Micro-droplet processing device and method of using the same

Abstract

A micro-droplet processing device is provided, comprising: a sample injection system for injecting aqueous and oil phase samples into a micro-droplet processing device, the sample injection system including at least a sample injection system I (41) for injecting an oil phase sample into the micro-droplet processing device and a sample injection system II (42) for injecting an aqueous phase sample into the micro-droplet processing device; a microfluidic chip system (46) including a substrate (4), channels (1a, 1b, 1c, 1d, 2, 3) formed in the substrate, a first detection window (9), and a second detection window (10); a temperature control system including a temperature sensor and a temperature control member; a droplet identification system including a laser light source (55) and a photoelectric sensor (56); a droplet detection system including an optical fiber (57), a spectrometer (58), and a halogen light source (59); and a control system (45) for controlling each system in the micro-droplet processing device. [Selected Figure] Figure 1

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic technology, and particularly relates to a micro-droplet processing device and a method for using the same. Specifically, the micro-droplet processing device can be a microorganism droplet culture device.

Background Art

[0002] Microfluidic chip technology has been widely used in sample preparation, reaction, separation, detection, etc. in processes such as biological, chemical, and medical analysis, and is one of the most rapidly developing frontier technologies and the most active research fields. Microfluidic chips for processing sample solutions have high requirements for sterilization and the stability of sample injection. The sample injection of microfluidic chips needs to be at a low speed, such as at the pL-nL / s level, and droplet microfluidics has higher requirements for the stability of sample injection, and even slight fluctuations will affect the stability and uniformity of droplets.

[0003] In conventional microorganism selection and breeding, usually, plate coating culture is used to obtain a single colony, and then it is verified by shake flask scale fermentation culture. General microorganisms usually take several months to several years until it is confirmed that they meet production needs through processes such as preliminary screening and re-screening. In the case of this selection and breeding method, the screening cycle becomes longer. And to adopt the conventional selection and breeding method, shake flask level fermentation culture and evaluation process, sufficient personnel, experimental space, and culture space are required, and the screening efficiency can only reach 10 1~2 per batch, so the screening throughput is low. Furthermore, the success of mutagenesis screening is closely related to the number of screenings, and a large number of screening samples are required to obtain target trait mutants, and the workload of research and development personnel is very large. Finally, since the conventional selection and breeding method is based on solid culture or a large amount of liquid culture, a large amount of materials and resources for culture and detection are consumed, and the experimental cost is high.

[0004] To solve the above problems, microplate culture and screening technologies, and microfluidic technologies have been developed. The microplate screening technology reduces the culture system from 50 to 100 milliliters at the shake flask level to several milliliters or even several tens of microliters, enabling simultaneous culture of multiple samples such as 24, 48, 96, 384, 1536, etc. In combination with the corresponding automatic monitoring device, online monitoring of specific process parameters can be realized. To improve the screening efficiency of microplates, specialized support devices including an automatic monoclonal selection device, an automatic sterilized medium preparation device, an automatic medium distribution system, and an automatic bacterial plate diluter have been developed for the multi-well plate system, significantly improving the working efficiency.

[0005] Microfluidic technology was developed in the field of analytical chemistry in the 1990s. This technology is a microanalysis experimental device that integrates the preparation, injection, reaction, separation, and detection of trace samples based on a microchannel network structure. Microfluidic technology has very high efficiency and a small structure, making it easy to integrate hundreds of microbial culture units on a chip at a time and saving a large amount of medium. When the operation is integrated by software, the entire experimental process operation on the chip can be simulated.

[0006] Patent Document CN103983794A disclosed a microfluidic chip in 2014. This microfluidic chip includes a detection window (detection hole), electrodes, a channel with a branching structure, a liquid injection pool (or liquid injection port), a finished product pool (or liquid discharge port), a waste liquid pool (or waste liquid port), etc. The liquid injection pool (or liquid injection port), the finished product pool (or liquid discharge port), and the waste liquid pool (or waste liquid port) are physically connected to the channel respectively. The detection window (or detection hole) is located near the channel, and the electrodes are fixed on both sides of the channel near the branching part. Thereby, five important problems in microfluidics, namely the identification of the position of droplets, the control of the volume of droplets, the segmentation of single samples in droplets, the separation of multiple samples in droplets, and the selection of droplet parameters, are structurally solved, realizing a material carrier for microfluidic technology. However, it could not realize the detection function and could not control the culture and screening of microbial droplets online.

[0007] Patent Document CN104007091A disclosed a high-throughput detection system based on a droplet microfluidic chip in 2014. This system mainly includes a droplet microfluidic chip system, an optical path system, and a data acquisition and analysis system. The droplet microfluidic chip system embeds the microorganisms to be detected to form independent single-droplet micro reaction chambers, and transmits the laser-induced fluorescence detection signals of the microbial samples in the single-droplet micro reaction chambers through the optical path system. The data acquisition and analysis system detects and analyzes the collected signals through the computer system, and can realize the high-efficiency screening of target enzymes and metabolites related to the production of various types of industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae, but it could not realize the online culture of microorganisms.

[0008] Since the selection and breeding of microorganisms are special biological processes, it is necessary to first sow and culture them, then detect and evaluate them, and finally select the target microorganisms. Therefore, there is a need for a device that can replace the conventional seeding, shaking flask culture, and conventional detection, realize the microvolume, high-throughput, long-term continuous culture, and real-time online detection of microorganisms, and also realize the selection and breeding of microorganisms based on growth performance and characteristics.

Summary of the Invention

[0009] To solve the above problems, the present invention provides a microdroplet processing device that can be used for culturing microorganism droplets. This device can carry hundreds of microdroplets with a volume of 0.5 - 10 μL containing microorganisms through a microfluidic chip, and realize the selection and breeding of microorganisms by continuous online culture, detection, and sorting.

[0010] The object of the present invention is realized by the following technical configuration. 1. A sample injection system for injecting aqueous and oil-phase samples into a microdroplet processing device, including at least a sample injection system I for injecting an oil-phase sample into the microdroplet processing device and a sample injection system II for injecting an aqueous-phase sample into the microdroplet processing device; A microfluidic chip system including a substrate, a pipeline formed in the substrate, a first detection window, and a second detection window; A temperature control system including a temperature sensor and a temperature control member; A droplet identification system including a laser light source and a photoelectric sensor; A droplet detection system including an optical fiber spectrometer and a halogen light source; and A control system for controlling each system in the microdroplet processing device A microdroplet processing device including the above. 2. The droplet identification system uses a photoelectric sensor and a laser light source to identify the aqueous-phase sample droplets passing through the first detection window of the microfluidic chip system; The droplet detection system uses an optical fiber spectrometer and a halogen light source to detect the spectral signal of the aqueous sample droplets passing through the second detection window of the microfluidic chip system. The device according to claim 1. 3. The sample injection system includes at least two sample injection systems I and one sample injection system II respectively. The device according to claim 1 or 2. 4. The sample injection system includes three sample injection systems I and three sample injection systems II. The device according to claim 3. 5. The sample injection system I includes a liquid container and a power source. The sample injection system II includes a liquid container, a power source, and a buffer bottle. The power source is a syringe pump, a peristaltic pump, a diaphragm pump, and / or a plunger pump, preferably a syringe pump. The liquid container contains a sample solution and a liquid that is immiscible and non-compressible with the sample solution to be injected. The power source drives the liquid to be sent into the buffer bottle via an input pipeline. The buffer bottle contains the sample solution. When driven by a power source, the liquid enters the buffer bottle via an input pipeline, and thereby the sample solution is pushed into the microfluidic chip system via an output pipeline. The liquid container, the power source, and the buffer bottle are communicated with each other via capillary tubes. The device according to claim 3 or 4. 6. The microfluidic chip system includes a substrate; and a first pipeline, a second pipeline, and a third pipeline formed in the substrate. a first detection window and a second detection window, which are transparent regions formed on the first pipeline. capillary tubes that are hermetically communicated with the first pipeline, the second pipeline, and the third pipeline. and includes The first pipeline includes a first connection port and a second connection port at both ends thereof, The second pipeline includes a third connection port at one end thereof, and the other end communicates with the first pipeline, The third pipeline includes a fourth connection port at one end thereof, and the other end communicates with the first pipeline, The first communication part between the first pipeline and the second pipeline is located upstream in the droplet movement direction of the second communication part between the third pipeline and the first pipeline, and the distance between the first communication part and the second communication part is 500 μm to 2000 μm, preferably 750 μm to 1800 μm, preferably 1000 μm to 1500 μm. The device according to any one of items 1 to 5. 7. Upstream of the first communication part, the first pipeline branches into a first pipeline a, a first pipeline b, a first pipeline c, and a first pipeline d, The first detection window and the second detection window are formed on the first pipeline a, The first pipeline a, the first pipeline b, the first pipeline c, and the first pipeline d are hermetically connected to the capillary through the first connection port and the second connection port thereon, The second pipeline and the third pipeline are hermetically connected to the capillary through the third connection port and the fourth connection port respectively, The device according to item 6. 8. The substrate, the first pipeline, the second pipeline, and the third pipeline are formed of any one or more selected from the group consisting of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and acrylonitrile-butadiene-styrene copolymer (ABS), and are preferably formed of polymethyl methacrylate (PMMA). The capillary is a hard tube, and more preferably, the capillary is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a copolymer tube of perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene (PFA tube), a polyether ether ketone tube (PEEK tube), a polycarbonate tube (PC tube), and a polystyrene tube (PS tube). More preferably, the cross-sectional areas of the first pipeline, the second pipeline, the third pipeline, and the capillary pipeline are 2.5×10 -3 mm 2 ~4 mm 2 , preferably 0.01~3 mm 2 , more preferably 0.1~2.5 mm 2 , still more preferably 0.25~1 mm 2 . The device according to item 6 or 7. 9. The device according to any one of claims 1 to 8, wherein the sample injection system and the microfluidic chip system form a closed-loop control structure sealed through a capillary pipeline and a connection port. 10. The sample injection system, the microfluidic chip system, the droplet identification system, and the droplet detection system are all provided in one box, the temperature inside the box is controlled by a temperature control system, more preferably, the temperature inside the box is controlled within the range of 10°C to 50°C, and the temperature fluctuation range is controlled within the range of ±0.5°C, The device according to item 1. 11. The device according to item 10, wherein the box further includes a sterilization device, and preferably the sterilization device is an ultraviolet lamp. 12. The control system includes a controller and a PC display control system, the controller is respectively connected to the sample injection system, the temperature control system, the droplet identification system, and the droplet detection system, and controls through the digital circuit of the control system, the PC display control system displays, stores, and analyzes the information of the sample injection system, the microfluidic chip system, the temperature control system, the droplet identification system, and the droplet detection system, The device according to any one of claims 1 to 11.

