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WO2019086019A1 - 微液滴检测装置 - Google Patents

微液滴检测装置 Download PDF

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Publication number
WO2019086019A1
WO2019086019A1 PCT/CN2018/113852 CN2018113852W WO2019086019A1 WO 2019086019 A1 WO2019086019 A1 WO 2019086019A1 CN 2018113852 W CN2018113852 W CN 2018113852W WO 2019086019 A1 WO2019086019 A1 WO 2019086019A1
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WO
WIPO (PCT)
Prior art keywords
micro
oil
droplet
component
microdroplet
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/113852
Other languages
English (en)
French (fr)
Inventor
荆高山
郭永
王博
苏世圣
白宇
朱修锐
付明珠
祝令香
刘宝霞
杨文军
王勇斗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Targetingone Corp
Original Assignee
Targetingone Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201711074989.0A external-priority patent/CN109746063B/zh
Priority claimed from CN201711074983.3A external-priority patent/CN109752353B/zh
Priority claimed from CN201711074957.0A external-priority patent/CN109746058B/zh
Application filed by Targetingone Corp filed Critical Targetingone Corp
Priority to EP18872941.2A priority Critical patent/EP3674393A4/en
Publication of WO2019086019A1 publication Critical patent/WO2019086019A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • 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
    • B01L2300/0883—Serpentine channels
    • 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream

Definitions

  • the invention relates to the field of micro-droplet digital PCR technology, and in particular to a micro-droplet detecting device.
  • Droplet digital PCR is a single-molecule PCR-based nucleic acid absolute quantitative analysis technique.
  • Microdroplet digital PCR technology is becoming the next revolutionary technology in the industry with its high sensitivity and high accuracy.
  • micro-droplet digital PCR technology has encountered the best opportunity to break through the technical bottleneck.
  • the technology uses a microfluidic chip to generate droplets having a diameter of several micrometers to hundreds of micrometers; the microdroplets encapsulate single molecules or single cells, and the reaction and detection are fully enclosed and fully integrated.
  • the micro-droplet digital PCR system works by first dividing the sample to be tested into a large number of nano-nano-scale (water-in-oil) micro-droplets with a diameter of several micrometers to hundreds of micrometers through a special micro-droplet generator. The number of droplets is on the order of a million. Since the number of microdroplets is sufficient, the microdroplets are separated from each other by the oil layer, so each microdroplet is equivalent to a "microreactor", and the microdroplet contains only the DNA single molecule of the sample to be tested; These microdroplets were transferred to an EP tube and reacted in a conventional PCR machine.
  • nano-nano-scale water-in-oil
  • the fluorescence signals of the droplets are detected one by one by a special micro-droplet analyzer, the droplets of the fluorescent signal are interpreted as 1, and the droplets without the fluorescent signal are interpreted as 0.
  • the Poisson distribution principle and the number and proportion of positive droplets the number of target DNA molecules of the sample to be tested can be obtained, and the absolute quantification of the nucleic acid sample can be realized.
  • a key step in the microdroplet digital PCR technique is to quickly and reliably determine the fluorescent signal of a microdroplet sample and count the number and proportion of positive microdroplets.
  • the determination of the fluorescence signal of the microdroplet sample relies on a core technology: the design and processing of the microdroplet fluorescence detection device, which uses the laser to excite the fluorescence signal of the product in the microdroplet to distinguish the negative microdroplet from the positive microdroplet.
  • a conventional procedure for microdroplet digital PCR technology is that the generated microdroplets are transferred to an EP tube and reacted in a conventional PCR machine.
  • the microdroplets subjected to the PCR amplification reaction are injected into a microdroplet fluorescence detecting device, and a special microdroplet analyzer is used for fluorescence signal detection.
  • microdroplet fluorescence detecting device is widely used and clinically tested, and the following principles are required: (1) an EP tube containing microdroplets subjected to PCR amplification reaction, and the microdroplet fluorescence detecting device is hermetically connected, without transfer or subtraction Small possible cross-contamination; (2) micro-droplets are injected into the micro-droplet fluorescence detecting device under the action of a special micro-droplet analyzer; when the micro-droplets flow through the laser detection zone, the single rows are neatly arranged, which is convenient Accurate detection of fluorescent signals; (3) Micro-droplets after detection are collected in a closed storage container to reduce possible cross-contamination; (4) Single-use, micro-droplet fluorescence detection device, material and processing costs Low, (5) is convenient for the operation of the micro-droplet fluorescence detecting device.
  • the micro-droplet detection chip based on microfluidic technology has great application prospects.
  • PDMS polydimethylsiloxane
  • the researchers used a soft lithography process (manually) to process PDMS microdroplets with micron levels.
  • the sample inlet and the micro-droplet generation outlet are punched by a mechanical processing process, and the sample introduction tube and the sample introduction tube are assembled.
  • the "oil phase” sample, "microdroplet” sample in the EP tube is manually inhaled into the syringe.
  • the "oil phase” sample, "microdroplet” sample sample is then injected into the PDMS microdroplet chip through a sample tube through an external syringe pump.
  • the optical detection system detects the fluorescence signal of the droplets one by one. Finally, the detected microdroplets are collected through a sample tube into a conventional laboratory consumable such as an EP tube.
  • a sample tube into a conventional laboratory consumable such as an EP tube.
  • the PDMS micro-droplet detection chip is open-connected with an EP tube containing a "micro-droplet” sample, which is liable to cause cross-contamination.
  • PDMS is a thermoelastic polymer material, which is not suitable for industrial grade injection molding and packaging processes. Manually processed PDMS microdroplet chips have poor reliability. PDMS microdroplet chips are expensive to process in batches.
  • PDMS micro-droplet chip sample injection and droplet collection are labor-intensive manual processes, which are not suitable for clinical testing applications.
  • micro-droplet detection device In view of the shortcomings of PDMS microdroplet chips, we designed and processed a micro-droplet detection device based on microfluidic technology.
  • the micro-droplet detecting device can detect the fluorescence signal of the micro-droplet sample quickly, reliably, conveniently, and without pollution; the material and processing cost of the micro-droplet detecting device is low, which is beneficial to the wide application of clinical detection.
  • the present invention provides a micro-droplet detecting device including a first member, a second member, and a third member, the first member being used for detecting microdroplets
  • the first component and the second component are each integrally molded.
  • the first component is provided with a card slot for securing the third component.
  • the third component comprises a microdroplet container and a microdroplet container upper cover
  • the microdroplet container is made of a hard plastic material
  • the microdroplet container upper cover is made of a soft plastic material.
  • the micro-droplet container upper cover cooperates with the card slot to realize detachable fixed connection of the first component and the third component.
  • a puncture needle protruding from a lower surface of the first member is disposed in the card slot, the puncture needle is hollow; when the first member and the third member are detached and fixed The puncture needle penetrates the upper cover of the microdroplet container.
  • the first component is provided with at least one sample of spacer oil and oil slick and a collection unit for detecting waste liquid, preferably four, eight and twelve; Provided with at least one micro-droplet detecting unit, preferably four, eight and twelve; the third component is a container comprising at least one micro-droplet for microdroplet PCR amplification, preferably four or eight And twelve; each spacer oil and upper oil loading and the collection unit of the waste liquid after detection and each of the corresponding micro-droplet detecting units and each of the micro-droplets for micro-droplet PCR amplification The container is matched to complete the detection of microdroplets after amplification.
