WO2024007622A1 - 一体式微液滴芯片及其微液滴多指标检测方法 - Google Patents

一体式微液滴芯片及其微液滴多指标检测方法 Download PDF

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Publication number
WO2024007622A1
WO2024007622A1 PCT/CN2023/082373 CN2023082373W WO2024007622A1 WO 2024007622 A1 WO2024007622 A1 WO 2024007622A1 CN 2023082373 W CN2023082373 W CN 2023082373W WO 2024007622 A1 WO2024007622 A1 WO 2024007622A1
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Prior art keywords
reaction chamber
micro
sample
detection
chamber
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PCT/CN2023/082373
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English (en)
French (fr)
Inventor
苏世圣
刘金伟
夏雷
王博
杨文军
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新羿制造科技（北京）有限公司
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Priority claimed from CN202221708244.1U external-priority patent/CN218250305U/zh
Priority claimed from CN202210781929.7A external-priority patent/CN115094120A/zh
Priority claimed from CN202210781877.3A external-priority patent/CN115181654A/zh
Priority claimed from CN202221708607.1U external-priority patent/CN218262530U/zh
Application filed by 新羿制造科技（北京）有限公司 filed Critical 新羿制造科技（北京）有限公司
Publication of WO2024007622A1 publication Critical patent/WO2024007622A1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control

Definitions

  • the invention belongs to the technical field of digital PCR analyzers, and specifically relates to an integrated micro-droplet chip and a micro-droplet multi-index detection method.
  • Digital PCR technology is called the third generation PCR technology. It has the advantages of absolute quantification and single-molecule detection sensitivity, and has important application prospects in the field of molecular diagnosis.
  • a mainstream technical route in digital PCR technology is the use of droplet microfluidic chips, in which the reaction system is divided into tens of thousands or even millions of uniformly sized droplets (also known as microdroplets). Each droplet independently performs nucleic acid amplification and fluorescence detection, and uses a mathematical model to calculate the precise copy number of the target molecule in the sample based on the fluorescence detection results.
  • digital PCR technology has the advantages of high detection sensitivity and absolute quantification, it still faces considerable challenges in low-concentration (and in some cases low-abundance) nucleic acid detection scenarios.
  • digital PCR technology is limited by the spectral distribution of fluorescent dyes. Only several fluorescent probes can be placed on one sample at the same time, corresponding to several detection targets. It is difficult to meet the requirement of distinguishing dozens of target sequences at the same time.
  • the technical problem to be solved by the present invention is to provide an integrated micro-droplet chip and a micro-droplet multi-index detection method thereof, so as to overcome the problem in the prior art that low-concentration detection samples are divided into too many copies during detection. Insufficiencies that lead to reduced detection sensitivity and inaccurate detection results.
  • the present invention provides an integrated multi-index detection microdroplet chip, which includes a chip body.
  • the chip body is constructed with at least one set of multiple mixing and reaction structures, and each of the multiple mixing and reaction structures.
  • the mixing and reaction structure corresponds to the micro-droplet generation structure and fluorescence detection area.
  • Each of the one-minute multi-mixing and reaction structures includes:
  • the primary reaction chamber is used to store the primary system and perform primary amplification, so that the detection sample with the first concentration in the primary system is amplified to form the detection sample with the second concentration, and the second concentration is high at the first concentration;
  • Multiple sample addition chambers are used to store the secondary system, and multiple sample addition chambers are in controllable communication with the same primary reaction chamber at the same time;
  • a plurality of secondary reaction chambers are controllably connected to a plurality of the sample adding chambers respectively, and are used to store the detection sample of the second concentration and the secondary system at the microdroplet generating structure. Generate microdroplets and perform secondary amplification;
  • the fluorescence detection area is used to detect microdroplets that have completed secondary amplification.
  • the primary reaction chamber has a plurality of first liquid outlet pipes connected in parallel, each of the sample adding chambers has a second liquid outlet pipe, and the second liquid outlet pipe of each of the sample adding chambers is The liquid outlet pipe and the plurality of first liquid outlet pipes are gathered in the mixing pipe in a one-to-one correspondence, and the plurality of mixing pipes are controllably connected to a plurality of the secondary reaction chambers in a one-to-one correspondence.
  • each of the one-minute multiple mixing and reaction structures further includes a plurality of fluid flow driving structures.
  • the number of the fluid flow driving structures is equal to the number of the mixing pipes.
  • Each of the fluid flow driving structures The driving structure includes a gas-liquid interface constructed on the chip body. Through the gas-liquid interface, the corresponding first-level reaction chamber and the second-level reaction chamber and the sampling chamber and the second-level reaction chamber can form a third A pressure difference is provided so that the fluids in the first-stage reaction chamber and the sampling chamber respectively flow into the corresponding mixing pipes under the action of the first pressure difference.
  • each of the fluid flow driving structures further includes an oil interface constructed on the chip body, and the oil interface intersects with the mixing pipe through an oil pipe to enable fluid flow in the When flowing through the intersection of the oil pipe and the mixing pipe, it is divided into a plurality of micro-droplets.
  • the present invention also provides a micro-droplet multi-index detection method using the above-mentioned integrated multi-index detection micro-droplet chip, which includes the following steps: a first-level amplification reaction step, controlling the amplification of the micro-droplets stored in the first-level reaction chamber.
  • the first-level system is heated to amplify the detection sample in the first-level system from a first concentration to a second concentration, and the second concentration is higher than the first concentration; the sample splitting and mixing steps control the first concentration.
  • the micro droplet generation step is to mix the detection sample with the second concentration in the micro droplet generation structure
  • the mixed detection sample is divided into multiple micro-droplets under the action of oil, and the formed micro-droplets are controlled to enter the secondary reaction chamber; in the secondary amplification reaction step, the control of the micro-droplets stored in the The micro-droplets in the secondary reaction chamber are heated to form secondary amplification; the micro-droplet fluorescence detection step is to perform multi-index fluorescence detection on the mixed detection sample after secondary amplification in the fluorescence detection area.
  • the step of generating micro droplets specifically includes: controlling the formation of a second pressure difference between the sampling chamber and the gas-liquid interface, and controlling the relationship between the oil-liquid interface and the gas-liquid interface.
  • a third pressure difference is formed between them, and the second pressure difference and the third pressure difference respectively drive the mixed detection sample and the generated oil in the mixing pipe to form the micro droplets at the intersection of the oil pipe and the mixing pipe. , and drive the formed microdroplets into the secondary reaction chamber.
  • a chip flipping step is included, which controls the chip body to flip up and down by 180°.
  • the chip body after controlling the chip body to flip up and down 180°, it also includes: a reverse flow control step of micro-droplets, controlling the injection of detection push oil into the secondary reaction chamber, so that the secondary reaction chamber
  • the microdroplets in the reaction chamber are driven to flow into the mixing pipe, and can be stored in the sample addition chamber after the microdroplets complete the microdroplet fluorescence detection step.
  • the invention also provides an integrated pre-amplification microdroplet chip, which includes a chip body.
  • the chip body is constructed with at least one set of mixing and reaction structures and a fluorescence detection area corresponding to each of the mixing and reaction structures.
  • each of the mixing and reaction structures includes: a primary reaction chamber, used to store the primary system and perform primary amplification, so that the detection sample with the first concentration in the primary system is amplified to form a second-level reaction chamber. concentration of the detection sample, the second concentration is higher than the first concentration; a sample addition chamber is used to store the secondary system, and the sample addition chamber forms a controllable connection with the primary reaction chamber at the same time; the secondary The reaction chamber is controllably connected to the sample addition chamber and the primary reaction chamber through a mixing pipe.
  • the detection sample of the second concentration and the secondary system are mixed in the mixing pipe to form micro droplets.
  • the form is stored in the secondary reaction chamber and undergoes secondary amplification; the fluorescence detection area is used to detect the microdroplets that have completed
  • the chip body also has a heating groove disposed adjacent to the first-level reaction chamber, and the first-level amplification heating module has a heating protrusion, and the heating protrusion can be inserted into the first-level reaction chamber. inside the heating groove.
  • the mixing tube has a plurality of continuous bends.
  • the secondary reaction chamber, the sample addition chamber, and the primary reaction chamber are all located on the first side, so The connection interface between the secondary reaction chamber and the first side of the chip body extends upward and forms a bell mouth that is smaller at the bottom and larger at the top.
  • a gas-liquid pipeline extending from bottom to top is also constructed in the secondary reaction chamber.
  • the lower opening of the gas-liquid pipeline is connected to the gas-liquid interface, and the upper opening of the gas-liquid pipeline is connected to the gas-liquid interface. Higher than the upper opening of the connection interface.
  • the invention provides an integrated micro-droplet chip and a micro-droplet multi-index detection method.
  • the chip body has at least one one-minute multi-mixing and reaction structure, and each one-minute multi-mixing and reaction structure respectively has The first-level reaction chamber, multiple sample addition chambers and multiple secondary reaction chambers allow the detection sample to be pre-amplified before generating micro-droplets, effectively increasing the concentration of the detection sample.
  • the increased concentration of the detection sample can be processed in one Under the action of multiple mixing and reaction structures, it is divided into more detection samples to ensure that each detection sample has a higher concentration, realizing multi-index detection of low-concentration samples, ensuring the sensitivity of sample detection, and improving The accuracy of the test results.
  • the primary reaction chamber, sample addition chamber and secondary reaction chamber in this technical solution are all integrated and constructed on the same chip body, thereby enabling primary amplification, mixing of samples and different systems,
  • the generation of micro-droplets, secondary amplification and droplet detection are integrated on the same chip.
  • the degree of integration and automation can be improved, which is an important technological breakthrough in the field of digital PCR.
  • Figure 1 is a schematic diagram of the principle of a reaction detection module of more than one minute (a specific example is one minute and four) in an integrated multi-index detection microdroplet chip according to an embodiment of the present invention
  • Figure 2 is a schematic three-dimensional structural diagram of an integrated multi-index detection microdroplet chip according to an embodiment of the present invention. The figure shows that there are two one-to-four reaction detection modules on the same chip body;
  • Figure 3 is a schematic diagram of a specific implementation of the mixing pipeline in the integrated multi-index detection microdroplet chip according to the embodiment of the present invention
  • Figure 4 is a schematic diagram of the internal structure of the integrated multi-index detection microdroplet chip in Figure 2;
  • Figure 5 is a schematic diagram of the micro droplet generation process according to the embodiment of the present invention.
  • Figure 6 is a schematic diagram of microdroplets being generated and stored in the secondary reaction chamber
  • Figure 7 is a schematic diagram of the state in the secondary reaction chamber after the integrated multi-index detection microdroplet chip in Figure 2 is flipped 180°;
  • Figure 8 is a diagram of the cooperation between the secondary amplification heating module and the secondary reaction chamber
  • Figure 9 is a schematic diagram of the state after passing oil into the secondary reaction chamber (that is, detecting the pushing oil) in the state of Figure 7;
  • Figure 10 is a schematic diagram of the microdroplet fluorescence detection process
  • Figure 11 is a schematic three-dimensional structural diagram of an integrated multi-index detection microdroplet chip according to another embodiment of the present invention, in which the secondary reaction chamber, the sample adding chamber and the primary reaction chamber are respectively located on both sides of the first side of the chip body;
  • Figure 12 is a schematic diagram of the principle of a one-to-two reaction detection module in another embodiment of the present invention.
  • Figure 13 is a schematic diagram of the principle of a one-to-eight reaction detection module in another embodiment of the present invention.
  • Figure 14 is a schematic diagram of the principle of an integrated pre-amplification microdroplet chip according to another embodiment of the present invention.
  • Figure 15 is a schematic diagram of the secondary reaction chamber of the integrated microdroplet chip in an embodiment of the present invention, in which a fluorescence detection area is integrated in the secondary reaction chamber.
  • Chip body 11. Primary reaction chamber; 111. Connection interface; 12. Sample addition chamber; 13. Secondary reaction chamber; 14. Gas-liquid interface; 15. Oil-liquid interface; 16. Fluorescence detection area; 2. Sealing cover; 31. First liquid outlet pipe; 32. Second liquid outlet pipe; 33. Mixing pipe; 34. Gas-liquid pipe; 35. Oil-liquid pipe; 36. Microdroplet observation area; 4. First-level amplification Heating module; 41. Heating protrusions; 5. Micro droplets; 6. Detection of pushing oil; 7. Secondary amplification heating module.
  • an integrated multi-index detection microdroplet chip including a chip body 1 , and at least one group of one-minute multi-mixing and reaction components are configured on the chip body 1 structure, a micro-droplet generation structure and a fluorescence detection area 16 corresponding to each one-minute multi-mixing and reaction structure.
  • Each one-minute multi-mixing and reaction structure includes: a first-level reaction chamber 11, which is used to store the first-level system and Perform primary amplification (also called pre-amplification) to amplify a detection sample with a first concentration (specifically, a nucleic acid sample) in the primary system to form a detection sample with a second concentration.
  • the second concentration is higher At the first concentration; multiple (at least two) sampling chambers 12 are used to store the secondary system, and multiple sampling chambers 12 simultaneously form controllable communication with the same primary reaction chamber 11; multiple (at least two) and the number is equal to the number of sampling chambers 12 in the same multi-mixing and reaction structure) the secondary reaction chambers 13 are in controllable communication with the plurality of sampling chambers 12 in one-to-one correspondence, and are used to store the samples from the first
  • the two-concentration detection sample and the secondary system generate micro-droplets 5 (specifically, water-in-oil droplets) at the micro-droplet generation structure and perform secondary amplification.
  • the fluorescence detection area 16 is used to detect the secondary amplification. Completed microdroplets 5.
  • the aforementioned first-level system contains the sample to be tested (specifically, a nucleic acid sample) and a pre-amplification system
  • the second-level system contains the digital PCR system and the stabilizer required for preparing microdroplets.
  • Different secondary systems that is, different primers and probes, can be added to the sample adding chamber 12 according to actual detection needs, thereby realizing the detection of different target sequences.
  • the aforementioned one-minute multiple mixing and reaction structure can be implemented in various forms, such as the one-minute two reaction detection module shown in Figure 12, or the one-minute four reaction detection module shown in Figure 1, or the one-minute four reaction detection module shown in Figure 13
  • the reaction detection module is one point eight, and it can be understood that it should be at least one point more and at least one point two.
  • the chip body 1 has at least one one-minute multi-mixing and reaction structure, and each one-minute multi-mixing and reaction structure has a primary reaction chamber 11, a plurality of sample addition chambers 12 and a plurality of secondary Reaction chamber 13, so that the detection sample first undergoes pre-amplification (that is, the aforementioned first-level amplification) before generating the microdroplets 5, effectively improving the detection sample
  • the concentration of the test sample after increasing the concentration can be divided into more test sample parts under the action of multi-mixing and reaction structure to ensure that each test sample has a higher concentration, realizing multiple low-concentration samples.
  • Indicator detection ensures the sensitivity of sample detection and improves the accuracy of detection results.
  • the first-level reaction chamber 11, the sample addition chamber 12 and the second-level reaction chamber 13 in this technical solution are all integrated into the same chip body 1 Therefore, the first-level amplification, the mixing of samples and different systems, the generation of micro-droplets, the second-level amplification and droplet detection can be integrated on the same chip.
  • the degree of integration and automation can be improved. It is a digital An important technological breakthrough in the field of PCR.
  • the chip body 1 also has a heating groove disposed adjacent to the primary reaction chamber 11, and the primary amplification heating module 4 has a heating protrusion 41, and the heating protrusion 41 can be inserted into the heating recess.
  • the heating protrusion 41 can extend into the groove and make close contact with the primary reaction chamber 11, thereby ensuring the heating efficiency of the primary amplification.
  • the first-level amplification heating module 4 can be implemented using a heating module in the industry, as long as it can be controlled and heated according to a preset cycle.
  • the specific implementation principle is not particularly limited in the present invention, as long as it has a
  • the structure of the heating protrusion 41 only needs to ensure that its heating heat can be quickly guided to the heating groove position and achieve efficient heating of the fluid in the primary reaction chamber 11 .
  • the aforementioned heating groove may specifically be a tapered groove to ensure a larger contact area with the heating protrusion 41 and achieve higher heat transfer efficiency.
