WO2021128113A1 - 一种高通量液滴微反应器检测系统及方法 - Google Patents

一种高通量液滴微反应器检测系统及方法 Download PDF

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WO2021128113A1
WO2021128113A1 PCT/CN2019/128504 CN2019128504W WO2021128113A1 WO 2021128113 A1 WO2021128113 A1 WO 2021128113A1 CN 2019128504 W CN2019128504 W CN 2019128504W WO 2021128113 A1 WO2021128113 A1 WO 2021128113A1
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sample
detection
droplet
microreactor
chamber
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PCT/CN2019/128504
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English (en)
French (fr)
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戴敬
张惠丹
丁志文
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苏州昊通仪器科技有限公司
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Publication of WO2021128113A1 publication Critical patent/WO2021128113A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/031—Multipass arrangements
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0325—Cells for testing reactions, e.g. containing reagents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells
    • G01N2021/035—Supports for sample drops
    • G01N2021/0353—Conveyor of successive sample drops
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0389—Windows

Definitions

  • the invention relates to a biological instrument and equipment, in particular to a high-throughput droplet microreactor detection system and method.
  • micro-droplet technology is a micro-nano technology that uses the interaction between the flow shear force and the surface tension to separate the continuous fluid into discrete nano-level and the following volume droplets. It is a kind of micro-nano technology in recent years. A brand-new technology for manipulating tiny liquid volumes has been developed.
  • Liquid-liquid micro-droplets have the advantages of small size, no diffusion between droplets and samples, avoiding cross-contamination between samples, stable reaction conditions, and rapid mixing under proper control; it is a very ideal microreactor. It has been used in the fields of chemistry and life sciences to study numerous reactions and processes under micro-scale conditions. Such as: chemical synthesis, microextraction, protein crystallization, enzyme synthesis and activity analysis, cell embedding, droplet PCR, etc.
  • Droplet microfluidics is the science and technology of generating and manipulating nanoliter to picoscale droplets in a closed microchannel network. Compared with traditional experimental technology, microfluidic droplet technology has shown significant advantages:
  • the diameter of the droplet is small, and the volume can be as small as picoliters or soaring, which greatly reduces the consumption of samples and reagents.
  • the specific surface area is larger and the reaction time is faster; the size of the droplets is relatively uniform, which is difficult for macroscopic experiments. Achieved; the monodispersity of the droplets is helpful for quantitative research;
  • the generated water-in-oil droplets and oil-in-water droplets can isolate the reactant from the outside world, so that it is not affected by changes in the external environment; the droplets are separated by another phase that is immiscible with each liquid. Both drops can be used as an independent microreactor;
  • droplet microfluidics is also called “droplet laboratory”, which is a microscopic chemical or biological reaction vessel.
  • the purpose of the present invention is to provide a high-throughput droplet microreactor detection system and method. With the aid of an area array detection device, the droplet microreactor flowing through the detection chamber is dynamically detected in real time to achieve precise optical quantitative analysis under the continuous flow of droplets.
  • the present invention provides a high-throughput droplet microreactor detection system, including a microfluidic chip and an area array detection device, which integrates the generation, reaction, control, and detection of the droplet microreactor, wherein:
  • the microfluidic chip includes an inflow port, a reaction chamber, a photoelectric detection chamber, an outflow port, and a plurality of flow channels connecting the inflow port, the reaction chamber, the photoelectric detection chamber, and the outflow port,
  • the inflow port is used to load the sample and the sample isolation medium, and enter the reaction chamber through the flow channel wrapped into a droplet microreactor,
  • the difference between the thickness of the detection cavity of the photoelectric detection chamber and the diameter of the droplet microreactor is within ⁇ 1/3 of the diameter of the droplet microreactor, and the detection window of the photoelectric detection chamber forms a non-zero angle with the ground plane.
  • the area array detection device is used to detect and record the reaction state and information in the droplet microreactor in real time through the detection window of the photoelectric detection chamber,
  • the outflow port is used to discharge bubbles, other substances lighter than the droplet microreactor, and waste liquid generated after detection.
  • the microfluidic chip is a vertical microfluidic chip, which is perpendicular to the ground plane.
  • the microfluidic chip further includes a chip information memory for storing the microfluidic chip information and sample information.
  • the chip information storage is also used as an identification mark of the manipulator gripping the chip.
  • the chip information storage includes, but is not limited to, an electronic erasable memory EEPROM, a flash memory FLASH, a ferroelectric memory FRAM, and a solid state hard disk SSD.
  • the inlet includes a sample loading port and a sample isolation medium loading port, which are respectively used for loading samples and sample isolation media,
  • the multiple flow channels include a sample micro flow channel, a sample isolation medium micro flow channel, a droplet micro reactor generating flow channel, and a reaction chamber sample discharge micro flow channel,
  • the sample microchannel is connected to the sample loading port, the sample isolation medium microchannel is connected to the sample isolation medium loading port, and the sample microchannel and the sample isolation medium microchannel merge in all
  • the droplet microreactor generation flow channel, the droplet microreactor generation flow channel is connected to the reaction chamber, and the sample discharge micro flow channel of the reaction chamber connects the reaction chamber and the photoelectricity with a U-shaped flow channel. Detection chamber.
  • the droplet microreactor generation flow channel includes a flow channel shrinkage and a droplet microreactor collection flow channel.
  • the sample micro flow channel and the sample isolation medium micro flow channel meet and enter the liquid through the flow channel shrinkage.
  • the drip micro-reactor collects the flow channel. Through the narrowing of the flow channel, the sample stream can be wrapped in the sample medium stream and cut into micro droplets to form a droplet microreactor.
  • the manner in which the sample microchannels and the sample isolation medium microchannels merge into a droplet microreactor to generate a channel include but are not limited to T-shaped, Y-shaped, cross-shaped, and spatial cross-shaped.
  • a heat conduction column for the reaction chamber is provided inside the reaction chamber for conducting the controlled temperature outside the microfluidic chip into the reaction chamber.
  • the heat-conducting column of the reaction chamber may be installed at the bottom of the reaction chamber and directly or indirectly contact with the droplet microreactor.
  • the heat conduction column of the reaction chamber is externally connected to a control device, and different temperature control programs can be set according to actual needs, and the heat conduction column of the reaction chamber provides a programmed temperature for the droplet microreactor in the reaction chamber. condition.
  • the heat conduction column of the reaction chamber is made of metal or non-metal material.
  • the photoelectric detection chamber electrode is placed outside the photoelectric detection chamber for ablation of the liquid droplet microreactor after detection.
  • the outflow port is connected to the photoelectric detection chamber and the waste liquid flow channel, and the waste liquid flow channel is used to discharge air bubbles mixed between the droplet microreactors, and other substances lighter than the droplet microreactors. And the waste liquid generated after the test. .