[0011] The present invention is a sample injection system composed of a sample injection system I for injecting an oil-phase sample and a sample injection system II for injecting an aqueous-phase sample; A microfluidic chip system comprising a substrate, a microchannel formed on the substrate, and a detection window; A culture system comprising a pipeline connected to the microfluidic chip; A temperature control system comprising a temperature sensor and a temperature control member; A droplet identification and detection system including an optical fiber spectrometer, a photoelectric sensor, a laser light source, and a halogen light source; and A control system including a controller that is respectively connected to a sample injection system, a temperature control system, and a detection system to control a digital circuit, and a PC display control system that displays, stores, and analyzes information of the sample injection system, the microfluidic chip system, the temperature control system, and the detection system A micro-droplet processing device comprising: The photoelectric sensor is used in combination with a laser light source to identify the state of micro-droplets at a first detection window of the microfluidic chip system, and the optical fiber spectrometer is used in combination with a halogen light source to detect a sample of the micro-droplets contained at a second detection window of the microfluidic chip system. The present invention relates to a micro-droplet processing device.

[0012] The sample injection system respectively includes at least two oil-phase sample injection systems and one water-phase sample injection system; the sample injection system may include a plurality of oil-phase sample injection systems and a plurality of water-phase sample injection systems; the sample injection power source is a pressure pump or a syringe pump; The microfluidic chip system can realize operations such as generation, division, fusion, detection, and separation of micro-droplets; The culture system is composed of soft hollow pipelines. One end of the pipeline is connected to the microchannel of the chip, and the other end of the pipeline may be connected to the microchannel of the chip or directly connected to the power device; the micro-droplets in the device can flow from the chip to the pipeline or from the pipeline to the chip; the pipeline of the culture system may be gas-permeable or gas-impermeable; the inner diameter of the pipeline used in the culture system is 0.1 - 2 mm; After culturing the micro-droplets containing microorganisms, quantitative division can be performed on the chip. The divided micro-droplets can fuse with new micro-droplets, and the newly formed fused micro-droplets can continue to be cultured; before, during, or after the division and fusion processes, the micro-droplets can be detected; The sample injection system, the microfluidic chip system, the culture system, and the attached power device are connected through pipelines, connection ports, etc. to form a sealed and sterile structure; The sample injection system, the microfluidic chip system, and the culture system are all arranged in a temperature-controllable box, and the temperature inside the box is controlled by a temperature control system; the temperature control system is composed of a temperature sensor and a temperature control member; the temperature of the temperature control member can reach at most 50 °C and at least 10 °C, and the temperature fluctuation range is controlled within the range of ±0.5 °C.

[0013] The temperature control box includes a sterilization device, and the sterilization device is an ultraviolet lamp.

[0014] A method for using a microorganism droplet culture device, including the following steps: 1) Turn on the power of the device and turn on the PC display control system; 2) Exhaust the pipelines of the device, turn on the temperature control system, and preheat for 5 - 30 minutes; 3) Use the PC display control system to initialize the pipeline liquid, the optical fiber spectrometer, and the photoelectric sensor; 4) Select the function mode, for example, select programs such as droplet generation, droplet splitting and fusion, droplet culture, and droplet sorting in the microfluidic chip system, set the device parameters, and start the operation; 5) Obtain the target droplets and export the data.

[0015] In the present invention, the culture of microorganisms can be continuously cultured for up to 90 days.

[0016] The technical effects of the present invention are as follows: With the sample injection system and the microfluidic chip system, in the micro-droplet processing device, it is possible to control the generation of 1 to 500 micro-droplets with a volume of 0.5 to 10 μL per droplet. When using such a micro-droplet processing device, the throughput is much higher and the consumption of the culture medium is less than that of a shaking flask or a deep well plate; the control system can realize the regular and quantitative replacement of fresh culture medium and the addition of chemical factors, which is much more convenient than the conventional subculture for collecting the seed solution, and can save time and labor. With the detection system, the growth of microorganisms in each micro-droplet can be detected online in real time, which is much more convenient than the conventional sampling detection, and can truly realize continuous culture without interruption; in addition, it can screen intelligently, set the screening characteristics, automatically screen the appropriate strains, and improve the efficiency.

Brief Description of the Drawings

[0017]

Figure 1

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Figure 3

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Figure 9

Figure 10

[0018] Explanation of symbols: 1 (1a, 1b, 1c, 1d) First pipeline; 2 Second pipeline; 3 Third pipeline; 4 Substrate; 5 Electrode; 6 Electrode; 7 Hole; 8 Hole; 11 (11’, 11’’, 11’’’) First connection port; 12 Second connection port; 21 Third connection port; 31 Fourth connection port; 13 First communication part; 14 Second communication part; 15 Branch part; 41 Sample injection system I; 42 Sample injection system II; 43 Temperature control system; 44 Detection system (including droplet identification system and droplet detection system); 45 Control system; 46 Microfluidic chip system; 47 Container bottle a; 48 Sample injection power source a; 49 Container bottle b; 50 Sample injection power source b; 51 Container bottle c; 52 EP tube; 53 Metal bath; 54 Temperature controller; 55 Laser light source; 56 Photoelectric sensor; 57 Optical fiber; 58 Spectrometer; 59 Halogen light source; 60 Oil bottle; 61 Sample injection buffer bottle; 62 Waste liquid bottle; 63 Syringe pump; 64 Cooling fan; 65 Rotary valve; 66 Ultraviolet lamp; 67 Temperature probe; 68 Illumination lamp.

Embodiments for Carrying Out the Invention

[0019] Hereinafter, specific embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Although specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. These embodiments are provided to better understand the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0020] In the specification and claims, specific terms are used to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. The specification and claims use the differences in the functions of the components as the criterion for distinction, rather than using the differences in terms as a way to distinguish components. The terms "comprising" or "including" mentioned throughout the specification and claims are open terms and should be construed as "including but not limited to". The following description in this specification is a preferred embodiment for implementing the present invention, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention. The protection scope of the present invention is considered to be defined by the appended claims.

[0021] To facilitate the understanding of the embodiments of the present invention, some specific embodiments will be further described by way of examples in conjunction with the drawings, and each drawing does not constitute a limitation to the embodiments of the present invention.