  • the loading unit of the spacer oil and the upper slick oil and the collection unit for the waste liquid after the detection include an upper slick oil sampling tank and an upper slick oil loading through hole provided on the upper surface above the first member.
  • a spacer oil sampling tank and a spacer oil loading through hole wherein the upper oil loading through hole and the interval oil loading through hole are respectively disposed in the upper floating oil sampling tank and the interval oil sampling tank a bottom oil; and an upper oil sampling pipe connected to the upper oil-loading through hole at a lower surface of the first member, and a spacer oil is connected to the space oil-adding through hole at a lower surface of the first member Injection pipe.
  • a waste liquid tank is disposed on a lower surface below the first member for collecting all waste liquid after the detection.
  • a structure for discharging residual air in the waste liquid tank is provided below the second member.
  • the micro-droplet detecting chip includes a central hole for injecting a plastic injection during the preparation of the micro-droplet detecting chip and mass production and/or batch fluorescence
  • the micro droplet detecting chip is transported during the detecting; and one or more micro-droplet detecting units are disposed on both sides of the center hole, and each micro-droplet detecting unit independently detects the micro-droplet.
  • the micro-droplet detecting unit comprises a micro-droplet detecting line, a spacer oil inlet, a spacer oil line, a micro-droplet container, a micro-droplet floating hole and a micro-droplet line;
  • the spacer oil inlet enters the interval oil line, and the micro droplets enter the microdroplet line from the microdroplet container through the microdroplet floating hole; the spacer oil line and the microdroplet line
  • a crisscross structure is formed prior to the microdroplet detection line such that the spacer oil separates the microdroplets in the microdroplet line.
  • a return flow resistance zone is disposed in the pipeline after the interval oil inlet, and after the interval oil flows through the return flow resistance zone, the two channels are respectively divided into two of the interval oil pipelines. .
  • a spacer oil filtration zone is disposed in the interval oil pipeline and/or an upper oil filtration zone, the interval oil filtration zone and/or the upper oil filtration zone are disposed in the upper oil pipeline. They are a set of columnar array structures.
  • the spacer oil line and/or the micro-droplet line is an arc-shaped pipe structure remote from the center hole; an auxiliary channel is disposed beside the micro-droplet detection line, such that The optical path of the detection system is positioned to the exact center of the microdroplet detection line.
  • the first component and the second component are thermoplastic materials, preferably polycarbonate materials, cyclic olefin copolymers or polymethyl methacrylate, polypropylene.
  • the volume of the upper oil loading tank and the spacer oil loading tank are respectively from 1 to 900 microliters, preferably from 5 to 500 microliters, more preferably from 100 to 200 microliters.
  • an observation window is further disposed on the second component for monitoring the generated micro-droplets in real time in conjunction with the optical system.
  • the micro-droplet detecting device further includes a fourth component through which external pressure is applied to the spacer oil and the upper slick oil in the first component.
  • the first component and the second component are respectively provided with positioning holes for facilitating the positioning of the two.
  • the first component and the second component are sealed and fixedly connected by dispensing or ultrasonic welding.
  • the crisscross structure is formed by two of the spacer oil lines and one of the microdroplet lines.
  • the micro-droplet detection system further includes a fourth component through which external pressure is applied to the spacer oil and the slick oil in the first component.
  • the micro-droplet detecting device of the present invention can achieve the following effects: (1) Fast, reliable, parallel detection of fluorescent signals of micron-sized "water-in-oil" microdroplet samples. (2) The first part and the second part of the device are processed by thermoplastic materials (such as PC, COP, PMMA), the material and batch processing cost are low, and (3) the external pressure source is used, and the micro-droplet detecting device is used. The detection rate of the microdroplet sample is high speed controllable. (4) Integrated micro-droplet chip design, the whole process is not easy to produce cross-contamination, the EP tube of the micro-droplet sample after PCR is directly connected with the chip, no need to open the cover, and the detected waste liquid will be collected into the waste collection tank. The whole process is not easy to produce cross-contamination. (5) The microdroplet fluorescence detecting chip is convenient to operate. It only needs to manually pre-add the interval oil and the floating oil, and then cooperate with the optical detection instrument to complete the whole inspection process conveniently.
  • the present invention proposes a micro-droplet detection chip based on a polymer material, which combines the mature optical disc preparation process and optical disc design specifications of the industry, adopts an innovative chip processing method and chip design, and is characterized by: (1) The micro droplets are arranged neatly in a single row at equal intervals in the chip.
  • FIG. 1 is a schematic structural view of a micro-droplet detecting device according to an embodiment of the present invention
  • FIG. 2 is a schematic structural view of a first component of an embodiment of the present invention.
  • FIG. 3 is a schematic view showing a structure in which a first component of the embodiment of the present invention is connected to a third component through a card slot;
  • FIG. 4 is a schematic structural view of a third component of an embodiment of the present invention.
  • Figure 5 is a schematic view showing the structure of a second member of an embodiment of the present invention.
  • Figure 6 is a schematic view showing the structure of a second member of another embodiment of the present invention.
  • Figure 7 is a schematic view of a spacer oil return type flow resistance zone according to an embodiment of the present invention.
  • Figure 8 is a schematic view of a filter zone of a spacer oil line according to an embodiment of the present invention.
  • Figure 9 is a schematic cross-sectional view of an embodiment of the present invention.
  • FIG. 10 is a schematic structural view of a detection pipeline according to an embodiment of the present invention.
  • Embodiment 1 The micro-droplet detecting device of the present invention
  • the micro-droplet generating device of the present invention comprises: a first member 1, a second member 2, and a third member 3, the first member 1 being a sample of the interval oil and the slick oil when the micro-droplet is detected and After the detection of the waste liquid collection device, the second component 2 is a micro-droplet detection chip, and the third component 3 is a container in which microdroplets perform PCR amplification of microdroplets.
  • the second member 2 is fixedly coupled to the upper surface of the first member 1; the third member 3 is detachably fixedly coupled to the lower surface of the first member 1.
  • the microdroplet generating device of the present invention further includes a fourth member 4 which is a spacer oil sampling tank for sealing the first member 1 and a top oil loading sample. groove.
  • the fourth component 4 is, for example, a water-tight gasket, for example made of silica gel, which may be provided with air holes for applying pressure therethrough, which serves to ensure a tight crimp between the external pressure source and the first component. Sexual and easy to apply external pressure.
  • locating holes are machined in the first component and/or the second component 2 to facilitate mating with each other.
  • the first member 1 is a sample for the separation of the interval oil and the upper oil and the collection of the waste liquid after the detection.
  • the first member 1 is provided with six spacer oils and a floating oil loading and a collecting unit for detecting the waste liquid, and the upper surface of the first member 1 is from left to right.
  • the loading of each spacer oil and the upper slick oil and the collection unit of the waste liquid after the detection are arranged in parallel on the first part 1 , the loading of the spacer oil and the slick oil, and each collection unit of the micro droplets after the detection.
  • the upper surface above the first component 1 is provided with an upper oil loading slot 11, an upper oil loading through hole 12, a spacer oil loading slot 13 and a spacer oil loading through hole 14, wherein the upper oil loading through hole 12 and the spacing
  • the oil application through holes 14 are respectively disposed at the center of the bottom of the upper oil sampling tank 11 and the spacer oil sampling tank 13; and are connected to the upper floating oil loading through hole 12 of the first member 1 on the lower surface of the first member 1.
  • the oil sampling pipe 15 is connected to the interval oil sampling pipe 16 at the interval of the oil application through hole 14 of the first member 1.