  • the area array detection device includes, but is not limited to, a CCD camera, a CMOS camera, and a PMT array, and its working frame rate is greater than or equal to the operating rate of the microreactor.
  • the detection window of the photoelectric detection chamber is perpendicular to the ground plane.
  • the present invention also provides a high-throughput droplet microreactor detection method, which includes: a plurality of droplet microreactors are automatically laid in parallel in a photoelectric detection chamber, and mixed bubbles are automatically discharged.
  • the flow area array detection device detects while using the area array detection device to continuously acquire images to detect and record the reaction state of the droplet microreactor in real time. After the detection, the droplet microreactor is electrically shocked by the electrode of the photoelectric detection chamber located outside the photoelectric detection chamber. Ablation, and discharge the photoelectric detection room.
  • the system is highly integrated, integrating the generation, reaction, control, and detection of the droplet microreactor, which can realize the one-button automatic operation process of intelligent control, and at the same time avoid the pollution and samples that may be caused by the transfer of the reaction system Or reagent loss.
  • the detection window of the photoelectric detection chamber is at a non-zero angle with the ground plane, that is, the photoelectric detection chamber is not parallel to the ground plane. Bubbles and other substances lighter than the droplet microreactor can be under the action of the buoyancy of the carrier liquid. , Gather to the upper part of the photoelectric detection room, and flow to the waste liquid pool through the waste liquid flow channel, improve the space utilization rate of the photoelectric detection room, reduce detection interference and false positive judgments, and improve the accuracy of photoelectric detection.
  • the photoelectric detection chamber is perpendicular to the ground plane. Since the droplet microreactor is in the carrier liquid, the density of the carrier liquid is greater than that of the microreactor. Under the action of buoyancy, the droplet microreactor will automatically move upward; The thickness of the cavity is the same as or slightly different from the diameter of the droplet microreactor (the difference range is preferably between ⁇ 1/3 of the diameter of the droplet microreactor), which can achieve the effect of quickly laying the droplet microreactor without external Power greatly simplifies the microfluidic control process and control method.
  • the droplet microreactors flow through one by one in order, and the detection channel is detected by the photomultiplier tube.
  • the bubble or impurities cannot be judged, which causes the false positive of the system.
  • the droplet microreactor is laid flat on the detection window of the photoelectric detection chamber, and the single frame flux is measured by the flow method, one droplet microreactor is detected at a time, which is significantly increased to tens of thousands of droplet microreactors.
  • the detection time is greatly shortened; at the same time, the area array detection can detect whether the droplet microreactor has physical defects or abnormal reactions by means of two-dimensional images, eliminating the influence of bubbles or impurities on the detection, such as protein crystallization caused by insufficient reaction. ,
  • the traditional one-dimensional inspection is powerless.
  • the microfluidic chips used in this application are arranged in a vertical array, which can greatly save chip placement space and facilitate the miniaturization of the instrument. At the same time, the detection throughput of the instrument will be significantly improved.
  • the single machine sample volume can achieve 1536 single machine volume (16 samples per chip, the instrument supports at least 96 chips). .
  • Figure 1 The structure diagram of a high-throughput droplet microreactor detection system of the present invention
  • Figure 2 The structure of the area array detection device of the present invention and a schematic diagram of area array detection
  • Fig. 3 The structure diagram of the generating flow channel of a cross-shaped droplet microreactor according to the present invention.
  • Figure 4 The structure diagram of the generating flow channel of a T-shaped droplet microreactor according to the present invention.
  • FIG. 5 Schematic diagram of the layout of the photoelectric detection chamber of the droplet microreactor of the present invention.
  • Figure 6 A schematic diagram of the electrode layout of the photoelectric detection chamber of the droplet microreactor of the present invention.
  • Fig. 7 A schematic diagram of the present invention arranging 4 chips at the same time for detection.
  • Fig. 8 A schematic diagram of the present invention arranging 8 chips for detection at the same time.
  • Fig. 9 is a schematic diagram of the present invention arranging multiple chips at the same time for area array detection.
  • 1 sample loading port
  • 2 sample isolation medium loading port
  • 3 sample micro flow channel
  • 4 sample isolation medium micro flow channel
  • 5 droplet micro reactor generating flow channel
  • 51 flow channel shrinkage
  • 52 droplet microreactor collection channel
  • 6 reaction chamber
  • 7 reaction chamber heat conduction column
  • 8 reaction chamber sample discharge microchannel
  • 9 photoelectric detection chamber
  • 10 waste liquid channel
  • 11 waste Liquid pool
  • 12 chip information memory
  • 13 photoelectric detection chamber electrode
  • 131 first photodetection chamber electrode
  • 132 second photodetection chamber electrode
  • 14 area array detection device
  • 141 camera
  • 142 first filter Light sheet
  • 143 second imaging lens
  • 144 second filter
  • 145 filter group
  • 146 exciting light source
  • 147 light source collimating lens group
  • 148 objective lens focusing ring
  • 149 objective lens
  • 15 Control device, 16-manipulator.
  • a high-throughput droplet microreactor detection system of the present invention integrates a microfluidic chip and an area array detection device 14.
  • the microfluidic chip is provided with a sample loading port 1. Isolation medium loading port 2, sample micro flow channel 3, sample isolation medium micro flow channel 4, droplet micro-reactor generation flow channel 5, reaction chamber 6, reaction chamber heat conduction column 7, reaction chamber sample discharge micro flow channel 8, photoelectric Detection chamber 9, waste liquid flow channel 10, waste liquid pool 11, chip information storage 12 and photoelectric detection chamber electrode 13.
  • the microfluidic chip shown in Figure 1 includes two inlets: the sample loading port 1 and the sample isolation medium loading port 2.
  • the sample flow enters the sample microchannel 3 through the sample loading port 1, and the sample medium flow is loaded through the sample isolation medium
  • the port 2 enters the sample isolation medium micro flow channel 4. More inlets can also be set, which is not limited here.
  • the flow of the liquid in the chip is driven by the continuous addition of the carrier liquid through the sample isolation medium loading port 2, and the flow or stop of the droplet microreactor is controlled by the injection or pause of the carrier liquid (such as stopping the process in the reaction chamber 6). reaction).
  • the number of sample isolation medium microchannels 4 can be one, two or more.
  • the droplet microreactor generation flow channel 5 merges the sample micro flow channel 3 and the sample isolation medium micro flow channel 4, and the confluence methods include but are not limited to T-shaped (see Figure 4), Y-shaped, and cross-shaped (see Figure 3) , Space cross shape.
  • the feature of the intersection between the flow channels is that the sample micro flow channel 3 and the sample isolation medium micro flow channel 4 cannot be on the same straight line, but must intersect at an angle that is not 0 degrees.
  • the sample micro flow channel 3 and the droplet micro The reactor generation flow channel 5, or the sample isolation medium micro flow channel 4 and the droplet micro reactor generation flow channel 5, may be on the same straight line, or intersect at an angle other than 0 degrees.