[0022] As shown in FIGS. 1 and 2, the present invention relates to a micro-droplet processing apparatus including at least a sample injection system I for injecting an oil-phase sample into the micro-droplet processing apparatus and a sample injection system II for injecting an aqueous-phase sample into the micro-droplet processing apparatus; a microfluidic chip system including a substrate, a pipeline formed in the substrate, a first detection window, and a second detection window; a temperature control system including a temperature sensor and a temperature control member; a droplet identification system including a laser light source and a photoelectric sensor; a droplet detection system including an optical fiber spectrometer and a halogen light source; and a control system for controlling each system in the micro-droplet processing apparatus.

[0023] Specifically, as shown in FIG. 1, the microbial droplet culture apparatus of the present invention includes a sample injection system, a microfluidic chip system, a temperature control system, a detection system (which may be a droplet identification system and a droplet detection system), and a control system.

[0024] The sample injection system consists of sample injection system I and sample injection system II. Sample injection system I injects an oil-phase sample, and sample injection system II injects a water-phase sample. Sample injection system I includes a container bottle a, a sample injection power source a, and interconnected conduits. Sample injection system II includes a container bottle b, a sample injection power source b, a container bottle c, and interconnected conduits. The container bottle b of sample injection system II contains a liquid that is incompatible, non-reactive, and non-compressible with the liquid in container bottle c. The sample injection power source a drives the liquid in container bottle a to be stably and controllably fed into the microfluidic chip system via the conduit. The bottom of container bottle c contains the biological sample to be injected. When driven by the sample injection power source b, the liquid in container bottle b enters container bottle c via the conduit, causing the hydraulic pressure to rise. As a result, the biological sample at the bottom of container bottle c stably and controllably enters the microfluidic chip. The power device is a sample injection power source, preferably a pressure pump or a syringe pump.

[0025] In the device of the present invention, the sample injection system includes at least two sample injection systems I (sometimes referred to as the oil-phase sample injection system in the present invention) and one sample injection system II (sometimes referred to as the water-phase sample injection system in the present invention). The sample injection system II includes a liquid container, a power source, and a buffer bottle; the sample injection system I includes an oil-phase container and a power source. The power source is a syringe pump, a peristaltic pump, a diaphragm pump, and / or a plunger pump, preferably a syringe pump. The liquid container contains a liquid that is incompatible and non-compressible with the sample solution to be injected. The power source drives the liquid to be sent into the buffer bottle via an inlet pipeline. The buffer bottle contains the sample solution. When driven by the power source, the liquid enters the buffer bottle via the inlet pipeline, thereby pushing the sample solution into the microfluidic chip system via an outlet pipeline. The liquid container, the power source, and the buffer bottle communicate with each other via capillary tubes. The oil-phase container contains a medium oil.

[0026] In one specific embodiment, the sample injection system of the device of the present invention includes three sample injection systems I and three sample injection systems II.

[0027] An example of the sample injection system II is shown in FIG. 7. As can be seen from FIG. 7, the sample injection system II includes a liquid container 71 (for example, the container bottle b) that contains a hydraulic liquid that is incompatible and incompressible with the sample solution. The liquid container 71 is connected to a power source 72 via a hydraulic liquid export pipeline, and the power source 72 drives the hydraulic liquid to push it into a buffer bottle 74 via an import pipeline 73. In the specific embodiment shown in FIG. 7, the density of the selected hydraulic liquid is lower than the density of the sample solution. The upper part of the buffer bottle 74 (for example, the container bottle c) contains the hydraulic liquid, and the bottom part contains the sample solution. The import pipeline 73 is connected to the mouth of the buffer bottle 74, and the export pipeline 75 is connected to the bottom of the buffer bottle 74. When the power source 72 is driven, the liquid enters the buffer bottle 74 via the import pipeline 73, thereby pushing the sample solution into the microfluidic chip system 76 via the export pipeline 75. Here, the hydraulic liquid may be an oil phase, and any of the export pipeline, import pipeline, etc. can adopt the capillary of the present invention described later.

[0028] In one specific embodiment, the above liquid container is made of a rigid material, and flexible changes of the liquid container can be avoided during the sample injection process. The rigid material is a plastic material or a metal material. The plastic material is any one or more selected from the group consisting of PC (polycarbonate), ABS (acrylonitrile-butadiene-styrene copolymer), PMMA (polymethyl methacrylate), and PS (polystyrene). The metal material may be a single metal, a metal oxide, or a metal alloy. The metal material is any one or more selected from the group consisting of iron, aluminum, copper, iron oxide, aluminum oxide, and copper oxide. It may also be an alloy formed by any one or more selected from the group consisting of iron, aluminum, copper, iron oxide, aluminum oxide, and copper oxide, or an alloy formed together with other materials.

[0029] The microfluidic chip system is composed of a substrate, a microchannel formed on the substrate, a first detection window, and a second detection window. The microfluidic chip system and the sample injection system form a sealed sterile structure through a pipeline.

[0030] FIG. 3 and FIG. 4 respectively show an example of a functional schematic diagram and a specific structural diagram of the microfluidic chip system used in the present invention. FIG. 3 shows a schematic structural diagram of the microfluidic chip of the present invention. The microfluidic chip includes at least a substrate 4, and a first pipeline 1, a second pipeline 2, and a third pipeline 3 formed in the substrate.

[0031] Specifically, in the microfluidic chip of the present invention, the first pipeline, the second pipeline, and the third pipeline formed in the substrate refer to the first pipeline, the second pipeline, and the third pipeline formed inside the substrate.

[0032] The substrate 4 is a microfluidic chip substrate, formed of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS). The first pipeline 1, the second pipeline 2, and the third pipeline 3 are formed inside the substrate 4. In one specific embodiment of the present invention, the substrate and the pipeline are integrally engraved and formed.

[0033] In this embodiment, the material of the pipeline is any one selected from the group consisting of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS). Preferably, the constituent material is polymethyl methacrylate. The cross-sectional shape of the pipeline is not limited, and may be any shape suitable for forming and the flow of droplets, such as circular, rectangular, elliptical, etc. The range of the cross-sectional area of the pipeline is 2.5×10 -3 mm 2 ~4mm 2 , preferably 0.01~3mm 2 , more preferably 0.1~2.5mm 2, more preferably 0.25 to 1 mm 2 That is. More preferably, the cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline are the same. Those skilled in the art can reasonably set the thickness of the pipeline according to the size of the chip substrate, the requirements of the droplets to be cultured, detected, and sorted.

[0034] In one specific embodiment, the cross-section of the pipeline of the present invention is square, and the side length is in the range of 0.5 to 2 mm.

[0035] In one specific embodiment, the cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline may be the same as or different from each other. The diameters of the first pipeline, the second pipeline, and the third pipeline may vary, that is, the cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline are not constant. In the present invention, it is only necessary that the range of the cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline satisfies the above limitations.

[0036] As shown in FIG. 3, a first pipeline 1, a second pipeline 2, and a third pipeline 3 are formed in the substrate 4. The first pipeline 1 includes a first connection port 11 and a second connection port 12 at both ends thereof. Before cutting and fusing, the first pipeline 1 accommodates the droplet a to be divided and the droplet b to be fused. When cutting and fusing, the first pipeline 1 can accommodate the second part of the droplet a and the new droplet c. The second pipeline 2 includes a third connection port 21 at one end thereof, and the other end communicates with the first pipeline 1. The second pipeline 2 accommodates the first part of the cut droplet a1. The third pipeline 3 includes a fourth connection port 31 at one end thereof, and the other end communicates with the first pipeline 1. The third pipeline 3 accommodates the second part of the cut droplet a2. The first communication part 13 between the first pipeline 1 and the second pipeline 2 is located upstream in the droplet movement direction of the second communication part 14 between the third pipeline 3 and the first pipeline 1, and the distance between the first communication part 13 and the second communication part 14 is 500 μm to 2000 μm, preferably 750 μm to 1800 μm, preferably 1000 μm to 1500 μm.

[0037] The distance between the first communication part 13 and the second communication part 14 is related to the size of the droplets and the cross-sectional area of the pipeline. In the present invention, the distance is 500 μm to 2000 μm, preferably 750 μm to 1800 μm, and preferably 1000 μm to 1500 μm. Specifically, the distance may be 600 μm, 700 μm, 800 μm, 900 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1600 μm, 1700 μm, 1900 μm.

[0038] The chip of the present invention further includes a first detection window 9 and a second detection window 10 formed on the first pipeline 1. There is no limitation on the specific form of the detection window as long as the first detection window 9 and the second detection window 10 can monitor and detect the micro-droplets moving in the chip pipeline at that position. When the chip and the pipeline itself are formed of a transparent material, the first detection window 9 and the second detection window 10 become two detection sites on the first pipeline 1. When the materials of the chip and the pipeline are not transparent, it is necessary to form two transparent parts on the pipeline itself as the first detection window 9 and the second detection window 10.