  • the upper oil sampling conduit 15 and the spacer oil sampling conduit 16 are machined from a lower surface above the first component 1 and then encapsulated with a sealing sheet to form a closed conduit.
  • a latching groove 17 of the third component 3 is disposed at a position below the upper oil sampling pipe 15 and the interval oil sampling pipe 16, and the latching groove 17 is detachable.
  • the third component 3 is fixedly connected; the puncture needle 18 protruding from the lower surface of the first component 1 is disposed in the latching groove 17 of the first component 1, the puncture needle 18 is hollow, and the upper oil passes through the third component 3.
  • a waste liquid tank 19 is disposed on the lower surface of the lower portion of the first member 1 for collecting all the waste liquid after the detection, and the micro-droplets detected by the fluorescence are sealed and recovered.
  • the third member 3 includes a microdroplet container 31 for accommodating microdroplets and an upper cap 32 pierceable by a puncture needle 18 provided on the first member.
  • the microdroplet container 31 is usually made of a hard plastic material.
  • the microdroplet container 31 is a commonly used centrifuge tube (EP tube)
  • the upper cover 32 is usually made of a soft plastic material for facilitating puncture with the puncture needle 18, for example, the upper cover 32 is made of silicone rubber.
  • Centrifuge tube cap is used.
  • each microdroplet container 31 cooperates with each spacer oil of the component 1 and the loading of the oil and the collection unit of the waste liquid after the detection.
  • each microdroplet container 31 is connected together.
  • six micro-droplet containers 31 are connected together, and six upper lids 32 are joined together; they are integrally processed.
  • FIG. 5 is a schematic structural view of a micro-droplet detecting chip of the second component 2, and six micro-droplet detecting units are arranged in parallel on the second component 2, and each micro-droplet detecting unit is equally spaced from left to right.
  • the loading of each spacer oil and the upper slick oil and the collection unit of the waste liquid after the detection are respectively matched with each micro-droplet detecting unit on the first component 1 for parallel detection of the micro-liquid drop.
  • micro-droplet detecting units are disposed in parallel on the second member 2; and a plurality of micro-droplets are disposed in parallel on the second member 2
  • the detection unit mainly utilizes the area of the micro-droplet detection chip to improve the detection efficiency.
  • each micro-droplet detecting unit includes a spacer oil inlet 211, two interval oil lines 212, an upper oil inlet 221, an upper oil-filling line 222, an upper oil hole 224, and a micro-droplet floating up. Hole 23, microdroplet line 24, microdroplet detection line 25, and microdroplet waste port 26.
  • a positioning assist channel 27 is provided alongside the microdroplet detection line 25, the auxiliary channel 27 being a closed channel in which no oil phase, water phase, or microdroplet sample flows, this region.
  • the imaging, light transmission/astigmatism properties are stable, and the optical path detection system is conveniently positioned to the center of the micro-droplet detection pipeline 25 to help accurately find the area to be detected.
  • venting opening 28 for discharging the residual air in the waste liquid tank when the waste liquid tank 19 collects the waste liquid
  • the venting opening 28 may be a passage on the second component.
  • the hole may not be a through hole.
  • the residual air in the waste liquid tank 19 is discharged from the exhaust hole 28 through an exhaust pipe communicating with the atmosphere, and one end of the exhaust pipe is a vent hole above the waste liquid tank. At the other end is a hole that communicates with the atmosphere.
  • the number of the vent holes 28 may be designed as needed, and may be one or plural.
  • a gas permeable film may be encapsulated on the upper surface of the hole connected to the atmosphere in the above case to reduce cross-contamination.
  • the first component 1 and the second component 2 are sealed and fixedly connected by means of dispensing or ultrasonic welding, for example, the first component 1 and the second component are dispensed around the upper oil sump inlet 221 of the second component 2 and the spacer oil inlet 211.
  • the component 2 is fixedly connected; the second component 2 is attached to the upper surface of the first component 1.
  • the third component 3 is detachably fixedly coupled to the first component 1 by a latching slot 17 in the first component 1.
  • the third component 3 and the first component 1 are detachably and fixedly coupled to the upper cover 32 through the latching slot 17, and the upper surface of the upper cover 32 is closely related to the lower surface of the first component latching slot 17. fit.
  • the upper surface of the first member 1 above the upper surface of the first member 1 and the upper oil inlet 221 of the second member are communicated by the upper oil sampling conduit 15; and the lower surface of the first member 1 is The spacer oil supply through-hole 14 of one component 1 and the interval oil inlet 211 of the second component are communicated by a spacer oil sampling conduit 16.
  • the upper slick oil and the interval oil are respectively added to the upper slick loading tank 11 and the spacer oil loading tank 13 of the first member 1.
  • the external pressure source is connected by a fourth component 4, which is a sealed gasket fitting.
  • the sealing rubber pad is a silica gel, and its function is to ensure the airtightness between the external pressure source and the first component 1.
  • the floating oil flows into the upper oil sampling pipe 15 through the upper oil filling through hole 12, then enters the upper oil floating pipe 222 through the upper oil inlet 221 on the second member 2, and finally passes through the upper oil passing hole 224.
  • the hollow of the puncture needle 18 enters the microdroplet container 31.
  • the spacer oil flows into the spacer oil sampling conduit 16 through the interval loading through hole 14, and then enters the two interval oil conduits 212 disposed in parallel on both sides of the spacer oil inlet through the spacer oil inlet 211 on the second member 2.
  • the two spacer oil lines 212 and the micro-droplet line 24 form a crisscross structure before the micro-droplet detection line 25.
  • the micro-droplets are extruded into a single row by spacer oil for optical detection.
  • each droplet's fluorescence signal is detected.
  • the microdroplets that have completed the detection are collected into the closed waste tank 19.
  • the first component 1 and the second component 2 are all prepared by a batch-integrated injection molding process.
  • the batch processing precision of the first component 1 and the second component 2 is high, and the consistency is good, which is advantageous for the stable connection of the external pneumatic power device.
  • the operator is easy and quick to operate, and the result is stable. It is suitable for the digital PCR technology based on microdroplet fluorescence detection to be applied to large-scale research and clinical testing applications.
  • the microdroplet detection work process of the present invention is as follows. First, the generated microdroplets are collected into a microdroplet container 31, such as an EP tube. Then, the EP tube was fitted with a closed EP tube cap 32 and placed in a conventional PCR machine for PCR amplification reaction. The microdroplet container 31 containing the amplified sample described above is then assembled to the first component 1 through the latching slot 17, and the puncture needle 18 completes the puncture of the upper cap 32 during the assembly and attachment process.
  • a microdroplet container 31 such as an EP tube.
  • the EP tube was fitted with a closed EP tube cap 32 and placed in a conventional PCR machine for PCR amplification reaction.
  • the microdroplet container 31 containing the amplified sample described above is then assembled to the first component 1 through the latching slot 17, and the puncture needle 18 completes the puncture of the upper cap 32 during the assembly and attachment process.
  • the slick oil is pre-loaded in the upper slick loading tank 11 of the first part 1. Under external pressure, the upper slick oil enters the upper slick line 222 from the upper slick inlet 221, and then enters the microdroplet container 31 through the upper sump hole 224 and the hollow portion of the lancet 18.