  • sample microchannel 3 and the sample isolation medium microchannel 4 intersect, they enter the droplet microreactor collection channel 52 through the channel shrinkage 51, and the channel shrinkage 51 is used to wrap the sample stream in the sample medium stream and cut it. Cut into micro droplets to generate droplet micro reactors.
  • the generated droplet microreactor enters the reaction chamber 6 through the droplet microreactor collection channel 52.
  • the number of sample isolation medium microchannels 4 can be one ( Figure 4), two ( Figure 3), or multiple.
  • the reaction chamber 6 is a container used to provide the reaction conditions of the droplet microreactor. It is equipped with a reaction chamber heat conduction column 7, which is installed at the bottom of the reaction chamber 6 in the figure, and is used to quickly and uniformly conduct the controlled temperature outside the chip to Inside the reaction chamber 6.
  • the heat conduction column 7 of the reaction chamber is made of metal or non-metal material, and it directly or indirectly contacts the droplet microreactor. After the droplet microreactor completes the reaction in the reaction chamber 6, it enters the photoelectric detection chamber 9 through the sample discharge micro flow channel 8 of the reaction chamber.
  • the photoelectric detection chamber 9 is used to flatten the droplet microreactor on the detection window, so that the area array detection device 14 can detect the results of physical and chemical reactions of substances in the droplet microreactor.
  • the thickness of the detection chamber of the photoelectric detection chamber 9 is the same as or slightly different from the diameter of the droplet microreactor (preferably ⁇ 1/3 of the diameter of the droplet microreactor), which can achieve the effect of quickly laying the droplet microreactor ( Figure 5) ), its angle with the earth's gravitational field is less than 90 degrees, that is, the angle between its detection window and the ground plane is not 0 degrees.
  • the detection window of the photoelectric detection chamber 9 is perpendicular to the ground plane.
  • the outflow port is connected to the photoelectric detection chamber 9 and the waste liquid flow channel 10.
  • the waste liquid flow channel 10 is used to discharge air bubbles and other substances with a lighter specific gravity mixed in the droplet microreactor, and/or to discharge the ablated liquid droplets. Reactor and other waste liquids generated after testing.
  • bubbles and other substances lighter than the droplet microreactor can accumulate to the upper part of the photoelectric detection chamber under the buoyancy of the carrier liquid, and flow to the waste liquid pool 11 through the waste liquid channel 10 to discharge the chip, which improves the photoelectric detection chamber.
  • the space utilization rate can reduce detection interference and false positive judgments, and improve the accuracy of photoelectric detection.
  • the chip information memory 12 stores microfluidic chip information and sample information, and serves as an identification mark for the manipulator 16 to pick up the chip (that is, each chip is equipped with a chip information memory 12).
  • the memory is an electronic information memory, which can be written and read, and stores microfluidic chip control information, single-cell sample preparation information, molecular tags, chip ID, and other information without power supply.
  • the software system can write or read relevant information in it.
  • the chip information memory 12 includes, but is not limited to, electronically erasable memory (EEPROM), flash memory (FLASH), ferroelectric memory (FRAM), and solid state hard disk (SSD).
  • the photoelectric detection chamber electrode 13 is used to ablate the liquid droplet microreactor after the detection, as shown in FIG. 6, includes a pair of electrodes externally placed outside the photodetection chamber 9: the first photodetection chamber electrode 131 and the second photodetection chamber electrode 131
  • the photoelectric detection chamber electrodes 132 are respectively loaded with high voltage at both ends of the two electrodes, and the droplet microreactors merge and disappear under the action of the electric field, thereby avoiding the overlapping problem of the new and old reactions of the detected microreactors.
  • Other feasible setting methods can also be used, as long as the above-mentioned ablation function can be realized, which is not limited here, and the ablated liquid enters the waste liquid pool 11 through the waste liquid flow channel 10.
  • the area array detection device 14 is used to detect and record the real-time information of the substances in the droplet microreactor after physical and chemical reactions in the photoelectric detection chamber 9. After the carrier liquid carrying the droplet microreactor flows into the photoelectric detection chamber 9, the droplets The micro-reactor is quickly laid on the plane of the detection window (as shown in FIG. 5), and images are continuously acquired by the area array detection device 14, and the detection is performed while flowing, and the reaction state information of the droplet reactor is detected and recorded in real time.
  • the area array detection device 14 such as a CMOS camera, has an operating frame rate greater than the operating rate of the microreactor.
  • the area array detection device can adopt the structure shown in Figure 2, including the imaging light path and the illuminating light path.
  • the camera 141 is used to take large-area microreactor images, and the computer counts the number of negative and positive microreactors, and calculates the concentration.
  • the illumination system includes four-color excitation light source 146 or expands to more light sources.
  • the wavelength of the light source corresponds to the fluorescent dye added to the microreactor. It is characterized by being able to detect multiple fluorescent dyes at the same time, or it is convenient to quickly switch to different excitation light sources.
  • the light beam emitted by the excitation light source 146 passes through the light source collimating lens group 147 and the second filter 144 and then becomes a parallel light beam. After being reflected by the filter group 145, it is irradiated to the droplet microreactor through the objective lens 149.
  • the imaging optical path and the illumination optical path share a high-resolution objective lens 149.
  • the objective lens 149 and the second imaging lens 143 are combined to form a clear upright magnified image on the camera target surface.
  • the interference beam is filtered by the first filter 142.
  • the objective lens The focus ring 148 helps focus to form a clear image.
  • the chip is clamped to a suitable position for detection with the help of the manipulator 16 (as shown in FIG. 2 and FIG. 9) above the outside, and each chip is detected in sequence. After the detection is completed, follow-up image processing and microreactor counting are performed, the number of negative and positive microreactors is counted, and the concentration is calculated.
  • the control device 15 is connected to the outside of the heat conduction column 7 of the reaction chamber, and different temperature control programs can be set according to actual needs.
  • the heat conduction column 7 of the reaction chamber provides programmed temperature conditions for the droplet microreactor in the reaction chamber 6.
  • the detection method of a high-throughput droplet microreactor of the present invention mainly includes: a plurality of droplet microreactors are automatically laid in parallel in the photoelectric detection chamber 9 and mixed bubbles are automatically discharged.
  • the side flow surface of the droplet microreactor The array detection device 14 detects while using the area array detection device 14 to continuously acquire images to detect and record the reaction state of the droplet microreactor in real time. After the detection, the droplet microreactor is ablated by the electrode 13 of the photoelectric detection chamber.
  • the liquid flow channel 10 is discharged out of the photoelectric detection chamber 9.
  • Embodiment 2 4 sample digital PCR integrated detection chip
  • the four single-sample detection structures 1-13 shown in Figure 1 can also be integrated on one chip to form a 4-sample digital PCR integrated detection chip.
