述多个微液滴进行高低温循环多次，直至所述多个微液滴平铺于所述微液滴容器底板。
  44. 如权利要求33所述的微液滴平铺方法，其特征在于，所述微液滴容器底板涂覆有疏油层。
  45. 一种温控装置，包括：
    柔性电路板(220)；
    与所述柔性电路板(220)间隔设置的加热基板(240)，所述加热基板(240)包括相对设置的第一表面(241)和第二表面(242)；
    多个半导体电偶对(230)，设置于所述柔性电路板(220)与所述第一表面(241)之间，所述多个半导体电偶对(230)相互串联、并联或者混合连接。
  46. 如权利要求45所述的温控装置，其特征在于，所述半导体电偶对(230)包括一个P型电偶(231)和一个与所述P型电偶(231)间隔设置的N型电偶(232)。
  47. 如权利要求46所述的温控装置，其特征在于，所述第一表面(241)包括多个间隔设置的第一电极片(243)，一个所述第一电极片(243)与一个所述半导体电偶对(230)对应，所述半导体电偶对(230)中的所述P型电偶(231)与所述N型电偶(232)通过所所述第一电极片(243)串联。
  48. 如权利要求47所述的温控装置，其特征在于，所述柔性电路板(220)包括多个间隔设置并相互串联的第二电极片(221)，相邻的两个所述半导体电偶对(230)通过一个所述第二电极片(221)串联。
  49. 如权利要求45所述的温控装置，其特征在于，还包括导热增强层(250)，设置于所述第二表面(242)。
  50. 如权利要求49所述的温控装置，其特征在于，所述导热增强层(250)的材料中包括石墨烯。
  51. 如权利要求48中任一项所述的温控装置，其特征在于，还包括第二控制器(210)，与所述多个半导体电偶对(230)电连接，用于控制电流大小。
  52. 如权利要求51所述的温控装置，其特征在于，进一步包括温度传感器(260)，设置于所述第二表面(242)并与所述第二控制器(210)电连接，用于检测所述第二表面(242)的温度并将该温度发送给所述第二控制器(210)。
  53. 如权利要求52所述的温控装置，其特征在于，所述第二控制器(210)包括：
    温度控制单元(212)，与所述温度传感器(260)连接，用以实时检测所述第二表面(242)的温度；
    控制电路(214)，与所述柔性电路板220连接，用以调控所述多个半导体电偶对(230)的温度变化。
  54. 如权利要求53所述的温控装置，其特征在于，所述柔性电路板(220)设置有第一电极(222)以及第二电极(223)，所述多个第二电极片(221)串联后与所述第一电极(222)和所述第二电极(223)串联，所述第一电极(222)与所述第二电极(223)分别与所述控制电路(214)连接。
  55. 如权利要求45所述的温控装置，其特征在于，所述温控装置还包括散热装置270，所述散热装置(270)包括基板(271)以及与所述基板(271)连接的散热片(272)，所述柔性电路板(220)设置于所述基板(271)的表面。
  56. 如权利要求55所述的温控装置，其特征在于，所述温控装置还包括风扇(273)设置于所述散热片(272)周围。
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