  • the first member 1 is hermetically connected to the upper cover 32 of the third member 3, and the upper oil hole 224 is disposed at a hollow portion corresponding to the puncture needle 18; the floating oil can flow only from the hollow portion of the puncture needle 18 into the microdroplet container 31. Since the density of the slick oil is greater than the density of the microdroplets in the microdroplet container 31, the microdroplets are always above the liquid level.
  • a void is formed around the puncture needle 18 passing through the upper cover 32, and the microdroplets in the microdroplet container 31 are floated up through the gap around the puncture needle 18 by the action of the floating oil. Since the first component 1 is tightly coupled to the second component 2, the floating droplets can only enter the microdroplet floating holes 23 on the second component 2 and then enter the microdroplet line 24.
  • the two spaced oil lines 212 and one of the microdroplet lines 24 form a crisscross structure for the purpose of spacing the closely spaced micro-droplets floating up through the spacer oil on both sides, and The signal interference between the droplets is reduced; at the same time, the "cross-over structure" design can control the distance between the single-layer arrangement of the micro-droplets by controlling the gas pressure of the oil on both sides.
  • the micro-droplet detecting line 25 after the "cross structure" after the micro-droplets are spaced apart, the flow of one-word flow is performed to perform optical detection.
  • microdroplets after the cross-over structure are separated by the interval oil, enter the micro-droplet detecting line 25, pass through the micro-droplet detecting line 25, and then enter the first part 1 through the micro-droplet waste port 26.
  • the lower surface of the lower portion is disposed in the waste liquid tank 19.
  • Embodiment 2 A micro-droplet detecting chip of the present invention
  • micro-droplet detecting units are arranged at equal intervals in a standard optical disc from left to right for parallel fluorescence. Detect microdroplets.
  • the traditional circular disc structure is not easy to position, the chip is processed into an octagonal structure, and two positioning holes are processed to facilitate the positioning and cooperation of the micro-droplet chip and related equipment.
  • Four identical micro-droplet detecting units are arranged at equal intervals on both sides of the center hole for detecting the micro-droplets in parallel.
  • each of the liquid droplet detecting units 1 includes a spacer oil inlet 211, a return flow resistance area 2111, two interval oil lines 2112, two interval oil filter areas 2113, and an oil floating inlet 221 from top to bottom. , an upper oil filter zone 2211 , an upper oil drain pipe 222 and an upper oil hole 224 , a micro droplet floating hole 23 , a micro droplet line 24 , a micro droplet detection line 25 and a micro droplet waste port 26 ;
  • the hole 224 is a through hole that connects the upper oil sump 222 and the third member 3 (lower, not shown).
  • an interval oil is injected into the interval oil inlet 211 by using an external air pump or a peristaltic pump.
  • a return flow resistance zone 2111 is provided to precisely control the interval oil injection amount.
  • the spacer oil will infiltrate the surface of the polymer material, and under the condition of no pressure applied, the interval oil will automatically flow into the micro-channel. In extreme cases, the interval oil continues to flow under capillary action.
  • the purpose of designing the return flow resistance zone 2111 is to precisely control the interval oil injection amount, and to minimize the continuous flow of the interval oil in the micro pipe under capillary action, so that the interval oil injection amount is controlled only by the external air pump or the peristaltic pump.
  • the interval oil passes through an oil phase split inlet, and is divided into two into the same design of the interval oil line 2112, and the function is that the interval oil and the floating droplet meet at the intersection to promote the movement of the micro droplet;
  • the flow effect squeezes the floating droplets into the center of the flow channel to facilitate detection of fluorescent signals within the microdroplets.
  • the two spacer oils each enter a spacer oil filter zone 2113.
  • the spacer oil filter zone 2113 is a group of columnar array structures.
  • the columnar array structure has a plurality of rows of columnar arrays interlaced. Impurities (particles, silk fibers, etc.) present in the spacer oil are blocked at the set of columnar structures, eliminating the effect of impurities on the fluorescence detection of the droplets.
  • the upper slick oil flows from the upper slick inlet 221 into the upper slick line 222 under the action of the external air pressure, flows down the upper sump hole 224, flows into the third part 3, and the micro droplets in the third part 3 are under the action of the floating oil.
  • the floating holes 23 are floated from the micro droplets.
  • the third member 3 is placed below the upper oil floating hole 224 and the micro droplet floating hole 23, and the pitch of the floating oil hole 224 and the micro droplet floating hole 23 is designed to refer to the size of the third member 3 if the third member 3 is an EP tube.
  • the spacing between the upper oil hole 224 and the microdroplet floating hole 23 is smaller than the width of the EP tube.
  • the spacer oil line 2112 and a microdroplet line 24 form a crisscross structure for the purpose of spacing the closely spaced micro-droplets floating up through the spacer oil on both sides, and making the micro-droplets
  • the signal interference between the two is reduced; at the same time, the "cross-over structure" design can control the distance between the single-layer arrangement of the micro-droplets by controlling the air pressure of the oil on both sides.
  • the micro-droplet detection line after the "cross structure" after the micro-droplets are spaced apart, the flow of the flow is discharged one by one to perform optical detection.
  • the microdroplets As shown in Fig. 10, after the "cross structure", the microdroplets have been arranged at equal intervals in the single row of the microdroplet detection pipeline, and at the same time, the fixed pitch is next to the microdroplet detection pipeline to be detected.
  • the position is provided with a closed auxiliary channel 27. There is no oil phase, water phase or microdroplet sample flow in the closed channel.
  • the imaging, light transmission/astigmatism properties of this region are stable, and the optical path detection system is conveniently positioned to the center of the microdroplet detection pipeline 25.
  • the micro-droplet detection system is provided with an exhaust inlet 281, an exhaust line 282, and an exhaust outlet 283 of the collection tank, and the exhaust inlet 281 is connected to the closed collection tank outlet.
  • the exhaust line 282 is connected to the exhaust outlet 283 for the purpose of releasing the pressure generated by the continuous inflow of liquid in the collecting tank while reducing cross-contamination.