  • this configuration takes into account
  • the specific implementation process of instrument cost and chip cost is as follows.
  • the sample solution is mixed with the sample isolation medium solution, the sample solution is entrapped in the sample isolation medium solution, and is cut into micro-droplets through the constriction 51 of the flow channel (refer to Figures 3 and 4), that is, droplets are formed Microreactor, the generated droplet microreactor enters the reaction chamber 6 through the droplet microreactor collection channel 52.
  • the reaction chamber heat-conducting column 7 is built in the reaction chamber 6, and the control device 15 provides the programmed temperature conditions required for the reaction in the droplet microreactor through the reaction chamber heat-conducting column 7.
  • a suitable temperature program can be set on the control device 15 to guide the work of the heat conduction column 7 in the reaction chamber.
  • the droplet microreactor enters the photoelectric detection chamber 9 through the sample discharge microchannel 8 of the reaction chamber.
  • the thickness of the detection chamber of the photoelectric detection chamber 9 is the same as or slightly different from the diameter of the droplet microreactor (preferably ⁇ 1/3 of the diameter of the droplet microreactor). After the reactor enters the photoelectric detection chamber 9, it is quickly laid flat on the detection window. The air bubbles entrapped in the carrier liquid and other substances lighter than the droplet microreactor will flow out of the photoelectric through the waste liquid channel 10 under the action of the buoyancy of the carrier liquid. Testing room 9.
  • the manipulator 16 clamps the chip to a suitable position during detection, so that the detection window of the photoelectric detection chamber 9 where the sample to be tested is located is directly in front of the objective lens 149 for imaging. After a sample is detected, the manipulator 16 Move the chip so that the detection window of the photoelectric detection chamber 9 where the next sample is located is directly in front of the objective lens 149, so that 4 samples are detected in sequence.
  • the detected droplet microreactor is ablated by the photoelectric detection chamber electrode 13, and discharged through the waste liquid channel 10, while the droplet microreactor in the reaction chamber 6 continues to enter the photoelectric detection chamber 9, repeat steps 5-7 , Until all the droplet microreactors have been tested.
  • Embodiment 3 8-sample digital PCR integrated detection chip
  • the sample solution is mixed with the sample isolation medium solution, the sample solution is entrapped in the sample isolation medium solution, and is cut into micro-droplets through the constriction 51 of the flow channel (refer to Figures 3 and 4), that is, droplets are formed Microreactor, the generated droplet microreactor enters the reaction chamber 6 through the droplet microreactor collection channel 52.
  • the reaction chamber heat-conducting column 7 is built in the reaction chamber 6, and the control device 15 provides the programmed temperature conditions required for the reaction in the droplet microreactor through the reaction chamber heat-conducting column 7.
  • a suitable temperature program can be set on the control device 15 to guide the work of the heat conduction column 7 in the reaction chamber.
  • the droplet microreactor enters the photoelectric detection chamber 9 through the sample discharge microchannel 8 of the reaction chamber.
  • the thickness of the detection chamber of the photoelectric detection chamber 9 is the same as or slightly different from the diameter of the droplet microreactor (preferably ⁇ 1/3 of the diameter of the droplet microreactor). After the reactor enters the photoelectric detection chamber 9, it is quickly laid flat on the detection window. The air bubbles entrapped in the carrier liquid and other substances lighter than the droplet microreactor will flow out of the photoelectric through the waste liquid channel 10 under the action of the buoyancy of the carrier liquid. Testing room 9.
  • the manipulator 16 clamps the chip to a suitable position during detection, so that the detection window of the photoelectric detection chamber 9 where the sample to be tested is located is directly in front of the objective lens 149 for imaging. After a sample is detected, the manipulator The chip is moved so that the detection window of the photoelectric detection chamber 9 where the next sample is located is directly in front of the objective lens 149, so that 8 samples are sequentially detected.
  • the detected droplet microreactor is ablated by the photoelectric detection chamber electrode 13, and discharged through the waste liquid channel 10, while the droplet microreactor in the reaction chamber 6 continues to enter the photoelectric detection chamber 9, repeat steps 5-7 , Until all the droplet microreactors have been tested.
  • the present invention can arrange multiple chips for detection at the same time according to actual needs, a single chip can be expanded to 16 samples at most, the instrument supports a maximum of 96 chips, and can be expanded to a maximum of 1536 single machines. In this embodiment, 12 8-sample chips or 6 16-sample chips are arranged for detection at the same time. In multi-chip detection, only structures 1 to 13 on the chip are correspondingly added, while the area array detection device 14 outside the chip structure does not follow Increase with the increase in the number of chips.
  • an external manipulator 16 (as shown in Figures 2 and 9) can be used to clamp the chip to be detected and carry it to a suitable detection position, so that the area array detection device can collect the droplet in the microreactor in the photoelectric detection chamber of the chip. Information.
  • the sample solution is mixed with the sample isolation medium solution, the sample solution is entrapped in the sample isolation medium solution, and is cut into micro-droplets through the constriction 51 of the flow channel (refer to Figures 3 and 4), that is, droplets are formed Microreactor, the generated droplet microreactor enters the reaction chamber 6 through the droplet microreactor collection channel 52.
  • the reaction chamber heat-conducting column 7 is built in the reaction chamber 6, and the control device 15 provides the programmed temperature conditions required for the reaction in the droplet microreactor through the reaction chamber heat-conducting column 7.
  • a suitable temperature program can be set on the control device 15 to guide the work of the heat conduction column 7 in the reaction chamber.
  • the droplet microreactor enters the photoelectric detection chamber 9 through the sample discharge microchannel 8 of the reaction chamber.
  • the thickness of the detection chamber of the photoelectric detection chamber 9 is the same as or slightly different from the diameter of the droplet microreactor (preferably ⁇ 1/3 of the diameter of the droplet microreactor). After the reactor enters the photoelectric detection chamber 9, it is quickly laid flat on the detection window. The air bubbles entrapped in the carrier liquid and other substances lighter than the droplet microreactor will flow out of the photoelectric through the waste liquid channel 10 under the action of the buoyancy of the carrier liquid. Testing room 9.
  • the manipulator 16 clamps the chip to a suitable position during detection, so that the detection window of the photoelectric detection chamber 9 where the sample to be tested is located is directly in front of the objective lens 149 for imaging. After a sample is detected, the manipulator The chip is moved so that the detection window of the photodetection chamber 9 where the next sample is located is directly in front of the objective lens 149, so that 8 or 16 samples of a single chip are sequentially detected.
  • the detected droplet microreactor is ablated by the photoelectric detection chamber electrode 13, and discharged through the waste liquid channel 10, while the droplet microreactor in the reaction chamber 6 continues to enter the photoelectric detection chamber 9, repeat steps 5-7 , Until all the droplet microreactors have been tested.
