Microdroplet processing device and method of using same

Abstract

To provide a micro liquid processing device that can realize microvolume, high throughput, long-term continuous culture, real-time online detection of microorganisms, and also realize selection and breeding of microorganisms based on their growth performance and characteristics. [Solution] The device includes a sample injection system for injecting aqueous and oil phase samples into a microdroplet processing device, the sample injection system including at least a sample injection system I41 for injecting an oil phase sample and a sample injection system II42 for injecting an aqueous phase sample into the microdroplet processing device; a microfluidic chip system 46 including a substrate, a channel 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, a spectrometer, and a halogen light source; and a control system 45 for controlling each system in the device.

Description

[Technical Field]

[0001] The present invention relates to the field of microfluidics, and in particular to a microdroplet processing device and a method for using the same. Specifically, the microdroplet processing device can be a microorganism droplet culture device. [Background technology]

[0002] Microfluidic chip technology is widely used for sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes, and is one of the rapidly developing frontier technologies and most active research fields. Microfluidic chips that process sample solutions have high requirements for sterilization and sample injection stability. Sample injection in microfluidic chips must be slow, for example, at the pL-nL / s level. Droplet microfluidics has higher requirements for sample injection stability, and even slight fluctuations can affect the stability and uniformity of droplets.

[0003] Traditional microbial selection and breeding typically involves obtaining single colonies using plate coating cultures and then verifying them through shake flask-scale fermentation culture. For common microorganisms, it usually takes several months to several years to go through processes such as preliminary selection and reselection before confirming that they meet production needs. This selection and breeding method results in a long selection cycle. Furthermore, adopting traditional selection and breeding methods, including shake flask-scale fermentation culture and evaluation processes, requires sufficient personnel, laboratory space, and culture space, and the screening efficiency is limited to 10 per batch. 1~2 This results in low screening throughput. Furthermore, the success of mutagenesis screening is closely related to the number of screenings, and obtaining target trait mutants requires a large number of screening samples, imposing a heavy workload on R&D personnel. Finally, traditional selection and breeding methods are based on solid culture or large-scale liquid culture, which consumes a large amount of materials and resources for cultivation and detection, resulting in high experimental costs.

[0004] To address these issues, microplate incubation and screening technologies and microfluidics have been developed. Microplate screening technology reduces incubation volumes from 50–100 ml in shake flasks to a few milliliters or even tens of microliters, enabling simultaneous incubation of multiple samples (e.g., 24, 48, 96, 384, 1536). Combined with corresponding automated monitoring equipment, it also enables online monitoring of specific process parameters. To enhance the efficiency of microplate screening, specialized supporting equipment has been developed around multiwell plate systems, including automated monoclonal screening equipment, automated sterilization medium preparation equipment, automated medium distribution systems, and automated bacterial plate diluters, significantly improving operational efficiency.

[0005] Microfluidic technology was developed in the field of analytical chemistry in the 1990s. This technology is a microanalytical laboratory device based on a microchannel network structure that integrates minute sample preparation, sample injection, reaction, separation, and detection. Microfluidic technology has high efficiency and a small structure, making it easy to assemble hundreds of microbial culture units on a chip at one time, saving a large amount of culture medium. By integrating operations through software, the entire experimental process operation can be simulated on the chip.

[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), and a waste liquid pool (or waste port). The liquid injection pool (or liquid injection port), finished product pool (or liquid discharge port), and waste liquid pool (or waste port) are physically connected to the channel, respectively. The detection window (or detection hole) is located near the channel, and electrodes are fixed on both sides of the channel near the branching section. This structurally solves five key problems in microfluidics: droplet position identification, droplet volume control, droplet segmentation, multiple sample separation within a droplet, and droplet parameter selection. This materialized a material carrier for microfluidic technology. However, it was unable to achieve detection functionality or online control of microbial droplet cultivation and screening.

[0007] Patent document CN104007091A, published in 2014, disclosed a high-throughput detection system based on a droplet microfluidic chip. This system mainly includes a droplet microfluidic chip system, an optical path system, and a data collection and analysis system. The droplet microfluidic chip system embeds the target microorganisms to be detected to form an independent single-droplet microreaction chamber. The laser-induced fluorescence detection signal of the microbial sample in the single-droplet microreaction chamber is transmitted via the optical path system. The data collection and analysis system detects and analyzes the collected signal via a computer system. This enabled 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 did not allow online microbial cultivation.

[0008] Microbial selection and breeding is a special biological process that requires seeding and cultivation, followed by detection and evaluation, and finally allows target microorganisms to be selected. Therefore, there is a need for a device that can replace traditional seeding, shake flask culture, and traditional detection, realize microvolume, high-throughput, long-term continuous culture, and real-time online detection of microorganisms, and also realize microbial selection and breeding 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 microbial droplet cultivation. This device can load hundreds of microdroplets containing microorganisms, each with a volume of 0.5 to 10 μL, via a microfluidic chip, and complete microbial selection and breeding through continuous online cultivation, detection, and screening.