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Abstract

一种微液滴检测装置，该检测装置包括第一部件（1）、第二部件（2）和第三部件（3），所述第一部件（1）是微液滴检测时用于间隔油和上浮油的加样以及检测后废液收集的装置，所述第二部件（2）是微液滴检测芯片，和所述第三部件（3）是装有微液滴进行微液滴PCR扩增的容器；所述第二部件（2）固定连接在所述第一部件（1）的上表面；和所述第三部件（3）可拆卸地固定连接在第一部件（1）的下表面。该微液滴检测装置，可以快速、可靠、便捷、以及无污染的检测微液滴样品的荧光信号；该微液滴检测装置的材料和加工成本低，有利于临床检测和其他生物检测的广泛应用。

Description

微液滴检测装置 技术领域
本发明涉及微液滴数字PCR技术领域，具体涉及一种微液滴检测装置。
背景技术
微液滴数字PCR技术(droplet digital PCR，ddPCR)是一种基于单分子PCR的核酸绝对定量分析技术。微液滴数字PCR技术以其高灵敏度、高准确性的优势正成为业界下一个革命性技术。近几年来，随着微纳米制造技术和微流体技术(micro-nanofabrication and microfluidics)的发展，微液滴数字PCR技术遇到了突破技术瓶颈的最佳契机。该技术借助微流控芯片，生成直径为数微米到数百微米的液滴；微液滴包裹单分子或单细胞，达到反应与检测全封闭，全集成。微液滴数字PCR系统工作原理是：首先通过特殊的微液滴生成仪将待测样品均分到大量纳升级(直径为数微米到数百微米)的“油包水”微液滴中，微液滴的数量在百万级别。由于微液滴数量足够多，微液滴之间被油层相互隔离，因此每个微液滴相当于一个“微型反应器”，微液滴中只含有待测样品的DNA单分子；然后，针对这些微液滴被转移到EP管中，在一个常规PCR仪中进行反应。经过PCR扩增反应的微液滴，通过特殊的微液滴分析仪逐个对液滴的荧光信号进行检测，有荧光信号的微滴判读为1，没有荧光信号的微滴判读为0。最后，根据泊松分布原理及阳性微滴的个数与比例即可得出待测样品的目标DNA分子数目，实现对核酸样本的绝对定量。微液滴数字PCR技术的一个关键步骤是快速、可靠的判定微液滴样品的荧光信号识别出来，统计阳性微液滴的个数和比例。
微液滴样品荧光信号的判定依赖一个核心技术：微液滴荧光检测装置的设计和加工，利用激光激发微液滴内产物的荧光信号高低来区分阴性微液滴和阳性微液滴。微液滴数字PCR技术的常规流程是：生成的微液滴被转移到EP管中，在一个常规PCR仪中进行反应。经过PCR扩增反应的微液滴， 被注入到一个微液滴荧光检测装置中，配合特殊的微液滴分析仪进行荧光信号检测。该微液滴荧光检测装置被广泛应用与临床检测需要具备以下原则：(1)含有经过PCR扩增反应的微液滴的EP管，与该微液滴荧光检测装置密闭连接，无需转移，减小可能的交叉污染；(2)微液滴在特殊微液滴分析仪作用下，被注入到该微液滴荧光检测装置中；微液滴流经激光检测区时，单排整齐排列，便于荧光信号的准确检测；(3)检测后的微液滴被收集到一个密闭的储存容器中，减小可能的交叉污染；(4)该微液滴荧光检测装置一次性使用，材料和加工成本低，(5)针对该微液滴荧光检测装置的操作便捷。针对以上原则，基于微流控技术的微液滴检测芯片具有很大的应用前景。
目前，基于聚二甲基硅氧烷(PDMS)的微流控芯片已被广泛用于检测微液滴。首先，研究人员利用软光刻工艺(人工操作)加工具备微米量级的PDMS微液滴芯片。当PDMS微液滴芯片制备成功后，在其样品入口、微液滴生成出口利用机械加工工艺打孔，装配进样管、出样管。EP管中的“油相”样品、“微液滴”样品通过手工方式吸入到注射器中。然后，通过外部注射泵将“油相”样品、“微液滴”样品样品经过进样管注入PDMS微液滴芯片中。在预先设计好的流道区域，光学检测系统逐个对液滴的荧光信号进行检测。最后，被检测的微液滴经过出样管被收集到常规实验耗材中，例如EP管。尽管PDMS微液滴芯片材料研发成本低、实验室加工工艺简单，但是其存在的不足包括：
(1)PDMS微液滴检测芯片与含有“微液滴”样品的EP管开放式连接，容易造成交叉污染。
(2)检测后的微液滴被收集到一个开放的EP管中，容易造成交叉污染。
(3)PDMS是热弹性聚合物材料，该类材料不适合于工业级注塑、封装工艺。手工加工的PDMS微液滴芯片可靠性差。PDMS微液滴芯片批量加工成本高昂。
(4)PDMS微液滴芯片样品注入、液滴收集为过程繁琐的人工操作流程，不适于临床检验应用。
针对PDMS微液滴芯片的不足，我们设计和加工了基于微流控技术的微液滴检测装置。该微液滴检测装置，可以快速、可靠、便捷、以及无污染的检测微液滴样品的荧光信号；该微液滴检测装置的材料和加工成本低，有利于临床检测的广泛应用。
发明内容
在一种实施方式中，本发明提供一种微液滴检测装置，所述微液滴检测装置包括第一部件、第二部件和第三部件，所述第一部件是微液滴检测时用于间隔油和上浮油的加样以及检测后废液收集的装置，所述第二部件是微液滴检测芯片，和所述第三部件是装有微液滴进行微液滴PCR扩增的容器；所述第二部件固定连接在所述第一部件的上表面；和所述第三部件可拆卸地固定连接在第一部件的下表面。
在一种实施方式中，所述第一部件和所述第二部件分别通过一体注塑而成。
在一种实施方式中，所述第一部件设置有用于固定所述第三部件的卡位槽。
在一种实施方式中，所述第三部件包括微液滴容器和微液滴容器上盖，所述微液滴容器由硬性塑料材质制成，微液滴容器上盖由软性塑料材料制成；固定时，所述微液滴容器上盖与所述卡位槽进行配合，实现所述第一部件和所述第三部件可拆卸地固定连接。
在一种实施方式中，在所述卡位槽中设置有从所述第一部件的下表面突出的穿刺针，所述穿刺针是中空的；所述第一部件和第三部件拆卸固定时，所述穿刺针穿透所述微液滴容器上盖。
在一种实施方式中，所述第一部件设置有至少一个间隔油和上浮油的加样以及检测后废液的收集单元，优选为四个、八个和十二个；所述第二部件设置有至少一个微液滴检测单元，优选为四个、八个和十二个；第三部件是包括至少一个装有微液滴进行微液滴PCR扩增的容器，优选为四个、八个和十二个；每个间隔油和上浮油的加样以及检测后废液的收集单元与其相对应的每一个微液滴检测单元和每一个装有微液滴进行微液滴PCR扩增的容器配合，完成扩增后微液滴的检测。
在一种实施方式中，所述间隔油和上浮油的加样以及检测后废液的收集单元包括在所述第一部件上方的上表面设置的上浮油加样槽、上浮油加样通孔、间隔油加样槽和间隔油加样通孔，其中所述上浮油加样通孔和所述间隔油加样通孔分别设置在所述上浮油加样槽和所述间隔油加样槽底部；和在第一部件下表面的所述上浮油加样通孔之处连接的上浮油进样管道，在所述第一部件下表面的所述间隔油加样通孔之处连接间隔油进样管道。
在一种实施方式中，在所述第一部件下方的下表面设置有废液槽，用于收集检测后的所有废液。
在一种实施方式中，在所述第二部件下方设置有将将废液槽内的残余空气排出的结构。
在一种实施方式中，所述微液滴检测芯片包括有一中心孔，所述中心孔用于所述微液滴检测芯片制备过程中注入注塑料以及批量化生产过程中和/或批量化荧光检测过程中微液滴检测芯片的转运；和所述中心孔两侧设置一个或多个微液滴检测单元，每个微液滴检测单元独立地检测微液滴。