[0039] In one specific embodiment, there is no specific limitation on the sizes of the formed first detection window 9 and second detection window 10. Since the two detection windows are one region on the first pipeline, the length of this region on the pipeline may be, for example, 200 μm to 1 mm, preferably 500 μm to 1 mm. Using a detection window with such a length can more effectively and accurately monitor and detect the micro-droplets moving in the pipeline.

[0040] When using the chip, when micro-droplets are transported into the chip, the first detection window 9 and the second detection window 10 can be used as a droplet identification site and a droplet detection site respectively. For example, the droplet identification site can detect whether a droplet passes through the detection window based on a laser system, and the droplet detection site can detect the spectral information of the droplet passing through the detection window through a spectroscopic system.

[0041] The first pipeline 1, the second pipeline 2, and the third pipeline 3 are formed inside the chip substrate 4, and the first connection port 11 and the second connection port 12 of the first pipeline, the third connection port 21 of the second pipeline, and the fourth connection port 31 of the third pipeline are all located at the edge of the substrate 4.

[0042] Of course, as can be fully understood by those skilled in the art, FIG. 3 only exemplarily shows an example of the chip according to the present invention, and each component in the chip can be constructed using any method known to those skilled in the art.

[0043] Specifically, the chip of the present invention can be used to realize the splitting and fusion operations of micro-droplets. Also, FIG. 3 shows the connection structure when the droplet a to be cut and the droplet b to be fused enter the first pipeline 1 from the first connection port 11 for cutting and fusing.

[0044] In one specific embodiment, for example, the first pipeline 1 communicates with a first power source outside the chip through the first connection port 11 and a capillary, and the first pipeline 1 communicates with a first valve outside the chip through the second connection port 12 and a capillary. The second pipeline 2 communicates with a second power source outside the chip and a second valve outside the chip respectively through the third connection port 21 and a capillary, and the third pipeline 3 communicates with a third valve outside the chip through the fourth connection port 31 and a capillary. In this embodiment, one end of the capillary is inserted and connected to the connection ports of the first pipeline, the second pipeline, and the third pipeline at the edge of the substrate, and the other end communicates with the power source and the valve. The specific communication method is shown in FIG. 6.

[0045] In the present invention, by using a power source, it is possible to achieve high-precision and stable liquid transportation without pulsation. In a specific embodiment of the present invention, the first power source and the second power source are each independently any one selected from a syringe pump, a pressure pump, a peristaltic pump, a diaphragm pump, and / or a plunger pump, and preferably, the first power source and the second power source are syringe pumps. In the present invention, there is no limitation on the size of the range of the power source, and those skilled in the art can select a syringe pump, a pressure pump, a peristaltic pump, a diaphragm pump, and / or a plunger pump having an appropriate range according to the number of samples to be injected.

[0046] In the present invention, by using a valve and changing the pressure in each sealed pipeline by opening and closing the valve, the flow direction of the droplets in each pipeline is controlled by the change in pressure. In the present invention, the first valve, the second valve, and the third valve are each independently any one selected from the group consisting of a solenoid valve, a rotary valve, a rocker valve, and a pinch-off valve. Preferably, the first valve, the second valve, and the third valve are rotary valves.

[0047] In some specific embodiments, those skilled in the art can understand that the valve can also be replaced with other forms of mechanisms or components. For example, as long as the pressure in the sealed pipeline can be changed by its opening and closing, a syringe pump as a power source can also be used as a valve.

[0048] In the present invention, the first pipeline, the second pipeline, and the third pipeline are only used to indicate different types of pipelines and are not intended to limit the number of pipelines. The first pipeline may be a plurality of first pipelines, the second pipeline may be a plurality of second pipelines, and the third pipeline may be a plurality of third pipelines.

[0049] In the present invention, the first valve, the second valve, and the third valve are used only to indicate valves that perform different functions, and are not intended to limit the number of valves. The first valve may be a plurality of first valves, the second valve may be a plurality of second valves, and the third valve may be a plurality of third valves.

[0050] In the present invention, the first power source and the second power source are used only to indicate power sources that perform different functions, and are not intended to limit the number of power sources. The first power source may be a plurality of first power sources, and the second power source may be a plurality of second power sources.

[0051] When using the chip of the present invention, the chip can be connected to the power source and the valve via the following capillary tube. In a specific embodiment of the present invention, as described below, the cross-sectional area of the capillary tube is 2.5×10 -3 mm 2 ~4 mm 2 , preferably 0.01~3 mm 2 , more preferably 0.1~2.5 mm 2 , even more preferably 0.25~1 mm 2 .

[0052] The connection port of the pipeline is connected to the capillary tube. After sealing the connection point, it communicates with the power source and / or the valve via the capillary tube. In order to stably conduct the pressure of the power source, the capillary tube is a hard tube, and there is no flexible change in the pipeline. More preferably, the capillary tube is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a copolymer tube of perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene (PFA tube), a polyether ether ketone tube (PEEK tube), a polycarbonate tube (PC tube), and a polystyrene tube (PS tube).

[0053] In one specific embodiment of the present invention, the first pipeline communicates with a first power source via a first connection port and a capillary pipeline. When the first power source is driven, pressure is generated in the pipeline through the first connection port of the first pipeline. Thereby, droplet a or droplet b is pushed into the first pipeline through the first connection port, and the movement of the droplets in the pipeline is controlled to culture the droplets.

[0054] The second pipeline communicates with a second valve via a third connection port and a capillary pipeline. When the first power source is driven and only the second valve is opened, the droplet a in the first pipeline can be controlled to flow into the second pipeline. The third pipeline communicates with a third valve via a fourth connection port and a capillary pipeline. When the first power source is driven and only the third valve is opened, the droplet a in the first pipeline can be controlled to flow into the third pipeline. By alternately opening the second valve and the third valve, the cutting of droplet a can be completed.

[0055] The second pipeline communicates with a second power source via a third connection port and a capillary pipeline. When the second power source is driven, pressure is generated in the pipeline through the third connection port of the second pipeline. Thereby, the droplet a1 to be fused is pushed into the droplet b remaining at the first communication portion between the first pipeline and the second pipeline, and the fusion of the droplets is completed.

[0056] The first pipeline communicates with a first valve via a second connection port and a capillary pipeline. When the first power source is driven and only the first valve is opened, the fused liquid c can be pushed out from the second connection port.

[0057] The first pipeline in the chip substrate is used, for example, to accommodate the droplet a to be divided and the droplet b to be fused with it. The droplet volumes of the droplet a to be divided and the droplet b to be fused with it are 0.5 to 10 μl, preferably 0.6 to 8 μl, more preferably 0.7 to 7 μl, still more preferably 0.8 to 6 μl, still more preferably 0.9 to 5 μl, and still more preferably 1 to 3 μl.

[0058] In one embodiment of the present invention, as shown in FIG. 4, there are two holes (7 and 8) on the chip substrate. The holes (7 and 8) are near the first communication portion 13 between the first pipeline 1 and the second pipeline 2. Their positions are not fixed and can be arranged on both sides of the second pipeline 2 or both sides of the first pipeline 1 respectively. The distance between the holes (7 and 8) and the first communication portion 13 between the first pipeline 1 and the second pipeline 2 is 0.1 mm to 1 cm, preferably 0.3 mm to 5 mm, and more preferably 0.5 mm to 2 mm. The holes (7 and 8) are used to accommodate the positive and negative electrodes of separately installed fusion electrodes. Those skilled in the art can determine the positions of the holes (7 and 8) within the above range according to the requirements of the chip design. The frequency of the voltage applied to the fusion electrodes is 0 to 20000 Hz, preferably 1000 to 10000 Hz, and the electrode voltage is 1 to 5000 V, preferably 500 to 1000 V. When the fusion electrodes are connected to a power source, an electric field acting on the droplets at the first communication portion is generated. This electric field can be either an alternating electric field or a constant electric field. The voltage applied to the electrodes is 1 to 5000 V, preferably 500 to 1000 V. By applying such an electric field, the fusion between the droplets a1 to be fused and the droplets b remaining at the first communication portion 13 between the first pipeline 1 and the second pipeline 2 can be further promoted.

[0059] In addition, in the present invention, there is no particular limitation on the specific shape and size of the holes (7 and 8) as long as they can be used to accommodate separately installed fusion electrodes. The fusion electrodes usually include a positive electrode and a negative electrode.