  • the temperature cycle process during the first-level amplification process is to first perform a pre-denaturation at 95°C for 10 minutes, followed by 15 temperature cycles, each cycle includes 95°C for 5 seconds and 60°C for 15 seconds. Finally keep warm at 4°C. In order to reduce evaporation, 30 microliters of low-density anti-volatilization reagent can be placed in the primary reaction chamber 11 in advance.
  • the primary reaction chamber 11 has a plurality of first liquid outlet pipes 31 connected in parallel, and each sample addition chamber 12 has a second liquid outlet pipe 32 respectively.
  • the second liquid outlet pipe of each sample addition chamber 12 32 and the plurality of first liquid outlet pipes 31 are respectively gathered in the mixing pipe 33 in a one-to-one correspondence.
  • the plurality of mixing pipes 33 are controllably connected to the plurality of secondary reaction chambers 13 in a one-to-one correspondence.
  • the mixing pipe 33 has a plurality of continuous bends, which are staggered along the flow direction of the fluid, forming a roughly S-shaped structure as shown in Figure 3 in appearance. , can increase the flow resistance of the mixing pipe 33, slow down the flow speed of the fluid and continuously change the flow direction of the fluid, so that the sample and the system are mixed more fully, and finally a mixed detection sample is formed.
  • the fluorescence detection area 16 is located on the mixing pipe 33.
  • the fluorescence detection area 16 is arranged on the mixing pipe 33 so that the microdroplets 5 that have completed secondary amplification in the secondary reaction chamber 13 can be transferred from the secondary reaction chamber 13 to the secondary reaction chamber 13. 13 performs fluorescence detection on the passing microdroplets 5 while flowing in the direction of the sample addition chamber 12.
  • the structural design is more reasonable, the direction reuse of the pipeline is realized, and the chip structure is more streamlined and compact.
  • each one-minute multi-mixing and reaction structure also includes a plurality of fluid flow driving structures.
  • the number of fluid flow driving structures is equal to the number of mixing pipes 33 and the two are in one-to-one correspondence.
  • Each fluid flow driving structure includes a gas-liquid interface 14 constructed on the chip body 1. Through the gas-liquid interface 14, the corresponding primary reaction chamber 11 and the secondary reaction chamber 13 can be connected, as well as the sampling chamber 12 and the secondary reaction chamber 13. A first pressure difference is formed between them, so that the fluids in the primary reaction chamber 11 and the sampling chamber 12 respectively flow into the corresponding mixing pipes 33 under the action of the first pressure difference.
  • the aforementioned first pressure The difference refers to the pressure difference between the primary reaction chamber 11 and the secondary reaction chamber 13, and between the sample addition chamber 12 and the secondary reaction chamber 13 when the sample is mixed.
  • both can be called the first pressure difference , but in actual operation, the actual values of the two pressure differences may be different.
  • the first pressure difference can be formed inside the primary reaction chamber 11 and the sampling chamber 12 by adjusting the specific pressure at the gas-liquid interface 14, thereby effectively driving the fluid flow, and the operation is simple and convenient.
  • the gas-liquid interface 14 is connected to the interior of the secondary reaction chamber 13 through the gas-liquid pipeline 34, so that on the one hand, one-minute-multiple mixing and mixing can be achieved by adjusting the gas pressure (such as negative pressure) at the gas-liquid interface 14.
  • the fluid flow drive of the reaction structure can introduce detection and push oil 6 (also called floating oil) into the secondary reaction chamber 13 through the gas-liquid interface 14 when needed, thereby realizing the secondary reaction chamber 13.
  • the microdroplets 5 that have completed amplification enter the mixing pipe 33 to realize fluorescence detection of these microdroplets 5 .
  • each fluid flow driving structure also includes an oil interface 15 constructed on the chip body 1, which is used to connect the detection separation oil.
  • the oil interface 15 intersects with the mixing pipe 33 through the oil pipe 35, so as to be able to
  • the fluid is divided into a plurality of microdroplets 5 when flowing through the intersection of the oil pipe 35 and the mixing pipe 33 .
  • the external pressure is controlled to drive and detect the pushing oil 6 from the gas-liquid interface 14 into the secondary reaction chamber 13, so that the micro droplets 5 in the secondary reaction chamber 13 flow out from the secondary reaction chamber 13 to the intersection position.
  • the external pressure drives the detection separation oil from the oil interface 15 into the aforementioned intersection position, and the detection separation oil will flow out from the secondary reaction chamber 13 to the micro droplets 5 at the intersection position to separate and form a queue, and enter the fluorescence detection area 16,
  • oil is provided to the gas-liquid interface 14 (that is, the detection push oil 6 is also called floating oil), and the amplified droplets in the secondary reaction chamber 13 are moved through the buoyancy of the oil.
  • the micro droplets 5 floats, and under the buoyancy of the oil, the micro droplets 5 flow out of the secondary reaction chamber 13 and enter the mixing pipe 33, and flow through the micro droplet observation area 36 into the intersection (that is, the aforementioned intersection position ), is detected in the fluorescence detection area 16, and then finally enters the sample addition chamber 12.
  • the sample addition chamber 12 is a waste liquid pool.
  • the secondary reaction chamber 13 , the sampling chamber 12 , and the primary reaction chamber 11 are all located on the first side.
  • the connection interface 111 between the first-stage reaction chamber 13 and the first side of the chip body 1 extends upward and forms a bell mouth that is smaller at the bottom and larger at the top.
  • the bell-shaped connection interface 111 can facilitate the micro-droplets 5 to enter the secondary reaction chamber 13 from the mixing pipe 33, and also facilitate the micro-droplets 5 to enter the mixing pipe 33 from the secondary reaction chamber 13, thereby preventing the micro-droplets 5 from being detained. .
  • the secondary reaction chamber 13 and the sample adding The cavities 12 are all located on the first side (specifically the top surface) of the chip body 1.
  • the microdroplets 5 entering the secondary reaction chamber 13 are all gathered at the connection interface 111.
  • the chip needs to be The body 1 is turned upside down as a whole, that is, turned 180°, so that the microdroplets 5 can be located in the reaction area of the secondary reaction chamber 13 .
  • the secondary reaction chamber 13 is also constructed with a gas-liquid pipeline 34 extending from bottom to top.
  • the lower opening of the gas-liquid pipeline 34 is connected to the gas-liquid interface 14 , and the upper opening of the gas-liquid pipeline 34 is higher than the connection.
  • the upper opening of the interface 111 can prevent the generated micro-droplets 5 from further flowing out of the gas-liquid pipe 34 after entering the secondary reaction chamber 13 when the pressure in the secondary reaction chamber 13 is negative.
  • micro droplet observation area 36 there is a micro droplet observation area 36 between the mixing pipe 33 and the connection interface 111.
  • the flow area of the micro droplet observation area 36 is much larger than the flow area of the mixing pipe 33, that is, the micro droplet observation area 36 It is an expanded area (width becomes larger) on the mixing pipe 33 to reduce the flow rate of the microdroplets 5 entering this area, which can facilitate external camera imaging, record the shape of the droplets, and determine whether the state of the droplet generation process is normal. .
  • the first-level reaction chamber 11 and the sampling chamber 12 are equipped with a sealing cover 2, so that the operator can achieve the sealing function after adding samples into the first-level reaction chamber 11 and the sampling chamber 12. Therefore, It will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully enclosed digital PCR process.
  • the sampling chamber 12 is provided with a filter membrane or an exhaust hole. When the sampling chamber 12 becomes a waste liquid pool (that is, when the droplet chip is turned upside down), a certain amount of air is removed to prevent the sampling chamber 12 from Internal pressure builds up.
  • FIG 11 another implementation of a fully integrated microdroplet chip is given.
  • the difference between it and the integrated microdroplet chip shown in Figure 2 is that the secondary reaction chamber 13 and the sample addition chamber 12 They are respectively located on two opposite sides of the chip body 1. Specifically, the sample loading chamber 12 is located on the first side, and the secondary reaction chamber 13 is located on the second side. The second side and the first side are opposite to the chip body 1. On both sides, at this time, the working principle and process of the integrated micro-droplet chip are basically the same as the previous integrated micro-droplet chip.
  • the secondary reaction chamber 13 is located on the chip body 1 On the bottom side (sampling chamber 12 is on the top side), when the microdroplets 5 enter the secondary reaction chamber 13, they will directly fall to the reaction area at the bottom of the secondary reaction chamber 13 and be collected in the reaction area. Therefore, The integrated micro-droplet chip does not need to be flipped 180° after the droplet generation is completed, and can directly enter the subsequent amplification process.
  • a micro-droplet multi-index detection method based on an integrated micro-droplet chip is also provided.
  • the integrated micro-droplet chip includes a chip body 1, and the chip body 1 is configured with at least one group of one-minute multi-mixing and The reaction structure, the micro-droplet generation structure and the fluorescence detection area 16 are provided corresponding to each of the one-minute multi-mixing and reaction structures.
  • Each one-minute multi-mixing and reaction structure includes a first-level reaction chamber 11 and a plurality of sample addition chambers. 12 and multiple secondary reaction chambers 13; the micro-droplet multi-index detection method includes the following steps:
  • the first-level amplification reaction step controls the heating of the first-level system stored in the first-level reaction chamber 11, so that the first-level system
  • the detection sample in the system is amplified from the first concentration to the second concentration, and the second concentration is higher than the first concentration;
  • a first pressure difference is formed between the primary reaction chamber 11 and the sample adding chamber 12 and the corresponding secondary reaction chamber 13 respectively, so that the test sample with the second concentration is separated from the sample adding chamber.
  • the secondary systems within 12 are mixed to form a mixed detection sample;
  • the mixed detection sample is divided into multiple micro-droplets 5 under the action of oil in the micro-droplet generation structure, and the formed micro-droplets 5 are controlled to enter the secondary reaction chamber 13;
  • the secondary amplification reaction step controls heating of the microdroplets 5 stored in the secondary reaction chamber 13 to form secondary amplification
  • microdroplet fluorescence detection step multi-index fluorescence detection is performed on the mixed detection sample after secondary amplification in the fluorescence detection area 16 .
  • the chip body 1 has at least one one-minute multi-mixing and reaction structure, and each one-minute multi-mixing and reaction structure has a primary reaction chamber 11, a plurality of sample addition chambers 12 and a plurality of secondary
  • the reaction chamber 13 allows the detection sample to first undergo pre-amplification (i.e. the aforementioned first-level amplification) before generating the microdroplets 5, effectively increasing the concentration of the detection sample.
  • the increased concentration of the detection sample can be used in more than one minute.
  • it is divided into more detection samples to ensure that each detection sample has a higher concentration, realizing multi-index detection of low-concentration samples, ensuring the sensitivity of sample detection, and improving the detection results.
  • the first-level reaction chamber 11, the sample addition chamber 12 and the second-level reaction chamber 13 in this technical solution are all integrated and constructed on the same chip body 1, thereby making the first-level amplification, sample and different systems Mixing, microdroplet generation, secondary amplification and droplet detection are integrated on the same chip.
  • the degree of integration and automation can be improved, which is an important technological breakthrough in the field of digital PCR.
  • each fluid flow driving structure also includes an oil interface 15 constructed on the chip body 1.
  • the oil interface 15 intersects the mixing pipe 33 through the oil pipe 35.
  • the micro droplet generation step specifically includes: A second pressure difference is formed between the sampling chamber 12 and the gas-liquid interface 14, and a third pressure difference is formed between the oil-liquid interface 15 and the gas-liquid interface 14. The second pressure difference and the third pressure difference drive the mixing pipe respectively.
  • the mixed detection sample and generated oil in 33 form micro droplets 5 at the intersection of the oil pipe 35 and the mixing pipe 33 , and drive the formed micro droplets 5 into the secondary reaction chamber 13 .
  • the second pressure difference is formed between the sampling chamber 12 and the gas-liquid interface 14 and the third pressure difference is formed between the oil-liquid interface 15 and the gas-liquid interface 14, so that the second pressure difference and the third pressure difference are respectively
  • the mixed sample in the mixing pipe 33 and the detection separation oil (also called generated oil) of the oil interface 15 are driven into the intersection of the oil pipe 35 and the mixing pipe 33, and the generated micro droplets 5 enter and are stored in the secondary reaction chamber.
  • oil is provided to the oil-liquid interface 15, and negative pressure is provided to the gas-liquid interface 14. Under the action of the negative pressure, the mixed sample in the mixing pipe 33 and the oil at the oil-liquid interface 15 are driven respectively. Along the mixing pipe 33 and the oil pipe 35, they converge at the intersection of the two.
  • the mixed sample forms micro droplets 5 of uniform size under the action of the oil fluid shear force and surface tension, and finally forms micro droplets 5 under negative pressure. do After use, the micro droplets 5 finally enter the secondary reaction chamber 13 for storage. It should be noted that when the micro droplets 5 enter the micro droplet observation area 36, the flow rate of the micro droplets 5 forms a dense droplet community. , convenient for camera imaging and recording.
  • the secondary reaction chamber 13 and the sampling chamber 12 are located on the first side.
  • the microdroplets are generated.
  • the step also includes: a chip flipping step, which controls the chip body 1 to flip up and down 180°. Specifically, the microdroplets 5 in the secondary reaction chamber 13 are flipped from the side close to the connection interface 111 (Fig. 6) to the side away from the connection interface 111. side (Fig. 7). At this time, the position of the secondary reaction chamber 13 corresponding to the microdroplet 5 is the reaction area of the secondary reaction chamber 13, and the reaction area will enter the secondary amplification heating module 7 (Fig.
  • the secondary amplification heating module 7 should be made of a material with good thermal conductivity. In this example, aluminum alloy material is used. The width of the middle groove of the secondary amplification heating module 7 should be able to tightly press the reaction area on the secondary reaction chamber 13 combination to ensure good heating efficiency.
  • the chip body 1 after controlling the chip body 1 to flip up and down 180°, it also includes: a micro droplet reverse flow control step, controlling the injection of detection push oil 6 into the secondary reaction chamber 13 so that the secondary reaction chamber 13
  • the microdroplets 5 inside are driven into the mixing pipe 33 and can be stored in the sampling chamber 12 after the microdroplets 5 complete the microdroplet fluorescence detection step.
  • the sampling chamber 12 at this time is used as waste liquid. pool.
  • the chip body 1 also has a heating groove disposed adjacent to the first-level reaction chamber 11, before the first-level amplification reaction step, it also includes: controlling the movement of the chip body 1 to make the heating groove cover the first-level amplification heating module. 4 has a heating protrusion 41, and controls the primary amplification heating module 4 to heat amplification according to a preset cycle.
  • the primary system contains the sample to be tested and the pre-amplification system.
  • the secondary system contains the digital PCR system and the stabilizer required to prepare droplets.
  • the secondary system added to each well of the 12 sample chambers is different, mainly in the primers and probes, which are used to detect different target sequences.
  • a chip contains two sets of one-to-four structures (i.e., the aforementioned one-to-four mixing and reaction structures, in this embodiment a one-to-four reaction detection module is used as an example), that is, it includes two primary reaction chambers 11 and Eight sample adding chambers 12 and two primary reaction chambers 11 are connected to eight groups of channels. Each group of channels includes a sample adding chamber 12, an oil-liquid interface 15, a gas-liquid interface 14, and a secondary reaction chamber 13.
  • the structure of the primary reaction chamber 11 should adopt a design with high heat transfer efficiency, such as using a tapered groove that can fit closely with the heating protrusion 41 to achieve higher heat transfer efficiency.
  • the temperature cycle process includes a pre-denaturation at 95°C for 10 minutes, then 15 temperature cycles, each cycle includes 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation.
  • 30 microliters of low-density anti-volatilization reagent can be placed in the primary reaction chamber 11 in advance.
  • a negative pressure is provided to the gas-liquid interface 14, and the pressure is -200 mBar, so that a pressure difference is formed between the gas-liquid interface 14 and the primary reaction chamber 11 and the sample addition chamber 12.
  • the chip design ensures the flow in each first liquid outlet pipe 31.