[0010] The object of the present invention is achieved by the following technical configuration. 1. A sample injection system for injecting aqueous and oil phase samples into a microdroplet processing device, comprising 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 conduit 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 a fiber optic spectrometer and a halogen light source; and Control system for controlling each system in a microdroplet processing device A microdroplet processing device comprising: 2. The droplet identification system uses a photoelectric sensor and a laser light source to identify sample droplets in the aqueous phase that pass through a first detection window of the microfluidic chip system; the droplet detection system uses a fiber optic spectrometer and a halogen light source to detect a spectral signal of the aqueous phase sample droplet that passes through a second detection window of the microfluidic chip system; Item 1. The device described in item 1. 3. The sample injection system includes at least two sample injection systems I and one sample injection system II; Item 1 or 2. The device according to item 1 or 2. 4. The sample injection system includes three sample injection systems I and three sample injection systems II; Item 3. The device according to item 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 liquid that is incompatible with the sample solution to be injected and is incompressible; The power source drives the liquid through an import line into a buffer bottle; The buffer bottle contains the sample solution, and when driven by a power source, the liquid enters the buffer bottle through an inlet pipe, thereby forcing the sample solution into the microfluidic chip system through an outlet pipe, and the liquid container, the power source, and the buffer bottle are in communication with each other through a capillary pipe. Item 3 or 4, the device according to item 3 or 4. 6. Microfluidic chip system substrate; and a first conduit, a second conduit, and a third conduit formed in the substrate; a first detection window and a second detection window, which are transparent regions formed on the first conduit; and a capillary passage in sealed communication with the first passage, the second passage, and the third passage; Including, the first conduit includes a first connection port and a second connection port at both ends thereof, the second conduit includes a third connection port at one end thereof and communicates with the first conduit at the other end thereof; the third conduit includes a fourth connection port at one end thereof and communicates with the first conduit at the other end thereof; The first communication part between the first conduit and the second conduit is located upstream of the second communication part between the third conduit and the first conduit in the droplet movement direction, 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, and preferably 1000 μm to 1500 μm. Item 6. The device according to any one of items 1 to 5. 7. The first pipe branched into a first pipe a, a first pipe b, a first pipe c, and a first pipe d upstream of the first communication portion; the first detection window and the second detection window are formed on the first conduit a; the first conduit a, the first conduit b, the first conduit c, and the first conduit d are sealedly connected to the capillary channel through the first connection port and the second connection port thereon; the second conduit and the third conduit are sealedly connected to the capillary conduit via the third connection port and the fourth connection port, respectively; Item 6. The device according to item 6. 8. The substrate and the first, second, and third ducts are made of one or more materials selected from the group consisting of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and acrylonitrile-butadiene-styrene copolymer (ABS), and are preferably made of polymethyl methacrylate (PMMA); The capillary channel is a rigid tube, and more preferably, the capillary channel is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene copolymer tube (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 area of ​​the first conduit, the second conduit, the third conduit, and the capillary conduit is less than 2.5×10 -3 mm 2 ~4mm 2 , preferably 0.01 to 3 mm 2 , more preferably 0.1 to 2.5 mm 2 , and more preferably 0.25 to 1 mm 2 That is, Item 6 or 7. 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 sealed closed-loop control structure via the capillary channel and the connection port. 10. The sample injection system, microfluidic chip system, droplet identification system, and droplet detection system are all installed 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 a range of 10°C to 50°C, and the temperature fluctuation range is controlled within a range of ±0.5°C. Item 1. The device described in item 1. Item 11. The device according to item 10, wherein the box further comprises a sterilizer, preferably an ultraviolet lamp. 12. The control system includes a controller and a PC display control system; The controller is connected to the sample injection system, the temperature control system, the droplet identification system, and the droplet detection system, respectively, and controls them through a digital circuit of the control system; The PC display control system displays, stores, and analyzes information from the sample injection system, the microfluidic chip system, the temperature control system, the droplet identification system, and the droplet detection system; Item 12. The device according to any one of items 1 to 11.

[0011] The present invention provides a sample injection system including 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 consisting of conduits connected to a microfluidic chip; a temperature control system comprising a temperature sensor and a temperature control member; a droplet identification and detection system including a fiber optic spectrometer, a photoelectric sensor, a laser light source, and a halogen light source; and a control system including a controller connected to the sample injection system, the temperature control system, and the detection system, respectively, and controlling digital circuits; and a PC display control system for displaying, storing, and analyzing information from the sample injection system, the microfluidic chip system, the temperature control system, and the detection system. A microdroplet processing device comprising: The microdroplet processing device relates to a microdroplet processing device in which the photoelectric sensor is used in combination with a laser light source to identify the state of the microdroplets in a first detection window of the microfluidic chip system, and the fiber optic spectrometer is used in combination with a halogen light source to detect a sample of the microdroplets contained in a second detection window of the microfluidic chip system.