[0060] In the process of implementation by those skilled in the art, according to the principle of the present invention, a plurality of first pipelines, second pipelines, and third pipelines can be provided on the chip for the cutting and fusion of different liquids. The number of the first connection ports of the first pipeline and the third connection ports of the second pipeline can be increased to be plural and connected to different power sources respectively, and different types of droplets a to be divided and different types of droplets b to be fused can be pushed into the first pipeline.

[0061] By using a plurality of first pipelines 1 and a plurality of second pipelines 2, droplets b to be fused with different types of divided droplets a can be pushed into the first pipeline 1 or the second pipeline 2. Those skilled in the art can design corresponding droplet cutting and fusing devices by using the connection principle of the cutting and fusing device described in the present invention according to the requirements of droplet cutting and fusing. Similarly, the above-mentioned first pipeline 1a, first pipeline 1b, and first pipeline 1c, second pipeline 2a, second pipeline 2b, and second pipeline ac are merely exemplary. The first pipeline 1 can be composed of n different branch pipelines, and the second pipeline can be composed of m different branch pipelines. Here, n and m may be the same or different, and n and m may each be an integer selected from 1 to 20.

[0062] Similarly, the divided droplets aa, divided droplets ab, divided droplets ac, fused droplets ba, fused droplets bb, and fused droplets bc are also merely exemplary. Based on n first pipelines and m second pipelines, they can be used for sample injection of n different divided droplets a and m different fused droplets b.

[0063] Furthermore, for m first pipelines 1 and n second pipelines 2, different droplets can be controlled and pushed respectively by using m first power sources and n second power sources. Of course, if the design is reasonable, it is also conceivable to combine some of the power sources to push n different divided droplets a and m different fused droplets b respectively.

[0064] In one specific embodiment of the present invention, the present invention relates to a microfluidic chip. As shown in FIG. 4, in the substrate 4, a first pipeline 1a, a first pipeline 1b, a first pipeline 1c, a first pipeline 1d, a second pipeline 2, and a third pipeline 3 are formed. The first pipeline 1a includes a first connection port 11 and a second connection port 12 at both ends thereof. The first pipeline 1b includes a first connection port 11' at one end thereof, and the other end merges with the first pipeline 1a at the branch portion 15. The first pipeline 1c includes a first connection port 11'' at one end thereof, and the other end merges with the first pipeline 1a at the branch portion 15. The first pipeline 1d includes a first connection port 11''' at one end thereof, and the other end merges with the first pipeline 1a at the branch portion 15. The second pipeline 2 includes a third connection port 21 at one end thereof, and the other end communicates with the first pipeline 1. The second pipeline 2 is used to accommodate the droplets a1 of the first cut portion. The third pipeline 3 includes a fourth connection port 31 at one end thereof, and the other end communicates with the first pipeline 1. The third pipeline 3 is used to accommodate the droplets a2 of the second cut portion. The first communication portion 13 between the first pipeline 1 and the second pipeline 2 is located upstream in the droplet movement direction of the second communication portion 14 between the third pipeline 3 and the first pipeline 1. The distance between the first communication portion 13 and the second communication portion 14 is 500 μm to 2000 μm, preferably 750 μm to 1800 μm, preferably 1000 μm to 1500 μm.

[0065] Furthermore, the branch portion 15 is located upstream of the first communication portion 13, and the distance between the branch portion 15 and the first communication portion 13 is 500 μm to 5000 μm, preferably 1000 μm to 3000 μm, preferably 2000 μm to 3000 μm.

[0066] In the above specific embodiment, as shown in FIG. 4, the first detection window 9 and the second detection window 10 are formed on the first pipeline 1a.

[0067] As shown in FIG. 4, the first connection ports 11', 11'', 11''', the third connection port 21, and the fourth connection port 31 can be connected to different power sources and valves respectively to realize the injection of oil-phase and water-phase samples. Here, the sample injection systems for the water phase and the oil phase can be switched. For example, when the chip is just started, the oil-phase sample injection system is connected to the above connection ports to fill the chip with the medium oil. When starting the injection of microbial droplets, some sample injection systems can inject water-phase samples, such as microbial droplets for culture, enzyme reaction systems for reaction, and fresh media, chemical factors, substrate reaction solutions, etc. for culture.

[0068] In one specific embodiment of the present invention, for example, the oil-phase sample injection system is connected to the first connection port 11', the water-phase sample injection system is connected to the first connection port 11'', the water-phase sample injection system is connected to the first connection port 11''', the water-phase sample injection system is connected to the third connection port 21, and the valve system is connected to the fourth connection port 31.

[0069] During the use of the chip and / or chip system of the present invention, for example, the oil-phase sample injection system connected to the first connection port 11' injects an oil-phase sample into the chip. At the same time, the water-phase sample injection system connected to the first connection port 11'' pulse-injects the water-phase sample at a predetermined sample injection time as needed, thereby forming minute water-in-oil droplets at the communication part between the first pipeline 1b and the first pipeline 1c, forming, for example, a fresh medium used for seeding a bacterial solution or a substrate solution for an enzyme reaction as can be seen by referring to FIGS. 4 and 8.

[0070] The power source continuously pushes the formed micro-droplets to the right and moves them to the communication part between the first pipeline 1d and the first pipeline 1b, and then stops pushing the micro-droplets. The power source connected to the first connection port 11''' pushes another aqueous solution (for example, a chemical factor solution for addition, or a solution containing another substrate for reaction) into the micro-droplets remaining in the first pipeline 1d and the first pipeline 1b to form the above-mentioned droplet b to be fused. Specifically, this droplet b to be fused can be a fresh medium added with a certain chemical factor, or a reactant solution combined with different reaction substrates (or matrices).

[0071] When starting to use the chip, an oil phase (for example, mineral oil used as a medium) is accommodated in the first pipeline 1b shown in FIG. 4, and an aqueous phase such as a bacterial solution to be seeded or an enzyme solution to react is accommodated in the second pipeline 2. The water-in-oil droplets, that is, the droplets a to be divided, for example, the seeded bacterial solution or the enzyme solution that reacts with the reactant, are formed at the first communication part 13 between the first pipeline 1a and the second pipeline 2.

[0072] When starting to use the chip and / or the chip system, first, the droplet a to be divided is formed, then it is cultured in the first pipeline, and then the droplet b to be fused is formed using the above structure of the present invention capable of realizing cutting and fusion, and then, through cutting and fusion, the droplet a to be divided is cut and fused to form a new droplet c. Also, the second pipeline 2 starts to accommodate the aqueous phase. After all the bacterial solutions have formed water-in-oil droplets, the oil phase is replenished, and the second pipeline performs cutting and fusion as the above-mentioned second pipeline during the operation of the chip.

[0073] Therefore, as described above, FIGS. 3 and 6 of the present invention show the basic chip structures for cutting and fusing, and FIGS. 4 and 8 show one specific embodiment of the chip and / or chip system of the present invention. As can be understood by those skilled in the art, in the chip shown in FIG. 4, its partial structure is the same as that of FIG. 3, and when its power source and valve structure can be realized by the structure shown in FIG. 6, droplet fusion and cutting can be realized. When operating using the chip shown in FIG. 4, either a droplet a to be cut, surrounded by medium oil first in the chip, or a droplet b to be fused, surrounded by medium oil, can be realized by the basic structures shown in FIGS. 3 and 6. After being realized, the chip shown in FIG. 4 can use the basic structures shown in FIGS. 3 and 6 to form a new droplet c surrounded by medium oil. By repeating the above process, several to hundreds of new droplets c can be formed.

[0074] Of course, the sample injection system and the valve system are connected via a capillary tube hermetically connected to the sample injection port as described above. As described above, the sample injection system usually mainly includes a container for accommodating the liquid to be injected, a pipeline and a power source for sample injection. The power source is, as described above, any one selected from the group consisting of a syringe pump, a pressure pump, a peristaltic pump, a diaphragm pump and / or a plunger pump, preferably a syringe pump. The valve is also, as described above, any one selected from the group consisting of a solenoid valve, a rotary valve, a rocker valve, and a pinch-off valve, preferably a rotary valve.

[0075] As can be understood by those skilled in the art, the above sample injection system and valve connected to the first connection ports 11', 11'', 11''', and the third connection port 21 and the fourth connection port 31 are merely examples. Depending on the state of the chip, the relationship between these sample injection systems and valves can be replaced.