  • the resistance consistency is controlled, and the pressure difference is controlled to drive the sample in the first-level reaction chamber 11 to flow uniformly to each first liquid outlet pipe 31. Therefore, 15 microliters of the first-level amplified sample will flow uniformly into each first liquid outlet pipe 31.
  • each second liquid outlet pipe 32 flows into a 30 microliter system, and the sample and system are in the mixing pipe 33, and then continue to move forward along the mixing pipe 33 driven by the first pressure difference.
  • the "S" shape position in the mixing pipe 33 Fig. 3, the "S" shape can make the fluid in the flow channel Continuously changing direction), so that the sample and system are mixed more fully, and finally a mixed sample is formed;
  • the generated oil required for droplet generation (that is, the aforementioned detection separation oil) is provided to the oil-liquid interface 15; a negative pressure is provided to the gas-liquid interface 14, and the pressure is -200mBar, so that the sampling chamber 12 and the oil-liquid interface 15 creates a pressure difference.
  • the sampling chamber 12 is connected to the mixing pipe 33
  • the oil-liquid interface 15 is connected to the oil-liquid pipe 35
  • the gas-liquid interface 14 is connected to the secondary reaction chamber 13 (through the gas-liquid pipe 34).
  • the oil pipeline 35 has two branches, located on both sides of the mixing pipe 33 , and both are connected to the oil interface 15 .
  • the mixing pipe 33 may include a micro-droplet observation area 36. The pipe in the observation area becomes wider and the flow rate decreases after the droplets enter, which can facilitate external camera imaging, record the shape of the droplets, and determine whether the state of the droplet generation process is normal.
  • the mixed sample enters the mixing pipe 33, and the generated oil enters the oil pipe 35, and meets at the cross structure (that is, the aforementioned intersection position).
  • the cross structure that is, the aforementioned intersection position.
  • Water-in-oil microdroplets of uniform size are formed5.
  • the channel depth at the intersection is approximately 70 microns, the width is 80 microns, and the microdroplet 5 size is approximately 100 microns.
  • the flow rate is reduced to form a dense droplet community, which facilitates camera imaging and recording.
  • the schematic diagram of the microdroplet generation process is shown in Figure 5.
  • connection interface 111 of the secondary reaction chamber 13 The bottom of the connection interface 111 has a slope structure (i.e., the aforementioned bell mouth). At the end of the droplet generation process, the pressure difference applied to the chip interface is removed. At this time, the microdroplets 5 should still be located below the gas-liquid pipe 34 ( Figure 6).
  • the secondary amplification heating module 7 should be made of a material with good thermal conductivity. In this example, aluminum alloy material is used.
  • the middle slot of the secondary amplification heating module 7 is The width should be able to tightly press the reaction area on the secondary reaction chamber 13 to ensure good heating efficiency, allowing the external system (i.e., the heating module) to heat and cool the reaction area from the left and right sides, not only ensuring the distance of temperature conduction Short, it also ensures a large contact area for efficient heat transfer.
  • the temperature cycle process includes a pre-denaturation at 95°C for 10 minutes, then 40 temperature cycles, each cycle includes 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation.
  • the secondary amplification reaction in the microdroplet 5 containing the template is also completed accordingly, and it is necessary to enter the droplet fluorescence detection step.
  • Inject detection oil that is, the above-mentioned detection push oil 6
  • the secondary reaction chamber 13 Fig. 9
  • a camera can also be used to perform bright field imaging on the droplet, thereby evaluating the state of the droplet after the amplification reaction.
  • the detection oil is injected into the oil interface 15.
  • the detection oil passes through the oil pipeline 35, merges with the droplet queue at the intersection position, and separates the closely arranged droplets into droplet queues with appropriate spacing.
  • the droplet queue passes through the fluorescence detection area 16 located in the mixing pipe 33 in sequence, as shown in FIG. 10 .
  • the corresponding position of the fluorescence detection area 16 is the fluorescence detection focus of the external system.
  • An external system focuses excitation light, such as lasers or LED narrowband light with wavelengths of 488nm, 532nm and 637nm, to the detection focus.
  • excitation light such as lasers or LED narrowband light with wavelengths of 488nm, 532nm and 637nm.
  • the fluorescence excited in the droplet including six kinds of fluorescence, including FAM, VIC, TAMRA, ROX, Cy5 and Q705
  • the fluorescence information of the droplets is used to delimit the signal threshold to distinguish negative and positive droplets, and the Poisson distribution model is used to calculate the copy number of the target molecule in the sample.
  • Each independent secondary reaction chamber 13 can detect 6 kinds of fluorescence, and each fluorescence corresponds to a target sequence.
  • there are 4 secondary reaction chambers 13, so one sample can detect 6*4 24 target molecules. , achieving high sensitivity and multi-index detection at the same time.
  • the droplets that have finally completed the fluorescence detection enter the sampling chamber 12. Since the sampling chamber 112 has been sealed by the sealing cover 2, it will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully enclosed digital PCR process.
  • the method of time-division multiplexing of the microdroplet generation structure is pioneered.
  • micro-droplet generation structures are used to achieve droplet generation.
  • the micro-droplet generation structure completes the separation of the droplet queue, ensuring the detection of droplet fluorescence signals.
  • this time-sharing multiplexing method for the first time on the flow-like digital PCR technology route, one chip can complete sample primary amplification, sample splitting and mixing, droplet generation, secondary amplification, and droplet detection.
  • the fully integrated and fully enclosed digital PCR process is an important technological breakthrough in the field of digital PCR.
  • the primary reaction chamber 11 is also connected to two first liquid outlet pipes 31 or eight first outlet pipes.
  • the liquid pipe 31 achieves a one-to-two or one-to-eight splitting effect (as shown in Figures 12 and 13).
  • the specific splitting method is the same as the above-mentioned one-to-four and will not be discussed in detail. But the number of detection indicators is different, one divided into two can detect 12 indicators, and one divided into eight can detect 48 indicators.
  • an integrated pre-amplification microdroplet chip including a chip body 1 on which at least one set of mixed and reaction structures, and a fluorescence detection area 16 corresponding to each mixing and reaction structure.
  • Each mixing and reaction structure includes: a first-level reaction chamber 11, which is used to store the first-level system and perform first-level amplification (also called (pre-amplification), so that the detection sample with the first concentration (specifically, a nucleic acid sample) in the primary system is amplified to form a detection sample with a second concentration, and the second concentration is higher than the first concentration; the sample addition chamber 12, It is used to store the secondary system, and the sample adding chamber 12 forms controllable communication with the primary reaction chamber 11 at the same time; the secondary reaction chamber 13 is controllably connected to the sample adding chamber 12 and the primary reaction chamber through the mixing pipe 33.
  • first-level reaction chamber 11 which is used to store the first-level system and perform first-level amplification (also called (pre-amplification), so that the detection sample with the first concentration (specifically, a nucleic acid sample) in the primary system is amplified to form a detection sample with a second concentration, and the second concentration is higher than the first concentration
  • the sample addition chamber 12 It is used to
  • the two-concentration test sample and the secondary system are mixed in the mixing pipe and stored in the secondary reaction chamber 13 in the form of microdroplets 5 (specifically, water-in-oil droplets) and undergo secondary amplification.
  • the fluorescence detection area 16 is used to detect microdroplets 5 after secondary amplification.
  • the aforementioned first-level system contains the sample to be tested (specifically, nucleic acid samples, for example) and the pre-amplification system
  • the second-level system contains the digital PCR system and the stabilizer required for preparing microdroplets
  • the sample addition chamber In 12 the secondary system can be added according to the actual detection needs, that is, the primers and probes required for the secondary reaction.
  • the aforementioned mixing and reaction structures can be implemented in various forms.
  • the chip body 1 has at least one mixing and reaction structure, and each mixing and reaction structure has a primary reaction chamber 11, a sample adding chamber 12 and a secondary reaction chamber 13 respectively, so that the detection sample can be generated during Microdroplets 5 are first pre-amplified (that is, the aforementioned first-level amplification), which effectively increases the concentration of the test sample.
  • the increased concentration of the test sample ensures the sensitivity of the test and improves the accuracy of the test results.
  • the primary reaction chamber 11, sample addition chamber 12 and secondary reaction chamber 13 in this technical solution are all integrated and constructed on the same chip body 1, thereby enabling primary amplification, mixing of samples and different systems, and mixing of micro droplets. Generation, secondary amplification and droplet detection are integrated on the same chip, and the degree of integration and automation can be improved, which is an important technological breakthrough in the field of digital PCR.
  • the chip body 1 also has a heating groove disposed adjacent to the primary reaction chamber 11, and the primary amplification heating module 4 has a heating protrusion 41, and the heating protrusion 41 can be inserted into the heating recess.
  • the heating protrusion 41 can extend into the groove and make close contact with the primary reaction chamber 11, thereby ensuring the heating efficiency of the primary amplification.
  • the first-level amplification heating module 4 can be implemented using a heating module in the industry, as long as it can be controlled and heated according to a preset cycle.
  • the specific implementation principle is not particularly limited in the present invention, as long as it has a
  • the structure of the heating protrusion 41 ensures that its heating heat can be quickly guided to the heating groove position and The fluid in the primary reaction chamber 11 only needs to be efficiently heated.
  • the aforementioned heating groove may specifically be a tapered groove to ensure a larger contact area with the heating protrusion 41 and achieve higher heat transfer efficiency.
  • the temperature cycle process during the first-level amplification process is to first perform a pre-denaturation at 95°C for 10 minutes, followed by 10 temperature cycles, each cycle includes 95°C for 5 seconds and 60°C for 15 seconds. Finally keep warm at 4°C. In order to reduce evaporation, 30 microliters of low-density anti-volatilization reagent can be placed in the primary reaction chamber 11 in advance.
  • the primary reaction chamber 11 has a first liquid outlet pipe 31
  • the sample adding chamber 12 has a second liquid outlet pipe 32
  • the second liquid outlet pipe 32 of the sample adding chamber 12 is combined with the first liquid outlet pipe 31
  • the aforementioned mixing pipe 33 is in controllable communication with the secondary reaction chamber 13.
  • the respective fluids in the two chambers are guided and collected through the first liquid outlet pipe 31 and the second liquid outlet pipe 32.
  • the mixing pipe 33 the primary system and the secondary system in the two chambers are mixed respectively in the mixing pipe 33. This mixing is realized during the flow process, which can have higher operating efficiency.
  • the mixing pipe 33 has a plurality of continuous bends, which are staggered along the flow direction of the fluid, forming a roughly S-shaped structure as shown in Figure 3 in appearance. , can increase the flow resistance of the mixing pipe 33, slow down the flow speed of the fluid and continuously change the flow direction of the fluid, so that the sample and the system are mixed more fully, and finally a mixed detection sample is formed.
  • the fluorescence detection area 16 is located on the mixing pipe 33.
  • the fluorescence detection area 16 is arranged on the mixing pipe 33 so that the microdroplets 5 that have completed secondary amplification in the secondary reaction chamber 13 can be transferred from the secondary reaction chamber 13 to the secondary reaction chamber 13. 13 performs fluorescence detection on the passing microdroplets 5 while flowing in the direction of the sample addition chamber 12.
  • the structural design is more reasonable, the direction reuse of the pipeline is realized, and the chip structure is more streamlined and compact.
  • each mixing and reaction structure also includes a micro-droplet generation structure.
  • Each micro-droplet generation structure includes a gas-liquid interface 14 constructed on the chip body 1, through which the gas-liquid interface 14 can be connected with it.
  • a first pressure difference is formed between the corresponding primary reaction chamber 11 and the secondary reaction chamber 13 and between the sampling chamber 12 and the secondary reaction chamber 13, so that the fluids in the primary reaction chamber 11 and the sampling chamber 12 are separated. It flows into the corresponding mixing pipe 33 under the action of the first pressure difference.
  • the aforementioned first pressure difference is respectively between the first-level reaction chamber 11 and the second-level reaction chamber 13, and between the sample adding chamber 12 and the second-level reaction chamber.
  • the first pressure difference can be formed inside the primary reaction chamber 11 and the sampling chamber 12 by adjusting the specific pressure at the gas-liquid interface 14, thereby effectively driving the fluid flow, and the operation is simple and convenient.
  • the gas-liquid interface 14 is connected to the interior of the secondary reaction chamber 13 through the gas-liquid pipeline 34, so that on the one hand, the mixing and reaction structure can be achieved by adjusting the gas pressure (such as negative pressure) at the gas-liquid interface 14.
  • Fluid flow drive can introduce detection and push oil 6 (also called floating oil) into the secondary reaction chamber 13 through the gas-liquid interface 14 when needed, thereby realizing secondary amplification in the secondary reaction chamber 13
  • detection and push oil 6 also called floating oil
  • the completed microdroplets 5 enter the mixing pipe 33 to realize fluorescence detection of these microdroplets 5 .
  • each microdroplet generation structure also includes an oil interface 15 constructed on the chip body 1, which is used to connect the detection separation oil.
  • the oil interface 15 intersects with the mixing pipe 33 through the oil pipe 35, so as to be able to When the fluid flows through the intersection of the oil pipe 35 and the mixing pipe 33 , it is divided into a plurality of microdroplets 5 .
  • the external pressure is controlled to drive and detect the pushing oil 6 from the gas-liquid interface 14 into the secondary reaction chamber 13, so that the micro droplets 5 in the secondary reaction chamber 13 flow out from the secondary reaction chamber 13 to the intersection position.
  • the external pressure drives the detection separation oil from the oil interface 15 into the aforementioned intersection position, and the detection separation oil will flow out from the secondary reaction chamber 13 to the micro droplets 5 at the intersection position to separate and form a queue, and enter the fluorescence detection area 16,
  • oil is provided to the gas-liquid interface 14 (that is, the detection push oil 6 is also called floating oil), and the amplified droplets in the secondary reaction chamber 13 are moved through the buoyancy of the oil.
  • the micro droplets 5 floats, and under the buoyancy of the oil, the micro droplets 5 flow out of the secondary reaction chamber 13 and enter the mixing pipe 33, and flow through the micro droplet observation area 36 into the intersection (that is, the aforementioned intersection position ), is detected in the fluorescence detection area 16, and then finally enters the sample addition chamber 12.
  • the sample addition chamber 12 is a waste liquid pool.
  • the secondary reaction chamber 13 , the sampling chamber 12 , and the primary reaction chamber 11 are all located on the first side.
  • the connection interface 111 between the first-stage reaction chamber 13 and the first side of the chip body 1 extends upward and forms a bell mouth that is smaller at the bottom and larger at the top.
  • the bell-shaped connection interface 111 can facilitate the micro-droplets 5 to enter the secondary reaction chamber 13 from the mixing pipe 33, and also facilitate the micro-droplets 5 to enter the mixing pipe 33 from the secondary reaction chamber 13, thereby preventing the micro-droplets 5 from being detained. .
  • the secondary reaction chamber 13 and the sampling chamber 12 are both located on the first side (specifically, the top surface) of the chip body 1 , and the microdroplets 5 entering the secondary reaction chamber 13 are all gathered at the connection At the interface 111, when performing a secondary amplification reaction, the entire chip body 1 needs to be turned upside down, that is, flipped 180°, so that the microdroplets 5 can be located in the reaction area of the secondary reaction chamber 13.
  • the secondary reaction chamber 13 is also constructed with a gas-liquid pipeline 34 extending from bottom to top.
  • the lower opening of the gas-liquid pipeline 34 is connected to the gas-liquid interface 14 , and the upper opening of the gas-liquid pipeline 34 is higher than the connection.
  • the upper opening of the interface 111 can prevent the generated micro-droplets 5 from further flowing out of the gas-liquid pipe 34 after entering the secondary reaction chamber 13 when the pressure in the secondary reaction chamber 13 is negative.
  • micro droplet observation area 36 there is a micro droplet observation area 36 between the mixing pipe 33 and the connection interface 111.
  • the flow area of the micro droplet observation area 36 is much larger than the flow area of the mixing pipe 33, that is, the micro droplet observation area 36 It is an expanded area (width becomes larger) on the mixing pipe 33 to reduce the flow rate of the microdroplets 5 entering this area, which can facilitate external camera imaging, record the shape of the droplets, and determine whether the state of the droplet generation process is normal. .