  • the manipulator 16 will put the chip back in place, clamp the next chip and repeat steps 5 to 8 until all the droplet microreactors of all chips are tested. .

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Abstract

一种高通量液滴微反应器检测系统及方法，检测系统包括微流控芯片和面阵检测装置（14），微流控芯片包括流入口（1, 2）、反应室（6）、光电检测室（9）、流出口以及连接流入口（1, 2）、反应室（6）、光电检测室（9）和流出口的多个流道（3, 4, 5, 8, 10）。该检测方法借助于面阵检测装置（14），对流经检测室（9）的液滴微反应器进行实时动态检测，以达到液滴连续流动状态下的精确光学定量分析。

Description

一种高通量液滴微反应器检测系统及方法 技术领域
本发明涉及一种生物仪器设备，具体涉及一种高通量液滴微反应器检测系统及方法。
背景技术
微液滴技术是在微尺度通道内，利用流动剪切力与表面张力之间的相互作用将连续流体分割分离成离散的纳升级及以下体积的液滴的一种微纳技术，它是近年来发展起来的一种全新的操纵微小液体体积的技术。
迄今为止，文献中报道的微液滴类型主要有气-液相液滴和液-液相液滴两种。液-液相微液滴由于体积小、液滴样品间无扩散、可避免样品间的交叉污染、反应条件稳定、适当操控下可实现迅速混合等优点；是一种十分理想的微反应器，已经被用于化学和生命科学等领域研究微尺度条件下众多的反应及其过程。如:化学合成、微萃取、蛋白质结晶、酶合成及其活性分析、细胞包埋、液滴PCR等。
液滴微流控是在封闭的微通道网络中生成和操控纳升至皮升级液滴的科学与技术。与传统的实验技术相比，微流控液滴技术已经呈现出显著优势：
1)液滴的直径微小，体积可小至皮升或飞升，大大降低了样品与试剂的消耗，比表面积较大，反应时间较快；液滴的大小较为均一，这是宏观实验途径很难实现的；液滴的单分散性，有助于定量研究；
2)生成的油包水液滴和水包油液滴能够把反应物和外界隔开，使之不受外界环境变化的影响；液滴被与之不互溶的另一相间隔，每个液滴皆可作为独立的微反应器；
3)短时间内可以生成大量的微反应器(最高可达数千赫兹)，适合高通量的生物和化学分析；
4)混合速度较连续流动的微流控系统明显加快，反应时间大大减少。
由于液滴的上述特征，液滴微流控又称“液滴实验室”也即一种微观的化学或生物反应容器。
发明内容
本发明的目的在于提供一种高通量液滴微反应器检测系统及方法。借助于面阵检测装置，对流经检测室的液滴微反应器进行实时动态检测，以达到液滴连续流动状态下的精确光学定 量分析。
为了实现上述目的，本发明采用以下技术方案：
本发明提供了一种高通量液滴微反应器检测系统，包括微流控芯片和面阵检测装置，集液滴微反应器的生成、反应、控制、以及检测为一体，其中，
所述微流控芯片包括流入口、反应室、光电检测室、流出口以及连接所述流入口、所述反应室、所述光电检测室和所述流出口的多个流道，
所述流入口用于加载样本和样本隔离介质，通过流道包裹成液滴微反应器进入所述反应室，
所述光电检测室的检测腔厚度与液滴微反应器直径相差范围为液滴微反应器直径的±1/3之间，光电检测室的检测窗与地平面呈非0度夹角，用于使液滴微反应器在载液浮力作用下平铺并使气泡及其他比液滴微反应器比重轻的物质在浮力作用下优先从所述流出口离开所述光电检测室，
所述面阵检测装置用于通过所述光电检测室的检测窗实时检测记录液滴微反应器中的反应状态与信息，
所述流出口用于排出气泡，其他比液滴微反应器比重轻的物质及检测后产生的废液。
优选地，所述微流控芯片为立式微流控芯片，与地平面垂直。
优选地，所述微流控芯片还包括芯片信息存储器，用于存储所述微流控芯片信息和样本信息。
优选地，在多样本实施方式中，所述芯片信息存储器还作为机械手夹取芯片的识别标志。
更优选地，所述芯片信息存储器包括但不限于电子可擦除存储器EEPROM、闪存存储器FLASH、铁电存储器FRAM、固态硬盘SSD。
优选地，
所述流入口包括样本加载口，样本隔离介质加载口，分别用于加载样本和样本隔离介质，
所述多个流道包括样本微流道、样本隔离介质微流道、液滴微反应器生成流道、反应室出样微流道，
所述样本微流道与所述样本加载口相连，所述样本隔离介质微流道与所述样本隔离介质加载口相连，所述样本微流道与所述样本隔离介质微流道汇合于所述液滴微反应器生成流道，所述液滴微反应器生成流道与所述反应室连接，所述反应室出样微流道以U型流道连接所述反应室和所述光电检测室。
优选地，所述样本隔离介质微流道可以有不少于一个。
优选地，所述液滴微反应器生成流道包括流道缩口和液滴微反应器收集流道，所述样本微流道和样本隔离介质微流道交汇后通过流道缩口进入液滴微反应器收集流道。通过流道缩口可将样本流包裹于样本介质流中并剪切成微液滴，生成液滴微反应器。
优选地，所述样本微流道和样本隔离介质微流道汇合成液滴微反应器生成流道的方式包括但不限于T形、Y形、十字交叉形、空间交叉形。
优选地，所述反应室内部设有反应室导热柱，用于将所述微流控芯片外的受控温度传导至所述反应室内。
优选地，所述反应室导热柱可安装于所述反应室的底部并和液滴微反应器直接或间接接触。
优选地，所述反应室导热柱外部连接控制装置，可以根据实际需求设定不同的温度控制程序，通过所述反应室导热柱为所述反应室中的液滴微反应器提供程序化的温度条件。
优选地，所述反应室导热柱为金属或非金属材质。
优选地，所述光电检测室外置光电检测室电极，用于消融检测完毕的液滴微反应器。
优选地，所述流出口连接所述光电检测室和废液流道，所述废液流道用于排出混于液滴微反应器间的气泡，其他比液滴微反应器比重轻的物质及检测后产生的废液。。
优选地，所述面阵检测装置包括但不限于CCD相机、CMOS相机、PMT阵列，其工作帧率大于或等于微反应器运的速率。
更优选地，所述光电检测室的检测窗与地平面垂直。
本发明还提供了一种高通量液滴微反应器检测方法，包括：多个液滴微反应器自动并行平铺于光电检测室中，并自动排出混杂的气泡，液滴微反应器边流动面阵检测装置边检测，以面阵检测装置连续获取图像，对液滴微反应器的反应状态实时进行检测记录，检测后的液滴微反应器通过光电检测室外置的光电检测室电极电击消融，并排出光电检测室外。
本发明的有益效果如下：