[0012] The sample injection systems each include at least two oil-phase sample injection systems and one aqueous-phase sample injection system; the sample injection systems may include a plurality of oil-phase sample injection systems and a plurality of aqueous-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 microdroplet generation, division, fusion, detection, and separation; The culture system is composed of a flexible hollow conduit, one end of which is connected to the microchannel of the chip, and the other end of which may be connected to the microchannel of the chip or directly to a power device; microdroplets in the device can flow from the chip to the conduit or from the conduit to the chip; the conduit of the culture system may be gas permeable or gas impermeable; the inner diameter of the conduit used in the culture system is 0.1 to 2 mm; After culturing the microdroplets containing the microorganisms, quantitative division can be performed on the chip, the divided microdroplets can be fused with new microdroplets, and the new fused microdroplets can be continuously cultivated; the microdroplets can be detected before or after the division and fusion process; The sample injection system, the microfluidic chip system, the culture system, and the attached power device are connected via conduits, connection ports, etc. to form a sealed and sterile structure; The sample injection system, the microfluidic chip system, and the incubation system are all placed 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 element; the temperature of the temperature control element can reach a maximum of 50°C and a minimum of 10°C, and the temperature fluctuation range is controlled within ±0.5°C.

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

[0014] A method of using a microbial droplet culture device, comprising the steps of: 1) Turn on the device and turn on the PC display control system; 2) Evacuate the equipment duct, turn on the temperature control system, and preheat for 5 to 30 minutes; 3) Use the PC display control system to initialize the pipe fluid, fiber optic spectrometer, and photoelectric sensor; 4) Select the function mode, for example, droplet generation in the microfluidic chip system, droplet splitting and fusion, droplet culture, droplet sorting, etc., and set the device parameters to start the operation; 5) Acquire the target droplet and export the data.

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

[0016] The technical effects of the present invention are as follows: The sample injection system and microfluidic chip system enable the microdroplet processor to control the generation of 1 to 500 microdroplets, each with a volume of 0.5 to 10 μL. This method allows for much higher throughput and less medium consumption than shake flasks or deep-well plates. The control system allows for regular and quantitative replacement of fresh medium and the addition of chemical factors, which is much more convenient than traditional subcultures that require seed solution collection, saving time and effort. The detection system allows for real-time online detection of microbial growth in each microdroplet, which is much more convenient than traditional sampling and enables truly uninterrupted cultivation. It also allows for intelligent screening, setting screening characteristics, and automatically selecting suitable strains, improving efficiency. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a functional schematic diagram of a microdroplet processing device according to the present invention. [Figure 2] 1 is a perspective view of an embodiment of a microdroplet processing apparatus of the present invention. [Figure 3] 1 is a functional schematic diagram of a microfluidic chip used in a microdroplet processing device of the present invention. FIG. [Figure 4] 1 is a schematic diagram of a microfluidic chip system used in the microdroplet processing device of the present invention. [Figure 5]1 is a structural schematic diagram of the function of the microfluidic chip of the present invention used for quantitative splitting and fusion of microdroplets. [Figure 6] 1 is a structural schematic diagram of the connection between the microfluidic chip system of the present invention and the power source and valves of the microdroplet processing device. FIG. [Figure 7] 1 is a schematic diagram of a sample injection system I of the present invention. [Figure 8] FIG. 1 is a schematic diagram showing the chip system and its connections to power sources and valves when used in a microdroplet processing device of the present invention. [Figure 9] FIG. 2 is a temperature change diagram of the microdroplet temperature control display of the present invention. [Figure 10] FIG. 1 is a 46-hour growth curve of Escherichia coli measured using the microbial droplet culture device of the present invention.

[0018] Symbols: 1 (1a, 1b, 1c, 1d) first conduit; 2 second conduit; 3 third conduit; 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 portion; 14 second communication portion; 15 branch portion; 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 bottle; 63 Syringe pump; 64 Cooling fan; 65 Rotating valve; 66 Ultraviolet lamp; 67 Temperature probe; 68 Illumination lamp. DETAILED DESCRIPTION OF THE INVENTION

[0019] Specific embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present invention are illustrated in the drawings, it should be understood that the present invention can be embodied in various forms and should not be limited to the embodiments set forth herein. These embodiments are provided to facilitate a better understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0020] It should be noted that the specification and claims use specific terms 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 do not use differences in terminology to distinguish between components, but rather use differences in the functions of the components as the basis for distinction. The terms "comprise" or "include" used throughout the specification and claims are open terms and should be interpreted as "including, but not limited to." The following description of preferred embodiments for carrying out the present invention is intended to illustrate the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention is deemed to be defined by the appended claims.

[0021] To facilitate understanding of embodiments of the present invention, some specific embodiments are further described by way of example in conjunction with drawings, each of which does not constitute a limitation on the embodiments of the present invention.