  • the first-level reaction chamber 11 and the sampling chamber 12 are equipped with a sealing cover 2, so that the operator can achieve the sealing function after adding samples into the first-level reaction chamber 11 and the sampling chamber 12. Therefore, It will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully enclosed digital PCR process.
  • the sample loading chamber 12 A filter membrane or exhaust hole is provided to remove a certain amount of air when the sampling chamber 12 becomes a waste liquid pool (that is, when the droplet chip is turned upside down) to prevent pressure from accumulating in the sampling chamber 12 .
  • FIG 11 another implementation of a fully integrated microdroplet chip is given.
  • the difference between it and the integrated microdroplet chip shown in Figure 2 is that the secondary reaction chamber 13 and the sample addition chamber 12 They are respectively located on two opposite sides of the chip body 1. Specifically, the sample loading chamber 12 is located on the first side, and the secondary reaction chamber 13 is located on the second side. The second side and the first side are opposite to the chip body 1. On both sides, at this time, the working principle and process of the integrated micro-droplet chip are basically the same as the previous integrated micro-droplet chip.
  • the secondary reaction chamber 13 is located on the chip body 1 On the bottom side (sampling chamber 12 is on the top side), when the microdroplets 5 enter the secondary reaction chamber 13, they will directly fall to the reaction area at the bottom of the secondary reaction chamber 13 and be collected in the reaction area. Therefore, The integrated micro-droplet chip does not need to be flipped 180° after the droplet generation is completed, and can directly enter the subsequent amplification process.
  • a detection method based on an integrated micro-droplet chip is also provided.
  • the integrated micro-droplet chip includes a chip body 1, and the chip body 1 is configured with at least one set of mixing and reaction structures, and each of the The mixing and reaction structures are provided with corresponding micro-droplet generation structures and fluorescence detection areas 16.
  • Each mixing and reaction structure includes a primary reaction chamber 11, a sample addition chamber 12 and a secondary reaction chamber 13; the micro-droplet detection method includes the following steps :
  • the first-level amplification reaction step controls heating of the first-level system stored in the first-level reaction chamber 11, so that the detection sample in the first-level system is amplified from the first concentration to a second concentration, and the second concentration is higher than the first concentration. concentration;
  • a first pressure difference is formed between the primary reaction chamber 11 and the sample adding chamber 12 and the corresponding secondary reaction chamber 13 respectively, so that the test sample with the second concentration is separated from the sample adding chamber.
  • the secondary systems within 12 are mixed to form a mixed detection sample;
  • the mixed detection sample is divided into multiple micro-droplets 5 under the action of oil in the micro-droplet generation structure, and the formed micro-droplets 5 are controlled to enter the secondary reaction chamber 13;
  • the secondary amplification reaction step controls heating of the microdroplets 5 stored in the secondary reaction chamber 13 to form secondary amplification
  • microdroplet fluorescence detection step multi-index fluorescence detection is performed on the mixed detection sample after secondary amplification in the fluorescence detection area 16 .
  • the chip body 1 has at least one mixing and reaction structure, and each mixing and reaction structure has a primary reaction chamber 11, a sample adding chamber 12 and a secondary reaction chamber 13 respectively, so that the detection sample can be Before generating the microdroplets 5, pre-amplification (that is, the aforementioned first-level amplification) is first performed, which effectively increases the concentration of the detection sample, ensures the sensitivity of sample detection, and improves the accuracy of the detection results.
  • pre-amplification that is, the aforementioned first-level amplification
  • the primary reaction chamber 11, sample addition chamber 12 and secondary reaction chamber 13 are all integrated and constructed on the same chip body 1, thereby enabling primary amplification, mixing of samples and different systems, generation of micro droplets, and secondary Amplification and droplet detection are integrated on the same chip, which is highly integrated and automatic.
  • the degree of automation can be improved, which is an important technological breakthrough in the field of digital PCR.
  • each fluid flow driving structure also includes an oil interface 15 constructed on the chip body 1.
  • the oil interface 15 intersects the mixing pipe 33 through the oil pipe 35.
  • the micro droplet generation step specifically includes: A second pressure difference is formed between the sampling chamber 12 and the gas-liquid interface 14, and a third pressure difference is formed between the oil-liquid interface 15 and the gas-liquid interface 14. The second pressure difference and the third pressure difference drive the mixing pipe respectively.
  • the mixed detection sample and generated oil in 33 form micro droplets 5 at the intersection of the oil pipe 35 and the mixing pipe 33 , and drive the formed micro droplets 5 into the secondary reaction chamber 13 .
  • the second pressure difference is formed between the sampling chamber 12 and the gas-liquid interface 14 and the third pressure difference is formed between the oil-liquid interface 15 and the gas-liquid interface 14, so that the second pressure difference and the third pressure difference are respectively
  • the mixed sample in the mixing pipe 33 and the detection separation oil (also called generated oil) of the oil interface 15 are driven into the intersection of the oil pipe 35 and the mixing pipe 33, and the generated micro droplets 5 enter and are stored in the secondary reaction chamber.
  • oil is provided to the oil-liquid interface 15, and negative pressure is provided to the gas-liquid interface 14. Under the action of the negative pressure, the mixed sample in the mixing pipe 33 and the oil at the oil-liquid interface 15 are driven respectively. Along the mixing pipe 33 and the oil pipe 35, they converge at the intersection of the two.
  • the mixed sample forms micro droplets 5 of uniform size under the action of the oil fluid shear force and surface tension, and finally forms micro droplets 5 under negative pressure. Under the action, the micro droplets 5 finally enter the secondary reaction chamber 13 for storage. It should be noted that when the micro droplets 5 enter the micro droplet observation area 36, the flow rate of the micro droplets 5 forms a dense droplet community. , convenient for camera imaging recording.
  • the secondary reaction chamber 13 and the sampling chamber 12 are located on the first side.
  • the microdroplets are generated.
  • the step also includes: a chip flipping step, which controls the chip body 1 to flip up and down 180°. Specifically, the microdroplets 5 in the secondary reaction chamber 13 are flipped from the side close to the connection interface 111 (Fig. 6) to the side away from the connection interface 111. side (Fig. 7). At this time, the position of the secondary reaction chamber 13 corresponding to the microdroplet 5 is the reaction area of the secondary reaction chamber 13, and the reaction area will enter the secondary amplification heating module 7 (Fig.
  • the secondary amplification heating module 7 should be made of a material with good thermal conductivity. In this example, aluminum alloy material is used. The width of the middle groove of the secondary amplification heating module 7 should be able to tightly press the reaction area on the secondary reaction chamber 13 combination to ensure good heating efficiency.
  • the chip body 1 after controlling the chip body 1 to flip up and down 180°, it also includes: a micro droplet reverse flow control step, controlling the injection of detection push oil 6 into the secondary reaction chamber 13 so that the secondary reaction chamber 13
  • the microdroplets 5 inside are driven into the mixing pipe 33 and can be stored in the sampling chamber 12 after the microdroplets 5 complete the microdroplet fluorescence detection step.
  • the sampling chamber 12 at this time is used as waste liquid. pool.
  • the chip body 1 also has a heating groove disposed adjacent to the first-level reaction chamber 11, before the first-level amplification reaction step, it also includes: controlling the movement of the chip body 1 to make the heating groove cover the first-level amplification heating module. 4 has a heating protrusion 41, and controls the primary amplification heating module 4 to heat amplification according to a preset cycle.
  • the secondary reaction chamber 13 has a flat cavity structure, and the fluorescence detection area 16 is constructed on the secondary reaction chamber 13.
  • the microdroplets 5 are formed by the interaction between the oil phase entering the secondary reaction chamber 13 and the mixed sample in the flat cavity structure; specifically, the corresponding secondary reaction chamber 13 can pre-store the generated oil, that is, The aforementioned oil phase, under the combined effect of the flat cavity structure, the incoming mixed sample, that is, the mixed sample, automatically undergoes interface rupture in the secondary reaction chamber 13, forming micro droplets 5, and forming a single layer tiled state.
  • micro-droplets 5 After the generation of micro-droplets 5 is completed, the micro-droplets 5 remain in a single-layer tiled state for secondary amplification; after the secondary amplification, the external fluorescence imaging module performs fluorescence excitation and imaging on the tiled micro-droplets 5, and Identify the imaging photos, determine the negative and positive microdroplets, and calculate the copy number in the sample based on the Poisson distribution.
  • the aforementioned oil phase may not be stored in advance, but may be introduced in real time.
  • the flat cavity structure has a plurality of micro pits 131 on the bottom wall of the cavity, and the micro droplets 5 are formed by the mixed sample entering the micro pits 131 under the physical separation of the micro pits 131 .
  • the micro-pits 131 are micron-sized pits.
  • the external fluorescence imaging module After the micro-droplets 5 in the micro-pit 131 undergo secondary amplification, the external fluorescence imaging module performs fluorescence excitation and imaging on the tiled micro-droplets 5, and identifies the imaging photos to determine whether the micro-droplets are negative or positive, and based on The Poisson distribution calculates the number of copies in a sample.
  • the generated oil has been pre-stored in the secondary reaction chamber 13 .
  • the operation process of using the integrated pre-amplification microdroplet chip of the present invention is further elaborated below in combination with the reagent system:
  • the primary reaction chamber 11 contains the sample to be tested and the pre-amplification system. Then, add 30 microliters of the secondary system into the sample addition chamber 12. The secondary system The volume contains the digital PCR system and the stabilizer required to prepare droplets. The secondary system, including primers and probes, is added to the 12 holes of the sample loading chamber.
  • the temperature cycle process includes a pre-denaturation at 95°C for 10 minutes, then 10 temperature cycles, each cycle includes 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation.
  • 30 microliters of low-density anti-volatilization reagent can be placed in the primary reaction chamber 11 in advance.
  • a negative pressure is provided to the gas-liquid interface 14, and the pressure is -200 mBar, so that a pressure difference is formed between the gas-liquid interface 14, the first-level reaction chamber 11 and the sample addition chamber 12, and the pressure difference is controlled to drive the sample in the first-level reaction chamber 11 to flow to the first
  • the liquid outlet pipe 31 simultaneously controls the pressure difference to drive the system (the aforementioned secondary system) in each sampling chamber 12 to flow to the second liquid outlet pipe 32.
  • the outlet pipe 32 flows into 30 microliters of the system.
  • the sample and the system are mixed in the mixing pipe 33, and then continue to move forward along the mixing pipe 33 driven by the first pressure difference.
  • the shape is in the correct position ( Figure 3, the "S" shape can continuously change the direction of the fluid in the flow channel), the sample and the system are mixed more fully, and finally a mixed sample is formed;
  • the generated oil required for droplet generation (that is, the aforementioned detection separation oil) is provided to the oil-liquid interface 15; a negative pressure is provided to the gas-liquid interface 14, and the pressure is -200mBar, so that the sampling chamber 12 and the oil-liquid interface 15 creates a pressure difference.
  • the sampling chamber 12 is connected to the mixing pipe 33
  • the oil-liquid interface 15 is connected to the oil-liquid pipe 35
  • the gas-liquid interface 14 is connected to the secondary reaction chamber 13 (through the gas-liquid pipe 34).
  • the oil pipeline 35 has two branches, located on both sides of the mixing pipe 33 , and both are connected to the oil interface 15 .
  • the mixing pipe 33 may include a micro-droplet observation area 36. The pipe in the observation area becomes wider and the flow rate decreases after the droplets enter, which can facilitate external camera imaging, record the shape of the droplets, and determine whether the state of the droplet generation process is normal.
  • the mixed sample enters the mixing pipe 33, and the generated oil enters the oil pipe 35, and meets at the cross structure (that is, the aforementioned intersection position).
  • the cross structure that is, the aforementioned intersection position.
  • Water-in-oil microdroplets of uniform size are formed5.
  • the channel depth at the intersection is approximately 70 microns, the width is 80 microns, and the microdroplet 5 size is approximately 100 microns.
  • the flow rate is reduced to form a dense droplet community, which facilitates camera imaging and recording.
  • the schematic diagram of the microdroplet generation process is shown in Figure 5.
  • connection interface 111 of the secondary reaction chamber 13 The bottom of the connection interface 111 has a slope structure (i.e., the aforementioned bell mouth). At the end of the droplet generation process, the pressure difference applied to the chip interface is removed. At this time, the microdroplets 5 should still be located below the gas-liquid pipe 34 ( Figure 6).
  • the secondary amplification heating module 7 should be made of a material with good thermal conductivity. In this example, aluminum alloy material is used. The width of the middle groove of the secondary amplification heating module 7 should be able to cover the reaction area on the secondary reaction chamber 13.
  • the external system i.e., the heating module
  • the temperature cycle process includes a pre-denaturation at 95°C for 10 minutes, then 40 temperature cycles, each cycle includes 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation.
  • the secondary amplification reaction in the microdroplet 5 containing the template is also completed accordingly, and it is necessary to enter the droplet fluorescence detection step.
  • Inject detection oil that is, the above-mentioned detection push oil 6
  • the secondary reaction chamber 13 Fig. 9
  • a camera can also be used to perform bright field imaging on the droplet, thereby evaluating the state of the droplet after the amplification reaction.
  • the detection oil is injected into the oil interface 15.
  • the detection oil passes through the oil pipeline 35, merges with the droplet queue at the intersection position, and separates the closely arranged droplets into droplet queues with appropriate spacing.
  • the droplet queue passes through the fluorescence detection area 16 located in the mixing pipe 33 in sequence, as shown in FIG. 10 .
  • the corresponding position of the fluorescence detection area 16 is the fluorescence detection focus of the external system.
  • An external system focuses excitation light, such as lasers or LED narrowband light with wavelengths of 488nm, 532nm and 637nm, to the detection focus.
  • excitation light such as lasers or LED narrowband light with wavelengths of 488nm, 532nm and 637nm.
  • the fluorescence excited in the droplet including six kinds of fluorescence, including FAM, VIC, TAMRA, ROX, Cy5 and Q705
  • the fluorescence information of the droplets is used to delimit the signal threshold to distinguish negative and positive droplets, and the Poisson distribution model is used to calculate the copy number of the target molecule in the sample.
  • the droplets that have finally completed the fluorescence detection enter the sampling chamber 12. Since the sampling chamber 112 has been sealed by the sealing cover 2, it will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully enclosed digital PCR process.
  • the method of time-division multiplexing of the microdroplet generation structure is pioneered.
  • micro-droplet generation structures are used to achieve droplet generation.
  • the micro-droplet generation structure completes the separation of the droplet queue, ensuring the detection of droplet fluorescence signals.
  • this time-sharing multiplexing method for the first time on the flow-like digital PCR technology route, one chip can complete sample primary amplification, sample splitting and mixing, droplet generation, secondary amplification, and droplet detection.
  • the fully integrated and fully enclosed digital PCR process is an important technological breakthrough in the field of digital PCR.
  • fluorescence imaging can be used to perform fluorescence detection on the microdroplets after secondary amplification.
  • the secondary reaction chamber 13 contains a fluorescence detection area, as shown in Figure 12.
  • the steps before microdroplet generation are the same as in the above embodiment.
  • the oil phase also relies on the interaction with the mixed sample to form microdroplets of uniform size. After the microdroplets are generated, the microdroplets enter the secondary reaction chamber to form a single layer of tiled droplets, and remain in the tiled state for secondary amplification.
  • the external fluorescence imaging module After the second-level amplification, the external fluorescence imaging module performs fluorescence excitation and imaging on the tiled microdroplets, identifies the imaging photos, determines the negative and positive microdroplets, and calculates the copy number in the sample based on the Poisson distribution.
  • fluorescence imaging can be used to perform fluorescence detection on the microdroplets after secondary amplification.
  • the secondary reaction chamber 13 contains a fluorescence detection area.
  • the steps before microdroplet generation are the same as in the above embodiment.
  • the generated oil has been pre-stored in the secondary reaction chamber 13. Relying on the pipeline structure, the mixed sample automatically undergoes interface rupture in the secondary reaction chamber 13 to form micro droplets and form a single layer. status. After the generation of microdroplets is completed, the microdroplets remain in a single-layer flat state for secondary amplification.