在一种实施方式中，所述微液滴检测单元包括微液滴检测管路、间隔油入口、间隔油管路、微液滴容器、微液滴上浮孔和微液滴管路；间隔油从所述间隔油入口进入所述间隔油管路，微液滴从所述微液滴容器通过微液滴上浮孔进入所述微液滴管路；所述间隔油管路和所述微液滴管路在所述微液滴检测管路之前形成十字交叉结构，从而间隔油将所述微液滴管路中微液滴隔开。
在一种实施方式中，所述间隔油入口之后的管路中设置有回型流阻区，间隔油流过所述回型流阻区后，分成两路分别进入二个所述间隔油管路。
在一种实施方式中，所述间隔油管路中设置有间隔油过滤区和/或所述上浮油管路中设置有上浮油过滤区，所述间隔油过滤区和/或所述上浮油过滤区分别是一组柱状阵列结构。
在一种实施方式中，所述间隔油管路和/或所述微液滴管路是远离所述中心孔的弧形管道结构；所述微液滴检测管路旁设置有辅助沟道，使得检测系统的光路定位到所述微液滴检测管路正中心。
在一种实施方式中，所述第一部件和所述第二部件是热塑性材料，优选为聚碳酸酯材料、环烯烃共聚物或聚甲基丙烯酸甲脂、聚丙烯。
在一种实施方式中，所述上浮油加样槽和所述间隔油加样槽的容积分别为1～900微升，优选为5～500微升，更优选为100～200微升。
在一种实施方式中，所述第二部件上还设置观察窗，用于配合光学系统实时监测生成的微液滴。
在一种实施方式中，所述微液滴检测装置还包括第四部件，并通过其给所述第一部件中间隔油和上浮油施加外部压力。
在一种实施方式中，所述第一部件和所述第二部件上还分别设置有便于 两者固定连接进行定位的定位孔。
在一种实施方式中，所述第一部件和所述第二部件通过点胶方式或超声焊接方式密封固定连接。
在一种实施方式中，通过二个所述间隔油管路和一个所述微液滴管路形成所述十字交叉结构。
在一种实施方式中，所述微液滴检测系统还包括第四部件，并通过其给所述第一部件中间隔油和上浮油施加外部压力。
本发明的微液滴检测装置，可以达到以下效果：(1)快速、可靠、并行检测微米量级“油包水”微液滴样本的荧光信号。(2)该装置的第一部件和第二部件采用热塑材料(例如PC、COP、PMMA)加工，材料和批量加工成本低廉，(3)配合外部压力源，使用该微液滴检测装置，微液滴样本的检测速率高速可控。(4)集成式微液滴芯片设计，整个过程不易产生交叉污染，盛装PCR后的微液滴样本的EP管直接与芯片连接，无需开盖，检测后的废液会收集到废液收集池中，整个过程不易产生交叉污染。(5)微液滴荧光检测芯片操作方便。只需要手动预加间隔油和上浮油，再配合光学检测仪器，就可以便捷的完成整个检测过程。
另外，本发明提出了一个基于聚合物材料的微液滴检测芯片，该微液滴芯片结合工业界成熟的光盘制备工艺和光盘设计规格，采用创新的芯片加工方法和芯片设计，其特点是：(1)微液滴在芯片内等间距单排整齐排布。微液滴样本流经激光检测区后的荧光信号的准确检测，(2)微液滴芯片采用热塑材料例如，聚碳酸酯、环烯烃共聚物、聚甲基丙烯酸甲酯和聚丙烯，材料和批量加工成本低廉，(3)对传统圆环形光盘结构进行修正，最大限度利用光盘的空间，并行检测微液滴荧光信息。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例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中记载的一些实施例，对于本领域普通技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1是本发明的一种实施方式的微液滴检测装置结构示意图；
图2是本发明的一种实施方式的第一部件结构示意图；
图3是本发明的一种实施方式的第一部件通过卡位槽与第三部件连接结构示意图；
图4是本发明的一种实施方式的第三部件结构示意图；
图5是本发明的一种实施方式的第二部件结构示意图；
图6是本发明的另一种实施方式的第二部件结构示意图；
图7是本发明的一种实施方式的间隔油回型流阻区示意图；
图8是本发明的一种实施方式的间隔油管路过滤区示意图；
图9本发明的一种实施方式的十字交叉结构示意图；和
图10本发明的一种实施方式的检测管路结构示意图
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结合实施例对本发明作进一步说明，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它实施例，都应当属于本申请保护的范围。下面结合附图及实施例对本发明作进一步描述。
实施例一 本发明的微液滴检测装置
一.微液滴检测装置结构
如图1所示，本发明的微液滴生成装置包括：第一部件1、第二部件2和第三部件3，第一部件1是微液滴检测时间隔油和上浮油的加样以及检测后废液收集装置，第二部件2是微液滴检测芯片，第三部件3是微液滴进行微液滴PCR扩增的容器。第二部件2固定连接在第一部件1的上表面；第三部件3可拆卸地固定连接在第一部件1的下表面。
如图1所示，在一些实施方式中，本发明的微液滴生成装置还包括第四部件4，第四部件4是用于密封第一部件1的间隔油加样槽和上浮油加样槽。第四部件4例如水密封胶垫，例如用硅胶制备，该水密封胶垫可以设置有气孔，用于通过其施加压力，其作用是保证外部压力源和第一部件之间的压接气密性，并且便于施加外部压力。
如图1所示，在一些实施方式中，在第一部件和/或第二部件2加工出定位孔，便于彼此之间的配合。
如图2所示，第一部件1是实现微液滴检测时间隔油和上浮油的加样以 及检测后废液的收集。如图2所示，在本实施例中，第一部件1上平行设置6个间隔油和上浮油的加样以及检测后废液的收集单元，从左到右，第一部件1的上表面上方每个间隔油和上浮油的加样以及检测后废液的收集单元等间距并行排布在第一部件1上，间隔油和上浮油的加样以及检测后微液滴的每个收集单元与第二部件2上每个微液滴检测单元相配合，用于微液滴检测单元的间隔油和油样样品的加入和检测后的微液滴废液收集。第一部件1上方的上表面设置有上浮油加样槽11、上浮油加样通孔12、间隔油加样槽13和间隔油加样通孔14，其中上浮油加样通孔12和间隔油加样通孔14分别设置在上浮油加样槽11和间隔油加样槽13底部中央；和在第一部件1下表面第一部件1的上浮油加样通孔12之处连接的上浮油进样管道15，在第一部件1的间隔油加样通孔14之处连接的间隔油进样管道16。
在一些实施方式中，上浮油进样管道15和间隔油进样管道16是在第一部件1上方的下表面加工出管道，然后用封装片进行封装形成密闭的管道。
如图3所示，在第一部件1的下表面，在上浮油进样管道15和间隔油进样管道16下方位置设置有第三部件3的卡位槽17，该卡位槽17可拆卸地固定连接第三部件3；在第一部件1卡位槽17中设置有从第一部件1的下表面突出的穿刺针18，该穿刺针18是中空的，上浮油通过其进入第三部件3。
在第一部件1下方的下表面设置有废液槽19，用于收集检测后的所有废液，将荧光检测过的微液滴进行密闭回收。