[0022] As shown in Figures 1 and 2, the present invention relates to a microdroplet processing device comprising: a sample injection system for injecting aqueous and oil phase samples into a microdroplet processing device, the sample injection system 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 channel 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 a fiber optic spectrometer and a halogen light source; and a control system for controlling each system in the microdroplet processing device.

[0023] Specifically, as shown in FIG. 1 , the microbial droplet culture device 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 comprises sample injection system I and sample injection system II, where sample injection system I injects an oil phase sample and sample injection system II injects an aqueous 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; container bottle b of sample injection system II contains a liquid that is immiscible, non-reactive, and incompressible with the liquid in container bottle c, and sample injection power source a drives the liquid in container bottle a to stably and controllably send it into the microfluidic chip system via the conduit; the bottom of container bottle c contains a biological sample to be injected, and when driven by sample injection power source b, the liquid in container bottle b enters container bottle c via the conduit, increasing the liquid pressure, thereby stably and controllably sending the biological sample at the bottom of container bottle c into the microfluidic chip; the power device is a sample injection power source, and is preferably a pressure pump or a syringe pump.

[0025] In the device of the present invention, the sample injection systems each include at least two sample injection systems I (sometimes referred to as oil-phase sample injection systems in the present invention) and one sample injection system II (sometimes referred to as aqueous-phase sample injection system in the present invention), and 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, and 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 with the sample solution to be injected and is incompressible. The power source drives the liquid to send it into the buffer bottle via an import pipeline. The buffer bottle contains the sample solution. When driven by the power source, the liquid enters the buffer bottle via the import pipeline, thereby forcing the sample solution into the microfluidic chip system via an export pipeline. The liquid container, the power source, and the buffer bottle are connected via a capillary line. 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 a 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 (e.g., the above-mentioned container bottle b) containing a hydraulic liquid that is incompatible with the sample solution and incompressible. The liquid container 71 is connected to a power source 72 via a hydraulic liquid export line, and the power source 72 drives the hydraulic liquid to push it into a buffer bottle 74 via an import line 73. In the specific embodiment shown in FIG. 7, the density of the selected hydraulic liquid is lower than that of the sample solution, and the top of the buffer bottle 74 (e.g., container bottle c) contains the hydraulic liquid, and the bottom contains the sample solution. The import line 73 is connected to the mouth of the buffer bottle 74, and the export line 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 line 73, thereby pushing the sample solution into a microfluidic chip system 76 via the export line 75. Here, the hydraulic liquid may be an oil phase, and the export pipeline and the import pipeline may both employ the capillary pipeline of the present invention described below.

[0028] In one specific embodiment, the liquid container is made of a rigid material to prevent the liquid container from becoming flexible during sample injection. 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 metal, a metal oxide, or a metal alloy. The metal material may be any one or more selected from the group consisting of iron, aluminum, copper, iron oxide, aluminum oxide, and copper oxide. The metal material may also be an alloy formed with any one or more selected from the group consisting of iron, aluminum, copper, iron oxide, aluminum oxide, and copper oxide, or an alloy formed 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, and the microfluidic chip system and the sample injection system form a sealed sterile structure via a conduit.

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

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

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

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

[0034] In one specific embodiment, the cross section of the conduit of the present invention is square, with the side length ranging from 0.5 to 2 mm.

[0035] In one specific embodiment, the cross-sectional areas of the first, second, and third conduits may be the same or different from one another. The diameters of the first, second, and third conduits may vary, i.e., the cross-sectional areas of the first, second, and third conduits are not constant. In the present invention, the ranges of the cross-sectional areas of the first, second, and third conduits may satisfy the above-mentioned restrictions.

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

[0037] The distance between the first communicating portion 13 and the second communicating portion 14 is related to the size of the droplets and the cross-sectional area of ​​the pipe. 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, or 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 channel 1. There are no limitations on the specific form of the first detection window 9 and the second detection window 10, as long as they are capable of monitoring and detecting microdroplets moving within the chip channel at their positions. If the chip and channel themselves are made of a transparent material, the first detection window 9 and the second detection window 10 become two detection sites on the first channel 1. If the chip and channel materials are not transparent, two transparent regions must be formed in the channel itself as the first detection window 9 and the second detection window 10.

[0039] In one specific embodiment, there is no specific limit to the size of the formed first detection window 9 and second detection window 10. Because 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, and preferably 500 μm to 1 mm. Using a detection window of such a length allows for more effective and accurate monitoring and detection of microdroplets moving within the pipeline.

[0040] When the chip is in use, when microdroplets 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 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 on the edge of the substrate 4.

[0042] Of course, as will be fully understood by those skilled in the art, FIG. 3 is merely an illustrative example of one example of a chip according to the present invention, and each component within 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 of microdroplets. Also, Figure 3 shows the connection structure when a droplet a to be split and a droplet b to be fused enter the first conduit 1 through the first connection port 11 for splitting and fusion.