  • the external fluorescence imaging module After the second-level amplification, the external fluorescence imaging module performs fluorescence excitation and imaging on the tiled micro-droplets, and identifies the imaging photos to determine the micro-droplets. Negative and positive, and calculate the copy number in the sample according to the Poisson distribution.
  • the generated oil has been pre-stored in the secondary reaction chamber.
  • fluorescence imaging can be used to perform fluorescence detection on the microdroplets after secondary amplification.
  • the secondary reaction chamber 13 contains a fluorescence detection area.
  • the steps before microdroplet generation are the same as in the above embodiment.
  • the secondary reaction chamber 13 contains a plurality of micro pits 131, and the micro pits have a micrometer scale.
  • the mixed sample fills the micropits in turn, and uses the micropits to form spatially separated independent microdroplets. After the microdroplets are generated, oil can be used to seal the micropits to further separate each microdroplet.
  • the external fluorescence imaging module After the micro-droplets in the micro-pit undergo secondary amplification, the external fluorescence imaging module performs fluorescence excitation and imaging on the tiled micro-droplets, and identifies the imaging photos to determine the negative and positive micro-droplets according to the Poisson distribution. Calculate the number of copies in the sample.
  • the generated oil has been pre-stored in the secondary reaction chamber 13 .

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Abstract

本申请提供一种一体式微液滴芯片及其微液滴多指标检测方法,属于数字PCR分析仪技术领域，该微液滴芯片包括芯片本体,芯片本体上构造有至少一组一分多混合及反应结构、微液滴生成结构及荧光检测区,每一一分多混合及反应结构包括:一级反应腔,使一级体系中具有第一浓度的检测样本扩增形成具有第二浓度的检测样本,第二浓度高于第一浓度;多个加样腔;多个二级反应腔,与多个加样腔分别一一对应可控连通,用于存储由第二浓度的检测样本及二级体系生成的微液滴并进行二级扩增。本申请有效提高检测样本的浓度,提高浓度后的检测样本被划分为更多检测样本份数而保证每份检测样本皆具有较高的浓度,实现低浓度样本的多指标检测,保证样本检测的灵敏度,提高检测结果的准确性。

Description

一体式微液滴芯片及其微液滴多指标检测方法
本发明要求于2022年07月05日提交中国专利局、申请号为202210781877.3、发明名称为“一体式多指标检测微液滴芯片”的中国专利申请的优先权、2022年07月05日提交中国专利局、申请号为202221708244.1、发明名称为“一体式预扩增微液滴芯片”的中国专利申请的优先权、2022年07月05日提交中国专利局、申请号为202210781929.7、发明名称为“一体式微液滴芯片的微液滴多指标检测方法”的中国专利申请的优先权、2022年07月05日提交中国专利局、申请号为202221708607.1、发明名称为“一体式多指标检测微液滴芯片”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于数字PCR分析仪技术领域，具体涉及一种一体式微液滴芯片及其微液滴多指标检测方法。
背景技术
数字PCR技术被称为第三代PCR技术，具有绝对定量和单分子检测灵敏度的优点，在分子诊断领域有着重要的应用前景。数字PCR技术中一种主流的技术路线为采用液滴微流控芯片，在该芯片内将反应体系分割成数万乃至数百万尺寸均一的液滴(也即微液滴)，每个液滴独立进行核酸扩增和荧光检测，通过荧光检测结果利用数学模型计算出样本中目标分子的精确拷贝数。
数字PCR技术虽然具有高检测灵敏度和绝对定量的优势，但在低浓度(在一些情况下也成为低丰度)核酸检测场景仍然面临着不小的挑战。例如在肿瘤液体活检、器官移植排斥监测、病原体超敏检测等领域，不仅要求在大量背景样本中检测罕见的目标序列，还要同时对多种可能的罕见目标序列进行区分。数字PCR技术受限于荧光染料的光谱分布限制，一个样本只能同时放置数种荧光探针，对应数种检测目标，难以满足同时区分数十种目标序列的要求。为了提高数字PCR的多指标检测，往往需要将样本分成多份进行检测，每一份检测数种不同的目标序列，从而满足指标数的要求。但分割低浓度的样本会带来灵敏度降低的问题，这会直接影响检测结果的准确性。
发明内容
因此，本发明要解决的技术问题在于提供一种一体式微液滴芯片及其微液滴多指标检测方法，以克服现有技术中低浓度的检测样本在检测时分割过多份数的检测样本导致检测灵敏度降低、检测结果不准确的不足。
为了解决上述问题，本发明提供一种一体式多指标检测微液滴芯片，包括芯片本体，所述芯片本体上构造有至少一组一分多混合及反应结构、与每个所述一分多混合及反应结构对应设置的微液滴生成结构及荧光检测区，每一所述一分多混合及反应结构包括：
一级反应腔，用于存储一级体系并进行一级扩增，以使所述一级体系中具有第一浓度的检测样本扩增形成具有第二浓度的检测样本，所述第二浓度高于所述第一浓度；
多个加样腔，用于存储二级体系，且多个所述加样腔同时与同一所述一级反应腔形成可控连通；
多个二级反应腔，与多个所述加样腔分别一一对应可控连通，用于存储由所述第二浓度的检测样本及所述二级体系在所述微液滴生成结构处生成的微液滴并进行二级扩增；
所述荧光检测区用于检测二级扩增完毕的微液滴。
在一些实施方式中，所述一级反应腔具有多个并联的第一出液管道，每个所述加样腔分别具有第二出液管道，每个所述加样腔的所述第二出液管道与多个所述第一出液管道分别一一对应地汇总于混合管道，多个所述混合管道分别与多个所述二级反应腔一一对应可控连通。
在一些实施方式中，每一所述一分多混合及反应结构还包括多个流体流动驱动结构，所述流体流动驱动结构的数量与所述混合管道的个数相等，每个所述流体流动驱动结构包括构造于所述芯片本体上的气液接口，通过所述气液接口能够将与其对应的所述一级反应腔与二级反应腔之间及加样腔与二级反应腔形成第一压力差，以使所述一级反应腔及加样腔内部的流体分别在所述第一压力差的作用下流入对应的所述混合管道内。
在一些实施方式中，每个所述流体流动驱动结构还包括构造于所述芯片本体上的油液接口，所述油液接口通过油液管道与所述混合管道形成交叉，以能够在流体在流经所述油液管道及所述混合管道的交叉位置时被分割成为多个所述微液滴。
本发明还提供一种如上述的一体式多指标检测微液滴芯片的微液滴多指标检测方法，包括如下步骤：一级扩增反应步骤，控制对存储于所述一级反应腔内的一级体系进行加热，以使所述一级体系中检测样本由第一浓度扩增为第二浓度，所述第二浓度高于所述第一浓度；样本分流及混合步骤，控制所述一级反应腔、所述加样腔分别与所述二级反应腔之间 形成第一压力差，以使所述具有第二浓度的检测样本与所述加样腔内的二级体系混合形成混合检测样本；微液滴生成步骤，于所述微液滴生成结构内将所述混合检测样本在油液的作用下分割为多个微液滴，并控制形成的所述微液滴进入所述二级反应腔内；二级扩增反应步骤，控制对存储于所述二级反应腔内的所述微液滴加热形成二级扩增；微液滴荧光检测步骤，于所述荧光检测区处对二级扩增后的所述混合检测样本进行多指标的荧光检测。
在一些实施方式中，所述微液滴生成步骤具体包括：控制所述加样腔与所述气液接口之间形成第二压力差，并控制所述油液接口与所述气液接口之间形成第三压力差，所述第二压力差以及第三压力差分别驱动所述混合管道内的混合检测样本及生成油在所述油液管道及混合管道的交叉位置形成所述微液滴，并驱动形成的所述微液滴进入所述二级反应腔内。
在一些实施方式中，在所述二级扩增反应步骤之前，在微液滴生成步骤之后还包括：芯片翻转步骤，控制所述芯片本体上下翻转180°。
在一些实施方式中，在控制所述芯片本体上下翻转180°后，还包括：微液滴反向流动控制步骤，控制向所述二级反应腔内注入检测推动油，以使所述二级反应腔内的微液滴被驱动流入所述混合管道内，并能够在所述微液滴完成微液滴荧光检测步骤之后存储于所述加样腔内。
本发明还提供一种一体式预扩增微液滴芯片，包括芯片本体，所述芯片本体上构造有至少一组混合及反应结构、与每个所述混合及反应结构对应设置的荧光检测区，每一所述混合及反应结构包括：一级反应腔，用于存储一级体系并进行一级扩增，以使所述一级体系中具有第一浓度的检测样本扩增形成具有第二浓度的检测样本，所述第二浓度高于所述第一浓度；加样腔，用于存储二级体系，且所述加样腔同时与所述一级反应腔形成可控连通；二级反应腔，与所述加样腔以及所述一级反应腔通过混合管道对应可控连通，所述第二浓度的检测样本及所述二级体系在所述混合管道中混合后以微液滴的形式存储于所述二级反应腔内并进行二级扩增；所述荧光检测区用于检测二级扩增完毕的微液滴。
在一些实施方式中，所述芯片本体上还具有与所述一级反应腔临近设置的加热凹槽，一级扩增加热模块具有加热凸起件，所述加热凸起件能够插装于所述加热凹槽内。
在一些实施方式中，所述混合管道具有多个连续的弯折部。
在一些实施方式中，以所述芯片本体的第一侧面处于水平方位为参照，所述二级反应腔与所述加样腔、所述一级反应腔皆处于所述第一侧面上，所述二级反应腔与所述芯片本体的所述第一侧面的连接接口向上延伸，且成下小上大的喇叭口。
在一些实施方式中，所述二级反应腔内还构造有自下而上延伸的气液管道，所述气液管道的下口与所述气液接口连通，所述气液管道的上口高于所述连接接口的上口。
本发明提供的一种一体式微液滴芯片及其微液滴多指标检测方法，所述芯片本体上至少具有一个一分多混合及反应结构，而每一一分多混合及反应结构则分别具有一级反应腔、多个加样腔及多个二级反应腔，从而使检测样本在生成微液滴之前首先进行预扩增，有效提高检测样本的浓度，提高浓度后的检测样本能够在一分多混合及反应结构的作用下被划分为更多检测样本份数而保证每份检测样本皆具有较高的浓度，实现了低浓度样本的多指标检测，保证了样本检测的灵敏度，提高了检测结果的准确性，同时，该技术方案中的一级反应腔、加样腔以及二级反应腔皆被集成构造于同一芯片本体上，从而使一级扩增、样本与不同体系的混合、微液滴的生成、二级扩增和液滴检测集成于同一个芯片上完成，集成度以及自动化程度都能够得到提高，是数字PCR领域的一个重要的技术突破。
附图说明
图1为本发明实施例的一体式多指标检测微液滴芯片中的一分多(具体示例出一分四)反应检测模块的原理示意图；
图2为本发明实施例的一体式多指标检测微液滴芯片的一种立体结构示意图，图中示出了同一芯片本体上具有两个一分四反应检测模块；
图3为本发明实施例的一体式多指标检测微液滴芯片中的混合管道的一种具体实现方式的示意图；
图4为图2中的一体式多指标检测微液滴芯片的内部结构示意图；
图5为本发明实施例的微液滴生成过程示意图；
图6为微液滴生成后存储于二级反应腔内的示意图；
图7为图2中的一体式多指标检测微液滴芯片翻转180°后二级反应腔内的状态示意；
图8为二级扩增加热模块与二级反应腔的配合状态图；
图9为图7状态下向二级反应腔内通入油液(也即检测推动油)后的状态示意；
图10为微液滴荧光检测过程示意图；
图11为本发明另一实施例的一体式多指标检测微液滴芯片的立体结构示意图，其中，二级反应腔与加样腔以及一级反应腔分别处于芯片本体第一侧面的两侧；
图12为本发明另一实施例中一分二反应检测模块的原理示意图；
图13为本发明另一实施例中一分八反应检测模块的原理示意图；
图14为本发明另一实施例的一体式预扩增微液滴芯片中的原理示意图；
图15为本发明实施例中的的一体式微液滴芯片的二级反应腔示意图，其中，二级反应腔中集成了荧光检测区。
附图标记表示为：
1、芯片本体；11、一级反应腔；111、连接接口；12、加样腔；13、二级反应腔；14、气液接口；15、油液接口；16、荧光检测区；2、密封盖；31、第一出液管道；32、第二出液管道；33、混合管道；34、气液管道；35、油液管道；36、微液滴观测区；4、一级扩增加热模块；41、加热凸起件；5、微液滴；6、检测推动油；7、二级扩增加热模块。
具体实施方式
实施例1：
结合参见图1至图13所示，根据本发明的实施例，提供一种一体式多指标检测微液滴芯片，包括芯片本体1，芯片本体1上构造有至少一组一分多混合及反应结构、与每个一分多混合及反应结构对应设置的微液滴生成结构及荧光检测区16，每一一分多混合及反应结构包括：一级反应腔11，用于存储一级体系并进行一级扩增(也可以称之为预扩增)，以使一级体系中具有第一浓度的检测样本(具体例如核酸样本)扩增形成具有第二浓度的检测样本，第二浓度高于第一浓度；多个(至少两个)加样腔12，用于存储二级体系，且多个加样腔12同时与同一一级反应腔11形成可控连通；多个(至少两个且数量与同一一分多混合及反应结构内的加样腔12的个数相等)二级反应腔13，与多个加样腔12分别一一对应可控连通，用于存储由第二浓度的检测样本及二级体系在微液滴生成结构处生成的微液滴5(具体例如为油包水液滴)并进行二级扩增，荧光检测区16用于检测二级扩增完毕的微液滴5。具体而言，前述的一级体系中含有待测样本(具体例如核酸样本)和预扩增体系，而二级体系中则含有数字PCR体系和制备微液滴所需的稳定剂，而在不同的加样腔12中可以根据实际的检测需求加入不同的二级体系，也即不同的引物与探针，从而实现不同目标序列的检测。前述的一分多混合及反应结构的实现形式可以是多样的，例如图12所示出的一分二反应检测模块，或者图1所示出的一分四反应检测模块，或者图13所示出的一分八反应检测模块，而能够理解的是，其至少应该是一分多至少应该是一分二。