如图4所示，第三部件3包括用于容纳微液滴的微液滴容器31和可用第一部件上设置的穿刺针18穿刺的上盖32，微液滴容器31通常用硬性塑料材质制成，例如，微液滴容器31是常用的离心管(EP管)，上盖32通常用软性塑料材料制成，便于用穿刺针18穿刺，例如上盖32是用有机硅橡胶制成的离心管盖。在一些实施方式中，每个微液滴容器31与部件1的每个间隔油和上浮油的加样以及检测后废液的收集单元相配合。在一些实施方式中，每个微液滴容器31连接在一起。在本实施方式中，6个微液滴容器31连接在一起，6个上盖32连接在一起；它们分别一体加工而成。
如图5所示，图5是第二部件2微液滴检测芯片结构示意图，第二部件2上平行设置6个微液滴检测单元，从左到右每个微液滴检测单元等间距并行排布在第二部件2上，每个间隔油和上浮油的加样以及检测后废液的收集单元与第一部件1上每个微液滴检测单元分别相配合，用于并行检测微液滴。
在一些实施方式中，第二部件2上平行设置8、10、12、14、16、18、20、22和24个微液滴检测单元；在第二部件2上平行设置多个微液滴检测单元，主要是充分利用微液滴检测芯片的面积，提高检测效率。
如图5所示，每个微液滴检测单元自上而下包括：间隔油入口211、两个间隔油管路212、上浮油入口221、上浮油管路222、上浮油孔224、微液滴上浮孔23、微液滴管路24、微液滴检测管路25和微液滴废液口26。
在一些实施方式中，在微液滴检测管路25旁边设置有定位辅助沟道27，辅助沟道27是封闭的沟道，其内没有油相、水相、微液滴样品流动，该区域成像、透光/散光性质稳定，便于光路检测系统定位到微液滴检测管路25正中心，帮助精准找到待检测区域。
在一些实施方式中，在第二部件下方设置有将废液槽19在收集废液时将废液槽内的残余空气排出的排气孔28，排气孔28可以是第二部件上的通孔，也可以不是通孔，例如，通过一个与大气连通的排气管道从排气孔28将废液槽19中的残余空气排出，该排气管道一端是与废液槽上方的排气孔，另一端是与大气连通的孔。排气孔28的数量可以根据需要进行设计，可以是1个，也可以是多个。为了防止污染，可以在上述情况中与大气连接的孔上表面封装透气薄膜，减小交叉污染。
第一部件1和第二部件2通过点胶方式或超声焊接方式密封固定连接，例如在第二部件2的上浮油入口221、间隔油入口211周围通过点胶方式将第一部件1和第二部件2固定连接；第二部件2连接在第一部件1的上表面。第三部件3通过第一部件1上的卡位槽17与第一部件1可拆卸地固定连接。在一种实施方式中，第三部件3与第一部件1通过卡位槽17与上盖32可拆卸地固定连接，上盖32的上表面与第一部件卡位槽17中的下表面紧密贴合。
第一部件1上方的下表面第一部件1的上浮油加样通孔12和第二部件的上浮油入口221之间通过上浮油进样管道15连通；和第一部件1上方的下表面第一部件1的间隔油加样通孔14和第二部件的间隔油入口211之间通过间隔油进样管道16连通。
上浮油和间隔油分别加入第一部件1的上浮油加样槽11和间隔油加样槽13。外部压力源通过第四部件4相连接，第四部件4是一个密封胶垫配件。密封胶垫是硅胶，其作用是保证外部压力源和第一部件1之间的压接气密性。然后，借助于外部压力，上浮油经过上浮油加样通孔12流入上浮油进样管 道15，然后通过第二部件2上的上浮油入口221进入上浮油管路222，最后通过上浮油通孔224和穿刺针18的中空进入微液滴容器31中。间隔油经过间隔加样通孔14流入间隔油进样管道16，然后通过第二部件2上的间隔油入口211进入间隔油入口处两边分别平行设置的两个间隔油管路212。
两个间隔油管路212与微液滴管路24在微液滴检测管路25之前形成十字交叉结构。在微液滴检测管路25中，微液滴被间隔油挤压成单排排布，便于光学检测。单排分布的微液滴经过光学检测区时，每个液滴荧光信号均被检测。最后，完成检测的微液滴被收集到密闭的废液槽19中。
在一些实施方式中，第一部件1、第二部件2均是通过批量一体式注塑工艺制备。这样，第一部件1、第二部件2的批量加工精度高，一致性好，利于外部气压动力装置稳定连接。同时操作人员操作简易快捷，结果稳定，适用于将以微液滴荧光检测为基础的数字PCR技术推向大规模研究、临床检验应用领域。
二、微液滴检测过程
本发明的微液滴检测工作过程如下。首先，生成的微液滴被收集到微液滴容器31中，例如为EP管。然后，EP管上装配密闭的EP管上盖32，放入常规PCR仪中进行PCR扩增反应。然后将上述装有扩增样品的微液滴容器31通过卡位槽17与第一部件1进行装配连接，在装配连接过程中穿刺针18完成对上盖32的穿刺。
上浮油被预先加样在第一部件1的上浮油加样槽11中。在外部压力作用下，上浮油从上浮油入口221进入上浮油管路222，然后通过上浮油孔224和穿刺针18的中空处进入微液滴容器31中。第一部件1与第三部件3的上盖32密闭连接，上浮油孔224设置在对应于穿刺针18的中空处；上浮油仅可从穿刺针18的中空处流入微液滴容器31中。由于上浮油的密度大于微液滴容器31中微液滴密度，微液滴一直处于液面上方。
在穿刺针18穿刺上盖32过程中，在穿过上盖32的穿刺针18周围形成空隙，微液滴容器31中的微液滴在上浮油的作用下，上浮通过穿刺针18周围的空隙，由于第一部件1与第二部件2紧密连接，上浮的液滴只能够进入第二部件2上的微液滴上浮孔23，然后进入微液滴管路24。
如图5所示，两个间隔油管路212和一个微液滴管路24形成十字交叉结构，目的是使上浮来的紧密排列的微液滴通过两侧的间隔油间隔开来，和 使得微液滴间的信号干扰降低；同时通过“十字交叉结构”设计可以通过控制两侧间隔油的气压来控制微液滴单层排布的间距距离。在“十字结构”后的微液滴检测管路25中，微液滴间隔开后，一字排开流动的流过进行光学检测。
通过上述十字交叉结构后的微液滴被间隔油间隔开，进入微液滴检测管路25，在微液滴检测管路25中通过，然后通过微液滴废液口26进入第一部件1下方的下表面设置的废液槽19中。
实施例二 本发明的一种微液滴检测芯片
如图6所示，在外圆直径为118mm、内圆直径为22mm的标准光盘中，从左到右，在一个标准光盘内等间距排布8个相同的微液滴检测单元，用于并行荧光检测微液滴。
在芯片中心有一中心孔，该中心孔来自于光盘加工工艺，用于注入注塑料以及批量化生产过程中基片的转运，以及用于批量化荧光检测过程中微液滴检测系统的转运。传统圆形光盘结构不易定位，将芯片加工为八边形结构，并加工出两个定位孔，便于微液滴芯片与相关设备的定位配合。在中心孔两侧各自等间距排布4个相同的微液滴检测单元，用于并行检测微液滴。
如图6所示，每个液滴检测单元1自上而下包括：间隔油入口211、回型流阻区2111、两个间隔油管路2112、两个间隔油过滤区2113、上浮油入口221、一个上浮油过滤区2211、上浮油管路222以及上浮油孔224、微液滴上浮孔23、微液滴管路24、微液滴检测管路25和微液滴废液口26；上浮油孔224是连接上浮油管路222与第三部件3(在下方，没有示出)的通孔。如图1所示的微液滴检测系统对常规光盘的标准结构进行了修正，可最大限度利用光盘的空间，并行排布微液滴检测流道。同时，利用精密注塑工艺加工的芯片，结合回型流阻区和过滤区设计，快速、可靠利用荧光检测均一的微米量级“油包水”微液滴。