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

[0045] In the present invention, the use of a power source enables high-precision, stable, and pulsation-free liquid transport. In a specific embodiment of the present invention, the first power source and the second power source are each independently 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 limit to the size of the range of the power source, and those skilled in the art can select a syringe pump, pressure pump, peristaltic pump, diaphragm pump, and / or plunger pump having an appropriate range depending on the number of samples to be injected.

[0046] In the present invention, the pressure in each sealed conduit is changed by using valves and opening and closing the valves, and the flow direction of droplets in each conduit 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 will appreciate that the valves can be replaced with other forms of mechanisms or components, for example, a syringe pump can be used as a power source as long as its opening and closing can change the pressure within the sealed conduit.

[0048] In the present invention, the terms "first conduit," "second conduit," and "third conduit" are used only to indicate different types of conduits and are not intended to limit the number of conduits. A first conduit may be a plurality of first conduits, a second conduit may be a plurality of second conduits, and a third conduit may be a plurality of third conduits.

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

[0050] In the present invention, the terms "first power source" and "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 multiple first power sources, and the second power source may be multiple second power sources.

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

[0052] The connection port of the conduit is connected to the capillary conduit, and after sealing the connection point, the capillary conduit communicates with the power source and / or the valve through the capillary conduit. To stably transmit the pressure of the power source, the capillary conduit is a rigid tube, and the conduit does not have any change in flexibility. More preferably, the capillary conduit is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene copolymer tube (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 conduit communicates with a first power source via a first connection port and a capillary channel, and when the first power source is driven, pressure is generated in the conduit via the first connection port of the first conduit, thereby forcing droplet a or droplet b into the first conduit via the first connection port, and controlling the movement of the droplet in the conduit to culture the droplet.

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

[0055] The second conduit is connected to a second power source via a third connection port and a capillary channel, and when the second power source is driven, pressure is generated in the conduit via the third connection port of the second conduit, thereby pushing the droplet a1 to be fused into the droplet b remaining in the first connection portion between the first conduit and the second conduit, thereby completing the fusion of the droplets.

[0056] The first conduit communicates with the first valve via the second connection port and the capillary channel, and when the first power source is activated to open only the first valve, the fused liquid c can be pushed out from the second connection port.

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

[0058] In one embodiment of the present invention, as shown in FIG. 4, two holes (7 and 8) are provided on the chip substrate. The holes (7 and 8) are located near the first connection 13 between the first conduit 1 and the second conduit 2. Their positions are not fixed and can be located on either side of the second conduit 2 or on either side of the first conduit 1. The distance between the holes (7 and 8) and the first connection 13 between the first conduit 1 and the second conduit 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 ranges according to the requirements of the chip design. The frequency of the voltage applied to the fusion electrode is 0 to 20,000 Hz, preferably 1,000 to 10,000 Hz, and the electrode voltage is 1 to 5,000 V, preferably 500 to 1,000 V. When the fusion electrode is connected to a power source, an electric field acting on the droplets in the first communication part is generated, and this electric field is either an AC electric field or a constant electric field. The voltage applied to the electrode is 1 to 5,000 V, preferably 500 to 1,000 V. Applying such an electric field can further promote the fusion of the droplets a1 to be fused and the droplets b remaining in the first communication part 13 between the first pipe line 1 and the second pipe line 2.

[0059] In addition, the present invention does not limit the specific shape and size of the holes (7 and 8) as long as they can be used to accommodate a separately installed fusion electrode. The fusion electrode usually includes a positive electrode and a negative electrode.

[0060] In the process of implementation by those skilled in the art, the principles of the present invention can be used to provide multiple first, second, and third pipelines on a chip for cutting and fusing different liquids, or the first connection port of the first pipeline and the third connection port of the second pipeline can be increased to multiple ports and connected to different power sources, respectively, so that different types of droplets a to be split and different types of droplets b to be fused can be pushed into the first pipeline.

[0061] By using multiple first conduits 1 and multiple second conduits 2, different types of droplets a to be split and b to be merged can be forced into the first conduits 1 or the second conduits 2. Those skilled in the art can design corresponding droplet cutting and fusion devices using the connection principles of the cutting and fusion device described in the present invention according to the droplet cutting and fusion requirements. Similarly, the above-mentioned first conduits 1a, 1b, and 1c, second conduits 2a, 2b, and second conduits ac are merely exemplary. The first conduit 1 can be composed of n different branch conduits, and the second conduit can be composed of m different branch conduits. Here, n and m can be the same or different, and n and m can each be an integer selected from 1 to 20.

[0062] Similarly, split droplet aa, split droplet ab, split droplet ac, fused droplet ba, fused droplet bb, and fused droplet bc are also exemplary. Based on n first conduits and m second conduits, n different split droplets a and m different fused droplets b can be used for sample injection.