该技术方案中，芯片本体1上至少具有一个一分多混合及反应结构，而每一一分多混合及反应结构则分别具有一级反应腔11、多个加样腔12及多个二级反应腔13，从而使检测样本在生成微液滴5之前首先进行预扩增(也即前述的一级扩增)，有效提高检测样本 的浓度，提高浓度后的检测样本能够在一分多混合及反应结构的作用下被划分为更多检测样本份数而保证每份检测样本皆具有较高的浓度，实现了低浓度样本的多指标检测，保证了样本检测的灵敏度，提高了检测结果的准确性，同时，该技术方案中的一级反应腔11、加样腔12以及二级反应腔13皆被集成构造于同一芯片本体1上，从而使一级扩增、样本与不同体系的混合、微液滴的生成、二级扩增和液滴检测集成于同一个芯片上完成，集成度以及自动化程度都能够得到提高，是数字PCR领域的一个重要的技术突破。
在一些实施方式中，芯片本体1上还具有与一级反应腔11临近设置的加热凹槽，一级扩增加热模块4具有加热凸起件41，加热凸起件41能够插装于加热凹槽内，通过该加热凹槽的设置，使加热凸起件41能够伸入其内并与一级反应腔11之间紧密接触，从而保证一级扩增加热效率。能够理解的是，一级扩增加热模块4可以采用业内的加热模块实现，其只要能够被控制的按照预设循环加热即可，其具体的实现原理本发明不做特别限定，其只要具有一个加热凸起件41的结构以保证其加热的热量能够被快速引导至加热凹槽位置并对一级反应腔11内的流体实现高效加热即可。前述的加热凹槽具体可以为锥度槽，以保证其与加热凸起件41之间具有更大的接触面积，实现较高的传热效率。在一个具体的实施例中，一级扩增过程中温度循环流程为先进行一个95℃10分钟的预变性，然后是15个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。为了减少蒸发，可在一级反应腔11内提前放置30微升低密度的防挥发试剂。
在一些实施方式中，一级反应腔11具有多个并联的第一出液管道31，每个加样腔12分别具有第二出液管道32，每个加样腔12的第二出液管道32与多个第一出液管道31分别一一对应地汇总于混合管道33，多个混合管道33分别与多个二级反应腔13一一对应可控连通，该技术方案中，通过第一出液管道31与第二出液管道32将两个腔室中分别的流体引导并汇总至混合管道33内，以在混合管道33内将两个腔室内分别的一级体系与二级体系混合，这种混合在流动过程中实现，能够具有更高的作业效率。作为一种较佳的实施例，混合管道33具有多个连续的弯折部，这些弯折部沿着流体的流动方向交错设置，在外观上形成如图3所示的大致呈S形的结构，能够提高混合管道33的流动阻力，使流体的流动速度变慢的同时不断改变流体的流动方向，使样本和体系混合更充分，最终形成混合检测样本。在一个实施例中，荧光检测区16处于混合管道33上，该荧光检测区16设置于混合管道33上能够在二级反应腔13中二级扩增完毕的微液滴5由二级反应腔13向加样腔12方向流动的过程中对流经的微液滴5进行荧光检测，结构设计更加合理，实现了管道的变向复用，芯片结构更加精简且紧凑。
在一个具体的实施例中，每一一分多混合及反应结构还包括多个流体流动驱动结构，流体流动驱动结构的数量与混合管道33的个数相等且两者一一对应，每个流体流动驱动结构包括构造于芯片本体1上的气液接口14，通过气液接口14能够将与其对应的一级反应腔11与二级反应腔13之间及加样腔12与二级反应腔13之间形成第一压力差，以使一级反应腔11及加样腔12内部的流体分别在第一压力差的作用下流入对应的混合管道33内，需要说明的是，前述的第一压力差分别针对于一级反应腔11与二级反应腔13之间、加样腔12与二级反应腔13之间在样本混合时的压差，两者虽然都可以被称之为第一压差，但是在实际的操作中，两个压差的实际取值可能存在不同。该技术方案中，通过气液接口14处的具体压力的调节能够在一级反应腔11及加样腔12内部形成第一压力差，进而实现对流体流动的有效驱动，操作简单、方便。需要说明的是，该气液接口14通过气液管道34与二级反应腔13的内部连通，这样一方面能够通过调整气液接口14处的气体压力(例如负压)实现一分多混合及反应结构的流体流动驱动，另一方面则可以在需要的时候通过气液接口14引入检测推动油6(也称上浮油)进入二级反应腔13内，从而实现二级反应腔13内的二级扩增完毕的微液滴5进入混合管道33内以实现对这些微液滴5的荧光检测。
进一步的，每个流体流动驱动结构还包括构造于芯片本体1上的油液接口15，其用于连接检测分隔油，油液接口15通过油液管道35与混合管道33形成交叉，以能够在流体在流经油液管道35及混合管道33的交叉位置时被分割成为多个微液滴5。参见图5所示，控制外部压力驱动检测推动油6从气液接口14进入二级反应腔13中，使得二级反应腔13中的微液滴5从二级反应腔13中流出至交叉位置，外部压力驱动检测分隔油从油液接口15进入前述的交叉位置，检测分隔油将从二级反应腔13中流出至交叉位置处的微液滴5分隔形成队列，并进入荧光检测区16，完成荧光检测，具体的，向气液接口14提供油液(也即检测推动油6，也称上浮油)，通过油液的浮力将二级反应腔13内的扩增反应后的微液滴5浮起，并能够在油液的浮力作用下，微液滴5流出二级反应腔13而进入混合管道33，并流经微液滴观测区36进入十字交叉口(也即前述的交叉位置)，在荧光检测区16进行检测，之后最终进入加样腔12内，此时，此时加样腔12为废液池。
在一些实施方式中，参见图2所示，以芯片本体1的第一侧面处于水平方位为参照，二级反应腔13与加样腔12、一级反应腔11皆处于第一侧面上，二级反应腔13与芯片本体1的第一侧面的连接接口111向上延伸，且成下小上大的喇叭口。成喇叭口的连接接口111能够利于微液滴5由混合管道33进入二级反应腔13，也利于微液滴5从二级反应腔13中进入混合管道33中，防止微液滴5的羁留。需要说明的是，此时的二级反应腔13及加样 腔12皆处于芯片本体1的第一侧面(具体为顶面)，进入二级反应腔13内的微液滴5皆集聚于连接接口111处，在进行二级扩增反应时，需要将芯片本体1整体倒置也即翻转180°，使微液滴5能够处于二级反应腔13的反应区内。
在一些实施方式中，二级反应腔13内还构造有自下而上延伸的气液管道34，气液管道34的下口与气液接口14连通，气液管道34的上口高于连接接口111的上口，这样能够防止在二级反应腔13内为负压时，生成的微液滴5在进入二级反应腔13后进一步从气液管道34流出。
在一些实施方式中，混合管道33与连接接口111之间具有微液滴观测区36，微液滴观测区36的通流面积远大于混合管道33的流通面积，也即微液滴观测区36为一个在混合管道33上扩大的区域(宽度变大)，以使进入该区域的微液滴5的流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
作为一种具体的实现方式，一级反应腔11和加样腔12配有密封盖2，以便于操作人员向一级反应腔11和加样腔12内加入样本后，可实现密封功能，因此不会与芯片外的环境接触，杜绝了气溶胶污染的可能，实现了全封闭的数字PCR流程。进一步的，加样腔12设置有滤膜或者排气孔，当加样腔12变成废液池的时候(也即在液滴芯片翻转倒置时)，排除一定的空气，防止加样腔12内积累压力。
如图11所示，给出了全集成式微液滴芯片的另一种实现方式，其与图2所示的一体式微液滴芯片的不同之处在于，二级反应腔13与加样腔12分别处于了芯片本体1的两个相对侧面上，具体的，加样腔12处于第一侧面上，二级反应腔13处于第二侧面上，第二侧面与第一侧面为芯片本体1的相对两侧，此时，一体式微液滴芯片工作原理和流程与前文的一体式微液滴芯片基本一致，不同的地方是，在液滴生成过程中，由于的二级反应腔13位于芯片本体1的底侧(加样腔12处于顶侧)，微液滴5进入到二级反应腔13之处时会直接下落至二级反应腔13底部的反应区，并在反应区中收集，也因此，一体式微液滴芯片在液滴生成结束后，不需要进行180°的翻转，直接进入后续的扩增环节即可。
根据本发明的实施例，还提供一种基于一体式微液滴芯片的微液滴多指标检测方法，一体式微液滴芯片包括芯片本体1，芯片本体1上构造有至少一组一分多混合及反应结构、与每个所述一分多混合及反应结构对应设置的微液滴生成结构及荧光检测区16，每一一分多混合及反应结构包括一级反应腔11、多个加样腔12以及多个二级反应腔13；微液滴多指标检测方法包括如下步骤：
一级扩增反应步骤，控制对存储于一级反应腔11内的一级体系进行加热，以使一级体 系中检测样本由第一浓度扩增为第二浓度，第二浓度高于第一浓度；
样本分流及混合步骤，控制一级反应腔11、加样腔12分别与前述两者相应的二级反应腔13之间形成第一压力差，以使具有第二浓度的检测样本与加样腔12内的二级体系混合形成混合检测样本；
微液滴生成步骤，于所述微液滴生成结构内将混合检测样本在油液的作用下分割为多个微液滴5，并控制形成的微液滴5进入二级反应腔13内；
二级扩增反应步骤，控制对存储于二级反应腔13内的微液滴5加热形成二级扩增；
微液滴荧光检测步骤，于所述荧光检测区16处对二级扩增后的混合检测样本进行多指标的荧光检测。
该技术方案中，芯片本体1上至少具有一个一分多混合及反应结构，而每一一分多混合及反应结构则分别具有一级反应腔11、多个加样腔12及多个二级反应腔13，从而使检测样本的在生成微液滴5之前首先进行预扩增(也即前述的一级扩增)，有效提高检测样本的浓度，提高浓度后的检测样本能够在一分多混合及反应结构的作用下被划分为更多检测样本份数而保证每份检测样本皆具有较高的浓度，实现了低浓度样本的多指标检测，保证了样本检测的灵敏度，提高了检测结果的准确性，同时，该技术方案中的一级反应腔11、加样腔12以及二级反应腔13皆被集成构造于同一芯片本体1上，从而使一级扩增、样本与不同体系的混合、微液滴的生成、二级扩增和液滴检测集成于同一个芯片上完成，集成度以及自动化程度都能够得到提高，是数字PCR领域的一个重要的技术突破。
在一些实施方式中，每个流体流动驱动结构还包括构造于芯片本体1上的油液接口15，油液接口15通过油液管道35与混合管道33形成交叉，微液滴生成步骤具体包括：控制加样腔12与气液接口14之间形成第二压力差，并控制油液接口15与气液接口14之间形成第三压力差，第二压力差以及第三压力差分别驱动混合管道33内的混合检测样本及生成油在油液管道35及混合管道33的交叉位置形成微液滴5，并驱动形成的微液滴5进入二级反应腔13内。具体的，控制加样腔12与气液接口14之间形成第二压力差、油液接口15与气液接口14之间形成第三压力差，以使第二压力差和第三压力差分别驱动混合管道33中的混合样本和油液接口15的检测分隔油(也称生成油)进入油液管道35与混合管道33的交叉位置，生成的微液滴5进入并存储于二级反应腔13中，具体的，向油液接口15提供油液，向气液接口14提供负压，在负压的作用下，混合管道33中的混合样本以及油液接口15处的油液被驱动分别沿着混合管道33、油液管道35汇聚于两者的交叉位置处，混合样本在油液流体剪切力与表面张力的作用下，形成尺寸均一的微液滴5，并最终在负压的作 用下，微液滴5最终进入二级反应腔13内存储，需要说明的是，微液滴5在进入微液滴观测区36中时，微液滴5的流速，形成密集的液滴群落，方便相机成像记录。
在一些实施方式中，以芯片本体1的第一侧面处于水平方位为参照，二级反应腔13与加样腔12处于第一侧面上，在二级扩增反应步骤之前，在微液滴生成步骤之后还包括：芯片翻转步骤，控制芯片本体1上下翻转180°，具体的，二级反应腔13内的微液滴5由接近连接接口111一侧(图6)翻置到远离连接接口111的一侧(图7)，此时微液滴5所对应的二级反应腔13的位置即为二级反应腔13的反应区，反应区会进入二级扩增加热模块7中(图8)，二级扩增加热模块7应为导热性好的材质，本实例中选用铝合金材料，二级扩增加热模块7中间槽的宽度应能将二级反应腔13上的反应区紧密压合，保证良好的加热效率。
在一些实施方式中，在控制芯片本体1上下翻转180°后，还包括：微液滴反向流动控制步骤，控制向二级反应腔13内注入检测推动油6，以使二级反应腔13内的微液滴5被驱动流入混合管道33内，并能够在微液滴5完成微液滴荧光检测步骤之后存储于加样腔12内，如前，此时的加样腔12作为废液池。在芯片本体1上还具有与一级反应腔11临近设置的加热凹槽时，在一级扩增反应步骤之前还包括：控制芯片本体1移动以使加热凹槽套于一级扩增加热模块4具有的加热凸起件41上，并控制一级扩增加热模块4按照预设循环加热扩增。
以下结合试剂体系对本发明的采用一体式多指标检测微液滴芯片的作业流程进一步阐述：
首先，将60微升一体体系加到一级反应腔11中，一级体系中含有待测样本和预扩增体系，然后在加样腔12各孔中，分别加入30微升的二级体系，二级体积含有数字PCR体系和制备液滴所需的稳定剂，加样腔12各孔中加入的二级体系有所不同，主要是引物和探针不同，用于检测不同的目标序列。一张芯片包含了两组一分四的结构(也即前述的一分多混合及反应结构，本实施例中以一分四反应检测模块为例)，即包含两个一级反应腔11和八个加样腔12，两个一级反应腔11连接了八组通道，每组通道都包含有加样腔12、油液接口15、气液接口14、二级反应腔13。
然后，将密封盖2盖紧或粘接，使一级反应腔11和加样腔12密封，一级反应腔11和加样腔12中最好有带滤膜或者口径小的排气孔。
然后将芯片本体1放置在一级扩增加热模块4上，一级扩增加热模块4上的加热凸起件41伸入芯片本体1相应的凹槽(也即前述的加热凹槽)内，与一级反应腔11外侧壁紧 密贴合，一级反应腔11的结构应采取热传递效率高的设计，比如采用能和加热凸起件41紧密贴合的锥度槽等，实现较高的传热效率。在本实施例中，温度循环流程为先进行一个95℃10分钟的预变性，然后是15个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。为了减少蒸发，可在一级反应腔11内提前放置30微升低密度的防挥发试剂。
接着向气液接口14提供负压，压力大小为-200mBar，使得气液接口14与一级反应腔11和加样腔12形成压力差，芯片设计保证各条第一出液管道31内的流阻一致性，控制压力差驱动一级反应腔11内样本均匀流向每条第一出液管道31，因此每条第一出液管道31内将均匀流入15微升的一级扩增后的样本，同时控制压力差驱动每个加样腔12内体系(前述的二级体系)流向第二出液管道32，每条第二出液管道32流入30微升的体系，样本和体系在混合管道33中进行混合，然后在第一压力差驱动下继续沿着混合管道33向前走，当走到混合管道33中“S”形状位置时(图3，“S”形可使流道中的流体不断的变向)，使样本和体系混合更充分，最终形成混合样本；
然后，向油液接口15提供液滴生成所需的生成油(也即前述的检测分隔油)；向气液接口14提供负压，压力大小为-200mBar，使得加样腔12和油液接口15形成压力差。其中，加样腔12与混合管道33相连，油液接口15与油液管道35相连，气液接口14均与二级反应腔13相连(通过气液管道34)。如图5所示，油液管道35有两条分支，位于混合管道33两侧，且均与油液接口15相连。混合管道33中可包含微液滴观测区36，观测区管道变宽，液滴进入后流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
在压力差的驱动下，混合样本进入混合管道33中，生成油进入油液管道35中，并在十字结构处(也即前述交叉位置)交汇，在流体剪切力与表面张力的作用下，形成尺寸均一的油包水微液滴5。交叉位置处的管道深度约为70微米、宽度为80微米，微液滴5尺寸约为100微米。微液滴5进入微液滴观测区36后降低流速，形成密集的液滴群落，方便相机成像记录，微液滴生成过程原理图如图5所示。
生成的微液滴5到达二级反应腔13的连接接口111。连接接口111的底部有斜坡结构(也即前述的喇叭口)，液滴生成流程结束时，撤掉施加在芯片接口处的压力差，此时微液滴5应仍位于气液管道34下方(图6)。