1、系统高度集成化，集液滴微反应器的生成、反应、控制、以及检测为一体，可实现智能控制的一键式自动运行过程，同时避免了反应体系转移时可能造成的污染及样本或试剂损耗。
2、光电检测室的检测窗与地平面呈非0度夹角，也即光电检测室不与地平面平行，气泡及其他比液滴微反应器比重轻的物质可以在载液浮力的作用下，向光电检测室上部集聚，通过废液流道流至废液池，提高光电检测室的空间利用率，降低检测干扰及假阳性判断，提高光电检测的准确度。
3、光电检测室垂直于地平面，由于液滴微反应器处于载液之中，载液密度大于微反应器的密度，在浮力的作用下，液滴微反应器会自动向上方移动；检测腔厚度与液滴微反应器直径相同或略有差异(相差范围优选在液滴微反应器直径的±1/3之间)，可以达到迅速平铺液滴微反应器的效果，而无需外部动力，极大简化了微流体控制过程与控制方法。
4、采用面阵检测方法。传统基于流式的方式，液滴微反应器按顺序一个个流过，检测通道，由光电倍增管检测其有无，不能对气泡或杂质进行判断，从而造成系统假阳性的误判。面阵检测方法下，液滴微反应器平铺于光电检测室检测窗，单帧通量由流式方法，一次检测一个液滴微反应器，显著提升至几万个液滴微反应器，检测时间大幅缩短；同时面阵检测可以以二维图像的方式检测液滴微反应器是否有物理缺陷或反应异常，杜绝了气泡或杂质对检测的影响，如反应不充分造成的蛋白结晶等现象，传统一维检测则无能为力。
5、与现有设备中微流控芯片需要水平放置不同，本申请采用的微流控芯片以立式阵列形式排布，可以极大结省芯片摆位空间，方便仪器小型化。同时，仪器的检测通量会显著提升。单机样本量可以做到1536单次上机量(每芯片16样本，仪器支持至少96个芯片)。。
附图说明
图1 本发明一种高通量液滴微反应器检测系统的结构图；
图2 本发明面阵检测装置结构及面阵检测示意图；
图3 本发明一种十字交叉形液滴微反应器生成流道的结构图；
图4 本发明一种T形液滴微反应器生成流道的结构图；
图5 本发明液滴微反应器光电检测室布局示意图；
图6 本发明液滴微反应器光电检测室电极布局示意图；
图7 本发明同时排列4个芯片进行检测的示意图。
图8 本发明同时排列8个芯片进行检测的示意图。
图9 本发明同时排列多个芯片进行面阵检测的示意图。
图中：1—样本加载口，2—样本隔离介质加载口，3—样本微流道，4—样本隔离介质微流道，5—液滴微反应器生成流道，51—流道缩口，52—液滴微反应器收集流道，6—反应室，7—反应室导热柱，8—反应室出样微流道，9—光电检测室，10—废液流道，11—废液池，12—芯片信息存储器，13—光电检测室电极，131-第一光电检测室电极，132-第二光电检测室电极，14—面阵检测装置，141—相机，142—第一滤光片，143—第二成像透镜，144—第二 滤光片，145—滤光片组，146—激发光源，147—光源准直透镜组，148—物镜对焦环，149—物镜，15—控制装置，16—机械手。
具体实施方式
下面结合附图及实施例对本发明做进一步的详细说明。
实施例1单样本检测结构
如图1和2所示，本发明一种高通量液滴微反应器检测系统集成了1个微流控芯片和面阵检测装置14，微流控芯片上设有样本加载口1、样本隔离介质加载口2、样本微流道3、样本隔离介质微流道4、液滴微反应器生成流道5、反应室6、反应室导热柱7、反应室出样微流道8、光电检测室9、废液流道10、废液池11、芯片信息存储器12和光电检测室电极13。
图1所示的微流控芯片包括两个流入口：即样本加载口1和样本隔离介质加载口2，样本流通过样本加载口1进入样本微流道3，样本介质流通过样本隔离介质加载口2进入样本隔离介质微流道4。流入口也可以设置更多，在此不做限定。此外，芯片内液体的流动是通过样本隔离介质加载口2持续加入载液来驱动的，由载液的注入或暂停注入来控制液滴微反应器的流动或停止(如停止在反应室6进行反应)。样本隔离介质微流道4的数量可以是一个，两个或多个。
液滴微反应器生成流道5汇合了样本微流道3及样本隔离介质微流道4，汇合方式包括但不限于T形(见图4)、Y形、十字交叉形(见图3)、空间交叉形。流道之间交汇的特征为，样本微流道3与样本隔离介质微流道4不能处于同一直线上，而是要以不为0度的夹角相交，样本微流道3与液滴微反应器生成流道5，或，样本隔离介质微流道4与液滴微反应器生成流道5，可以处于同一直线上，或，以不为0度的夹角相交。样本微流道3与样本隔离介质微流道4交汇后通过流道缩口51进入液滴微反应器收集流道52，流道缩口51用于将样本流包裹于样本介质流中并剪切成微液滴，生成液滴微反应器。生成的液滴微反应器通过液滴微反应器收集流道52进入反应室6。样本隔离介质微流道4的数量可以是一个(图4)，两个(图3)，也可以是多个。
反应室6，用于提供液滴微反应器反应条件的容器，内部设有反应室导热柱7，图中安装于反应室6的底部，用于把芯片外的受控温度快速、均匀传导至反应室6内。反应室导热柱7为金属或非金属材质，其和液滴微反应器直接或间接接触。液滴微反应器在反应室6内反应完成后，通过反应室出样微流道8进入光电检测室9。
光电检测室9，用于将液滴微反应器平铺于检测窗，便于面阵检测装置14对液滴微反应 器中物质的物理、化学反应后的结果进行检测。光电检测室9检测腔厚度与液滴微反应器直径相同或略有差异(优选液滴微反应器直径的±1/3)，可以达到迅速平铺液滴微反应器的效果(如图5)，其与地球重力场相夹角小于90度，也即其检测窗与地平面夹角不为0度，作为系统设计优选，光电检测室9的检测窗与地平面垂直。
流出口连接光电检测室9和废液流道10，废液流道10用于排出混于液滴微反应器间的气泡及其他比重较轻的物质，和/或，排出消融的液滴微反应器等其他检测后产生的废液。
检测时，气泡及其他比液滴微反应器比重轻的物质可以在载液浮力作用下，向光电检测室上部集聚，通过废液流道10流至废液池11排出芯片，提高光电检测室的空间利用率，降低检测干扰及假阳性判断，提高光电检测的准确度。
芯片信息存储器12，储存微流控芯片信息和样本信息，并作为机械手16夹取芯片的识别标志(即每个芯片上都装有芯片信息存储器12)。具体地，该存储器为电子信息存储器，可以写入与读出，没有电源的状态下存储微流控芯片控制信息、单细胞样本制备信息、分子标签、芯片ID等信息。当微流控芯片置于芯片加载模块中时，软件系统可以写入或读出其中的相关信息。芯片信息存储器12包括但不限于电子可擦除存储器(EEPROM)、闪存存储器(FLASH)、铁电存储器(FRAM)、固态硬盘(SSD)。