[0063] Furthermore, m first power sources and n second power sources can be used to control and push different droplets for m first conduits 1 and n second conduits 2. Of course, if the design is reasonable, some of the power sources can be combined to push n different droplets a that are split and m different droplets b that are fused.

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

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

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

[0067] As shown in Figure 4, the first connection ports 11', 11'', and 11''', the third connection port 21, and the fourth connection port 31 can be connected to different power sources and valves to inject oil and aqueous phase samples. The aqueous and oil phase sample injection systems can be switched between them. For example, when the chip is first powered up, the oil phase sample injection system is connected to the above connection ports to fill the chip with oil. When injecting microbial droplets, some sample injection systems can inject aqueous phase samples, such as microbial droplets for cultivation, enzyme reaction systems for reactions, and fresh culture media, chemical factors, and substrate reaction solutions.

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

[0069] During 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, and at the same time, the aqueous phase sample injection system connected to the first connection port 11'' pulse-injects an aqueous phase sample at a predetermined sample injection time, as needed, thereby forming water-in-oil microdroplets at the junction between the first conduit 1b and the first conduit 1c, and forming, for example, a fresh medium used to seed a bacterial solution or a substrate solution for an enzyme reaction, as can be seen in Figures 4 and 8.

[0070] The power source continues to push the formed microdroplets to the right, moving them to the connecting portion between first conduit 1d and first conduit 1b, and then stops pushing the microdroplets, and the power source connected to first connection port 11''' pushes another aqueous phase solution (e.g., a chemical factor solution for addition, or a solution containing another substrate for reaction) into the microdroplets remaining in first conduit 1d and first conduit 1b, forming the fused droplets b. Specifically, this fused droplets b can be a fresh medium to which a chemical factor has been added, or a reactant solution combining different reaction substrates (or matrices).

[0071] When the chip is first used, an oil phase (e.g., mineral oil used as a medium) is contained in the first conduit 1b shown in Figure 4, and an aqueous phase, such as a bacterial solution to be seeded or an enzyme solution to be reacted, is contained in the second conduit 2. Water-in-oil droplets, i.e., droplets a to be divided, such as the bacterial solution to be seeded or the enzyme solution to be reacted with a reactant, are formed at a first communication part 13 between the first conduit 1a and the second conduit 2.

[0072] When the chip and / or chip system is started to be used, droplet a to be split is first formed, then it is cultured in the first channel, and then droplet b to be fused is formed using the above-mentioned structure of the present invention that can realize cutting and fusion, and then, by cutting and fusion, droplet b to be split is cut and fused with droplet a to form new droplet c. In addition, second channel 2 starts to contain the aqueous phase, and after all the bacterial solution has formed water-in-oil droplets, the oil phase is replenished, and the second channel performs cutting and fusion as the above-mentioned second channel during the operation of the chip.

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

[0074] Of course, the sample injection system and the valve system are connected via a capillary channel that is hermetically connected to the sample injection port as described above. As described above, the sample injection system typically mainly includes a container for containing the liquid to be injected, a conduit for sample injection, and a power source. As described above, the power source is 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. As described above, the valve is 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 systems and valves connected to the first connection ports 11′, 11″, 11′″, 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 interchanged.

[0076] In the present invention, a culture system composed of capillary channels connected to a microfluidic chip can be considered to exist within the device. For example, in the chip of the present invention shown in Figure 4, the first connection port 11 is connected to the first culture channel via a capillary channel, and the second connection port 12 is connected to the second culture channel via a capillary channel. The first and second culture channels may be composed of capillary channels, and the lengths of the first and second culture channels can be designed according to the culture and reaction time requirements of the culture and reaction system.

[0077] At the same time, by controlling a power source connected to the chip of the present invention, the direction of the microdroplets flowing within the chip can be reversed. As described above, several to several hundred new droplets c formed can be cultured in the first culture channel, and the direction of movement of the microdroplets can be reversed by controlling the power source as needed, thereby realizing the reciprocating motion of the microdroplets within the channel.

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

[0079] Furthermore, the distance between the first detection window 9 and the second detection window 10 on the first conduit 1a is not particularly limited as long as it is possible to identify and detect microdroplets flowing through the conduit. For example, the distance between the first detection window 9 and the second detection window 10 (e.g., the distance along the conduit) may be 1 cm to 10 cm, and preferably 3 cm to 5 cm. The first detection window 9 and the second detection window 10 can be used as a microdroplet identification window and a microdroplet detection window, respectively.

[0080] In one specific embodiment, the first detection window 9 is used as a microdroplet discrimination window and the second detection window 10 is used as a microdroplet optical detection window.

[0081] As described above, microdroplet identification can be achieved in cooperation with a laser system installed outside the chip (e.g., installed on the microdroplet processing device of the present invention). When the laser system continuously emits a laser to irradiate the first detection window 9, when a microdroplet passes through the first detection window 9, the laser beam is temporarily blocked, and an external recording system can record the change in the laser beam.