[0076] In the present invention, it can also be considered that a culture system composed of a capillary tube connected to a microfluidic chip is within the apparatus. For example, in the chip of the present invention shown in FIG. 4, the first connection port 11 is connected to the first culture pipeline via a capillary tube, and the second connection port 12 is connected to the second culture pipeline via a capillary tube. The first culture pipeline and the second culture pipeline may be composed of capillary tubes, and the lengths of the first culture pipeline and the second culture pipeline can be designed according to the requirements of the culture and reaction systems for the culture time and reaction time.

[0077] At the same time, by controlling a power source or the like connected to the chip of the present invention, the direction of the micro-droplets flowing in the chip can be reversed. As described above, several to several hundred newly formed droplets c can be cultured in the first culture pipeline. If necessary, the moving direction of the micro-droplets can be reversed by controlling the power source, thereby realizing the reciprocating movement of the micro-droplets in the pipeline.

[0078] As shown in FIG. 4, in one specific embodiment, a first detection window 9 and a second detection window 10 are provided on the first pipeline 1a of the chip of the present invention, and the descriptions of the first detection window 9 and the second detection window 10 are as described above.

[0079] Also, the distance between the first detection window 9 and the second detection window 10 on the first pipeline 1a is not particularly limited as long as the identification and detection of the micro-droplets flowing in the pipeline can be realized respectively. For example, the distance between the first detection window 9 and the second detection window 10 (taking the distance along the pipeline as an example) may be 1 cm to 10 cm, preferably 3 cm to 5 cm. The first detection window 9 and the second detection window 10 can be used as a micro-droplet identification window and a micro-droplet detection window respectively.

[0080] In one specific embodiment, the first detection window 9 is used as a micro-droplet identification window, and the second detection window 10 is used as an optical detection window for micro-droplets.

[0081] As described above, the identification of the micro-droplets can be achieved in cooperation with a laser system installed outside the chip (for example, installed on the micro-droplet processing apparatus of the present invention). When the laser system continuously emits a laser and irradiates the first detection window 9, when the micro-droplets pass through the first detection window 9, the laser beam is temporarily blocked, and the external recording system can record the change of the laser beam.

[0082] The detection of the micro-droplets can be achieved in cooperation with a spectroscopic system installed outside the chip (for example, installed on the micro-droplet processing apparatus of the present invention). For example, a spectroscopic detector can be provided to detect the absorbance of the micro-droplets passing through the second detection window 10. This absorbance can reflect, for example, the degree of growth of microorganisms in the microorganism culture system, or can reflect the color change of a reaction system accompanied by a color change.

[0083] FIG. 5 is a schematic diagram of the microfluidic chip system of the present invention. In FIG. 5, the pipeline outside the substrate of the chip shows a capillary pipeline. Similar to the structure shown in FIG. 4, the first pipeline branches into a first pipeline a, a first pipeline b, a first pipeline c, and a first pipeline d upstream of the first communication part, and the first detection window and the second detection window are formed on the first pipeline a. The first pipeline a, the first pipeline b, the first pipeline c, and the first pipeline d are hermetically connected to the capillary pipeline through the first connection port and the second connection port thereon. The second pipeline and the third pipeline are hermetically connected to the capillary pipeline through the third connection port and the fourth connection port.

[0084] The cross-sectional areas of the first pipeline, the second pipeline, the third pipeline, and the capillary pipeline are 2.5×10 -3 mm 2 ~4 mm 2 Preferably 0.01~3 mm 2 More preferably 0.1~2.5 mm 2 Even more preferably 0.25~1 mm 2Moreover, more preferably, the cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline are the same. The sealed connection parts of the capillary pipelines hermetically connected to the first pipeline, the second pipeline, and the third pipeline are located inside the substrate.

[0085] In the present invention, the above-mentioned sealed connection is not particularly limited. For example, after the design of the chip structure, it is processed by a engraving machine, and then the chip substrate is pressed by a hot press to form a capillary pipeline in a deep hole on the side surface of the chip, and an adhesive is injected for sealed bonding.

[0086] The capillary pipeline in the chip system of the present invention is a hard tube. More preferably, the capillary pipeline is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a copolymer tube of perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene (PFA tube), a polyether ether ketone tube (PEEK tube), a polycarbonate tube (PC tube), and a polystyrene tube (PS tube).

[0087] Furthermore, the temperature control system includes a temperature sensor and a temperature control member. The temperature control member further includes a heating member and a cooling member. The heating member includes a temperature controller and a metal bath. The container bottle and / or the hydraulic buffer bottle are placed in the metal bath, and a fan is attached to the temperature controller.

[0088] In one specific embodiment of the present invention, the heating member includes a temperature controller and a metal bath. The container bottle and / or the hydraulic buffer bottle are placed in the metal bath, and a fan is attached to the temperature controller. A fan, which is a cooling device for lowering the temperature of the operating environment of the equipment and further lowering the temperature of the microorganism culture, is arranged in the temperature control system.

[0089] In one specific embodiment of the present invention, the heating and cooling member may further include a drainage temperature control system. The drainage temperature control plate as the drainage temperature control system is provided in the micro-droplet treatment device. The water inlet and the drainage outlet of the drainage temperature control plate communicate with an external constant temperature water bath device. The temperature of the drainage temperature control plate is controlled by the constant temperature principle of the constant temperature water bath device to perform heating and constant temperature control of the system, thereby achieving the effect of adjusting the temperature inside the device.

[0090] The temperature sensor can measure the temperature of the culture system of the present invention online. When the temperature control switch is turned on through the host computer software of the control system, the circuit board program turns on the temperature control function and turns on the fan. After the temperature is detected, it starts to heat step by step. When it is detected that the temperature has reached the target value, some parameters are automatically adjusted, the automatic control function of the fan is turned on, and it functions in combination with the temperature sensor to keep the temperature within the range of the target value of (10 - 50) °C ± 0.2 °C. The temperature measured at 40 °C is shown in Figure 9.

[0091] The present invention can control the generation of 1 - 500 micro-droplets, preferably 5 - 400 micro-droplets, preferably 10 - 300 micro-droplets, preferably 20 - 200 micro-droplets. The volume of each micro-droplet is 0.5 - 10 μL, and the throughput is much higher than that in the case of a shaking flask or a deep well plate, and the consumption of the culture medium is small; it can realize the regular and quantitative replacement of fresh culture medium and the addition of chemical factors, which is much more convenient than the conventional subculture for extracting the seed solution, and can save time and labor; the growth of microorganisms in each micro-droplet can be detected online in real time, which is much more convenient than the conventional sampling detection, and can truly realize continuous culture; in addition, it can screen intelligently, set screening characteristics, automatically screen appropriate strains, and improve efficiency.

[0092] Furthermore, the droplet identification system includes a laser light source and a photoelectric sensor, and the droplet detection system includes an optical fiber spectrometer and a halogen light source.

[0093] Note that the droplet identification system uses a photoelectric sensor and a laser light source to identify aqueous sample droplets passing through the first detection window of the microfluidic chip system, and the droplet detection system uses an optical fiber spectrometer and a halogen light source to detect the spectral signals of aqueous sample droplets passing through the second detection window of the microfluidic chip system.

[0094] In one embodiment of the present invention, as shown in FIG. 2, the sample injection system, the microfluidic chip system, the temperature control system, the droplet identification system, and the droplet detection system are all provided in one box. As the heating and cooling members in the temperature control system, an air heater and a fan are used, and both the air heater and the fan are installed inside the box. In another embodiment of the present invention (not shown), the sample injection system, the microfluidic chip system, the droplet identification system, and the droplet detection system are all provided in one box. As the heating and cooling members in the temperature control system, a drain temperature control system and a fan are used. The drain temperature control plate is installed inside the box, and the water inlet and outlet of the drain temperature control plate communicate with an external constant temperature water bath device respectively. The temperature of the drain temperature control plate is controlled through the external constant temperature water bath device, thereby adjusting the temperature inside the box. The fan is installed inside the box to lower the temperature of the operating environment of the device.

[0095] The temperature inside the box is controlled by the temperature control system. More preferably, the temperature inside the box is controlled within the range of 10°C to 50°C, and the temperature fluctuation range is controlled within the range of ±0.5°C. The box further includes a sterilization device, and preferably the sterilization device is an ultraviolet lamp.

[0096] In the device of the present invention, the control system controls each system in the micro-droplet processing device.