之后，将芯片上下翻转180°，使液滴从连接接口111转移至反应区(也即远离连接接口111)(图7)，接着将二级反应腔13插入二级扩增加热模块7中(图8)，二级扩增加热模块7应为导热性好的材质，本实例中选用铝合金材料，二级扩增加热模块7中间槽的 宽度应能将二级反应腔13上的反应区紧密压合，保证良好的加热效率，让外部系统(也即加热模块)从左右两侧给反应区进行加热和制冷，不仅保证温度传导的距离短，还保证较大的接触面积，从而实现高效的热传递。在本实施例中，温度循环流程为先进行一个95℃10分钟的预变性，然后是40个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。
在温度循环结束后，包含有模板的微液滴5内的二级扩增反应也相应完成了，需要进入液滴荧光检测环节。向气液接口14中注入检测油(也即上述的检测推动油6)，不断充满二级反应腔13(图9)，然后经过连接接口111的斜坡引导，最终进入到之前的微液滴观测区36中，并继续沿着管道往前走。
微液滴经过微液滴观测区36时，也可利用相机对液滴进行明场成像，从而对扩增反应后的液滴状态进行评估。同时，向油液接口15中注入检测油，检测油经由油液管道35，在交叉位置与液滴队列汇合，并将紧密排列的液滴分隔成有合适间距的液滴队列。液滴队列依次通过位于混合管道33的荧光检测区16，如图10所示。荧光检测区16对应的位置，是外部系统的荧光检测焦点。外部系统将激发光，如波长为488nm、532nm和637nm的激光或LED窄带光，聚焦到检测焦点。微液滴5在依次通过检测焦点的过程中，液滴内激发出的荧光，包括FAM、VIC、TAMRA、ROX、Cy5和Q705六种荧光，同样会被外部系统的采集光路接收，从而获取每个液滴的荧光信息。利用液滴的荧光信息，划定信号阈值，对液滴的阴阳性进行区分，并利用泊松分布模型，计算出样本中目标分子的拷贝数。
每个独立的二级反应腔13可检测6种荧光，每种荧光对应一种目标序列，而本实施例有4个二级反应腔13，因此一个样本可检测6*4＝24中目标分子，同时实现了高灵敏度和多指标检测。
最后完成荧光检测的液滴进入加样腔12中，由于加样腔112已经被密封盖2密封，因此不会与芯片外的环境接触，杜绝了气溶胶污染的可能，实现了全封闭的数字PCR流程。
在这个实施例中，开创性的采用了对微液滴生成结构进行分时复用的方法。在液滴生成时，利用微液滴生成结构实现液滴生成。而在液滴荧光检测时，微液滴生成结构完成了对液滴队列的分隔，保证了液滴荧光信号检测。采用这种分时复用的方法，在类流式的数字PCR技术路线上首次实现了一张芯片完成样本一级扩增、样本分流和混合、液滴生成，二级扩增，液滴检测全集成和全封闭的数字PCR流程，是数字PCR领域的一个重要的技术突破。
在另一种实施例中，也会将一级反应腔11连通两路第一出液管道31或者八路第一出 液管道31，实现一分二或者一分八的分流效果(如图12和图13)，其具体分流方法与上述一分四相同，不再做详细论述。但检测指标数不同，一分二的可检测12指标，一分八的可检测48指标。
实施例2：
结合参见图14、15及图2至图11所示，根据本发明的实施例，提供一种一体式预扩增微液滴芯片，包括芯片本体1，芯片本体1上构造有至少一组混合及反应结构、与每个混合及反应结构对应设置的荧光检测区16，每一混合及反应结构包括：一级反应腔11，用于存储一级体系并进行一级扩增(也可以称之为预扩增)，以使一级体系中具有第一浓度的检测样本(具体例如核酸样本)扩增形成具有第二浓度的检测样本，第二浓度高于第一浓度；加样腔12，用于存储二级体系，且加样腔12同时与一级反应腔11形成可控连通；二级反应腔13，与加样腔12以及一级反应腔通过混合管道33对应可控连通，第二浓度的检测样本及二级体系在混合管道中混合后以微液滴5(具体例如为油包水液滴)的形式存储于二级反应腔13内并进行二级扩增，荧光检测区16用于检测二级扩增完毕的微液滴5。具体而言，前述的一级体系中含有待测样本(具体例如核酸样本)和预扩增体系，而二级体系中则含有数字PCR体系和制备微液滴所需的稳定剂，加样腔12中可以根据实际的检测需求加入二级体系，即二级反应所需的引物与探针。前述的混合及反应结构的实现形式可以是多样的。
该技术方案中，芯片本体1上至少具有一个混合及反应结构，而每一混合及反应结构则分别具有一级反应腔11、加样腔12及二级反应腔13，从而使检测样本在生成微液滴5之前首先进行预扩增(也即前述的一级扩增)，有效提高检测样本的浓度，提高浓度后的检测样本保证了检测的灵敏度，提高了检测结果的准确性，同时，该技术方案中的一级反应腔11、加样腔12以及二级反应腔13皆被集成构造于同一芯片本体1上，从而使一级扩增、样本与不同体系的混合、微液滴的生成、二级扩增和液滴检测集成于同一个芯片上完成，集成度以及自动化程度都能够得到提高，是数字PCR领域的一个重要的技术突破。
在一些实施方式中，芯片本体1上还具有与一级反应腔11临近设置的加热凹槽，一级扩增加热模块4具有加热凸起件41，加热凸起件41能够插装于加热凹槽内，通过该加热凹槽的设置，使加热凸起件41能够伸入其内并与一级反应腔11之间紧密接触，从而保证一级扩增加热效率。能够理解的是，一级扩增加热模块4可以采用业内的加热模块实现，其只要能够被控制的按照预设循环加热即可，其具体的实现原理本发明不做特别限定，其只要具有一个加热凸起件41的结构以保证其加热的热量能够被快速引导至加热凹槽位置并对 一级反应腔11内的流体实现高效加热即可。前述的加热凹槽具体可以为锥度槽，以保证其与加热凸起件41之间具有更大的接触面积，实现较高的传热效率。在一个具体的实施例中，一级扩增过程中温度循环流程为先进行一个95℃10分钟的预变性，然后是10个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。为了减少蒸发，可在一级反应腔11内提前放置30微升低密度的防挥发试剂。
在一些实施方式中，一级反应腔11具有第一出液管道31，加样腔12具有第二出液管道32，加样腔12的第二出液管道32与第一出液管道31汇总于前述的混合管道33，与二级反应腔13对应可控连通，该技术方案中，通过第一出液管道31与第二出液管道32将两个腔室中分别的流体引导并汇总至混合管道33内，以在混合管道33内将两个腔室内分别的一级体系与二级体系混合，这种混合在流动过程中实现，能够具有更高的作业效率。作为一种较佳的实施例，混合管道33具有多个连续的弯折部，这些弯折部沿着流体的流动方向交错设置，在外观上形成如图3所示的大致呈S形的结构，能够提高混合管道33的流动阻力，使流体的流动速度变慢的同时不断改变流体的流动方向，使样本和体系混合更充分，最终形成混合检测样本。在一个实施例中，荧光检测区16处于混合管道33上，该荧光检测区16设置于混合管道33上能够在二级反应腔13中二级扩增完毕的微液滴5由二级反应腔13向加样腔12方向流动的过程中对流经的微液滴5进行荧光检测，结构设计更加合理，实现了管道的变向复用，芯片结构更加精简且紧凑。
在一个具体的实施例中，每一混合及反应结构还包括微液滴生成结构，每个微液滴生成结构包括构造于芯片本体1上的气液接口14，通过气液接口14能够将与其对应的一级反应腔11与二级反应腔13之间及加样腔12与二级反应腔13之间形成第一压力差，以使一级反应腔11及加样腔12内部的流体分别在第一压力差的作用下流入对应的混合管道33内，需要说明的是，前述的第一压力差分别针对于一级反应腔11与二级反应腔13之间、加样腔12与二级反应腔13之间在样本混合时的压差，两者虽然都可以被称之为第一压差，但是在实际的操作中，两个压差的实际取值可能存在不同。该技术方案中，通过气液接口14处的具体压力的调节能够在一级反应腔11及加样腔12内部形成第一压力差，进而实现对流体流动的有效驱动，操作简单、方便。需要说明的是，该气液接口14通过气液管道34与二级反应腔13的内部连通，这样一方面能够通过调整气液接口14处的气体压力(例如负压)实现混合及反应结构的流体流动驱动，另一方面则可以在需要的时候通过气液接口14引入检测推动油6(也称上浮油)进入二级反应腔13内，从而实现二级反应腔13内的二级扩增完毕的微液滴5进入混合管道33内以实现对这些微液滴5的荧光检测。
进一步的，每个微液滴生成结构还包括构造于芯片本体1上的油液接口15，其用于连接检测分隔油，油液接口15通过油液管道35与混合管道33形成交叉，以能够在流体在流经油液管道35及混合管道33的交叉位置时被分割成为多个微液滴5。参见图5所示，控制外部压力驱动检测推动油6从气液接口14进入二级反应腔13中，使得二级反应腔13中的微液滴5从二级反应腔13中流出至交叉位置，外部压力驱动检测分隔油从油液接口15进入前述的交叉位置，检测分隔油将从二级反应腔13中流出至交叉位置处的微液滴5分隔形成队列，并进入荧光检测区16，完成荧光检测，具体的，向气液接口14提供油液(也即检测推动油6，也称上浮油)，通过油液的浮力将二级反应腔13内的扩增反应后的微液滴5浮起，并能够在油液的浮力作用下，微液滴5流出二级反应腔13而进入混合管道33，并流经微液滴观测区36进入十字交叉口(也即前述的交叉位置)，在荧光检测区16进行检测，之后最终进入加样腔12内，此时，此时加样腔12为废液池。
在一些实施方式中，参见图2所示，以芯片本体1的第一侧面处于水平方位为参照，二级反应腔13与加样腔12、一级反应腔11皆处于第一侧面上，二级反应腔13与芯片本体1的第一侧面的连接接口111向上延伸，且成下小上大的喇叭口。成喇叭口的连接接口111能够利于微液滴5由混合管道33进入二级反应腔13，也利于微液滴5从二级反应腔13中进入混合管道33中，防止微液滴5的羁留。需要说明的是，此时的二级反应腔13及加样腔12皆处于芯片本体1的第一侧面(具体为顶面)，进入二级反应腔13内的微液滴5皆集聚于连接接口111处，在进行二级扩增反应时，需要将芯片本体1整体倒置也即翻转180°，使微液滴5能够处于二级反应腔13的反应区内。
在一些实施方式中，二级反应腔13内还构造有自下而上延伸的气液管道34，气液管道34的下口与气液接口14连通，气液管道34的上口高于连接接口111的上口，这样能够防止在二级反应腔13内为负压时，生成的微液滴5在进入二级反应腔13后进一步从气液管道34流出。
在一些实施方式中，混合管道33与连接接口111之间具有微液滴观测区36，微液滴观测区36的通流面积远大于混合管道33的流通面积，也即微液滴观测区36为一个在混合管道33上扩大的区域(宽度变大)，以使进入该区域的微液滴5的流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
作为一种具体的实现方式，一级反应腔11和加样腔12配有密封盖2，以便于操作人员向一级反应腔11和加样腔12内加入样本后，可实现密封功能，因此不会与芯片外的环境接触，杜绝了气溶胶污染的可能，实现了全封闭的数字PCR流程。进一步的，加样腔12 设置有滤膜或者排气孔，当加样腔12变成废液池的时候(也即在液滴芯片翻转倒置时)，排除一定的空气，防止加样腔12内积累压力。
如图11所示，给出了全集成式微液滴芯片的另一种实现方式，其与图2所示的一体式微液滴芯片的不同之处在于，二级反应腔13与加样腔12分别处于了芯片本体1的两个相对侧面上，具体的，加样腔12处于第一侧面上，二级反应腔13处于第二侧面上，第二侧面与第一侧面为芯片本体1的相对两侧，此时，一体式微液滴芯片工作原理和流程与前文的一体式微液滴芯片基本一致，不同的地方是，在液滴生成过程中，由于的二级反应腔13位于芯片本体1的底侧(加样腔12处于顶侧)，微液滴5进入到二级反应腔13之处时会直接下落至二级反应腔13底部的反应区，并在反应区中收集，也因此，一体式微液滴芯片在液滴生成结束后，不需要进行180°的翻转，直接进入后续的扩增环节即可。
根据本发明的实施例，还提供一种基于一体式微液滴芯片的检测方法，一体式微液滴芯片包括芯片本体1，芯片本体1上构造有至少一组混合及反应结构、与每个所述混合及反应结构对应设置的微液滴生成结构及荧光检测区16，每一混合及反应结构包括一级反应腔11、加样腔12以及二级反应腔13；微液滴检测方法包括如下步骤：
一级扩增反应步骤，控制对存储于一级反应腔11内的一级体系进行加热，以使一级体系中检测样本由第一浓度扩增为第二浓度，第二浓度高于第一浓度；
样本分流及混合步骤，控制一级反应腔11、加样腔12分别与前述两者相应的二级反应腔13之间形成第一压力差，以使具有第二浓度的检测样本与加样腔12内的二级体系混合形成混合检测样本；
微液滴生成步骤，于所述微液滴生成结构内将混合检测样本在油液的作用下分割为多个微液滴5，并控制形成的微液滴5进入二级反应腔13内；
二级扩增反应步骤，控制对存储于二级反应腔13内的微液滴5加热形成二级扩增；
微液滴荧光检测步骤，于所述荧光检测区16处对二级扩增后的混合检测样本进行多指标的荧光检测。
该技术方案中，芯片本体1上至少具有一个混合及反应结构，而每一混合及反应结构则分别具有一级反应腔11、加样腔12及二级反应腔13，从而使检测样本的在生成微液滴5之前首先进行预扩增(也即前述的一级扩增)，有效提高检测样本的浓度，保证了样本检测的灵敏度，提高了检测结果的准确性，同时，该技术方案中的一级反应腔11、加样腔12以及二级反应腔13皆被集成构造于同一芯片本体1上，从而使一级扩增、样本与不同体系的混合、微液滴的生成、二级扩增和液滴检测集成于同一个芯片上完成，集成度以及自动 化程度都能够得到提高，是数字PCR领域的一个重要的技术突破。
在一些实施方式中，每个流体流动驱动结构还包括构造于芯片本体1上的油液接口15，油液接口15通过油液管道35与混合管道33形成交叉，微液滴生成步骤具体包括：控制加样腔12与气液接口14之间形成第二压力差，并控制油液接口15与气液接口14之间形成第三压力差，第二压力差以及第三压力差分别驱动混合管道33内的混合检测样本及生成油在油液管道35及混合管道33的交叉位置形成微液滴5，并驱动形成的微液滴5进入二级反应腔13内。具体的，控制加样腔12与气液接口14之间形成第二压力差、油液接口15与气液接口14之间形成第三压力差，以使第二压力差和第三压力差分别驱动混合管道33中的混合样本和油液接口15的检测分隔油(也称生成油)进入油液管道35与混合管道33的交叉位置，生成的微液滴5进入并存储于二级反应腔13中，具体的，向油液接口15提供油液，向气液接口14提供负压，在负压的作用下，混合管道33中的混合样本以及油液接口15处的油液被驱动分别沿着混合管道33、油液管道35汇聚于两者的交叉位置处，混合样本在油液流体剪切力与表面张力的作用下，形成尺寸均一的微液滴5，并最终在负压的作用下，微液滴5最终进入二级反应腔13内存储，需要说明的是，微液滴5在进入微液滴观测区36中时，微液滴5的流速，形成密集的液滴群落，方便相机成像记录。
在一些实施方式中，以芯片本体1的第一侧面处于水平方位为参照，二级反应腔13与加样腔12处于第一侧面上，在二级扩增反应步骤之前，在微液滴生成步骤之后还包括：芯片翻转步骤，控制芯片本体1上下翻转180°，具体的，二级反应腔13内的微液滴5由接近连接接口111一侧(图6)翻置到远离连接接口111的一侧(图7)，此时微液滴5所对应的二级反应腔13的位置即为二级反应腔13的反应区，反应区会进入二级扩增加热模块7中(图8)，二级扩增加热模块7应为导热性好的材质，本实例中选用铝合金材料，二级扩增加热模块7中间槽的宽度应能将二级反应腔13上的反应区紧密压合，保证良好的加热效率。
在一些实施方式中，在控制芯片本体1上下翻转180°后，还包括：微液滴反向流动控制步骤，控制向二级反应腔13内注入检测推动油6，以使二级反应腔13内的微液滴5被驱动流入混合管道33内，并能够在微液滴5完成微液滴荧光检测步骤之后存储于加样腔12内，如前，此时的加样腔12作为废液池。在芯片本体1上还具有与一级反应腔11临近设置的加热凹槽时，在一级扩增反应步骤之前还包括：控制芯片本体1移动以使加热凹槽套于一级扩增加热模块4具有的加热凸起件41上，并控制一级扩增加热模块4按照预设循环加热扩增。