[0082] The detection of the microdroplets can be achieved in cooperation with a spectroscopic system installed outside the chip (for example, installed on the microdroplet processing device of the present invention). For example, a spectroscopic detector can be provided to detect the absorbance of the microdroplets passing through the second detection window 10. This absorbance can reflect, for example, the degree of growth of microorganisms in a microbial culture system, or the color change of a reaction system involving a color change.

[0083] 5 is a schematic diagram of a microfluidic chip system of the present invention, in which the channels outside the chip substrate are capillary channels. Similar to the structure shown in FIG. 4, the first channel branches into first channel a, first channel b, first channel c, and first channel d upstream of the first connecting portion. A first detection window and a second detection window are formed on first channel a. First channel a, first channel b, first channel c, and first channel d are hermetically connected to the capillary channel via first and second connection ports thereon, and the second and third channels are hermetically connected to the capillary channel via third and fourth connection ports.

[0084] The cross-sectional areas of the first channel, the second channel, the third channel, and the capillary channel are 2.5×10 -3 mm 2 ~4mm 2 , preferably 0.01 to 3 mm 2 , more preferably 0.1 to 2.5 mm 2 , and more preferably 0.25 to 1 mm 2and more preferably, the first, second, and third conduits have the same cross-sectional area. The sealed connections of the capillary conduits sealed to the first, second, and third conduits are located within the substrate.

[0085] In the present invention, there is no particular limitation on the above-mentioned hermetic connection. For example, after the chip structure is designed, it is processed using an engraving machine, and then the chip substrate is pressed using a hot press to form a capillary channel in a deep hole on the side of the chip, and then an adhesive is injected to form a hermetic bond.

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

[0087] The temperature control system further includes a temperature sensor and a temperature control member, the temperature control member further includes a temperature raising member and a temperature lowering member, the temperature raising 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 the temperature controller is equipped with a fan.

[0088] In one specific embodiment of the present invention, the temperature raising 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 the temperature controller is equipped with a fan. The temperature control system is equipped with a fan, which is a temperature lowering device for lowering the temperature of the operating environment of the equipment and further lowering the temperature of the microbial culture.

[0089] In one specific embodiment of the present invention, the heating and cooling members can further include a drain temperature control system, which is a drain temperature control plate installed in the micro-droplet processing device, with the water inlet and outlet of the drain temperature control plate connected to an external constant temperature water bath, and the temperature of the drain temperature control plate is controlled according to the constant temperature principle of the constant temperature water bath to perform constant temperature control of the system, thereby achieving the effect of regulating 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 via the control system's host computer software, the circuit board program turns on the temperature control function and turns on the fan, and after the temperature is detected, it begins to heat up in stages. When it detects that the temperature has reached the target value, it automatically adjusts some parameters and turns on the automatic fan control function, which works in conjunction with the temperature sensor to maintain the temperature within the target value of (10 to 50) °C ± 0.2 °C. The temperature measured at 40 °C is shown in Figure 9.

[0091] The present invention can control the production of 1 to 500 microdroplets, preferably 5 to 400 microdroplets, preferably 10 to 300 microdroplets, preferably 20 to 200 microdroplets, with each microdroplet having a volume of 0.5 to 10 μL. This allows for much higher throughput and less medium consumption than shake flasks or deep-well plates; it allows for regular and quantitative replacement of fresh medium and addition of chemical factors, which is much more convenient than traditional subculture using seed solution extraction and saves time and effort; it can detect the growth of microorganisms in each microdroplet online in real time, which is much more convenient than traditional sampling detection and enables truly uninterrupted cultivation; and it can also perform intelligent screening, set screening characteristics, and automatically screen for suitable strains, improving efficiency.

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

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

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

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

[0096] In the apparatus of the present invention, the control system controls each system in the microdroplet processing apparatus.

[0097] The control system includes a controller and a PC display control system. The controller is connected to the sample injection system, the temperature control system, the droplet identification system, and the droplet detection system, respectively, and controls them through the digital circuits of the control system. The PC display control system displays, stores, and analyzes information from 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, second, and third conduits are conduits formed in a chip substrate, the size of which is 3 cm * 5 cm * 4 mm (length * width * thickness), the material of which is PMMA, and the first, second, and third conduits formed in the chip substrate are conduits with square cross sections and cross-sectional areas of 1 mm. 2 The first pipeline is connected to the second pipeline and the third pipeline, respectively, and the first connecting portion between the first pipeline and the second pipeline is located upstream of the second connecting portion between the first pipeline and the third pipeline, and the distance between the first connecting portion and the second connecting portion is 1.5 mm. The pipeline is filled with medium oil.