[0097] The control system includes a controller and a PC display control system. The controller is respectively connected to a sample injection system, a temperature control system, a droplet identification system, and a droplet detection system, and controls them via the digital circuit of the control system. The PC display control system displays, stores, and analyzes the information of the sample injection system, the microfluidic chip system, the temperature control system, the droplet identification system, and the droplet detection system. Example

[0098] Example 1 The first pipeline, the second pipeline, and the third pipeline are pipelines formed in the chip substrate. The size of the chip substrate is 3 cm * 5 cm * 4 mm (length * width * thickness), the material of the chip substrate is PMMA, and the first pipeline, the second pipeline, and the third pipeline formed in the chip substrate are pipelines with a square cross-section, and the cross-sectional area thereof is 1 mm 2 (the length of one side is 1 mm). The first pipeline communicates with the second pipeline and the third pipeline respectively. The first communication part between the first pipeline and the second pipeline is upstream of the second communication part between the first pipeline and the third pipeline, and the distance between the first communication part and the second communication part is 1.5 mm. The pipeline is filled with medium oil.

[0099] A syringe pump A is connected to the first connection port of the first pipeline, a first rotary valve is connected to the second connection port, a syringe pump B and a second rotary valve are connected to the third connection port of the second pipeline, and a third rotary valve is connected to the fourth connection port of the third pipeline. The pipeline, the syringe pump, and the rotary valve are connected via capillary tubes. The inner diameter of the capillary tube is 1.0 mm, and the material is polytetrafluoroethylene.

[0100] In this example, both the used syringe pump A and syringe pump B are industrial syringe pumps, and the valves attached to the syringe pumps are 3-port valves. The first valve, the second valve, and the third valve are all high-pressure 2-port valves.

[0101] Turn on the syringe pump A, open the first rotary valve, and push the droplet a with a volume of 2 μl to be divided and the droplet b with a volume of 2 μl to be fused into the first pipeline. The droplet a and the droplet b were interrupted by the oily medium oil phase in the pipeline. In this example, the droplet to be divided is a microbial culture solution containing Escherichia coli BL21, and the droplet b is a fresh LB medium. The medium oil phase is mineral oil.

[0102] Close the first rotary valve, open the second rotary valve, and continue to drive the syringe pump A to push the droplet a to move to the first communication part between the first pipeline and the second pipeline. 70% of a is the droplet a1, that is, about 1.4 milliliters entered the second pipeline. At this time, close the second rotary valve, open the third rotary valve, and continue to drive the syringe pump A. The remaining 30% of the droplet a, that is, about 0.6 milliliters of the droplet a2, all entered the third pipeline. Finally, the droplet a was cut into two parts, the droplet a1 and the droplet a2. The droplet a1 was held in the second pipeline, the droplet a2 entered the third pipeline, and the third rotary valve was closed to complete the division of the droplet. The division of the droplet a mainly cuts the droplet a so that the bottom surface of the droplet a is parallel to the pipeline A, reduces the amount of the oil phase in the middle interval, and then quantitatively injects a part of the solution of the cut droplet a into the droplet b to be fused, thereby realizing high precision of the injection amount. Since the droplet a was cut by the oil phase in the pipeline, one end of the droplet b and the channel of the first pipeline are aligned, laying the foundation for accurate quantitative sample injection later.

[0103] Open the first rotary valve, continue to drive the syringe pump A to push the droplet b to move to the joint between the second pipeline and the first pipeline, then stop pushing, drive the syringe pump B to quantitatively push a part (a1) of the droplet a into the droplet b to form a new droplet c.

[0104] Turn on the syringe pump A, push the new droplet c to move forward, and the remaining part of the droplet a1 stays in the B channel, and the quantitative material exchange between the droplet a and the droplet b is completed.

[0105] The above steps were repeated such that a plurality of culture solutions containing Escherichia coli BL21 were seeded into fresh LB medium.

[0106] Example 2 As shown in FIG. 4, a microfluidic chip was formed using the same materials as in Example 1. In Example 2, the length of the substrate was 7.5 cm and the width was 5 cm.

[0107] Also, in Example 2, as shown in FIG. 8, the configuration of the sample injection system was such that three sample injection systems I and three sample injection systems II were connected to the microfluidic chip, and the power source and valves were adopted in the same manner as in Example 1.

[0108] In Example 2, before using the chip, the sample injection system I filled the chip with an oil phase, and the sample injection system II connected to the second pipeline 2 injected a bacterial solution for seeding into the second pipeline 2 under sterile conditions, thereby forming water-in-oil microdroplets at the first communication part 13. After the sample injection was completed, the second pipeline was filled with the oil phase again. Next, due to the action of the power source of the sample injection system I, the water-in-oil microdroplets formed at the first communication part 13 reciprocated within the first pipeline 1a, and bacterial culture in the droplets was performed. After the culture, the droplets were divided into droplets a. The first detection window 9 served as a droplet identification site, and the second detection window 10 served as a droplet detection site for OD detection of the bacterial concentration.

[0109] Then, the oil phase was introduced into the chip from the first connection port 11' through the sample injection system I, and the sample injection time zone was controlled through the first connection port 11'' by the sample injection system II as needed. Thereby, the aqueous medium was intermittently injected into the chip to form water-in-oil medium droplets at the communication part between the first pipeline 1b and the first pipeline 1c. The following steps can be selected. That is, after the micro-droplets move to the right and reach the communication part between the first pipeline 1d and the first pipeline 1b, stop pushing the micro-droplets. The sample injection system II connected to the first connection port 11''' pushes the sodium chloride solution into the medium droplets remaining in the first pipeline 1b and the first pipeline 1d (changing the concentration of the medium factors in the medium) to form the droplet b to be fused, let it enter the first pipeline, and position it on the left side of the droplet a to be divided. In the first pipeline 1a, the liquid was pushed to move from the first connection port 11 to the second connection port 12.

[0110] For the droplet a to be divided and the droplet b to be fused, the splitting and fusing processes in the above Example 1 were repeated. After the splitting and fusing of all droplets were completed, a new droplet culture of microorganisms could be carried out again, and the OD value of the droplets could be detected again through the second detection window 10.

[0111] Example 3 In Example 3, as shown in Figure 2, the sample injection system, the microfluidic chip system, the culture system, the temperature control system, the droplet identification system, and the droplet detection system were all arranged in one box. By the installation method shown in Figure 2, using the microfluidic chip system of Example 2, droplet splitting, fusing, and a new droplet culture of microorganisms were realized.

[0112] Among them, as shown in FIG. 8, the sample injection system includes three sample injection systems I, three sample injection systems II, a valve, and a waste liquid bottle. The sample injection system I consists of a power pump and an oil phase bottle. The sample injection system II consists of a power pump, an oil phase bottle, and a sample injection buffer bottle. The oil phase medium contained in the oil phase bottle used in the sample injection system I is the same as the oil phase medium contained in the oil phase bottle of the sample injection system II. In a specific embodiment, the oil phase bottles can be shared. In this embodiment, as shown in FIG. 2, the three sample injection systems I and the three sample injection systems II share an oil bottle 60 for containing the oil phase medium. The three sample injection buffer bottles 61 respectively contain an aqueous bacterial solution, a culture medium, and chemical factors for addition. The six syringe pumps 63 are the power sources of the sample injection system. All six syringe pumps 63 are connected to the oil bottle 60. Three of them push the oil phase medium in the oil bottle 60 into the microfluidic chip system 46 to form the three sample injection systems I shown in FIG. 8. The other three syringe pumps push the oil phase medium in the oil bottle 60 into the three sample injection buffer bottles 61 respectively, and the aqueous liquid in the sample injection buffer bottle 61 is pushed into the microfluidic chip system 46 by the liquid pressure of the oil phase medium to form the three sample injection systems II shown in FIG. 8. The syringe pumps 63, the oil bottle 60, the sample injection buffer bottles 61, the rotary valve 65, the waste liquid bottle 62, and the microfluidic chip system 46 are connected via capillary tubes (not shown) according to the connection method of Example 2. Through the cooperation of the control of the above sample injection system and the valve, the cultured bacterial solution is divided and fused in the microfluidic chip system 46 to form a plurality of water-in-oil microbial droplets, and is cultured reciprocally in the pipeline of the microfluidic chip system 46 under the action of the syringe pumps 63.

[0113] The temperature control system includes a heating member, a cooling member, and a temperature control member. As shown in Figure 2, the heating member includes an air heater 54 and a metal bath 53. The bottle 61 for sample injection buffer is placed in the metal bath 53, and the air heater 54 heats the culture environment of microorganisms inside the device box through a heating fan. The cooling member includes two fans 64 for the temperature drop of the operating environment of the device and the temperature drop of the culture environment of microorganisms respectively. The temperature probe 67 measures the temperature of the culture environment of microorganisms inside the box and feeds back to the control system of the device, thereby controlling the operations of the heating member and the cooling member, and heating the droplets in the microfluidic chip and performing constant temperature control. In this embodiment, the temperature control curve is shown in Figure 9.