光电检测室电极13，用于对检测完毕的液滴微反应器进行消融，如图6所示，包括外置于光电检测室9外部的成对电极：第一光电检测室电极131和第二光电检测室电极132，分别在两个电极两端加载高压电，在电场作用下液滴微反应器彼此融合、消失，从而避免了检测的微反应器新旧反应互叠问题。也可采用其他可行的设置方式，只要能够实现上述消融功能即可，在此不做限定，消融后的液体通过废液流道10进入废液池11。
面阵检测装置14，用于检测记录光电检测室9中经过物理、化学反应后的液滴微反应器内物质的实时信息，载液携液滴微反应器流入光电检测室9后，液滴微反应器迅速平铺于检测窗平面(如图5)，以面阵检测装置14连续获取图像，边流动边检测，对液滴反应器的反应状态信息进行实时检测记录。
面阵检测装置14如CMOS相机，其工作帧率大于微反应器运的的速率。具体实施时，面阵检测装置可以采用如图2中的结构，包含成像光路和照明光路，用相机141拍摄大面积的微反应器图像，计算机统计阴性和阳性的微反应器数，并计算浓度。其中照明系统包含四色激发光源146或者扩展到更多光源，光源的波段根据微反应器所加的荧光染料对应，其特点在于可以同时检测多种荧光染料，或者方便快速切换到不同激发光源。激发光源146发出光束经光源准直透镜组147和第二滤光片144后变成平行光束，经由滤光片组145反射后通 过物镜149照射到液滴微反应器。成像光路与照明光路共用一款高分辨率的物镜149，物镜149和第二成像透镜143组合，在相机靶面上成清晰的正立放大像，由第一滤光片142过滤干扰光束，物镜对焦环148帮助对焦形成清晰图像。借助外部上方的机械手16(如图2、图9)夹持芯片至合适的位置进行检测，每一张芯片按顺序依次分别进行检测。完成检测后，进行后续的图像处理和微反应器的计数，统计阴性和阳性的微反应器个数，并计算浓度。
控制装置15，连接于反应室导热柱7外部，可以根据实际需求设定不同的温度控制程序，通过反应室导热柱7为反应室6中的液滴微反应器提供程序化的温度条件。
本发明一种高通量液滴微反应器检测方法主要包括：多个液滴微反应器自动并行平铺于光电检测室9中，并自动排出混杂的气泡，液滴微反应器边流动面阵检测装置14边检测，以面阵检测装置14连续获取图像，对液滴微反应器的反应状态实时进行检测记录，检测后的液滴微反应器由光电检测室电极13电击消融，从废液流道10排出光电检测室9外。
实施例2 4样本数字PCR一体化检测芯片
如图7所示，4个如图1所示的单样本检测结构1～13还可以集成于一个芯片之上，形成4样本数字PCR一体化检测芯片，对于科研应用来说，此配置兼顾了仪器成本和芯片成本，具体实施过程如下。
1、通过样本加载口1和样本隔离介质加载口2分别注入样本液和样本隔离介质液，样本微流道3中的样本液与样本隔离介质微流道4中的样本隔离介质液交汇于液滴微反应器生成流道5。
2、样本液与样本隔离介质液混合后，样本液裹挟于样本隔离介质液中，通过流道缩口51(可参考图3和图4)被剪切成微液滴，即生成了液滴微反应器，生成的液滴微反应器通过液滴微反应器收集流道52进入反应室6中。
3、反应室6中内置反应室导热柱7，控制装置15通过反应室导热柱7提供液滴微反应器内进行反应所需要的程序化的温度条件。
4、可根据具体的样本情况，在控制装置15上设定合适的温度程序来指导反应室导热柱7的工作。
5、反应完成后的液滴微反应器通过反应室出样微流道8进入光电检测室9。
6、光电检测室9的检测腔厚度与液滴微反应器直径相同或略有差异(优选液滴微反应器直径的±1/3)，与地平面呈非0度夹角，液滴微反应器进入光电检测室9后，迅速平铺于检测窗，载液中裹挟的气泡和其他比液滴微反应器比重轻的物质在载液浮力的作用下，通过废 液流道10流出光电检测室9。
7、结合图2，检测时由机械手16夹持芯片至合适的位置，使待检测样本所处的光电检测室9的检测窗位于物镜149的正前方便于成像，一个样本检测完毕后，由机械手16移动芯片使下一个样本所处的光电检测室9的检测窗位于物镜149的正前方，如此依次检测完毕4个样本。
8、检测完毕的液滴微反应器由光电检测室电极13进行消融，通过废液流道10排出，同时反应室6内的液滴微反应器继续进入光电检测室9，重复步骤5～7，直至所有液滴微反应器全部检测完毕。
实施例3 8样本数字PCR一体化检测芯片
如图8所示，8个如图1所述的单样本检测结构1～13可以集成于一个芯片之上，形成8样本数字PCR一体化检测芯片，对于标准样本前处理流程应用来说，一般以8的倍数为单位，所以这种配置较为适合作为配置多芯片检测的基础结构，具体实施过程如下。
1、通过样本加载口1和样本隔离介质加载口2分别注入样本液和样本隔离介质液，样本微流道3中的样本液与样本隔离介质微流道4中的样本隔离介质液交汇于液滴微反应器生成流道5。
2、样本液与样本隔离介质液混合后，样本液裹挟于样本隔离介质液中，通过流道缩口51(可参考图3和图4)被剪切成微液滴，即生成了液滴微反应器，生成的液滴微反应器通过液滴微反应器收集流道52进入反应室6中。
3、反应室6中内置反应室导热柱7，控制装置15通过反应室导热柱7提供液滴微反应器内进行反应所需要的程序化的温度条件。
4、可根据具体的样本情况，在控制装置15上设定合适的温度程序来指导反应室导热柱7的工作。
5、反应完成后的液滴微反应器通过反应室出样微流道8进入光电检测室9。
6、光电检测室9的检测腔厚度与液滴微反应器直径相同或略有差异(优选液滴微反应器直径的±1/3)，与地平面呈非0度夹角，液滴微反应器进入光电检测室9后，迅速平铺于检测窗，载液中裹挟的气泡和其他比液滴微反应器比重轻的物质在载液浮力的作用下，通过废液流道10流出光电检测室9。
7、结合图2，检测时由机械手16夹持芯片至合适的位置，使待检测样本所处的光电检测 室9的检测窗位于物镜149的正前方便于成像，一个样本检测完毕后，由机械手移动芯片使下一个样本所处的光电检测室9的检测窗位于物镜149的正前方，如此依次检测完毕8个样本。
8、检测完毕的液滴微反应器由光电检测室电极13进行消融，通过废液流道10排出，同时反应室6内的液滴微反应器继续进入光电检测室9，重复步骤5～7，直至所有液滴微反应器全部检测完毕。
实施例4单机96样本检测
本发明可以根据实际需要同时排列多个芯片进行检测，单一芯片最大可扩展至16样本，仪器最大支持96个芯片，最高可扩展至1536单次上机量。本实施例同时排列12个8样本芯片或6个16样本芯片进行检测，多芯片检测时，只相应增加位于芯片上的结构1～13，而位于芯片结构之外的面阵检测装置14并不随着芯片数量的增加而增加。进行面阵检测时，可借助外部的机械手16(如图2、图9)，夹持待检测芯片携至合适的检测位置，以便面阵检测装置采集该芯片光电检测室内液滴微反应器中的信息。