在一种实施方式中，在图6的8个液滴检测单元1中，各个间隔油入口211之间的距离、上浮油入口221之间的距离和微液滴上浮孔23之间的距离，是相等的，该距离等于标准八通道移液器吸头之间的距离。
如图7所示，首先利用外部气泵或者蠕动泵，间隔油被注入到间隔油入口211。为了精确控制间隔油相样品进样量，在一种实施方式中，设置了回型流阻区2111，精确控制间隔油进样量。间隔油会浸润在聚合物材料表面， 在未施加压力条件下，经过毛细作用，间隔油自动流入微管道中。极端情况下，间隔油在毛细作用下，持续流动。设计回型流阻区2111的目的是精确控制间隔油进样量，尽量减少间隔油在毛细作用下在微管道中的持续流动，使得间隔油进样量仅由外部气泵或者蠕动泵控制。
然后，间隔油经过一个油相分流入口，一分为二的进入相同设计的间隔油管路2112中，其作用是间隔油与上浮液滴在十字交叉处交汇，推动微液滴运动；并借助鞘流效应，将上浮液滴挤压到流道中央，便于检测微液滴内的荧光信号。如图8所示，两路间隔油各进入一个间隔油过滤区2113，间隔油过滤区2113是一组柱状阵列结构，如图8所示，柱状阵列结构有多排柱状阵列交错组成。间隔油中存在的杂质(颗粒、丝絮纤维等)被阻挡在这组柱状结构处，消除杂质对于液滴荧光检测的影响。
上浮油在外界气压作用下，从上浮油入口221流入上浮油管路222，在上浮油孔224处流下，流入第三部件3中，同时第三部件3内的微液滴在上浮油作用下，从微液滴上浮孔23上浮。第三部件3放置在上浮油孔224和微液滴上浮孔23下方，上浮油孔224和微液滴上浮孔23的间距设计是参考第三部件3的尺寸，如果第三部件3是EP管，上浮油孔224和微液滴上浮孔23之间的间距是小于EP管的宽度。
如图9所示，间隔油管路2112和一个微液滴管路24形成十字交叉结构，目的是使上浮来的紧密排列的微液滴通过两侧的间隔油间隔开来，和使得微液滴间的信号干扰降低；同时通过“十字交叉结构”设计可以通过控制两侧间隔油的气压来控制微液滴单层排布的间距距离。在“十字结构”后的微液滴检测管路中，微液滴间隔开后，一字排开流动的流过进行光学检测。
如图10所示在“十字交叉结构”后，此时微液滴已经在微液滴检测管路单排等间距排布开来，同时在待检测的微液滴检测管路旁固定间距的位置设置有一个封闭的辅助沟道27。封闭的沟道内没有油相、水相、微液滴样品流动，该区域成像、透光/散光性质稳定，便于光路检测系统定位到微液滴检测管路25正中心。
随着大量的微液滴检测后，将检测完信号的微液滴从微液滴废液口26流出，流入一个外部的封闭收集槽入口中。收集槽封闭的目的是防止微液滴废液引起的交叉污染。如图5所示，在一些实施方式中，在微液滴检测系统设置有收集槽的排气入口281、排气管路282和排气出口283，排气入口281 与封闭收集槽出口连接，经过排气管路282与排气出口283连接，目的是为了释放收集槽中持续流入液体产生的压力，同时减少交叉污染。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。

Claims (14)

  1. 一种微液滴检测装置，其特征在于：所述微液滴检测装置包括第一部件、第二部件和第三部件，所述第一部件是微液滴检测时用于间隔油和上浮油的加样以及检测后废液收集的装置，所述第二部件是微液滴检测芯片，和所述第三部件是装有微液滴进行微液滴PCR扩增的容器；所述第二部件固定连接在所述第一部件的上表面；和所述第三部件可拆卸地固定连接在第一部件的下表面。
  2. 根据权利要求1所述的微液滴检测装置，其特征在于：所述第一部件和所述第二部件分别通过一体注塑而成。
  3. 根据权利要求1所述的微液滴检测装置，其特征在于：所述第一部件设置有用于固定所述第三部件的卡位槽。
  4. 根据权利要求3所述的微液滴检测装置，其特征在于：所述第三部件包括微液滴容器和微液滴容器上盖，所述微液滴容器由硬性塑料材质制成，微液滴容器上盖由软性塑料材料制成；固定时，所述微液滴容器上盖与所述卡位槽进行配合，实现所述第一部件和所述第三部件可拆卸地固定连接。
  5. 根据权利要求4所述的微液滴检测装置，其特征在于：在所述卡位槽中设置有从所述第一部件的下表面突出的穿刺针，所述穿刺针是中空的；所述第一部件和第三部件拆卸固定时，所述穿刺针穿透所述微液滴容器上盖。
  6. 根据权利要求1所述的微液滴检测装置，其特征在于：所述第一部件设置有至少一个间隔油和上浮油的加样以及检测后废液的收集单元，优选为四个、八个和十二个；所述第二部件设置有至少一个微液滴检测单元，优选为四个、八个和十二个；第三部件是包括至少一个装有微液滴进行微液滴PCR扩增的容器，优选为四个、八个和十二个；每个间隔油和上浮油的加样以及检测后废液的收集单元与其相对应的每一个微液滴检测单元和每一个装有微液滴进行微液滴PCR扩增的容器配合，完成扩增后微液滴的检测。
  7. 根据权利要求6所述的微液滴检测装置，其特征在于：所述间隔油和上浮油的加样以及检测后废液的收集单元包括在所述第一部件上方的上表面设置的上浮油加样槽、上浮油加样通孔、间隔油加样槽和间隔油加样通孔， 其中所述上浮油加样通孔和所述间隔油加样通孔分别设置在所述上浮油加样槽和所述间隔油加样槽底部；和在第一部件下表面的所述上浮油加样通孔之处连接的上浮油进样管道，在所述第一部件下表面的所述间隔油加样通孔之处连接间隔油进样管道。
  8. 根据权利要求7所述的微液滴检测装置，其特征在于：在所述第一部件下方的下表面设置有废液槽，用于收集检测后的所有废液。
  9. 根据权利要求8所述的微液滴检测装置，其特征在于：在所述第二部件下方设置有将将废液槽内的残余空气排出的结构。
  10. 根据权利要求1所述的微液滴检测装置，其特征在于：所述微液滴检测芯片包括有一中心孔，所述中心孔用于所述微液滴检测芯片制备过程中注入注塑料以及批量化生产过程中和/或批量化荧光检测过程中微液滴检测芯片的转运；和所述中心孔两侧设置一个或多个微液滴检测单元，每个微液滴检测单元独立地检测微液滴。
  11. 根据权利要求10所述的微液滴检测装置，其特征在于：所述微液滴检测单元包括微液滴检测管路、间隔油入口、间隔油管路、微液滴容器、微液滴上浮孔和微液滴管路；间隔油从所述间隔油入口进入所述间隔油管路，微液滴从所述微液滴容器通过微液滴上浮孔进入所述微液滴管路；所述间隔油管路和所述微液滴管路在所述微液滴检测管路之前形成十字交叉结构，从而间隔油将所述微液滴管路中微液滴隔开。
  12. 根据权利要求11所述的微液滴检测装置，其特征在于：所述间隔油入口之后的管路中设置有回型流阻区，间隔油流过所述回型流阻区后，分成两路分别进入二个所述间隔油管路。
  13. 根据权利要求11述的微液滴检测芯片，其特征在于：所述间隔油管路中设置有间隔油过滤区和/或所述上浮油管路中设置有上浮油过滤区，所述间隔油过滤区和/或所述上浮油过滤区分别是一组柱状阵列结构。
  14. 根据权利要求11所述的微液滴检测芯片，其特征在于：所述间隔油管路和/或所述微液滴管路是远离所述中心孔的弧形管道结构；所述微液滴检测管路旁设置有辅助沟道，使得检测系统的光路定位到所述微液滴检测管路正中心。
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