参见图12所示，在一个具体实施例中，二级反应腔13为扁平腔体结构，且荧光检测区16构造于二级反应腔13上，第二浓度的检测样本及二级体系混合后形成混合样本，微液滴5由进入二级反应腔13内的油相与混合样本在扁平腔体结构内相互作用形成；具体而言，对应的二级反应腔13内可预存生成油也即前述的油相，在扁平腔体结构的综合作用下，进入的混合后的样本也即混合样本在二级反应腔13自动发生界面破裂，形成微液滴5，并形成单层平铺的状态。在微液滴5生成结束后，微液滴5保持单层平铺状态进行二级扩增；二级扩增后，外部荧光成像模块对平铺的微液滴5进行荧光激发和成像，并对成像照片进行识别，确定微液滴阴阳性，并依据泊松分布计算样本中的拷贝数。前述的油相还可以不预存，而是被实时的引入。
或者，扁平腔体结构的腔底壁上具有多个微坑131，微液滴5由进入微坑131内的混合样本在微坑131的物理间隔作用下形成。具体而言，二级反应腔13内具有的微坑131有数千乃至数万个，且该微坑131为微米级小坑。微坑131中的微液滴5进行二级扩增后，外部荧光成像模块对平铺的微液滴5进行荧光激发和成像，并对成像照片进行识别，确定微液滴阴阳性，并依据泊松分布计算样本中的拷贝数。在这个实施例中，生成油已预存于二级反应腔13中。以下结合试剂体系对本发明的采用一体式预扩增微液滴芯片的作业流程进一步阐述：
首先，将15微升一体体系加到一级反应腔11中，一级体系中含有待测样本和预扩增体系，然后在加样腔12中，加入30微升的二级体系，二级体积含有数字PCR体系和制备液滴所需的稳定剂，加样腔12孔中加入二级体系，包括引物和探针。
然后，将密封盖2盖紧或粘接，使一级反应腔11和加样腔12密封，一级反应腔11和加样腔12中最好有带滤膜或者口径小的排气孔。
然后将芯片本体1放置在一级扩增加热模块4上，一级扩增加热模块4上的加热凸起件41伸入芯片本体1相应的凹槽(也即前述的加热凹槽)内，与一级反应腔11外侧壁紧密贴合，一级反应腔11的结构应采取热传递效率高的设计，比如采用能和加热凸起件41紧密贴合的锥度槽等，实现较高的传热效率。在本实施例中，温度循环流程为先进行一个95℃10分钟的预变性，然后是10个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。为了减少蒸发，可在一级反应腔11内提前放置30微升低密度的防挥发试剂。
接着向气液接口14提供负压，压力大小为-200mBar，使得气液接口14与一级反应腔11和加样腔12形成压力差，控制压力差驱动一级反应腔11内样本流向第一出液管道31，同时控制压力差驱动每个加样腔12内体系(前述的二级体系)流向第二出液管道32，第二 出液管道32流入30微升的体系，样本和体系在混合管道33中进行混合，然后在第一压力差驱动下继续沿着混合管道33向前走，当走到混合管道33中“S”形状位置时(图3，“S”形可使流道中的流体不断的变向)，使样本和体系混合更充分，最终形成混合样本；
然后，向油液接口15提供液滴生成所需的生成油(也即前述的检测分隔油)；向气液接口14提供负压，压力大小为-200mBar，使得加样腔12和油液接口15形成压力差。其中，加样腔12与混合管道33相连，油液接口15与油液管道35相连，气液接口14均与二级反应腔13相连(通过气液管道34)。如图5所示，油液管道35有两条分支，位于混合管道33两侧，且均与油液接口15相连。混合管道33中可包含微液滴观测区36，观测区管道变宽，液滴进入后流速降低，可方便外部相机成像，记录液滴形态，判断液滴生成过程的状态是否正常。
在压力差的驱动下，混合样本进入混合管道33中，生成油进入油液管道35中，并在十字结构处(也即前述交叉位置)交汇，在流体剪切力与表面张力的作用下，形成尺寸均一的油包水微液滴5。交叉位置处的管道深度约为70微米、宽度为80微米，微液滴5尺寸约为100微米。微液滴5进入微液滴观测区36后降低流速，形成密集的液滴群落，方便相机成像记录，微液滴生成过程原理图如图5所示。
生成的微液滴5到达二级反应腔13的连接接口111。连接接口111的底部有斜坡结构(也即前述的喇叭口)，液滴生成流程结束时，撤掉施加在芯片接口处的压力差，此时微液滴5应仍位于气液管道34下方(图6)。
之后，将芯片上下翻转180°，使液滴从连接接口111转移至反应区(也即远离连接接口111)(图7)，接着将二级反应腔13插入二级扩增加热模块7中(图8)，二级扩增加热模块7应为导热性好的材质，本实例中选用铝合金材料，二级扩增加热模块7中间槽的宽度应能将二级反应腔13上的反应区紧密压合，保证良好的加热效率，让外部系统(也即加热模块)从左右两侧给反应区进行加热和制冷，不仅保证温度传导的距离短，还保证较大的接触面积，从而实现高效的热传递。在本实施例中，温度循环流程为先进行一个95℃10分钟的预变性，然后是40个温度循环，每个循环中95℃5秒，60℃15秒，最后4℃保温。
在温度循环结束后，包含有模板的微液滴5内的二级扩增反应也相应完成了，需要进入液滴荧光检测环节。向气液接口14中注入检测油(也即上述的检测推动油6)，不断充满二级反应腔13(图9)，然后经过连接接口111的斜坡引导，最终进入到之前的微液滴观测区36中，并继续沿着管道往前走。
微液滴经过微液滴观测区36时，也可利用相机对液滴进行明场成像，从而对扩增反应后的液滴状态进行评估。同时，向油液接口15中注入检测油，检测油经由油液管道35，在交叉位置与液滴队列汇合，并将紧密排列的液滴分隔成有合适间距的液滴队列。液滴队列依次通过位于混合管道33的荧光检测区16，如图10所示。荧光检测区16对应的位置，是外部系统的荧光检测焦点。外部系统将激发光，如波长为488nm、532nm和637nm的激光或LED窄带光，聚焦到检测焦点。微液滴5在依次通过检测焦点的过程中，液滴内激发出的荧光，包括FAM、VIC、TAMRA、ROX、Cy5和Q705六种荧光，同样会被外部系统的采集光路接收，从而获取每个液滴的荧光信息。利用液滴的荧光信息，划定信号阈值，对液滴的阴阳性进行区分，并利用泊松分布模型，计算出样本中目标分子的拷贝数。
最后完成荧光检测的液滴进入加样腔12中，由于加样腔112已经被密封盖2密封，因此不会与芯片外的环境接触，杜绝了气溶胶污染的可能，实现了全封闭的数字PCR流程。
在这个实施例中，开创性的采用了对微液滴生成结构进行分时复用的方法。在液滴生成时，利用微液滴生成结构实现液滴生成。而在液滴荧光检测时，微液滴生成结构完成了对液滴队列的分隔，保证了液滴荧光信号检测。采用这种分时复用的方法，在类流式的数字PCR技术路线上首次实现了一张芯片完成样本一级扩增、样本分流和混合、液滴生成，二级扩增，液滴检测全集成和全封闭的数字PCR流程，是数字PCR领域的一个重要的技术突破。
在另一种实施例中，可采用荧光成像的方式对二级扩增后的微液滴进行荧光检测。这种实施例中，二级反应腔13中包含了荧光检测区，如图12。在这个实施例中，在微液滴生成之前的步骤与上述实施例相同。在微液滴生成步骤，同样依靠油相与混合后的样本相互作用，形成尺寸均一的微液滴。在微液滴生成之后，微液滴进入二级反应腔，形成单层平铺的液滴层，并保持平铺状态，进行二级扩增。二级扩增后，外部荧光成像模块对平铺的微液滴进行荧光激发和成像，并对成像照片进行识别，确定微液滴阴阳性，并依据泊松分布计算样本中的拷贝数。
在另一种实施例中，可采用荧光成像的方式对二级扩增后的微液滴进行荧光检测。这种实施例中，二级反应腔13中包含了荧光检测区。在这个实施例中，在微液滴生成之前的步骤与上述实施例相同。在微液滴生成步骤，生成油已预存在二级反应腔13中，依靠管道结构，让混合后的样本在二级反应腔13自动发生界面破裂，形成微液滴，并形成单层平铺的状态。微液滴生成结束后，微液滴保持单层平铺状态进行二级扩增。二级扩增后，外部荧光成像模块对平铺的微液滴进行荧光激发和成像，并对成像照片进行识别，确定微液滴 阴阳性，并依据泊松分布计算样本中的拷贝数。在这个实施例中，生成油已预存与二级反应腔中。
在另一种实施例中，可采用荧光成像的方式对二级扩增后的微液滴进行荧光检测。这种实施例中，二级反应腔13中包含了荧光检测区。在这个实施例中，在微液滴生成之前的步骤与上述实施例相同。在二级反应腔13中包含了多个微坑131，微坑具有微米尺度。在微液滴生成步骤，混合后的样本依次填满微坑，并利用微坑形成空间分隔的独立微液滴。微液滴生成后，可使用油对微坑密封，进行进一步分隔每个微液滴。微坑中的微液滴进行二级扩增后，外部荧光成像模块对平铺的微液滴进行荧光激发和成像，并对成像照片进行识别，确定微液滴阴阳性，并依据泊松分布计算样本中的拷贝数。在这个实施例中，生成油已预存与二级反应腔13中。
本领域的技术人员容易理解的是，在不冲突的前提下，上述各有利方式可以自由地组合、叠加。
以上仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (13)

  1. 一种一体式多指标检测微液滴芯片，其特征在于，包括芯片本体(1)，所述芯片本体(1)上构造有至少一组一分多混合及反应结构、与每个所述一分多混合及反应结构对应设置的微液滴生成结构及荧光检测区(16)，每一所述一分多混合及反应结构包括：
    一级反应腔(11)，用于存储一级体系并进行一级扩增，以使所述一级体系中具有第一浓度的检测样本扩增形成具有第二浓度的检测样本，所述第二浓度高于所述第一浓度；
    多个加样腔(12)，用于存储二级体系，且多个所述加样腔(12)同时与同一所述一级反应腔(11)形成可控连通；
    多个二级反应腔(13)，与多个所述加样腔(12)分别一一对应可控连通，用于存储由所述第二浓度的检测样本及所述二级体系在所述微液滴生成结构处生成的微液滴(5)并进行二级扩增；
    所述荧光检测区(16)用于检测二级扩增完毕的微液滴(5)。
  2. 根据权利要求1所述的一体式多指标检测微液滴芯片，其特征在于，所述一级反应腔(11)具有多个并联的第一出液管道(31)，每个所述加样腔(12)分别具有第二出液管道(32)，每个所述加样腔(12)的所述第二出液管道(32)与多个所述第一出液管道(31)分别一一对应地汇总于混合管道(33)，多个所述混合管道(33)分别与多个所述二级反应腔(13)一一对应可控连通。
  3. 根据权利要求2所述的一体式多指标检测微液滴芯片，其特征在于，每一所述一分多混合及反应结构还包括多个流体流动驱动结构，所述流体流动驱动结构的数量与所述混合管道(33)的个数相等，每个所述流体流动驱动结构包括构造于所述芯片本体(1)上的气液接口(14)，通过所述气液接口(14)能够将与其对应的所述一级反应腔(11)与二级反应腔(13)之间及加样腔(12)与二级反应腔(13)形成第一压力差，以使所述一级反应腔(11)及加样腔(12)内部的流体分别在所述第一压力差的作用下流入对应的所述混合管道(33)内。
  4. 根据权利要求3所述的一体式多指标检测微液滴芯片，其特征在于，每个所述流体流动驱动结构还包括构造于所述芯片本体(1)上的油液接口(15)，所述油液接口(15)通过油液管道(35)与所述混合管道(33)形成交叉，以能够在流体在流经所述油液管道 (35)及所述混合管道(33)的交叉位置时被分割成为多个所述微液滴(5)。
  5. 一种如权利要求4所述的一体式多指标检测微液滴芯片的微液滴多指标检测方法，包括如下步骤：
    一级扩增反应步骤，控制对存储于所述一级反应腔(11)内的一级体系进行加热，以使所述一级体系中检测样本由第一浓度扩增为第二浓度，所述第二浓度高于所述第一浓度；
    样本分流及混合步骤，控制所述一级反应腔(11)、所述加样腔(12)分别与所述二级反应腔(13)之间形成第一压力差，以使所述具有第二浓度的检测样本与所述加样腔(12)内的二级体系混合形成混合检测样本；
    微液滴生成步骤，于所述微液滴生成结构内将所述混合检测样本在油液的作用下分割为多个微液滴(5)，并控制形成的所述微液滴(5)进入所述二级反应腔(13)内；
    二级扩增反应步骤，控制对存储于所述二级反应腔(13)内的所述微液滴(5)加热形成二级扩增；
    微液滴荧光检测步骤，于所述荧光检测区(16)处对二级扩增后的所述混合检测样本进行多指标的荧光检测。
  6. 根据权利要求5所述的微液滴多指标检测方法，其特征在于，所述微液滴生成步骤具体包括：
    控制所述加样腔(12)与所述气液接口(14)之间形成第二压力差，并控制所述油液接口(15)与所述气液接口(14)之间形成第三压力差，所述第二压力差以及第三压力差分别驱动所述混合管道(33)内的混合检测样本及生成油在所述油液管道(35)及混合管道(33)的交叉位置形成所述微液滴(5)，并驱动形成的所述微液滴(5)进入所述二级反应腔(13)内。
  7. 根据权利要求6所述的微液滴多指标检测方法，其特征在于，在所述二级扩增反应步骤之前，在微液滴生成步骤之后还包括：
    芯片翻转步骤，控制所述芯片本体(1)上下翻转180°。
  8. 根据权利要求7所述的微液滴多指标检测方法，其特征在于，在控制所述芯片本体(1)上下翻转180°后，还包括：
    微液滴反向流动控制步骤，控制向所述二级反应腔(13)内注入检测推动油(6)，以 使所述二级反应腔(13)内的微液滴(5)被驱动流入所述混合管道(33)内，并能够在所述微液滴(5)完成微液滴荧光检测步骤之后存储于所述加样腔(12)内。
  9. 一种一体式预扩增微液滴芯片，包括芯片本体(1)，其特征在于，所述芯片本体(1)上构造有至少一组混合及反应结构、与每个所述混合及反应结构对应设置的荧光检测区(16)，每一所述混合及反应结构包括：
    一级反应腔(11)，用于存储一级体系并进行一级扩增，以使所述一级体系中具有第一浓度的检测样本扩增形成具有第二浓度的检测样本，所述第二浓度高于所述第一浓度；
    加样腔(12)，用于存储二级体系，且所述加样腔(12)同时与所述一级反应腔(11)形成可控连通；
    二级反应腔(13)，与所述加样腔(12)以及所述一级反应腔(11)通过混合管道(33)对应可控连通，所述第二浓度的检测样本及所述二级体系在所述混合管道(33)中混合后以微液滴(5)的形式存储于所述二级反应腔(13)内并进行二级扩增；
    所述荧光检测区(16)用于检测二级扩增完毕的微液滴(5)。
  10. 根据权利要求1所述的一体式多指标检测微液滴芯片或者权利要求9所述的一体式预扩增微液滴芯片，其特征在于，所述芯片本体(1)上还具有与所述一级反应腔(11)临近设置的加热凹槽，一级扩增加热模块(4)具有加热凸起件(41)，所述加热凸起件(41)能够插装于所述加热凹槽内。
  11. 根据权利要求2所述的一体式多指标检测微液滴芯片或者权利要求9所述的一体式预扩增微液滴芯片，其特征在于，所述混合管道(33)具有多个连续的弯折部。
  12. 根据权利要求2所述的一体式多指标检测微液滴芯片或者权利要求9所述的一体式预扩增微液滴芯片，其特征在于，以所述芯片本体(1)的第一侧面处于水平方位为参照，所述二级反应腔(13)与所述加样腔(12)、所述一级反应腔(11)皆处于所述第一侧面上，所述二级反应腔(13)与所述芯片本体(1)的所述第一侧面的连接接口(111)向上延伸，且成下小上大的喇叭口。
  13. 根据权利要求1所述的一体式多指标检测微液滴芯片或者权利要求9所述的一体式预扩增微液滴芯片，其特征在于，所述二级反应腔(13)内还构造有自下而上延伸的气液管道(34)，所述气液管道(34)的下口与所述气液接口(14)连通，所述气液管道(34) 的上口高于所述连接接口(111)的上口。
PCT/CN2023/082373 2022-07-05 2023-03-18 一体式微液滴芯片及其微液滴多指标检测方法 WO2024007622A1 (zh)

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