[0114] The droplet identification system includes a laser light source 55 and a photoelectric sensor 56. As shown in Figure 2, the laser light source 55 irradiates the first detection window of the microfluidic chip system 46, identifies the aqueous sample droplets passing through the first detection window, and sends back the position information of the droplets to the control device of the device through the photoelectric sensor 56.

[0115] The droplet detection system includes an optical fiber 57, a spectrometer 58, and a halogen light source 59. As shown in Figure 2, the optical fiber 57 is aligned with the second detection window of the microfluidic chip system 46, and through the spectrometer 58, detects the spectral signal of the aqueous sample droplets passing through the second detection window of the microfluidic chip system.

[0116] In the detection system, the photoelectric sensor 56 and the laser light source are used in combination to identify the state of the micro-droplets at the first detection window of the microfluidic chip system 46, thereby controlling the reciprocation and culture of the droplets. The optical fiber 57, the spectrometer 58, and the halogen light source 59 are used in combination to detect the sample in the contained micro-droplets at the second detection window of the microfluidic chip system 46. The EP tube 52 contains the droplets of the cultured microorganisms, and the ultraviolet lamp 66 can sterilize the culture environment of the microorganisms inside the device.

[0117] The steps of microorganism culture are as follows: 1) The equipment was sterilized by turning on the ultraviolet lamp for 30 minutes; 2) The device pipeline was exhausted, and the temperature control system was turned on and preheated for 30 minutes; 3) 1 mL of the inoculated bacterial solution (E. coli BL21 as described above) was added to one sample injection buffer bottle, placed in a vacuum degassing box for 30 minutes of degassing treatment, fresh culture medium for culture (LB medium) was placed in one sample injection buffer bottle, and the chemical factor for addition, namely ascorbic acid solution, was placed in another sample injection buffer bottle; 4) The chip was attached to the bracket and connected to the inlets of the sample injection buffer bottle and the oil bottle; 5) The equipment software was opened, the "growth curve" measurement mode was selected, and the basic parameters were set such as the detection cycle of 0.5 hours, the number of droplets of 100, and the detection wavelength of 620 nm; 6) The execute button was clicked; 7) After running for 46 hours, the data was acquired, and the results are shown in Figure 10.

[0118] The present invention can achieve accurate temperature control, improve the reliability of results, enable smooth growth of microorganisms, and realize continuous culture for up to 90 days. Industrial Applicability

[0119] The fully automatic high-throughput microorganism droplet culture device and usage method of the present invention can be manufactured and used in the field of high-throughput microorganism culture.

[0120] Regarding the embodiments of the present invention, they have been described in combination with the accompanying drawings, but the present invention is not limited to the above specific embodiments and application fields. The above specific embodiments are merely illustrative, guiding, and not limiting. Under the teachings of this specification, those skilled in the art can form many forms without departing from the protection scope of the claims of the present invention, and all of them are within the protection scope of the present invention.

Claims

1. A sample injection system for injecting samples of an oil phase and an aqueous phase into a micro-droplet processing device, comprising at least a sample injection system I for injecting a sample of the oil phase into the micro-droplet processing device and a sample injection system II for injecting a sample of the aqueous phase into the micro-droplet processing device; A microfluidic chip system comprising a substrate, a pipeline formed in the substrate, a first detection window, and a second detection window; A temperature control system comprising a temperature sensor and a temperature control member; A droplet identification system comprising a laser light source and a photoelectric sensor; A droplet detection system comprising an optical fiber spectrometer and a halogen light source; and A control system for controlling each system in the micro-droplet processing device comprising, wherein the microfluidic chip system comprises a substrate; and a first pipeline, a second pipeline, and a third pipeline formed in the substrate, a first detection window and a second detection window, which are transparent regions formed on the first pipeline, and a capillary tube hermetically communicating with the first pipeline, the second pipeline, and the third pipeline comprising, the first pipeline includes a first connection port and a second connection port at both ends thereof, one end of the second pipeline includes a third connection port, and the other end communicates with the first pipeline, one end of the third pipeline includes a fourth connection port, and the other end communicates with the first pipeline, a first communication part between the first pipeline and the second pipeline is located upstream in the droplet movement direction of a second communication part between the third pipeline and the first pipeline, and the distance between the first communication part and the second communication part is 1000 μm to 1500 μm, The cross-sectional areas of the first pipeline, the second pipeline, and the third pipeline are 0.25 to 1 mm ２ respectively, the droplet volumes of the droplet a to be divided and the droplet b to be fused accommodated in the micro-droplet processing device are 0.9 to 5 μl, before the first pipeline cuts and fuses, it accommodates the droplet a to be divided and the droplet b to be fused, and when cutting and fusing, the first pipeline is controlled to accommodate the droplet a2 of the second part and the new droplet c, the second pipeline is controlled to accommodate the first part of the droplet a1 after being cut, the first part of the droplet a1 and the droplet b to be fused are controlled to fuse at the first communication part, a micro-droplet processing device, wherein the third pipeline is controlled to accommodate the second part of the droplet a2 after being cut.

2. The droplet identification system identifies aqueous sample droplets passing through a first detection window of a microfluidic chip system by means of a photoelectric sensor and a laser light source; The droplet detection system detects spectral signals of aqueous sample droplets passing through a second detection window of a microfluidic chip system by means of an optical fiber spectrometer and a halogen light source. The device according to claim 1.

3. The sample injection system includes at least two sample injection systems I and one sample injection system II respectively. The device according to claim 1 or 2.

4. The sample injection system includes three sample injection systems I and three sample injection systems II. The device according to claim 3.

5. The sample injection system I includes a liquid container and a power source. The sample injection system II includes a liquid container, a power source, and a buffer bottle. The power source is a syringe pump, a peristaltic pump, a diaphragm pump, and / or a plunger pump. The liquid container contains a liquid that is incompatible and non-compressible with the sample solution to be injected. The power source drives the liquid to be sent into the buffer bottle via an input pipeline. The buffer bottle contains the sample solution. When driven by a power source, the liquid enters the buffer bottle via an input pipeline, and thereby the sample solution is pushed into the microfluidic chip system via an output pipeline. The liquid container, the power source, and the buffer bottle are connected in communication via capillary tubes. The device according to claim 3 or 4.

6. The first pipeline branches into a first pipeline a, a first pipeline b, a first pipeline c, and a first pipeline d upstream of the first communication part. The first detection window and the second detection window are formed on the first pipeline a. The first pipeline a, the first pipeline b, the first pipeline c, and the first pipeline d are hermetically connected to the capillary tube via a first connection port and a second connection port thereon. The second pipeline and the third pipeline are hermetically connected to the capillary tube via a third connection port and a fourth connection port. The device according to claim 1.

7. The substrate, the first pipeline, the second pipeline, and the third pipeline are formed of any one or more selected from the group consisting of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and acrylonitrile-butadiene-styrene copolymer (ABS). The device according to claim 6, wherein the capillary pipeline is a rigid tube.

8. The substrate, the first pipeline, the second pipeline, and the third pipeline are formed of polymethyl methacrylate (PMMA), or The capillary pipeline of the device according to claim 7 is any one selected from the group consisting of polytetrafluoroethylene tubes (PTFE tubes), copolymer tubes of perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene (PFA tubes), polyether ether ketone tubes (PEEK tubes), polycarbonate tubes (PC tubes), and polystyrene tubes (PS tubes).

9. The cross-sectional area of the capillary tube is 2.5×10 －３ mm ２ to 4 mm ２ The device according to claim 7.

10. The sample injection system and the microfluidic chip system are connected via a capillary pipeline and a connection port to form a sealed closed-loop control structure. The device according to any one of claims 1 to 9.

11. The sample injection system, the microfluidic chip system, the droplet identification system, and the droplet detection system are all provided in one box. The temperature inside the box is controlled by a temperature control system. The device according to claim 1.

12. The temperature inside the box is controlled within the range of 10°C to 50°C, and the temperature fluctuation range is controlled within the range of ±0.5°C. The device according to claim 11.

13. The box further includes a sterilization device. The device according to claim 11 or 12.

14. The control system includes a controller and a PC display control system. The controller is respectively connected to the sample injection system, the temperature control system, the droplet identification system, and the droplet detection system, and controls them via the digital circuit of the control system. The PC display control system displays, stores, and analyzes the information of the sample injection system, the microfluidic chip system, the temperature control system, the droplet identification system, and the droplet detection system. The device according to any one of claims 1 to 13.

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