具体实施过程如下：
1、通过样本加载口1和样本隔离介质加载口2分别注入样本液和样本隔离介质液，样本微流道3中的样本液与样本隔离介质微流道4中的样本隔离介质液交汇于液滴微反应器生成流道5。
2、样本液与样本隔离介质液混合后，样本液裹挟于样本隔离介质液中，通过流道缩口51(可参考图3和图4)被剪切成微液滴，即生成了液滴微反应器，生成的液滴微反应器通过液滴微反应器收集流道52进入反应室6中。
3、反应室6中内置反应室导热柱7，控制装置15通过反应室导热柱7提供液滴微反应器内进行反应所需要的程序化的温度条件。
4、可根据具体的样本情况，在控制装置15上设定合适的温度程序来指导反应室导热柱7的工作。
5、反应完成后的液滴微反应器通过反应室出样微流道8进入光电检测室9。
6、光电检测室9的检测腔厚度与液滴微反应器直径相同或略有差异(优选液滴微反应器直径的±1/3)，与地平面呈非0度夹角，液滴微反应器进入光电检测室9后，迅速平铺于检测窗，载液中裹挟的气泡和其他比液滴微反应器比重轻的物质在载液浮力的作用下，通过废液流道10流出光电检测室9。
7、结合图2，检测时由机械手16夹持芯片至合适的位置，使待检测样本所处的光电检测室9的检测窗位于物镜149的正前方便于成像，一个样本检测完毕后，由机械手移动芯片使下一个样本所处的光电检测室9的检测窗位于物镜149的正前方，如此依次检测完毕单张芯片的8个或16个样本。
8、检测完毕的液滴微反应器由光电检测室电极13进行消融，通过废液流道10排出，同时反应室6内的液滴微反应器继续进入光电检测室9，重复步骤5～7，直至所有液滴微反应器全部检测完毕。
9、单张芯片的8个或16个样本检测完毕后，由机械手16将芯片放回原位，夹持下一张芯片重复步骤5～8，直至所有芯片的液滴微反应器全部检测完毕。

Claims (10)

  1. 一种高通量液滴微反应器检测系统，包括微流控芯片和面阵检测装置(14)，其中，
    所述微流控芯片包括流入口、反应室(6)、光电检测室(9)、流出口以及连接所述流入口、所述反应室(6)、所述光电检测室(9)和所述流出口的多个流道，
    所述流入口用于加载样本和样本隔离介质，通过流道包裹成液滴微反应器进入所述反应室(6)，
    所述光电检测室(9)的检测腔厚度与液滴微反应器直径相差范围在液滴微反应器直径的±1/3之间，光电检测室(9)的检测窗与地平面呈非0度夹角，用于使液滴微反应器在载液浮力作用下平铺并使气泡及其他比液滴微反应器比重轻的物质在浮力作用下优先从所述流出口离开所述光电检测室(9)，
    所述面阵检测装置(14)用于通过所述光电检测室(9)的检测窗实时检测记录液滴微反应器中的反应状态与信息，
    所述流出口用于排出气泡，其他比液滴微反应器比重轻的物质及检测后产生的废液。
  2. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，所述微流控芯片还包括芯片信息存储器(12)，用于存储所述微流控芯片信息和样本信息。
  3. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，
    所述流入口包括样本加载口(1)，样本隔离介质加载口(2)，分别用于加载样本和样本隔离介质，
    所述多个流道包括样本微流道(3)、样本隔离介质微流道(4)、液滴微反应器生成流道(5)、反应室出样微流道(8)，
    所述样本微流道(3)与所述样本加载口(1)相连，所述样本隔离介质微流道(4)与所述样本隔离介质加载口(2)相连，所述样本微流道(3)与所述样本隔离介质微流道(4)汇合于所述液滴微反应器生成流道(5)，所述液滴微反应器生成流道(5)与所述反应室(6)连接，所述反应室出样微流道(8)以U型流道连接所述反应室(6)和所述光电检测室(9)。
  4. 根据权利要求3所述的一种高通量液滴微反应器检测系统，其特征在于，所述液滴微反应器生成流道(5)包括流道缩口(51)和液滴微反应器收集流道(52)，所述样本微流道(3)和样本隔离介质微流道(4)交汇后通过流道缩口(51)进入液滴微反应器收集流道(52)。
  5. 根据权利要求3所述的一种高通量液滴微反应器检测系统，其特征在于，所述样本微流道(3)和所述样本隔离介质微流道(4)汇合成所述液滴微反应器生成流道(5)的方式包括T形、Y形、十字交叉形、空间交叉形。
  6. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，所述反应室(6)内部设有反应室导热柱(7)，用于将所述微流控芯片外的受控温度传导至所述反应室(6)内。
  7. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，所述光电检测室(9)外置光电检测室电极(13)，用于消融检测完毕的液滴微反应器。
  8. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，所述流出口连接所述光电检测室(9)和废液流道(10)，所述废液流道(10)用于排出混于液滴微反应器间的气泡，其他比液滴微反应器比重轻的物质及检测后产生的废液。
  9. 根据权利要求1所述的一种高通量液滴微反应器检测系统，其特征在于，所述面阵检测装置(14)包括CCD相机、CMOS相机、PMT阵列，其工作帧率大于或等于微反应器运动的速率，实现边流动边实时检测记录。
  10. 一种高通量液滴微反应器检测方法，通过权利要求1-9任意一项所述的一种高通量液滴微反应器检测系统实现，包括：多个液滴微反应器自动并行平铺于光电检测室(9)中，并自动排出混杂的气泡，液滴微反应器边流动面阵检测装置(14)边检测，以面阵检测装置(14)连续获取图像，对液滴微反应器的反应状态实时进行检测记录，检测后的液滴微反应器通过光电检测室(9)外置的光电检测室电极电击消融，并排出光电检测室(9)外。
PCT/CN2019/128504 2019-12-24 2019-12-26 一种高通量液滴微反应器检测系统及方法 WO2021128113A1 (zh)

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