[0099] A syringe pump A is connected to a first connection port of the first conduit, a first rotary valve is connected to a second connection port, a syringe pump B and a second rotary valve are connected to a third connection port of the second conduit, and a third rotary valve is connected to a fourth connection port of the third conduit. The conduits, syringe pumps, and rotary valves are connected via a capillary channel, which has an inner diameter of 1.0 mm and is made of polytetrafluoroethylene.

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

[0101] Syringe pump A was turned on, and the first rotary valve was opened to push 2 μL of droplet a to be split and 2 μL of droplet b to be fused into the first conduit. Droplets a and b were suspended by an oily medium oil phase in the conduit. In this example, the droplet to be split was a microbial culture medium containing E. coli BL21, and droplet b was fresh LB medium. The medium oil phase was mineral oil.

[0102] The first rotary valve was closed, the second rotary valve was opened, and syringe pump A was continued to operate, forcing droplet a into the first connection between the first and second conduits. Seventy percent of droplet a was droplet a1, i.e., approximately 1.4 milliliters, entered the second conduit. At this time, the second rotary valve was closed, the third rotary valve was opened, and syringe pump A was continued to operate. The remaining 30% of droplet a, i.e., approximately 0.6 milliliters of droplet a2, entered the third conduit. Finally, droplet a was split into two parts: droplet a1 and droplet a2. Droplet a1 remained in the second conduit, and droplet a2 entered the third conduit. The third rotary valve was then closed, completing the droplet split. The division of droplet a is primarily achieved by cutting droplet a so that its bottom is parallel to channel A, reducing the amount of oil in the intermediate space, and then quantitatively injecting a portion of the solution from the cut droplet a into the fused droplet b, thereby achieving high accuracy of the injection amount. Because droplet a is cut by the oil in the channel, one end of droplet b is aligned with the channel of the first channel, laying the foundation for subsequent accurate quantitative sample injection.

[0103] The first rotary valve was opened, and syringe pump A was continued to be driven to push droplet b to the junction between the second and first conduits. Then, the pushing was stopped, and syringe pump B was driven to quantitatively push a portion (a1) of droplet a into droplet b to form a new droplet c.

[0104] Syringe pump A was turned on, pushing new droplet c to move forward, and the remaining droplet a1 portion remained in channel B, completing the quantitative mass exchange between droplet a and droplet b.

[0105] The above steps were repeated to inoculate fresh LB medium with multiple cultures containing E. coli BL21.

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

[0107] In addition, in Example 2, the configuration of the sample injection system was as shown in Figure 8, with three sample injection systems I and three sample injection systems II connected to the microfluidic chip, and the power source and valves were the same as those in Example 1.

[0108] In Example 2, before using the chip, the sample injection system I filled the chip with an oil phase. Then, the sample injection system II connected to the second conduit 2 injected the inoculation bacterial solution into the second conduit 2 under sterile conditions, thereby forming water-in-oil microdroplets in the first communication section 13. After sample injection was completed, the second conduit was filled with oil again. Next, by operating the power source of the sample injection system I, the water-in-oil microdroplets formed in the first communication section 13 traveled back and forth within the first conduit 1a, where bacteria were cultured. After the culture, they became droplets a, which were then split. The first detection window 9 served as a droplet identification site, and the second detection window 10 served as a droplet detection site for detecting the OD of the bacterial concentration.

[0109] Then, the oil phase was introduced into the chip through the first connection port 11' via the sample injection system I, and the sample injection time period was controlled as needed via the first connection port 11'' using the sample injection system II, which intermittently injected the aqueous medium into the chip to form water-in-oil medium droplets at the junction between the first conduit 1b and the first conduit 1c. The next step was selected: after the microdroplets moved to the right and reached the junction between the first conduit 1d and the first conduit 1b, the microdroplets were no longer pushed, and the sample injection system II connected to the first connection port 11''' pushed the sodium chloride solution into the medium droplets (which changed the concentration of medium factors in the medium) remaining in the first conduit 1b and the first conduit 1d, forming droplet b to be fused. This droplet entered the first conduit and was positioned to the left of droplet a to be split. In the first conduit 1a, the liquid was pushed from the first connection port 11 to the second connection port 12.

[0110] The process of splitting and fusing droplets a and b in Example 1 was repeated. After all droplets were split and fused, new droplet cultures of microorganisms were again prepared, and the OD values ​​of the droplets were again detected through the second detection window 10.

[0111] Example 3 In Example 3, the sample injection system, microfluidic chip system, incubation system, temperature control system, droplet identification system, and droplet detection system are all arranged in a single box, as shown in Figure 2. Using the installation method shown in Figure 2, droplet division, fusion, and novel droplet cultivation of microorganisms were realized using the microfluidic chip system of Example 2.

[0112] As shown in Figure 8, the sample injection system includes three sample injection systems I, three sample injection systems II, a valve, and a waste bottle. Sample injection system I includes a power pump and an oil phase bottle. Sample injection system II includes 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 sample injection system I is the same as the oil phase medium contained in the oil phase bottle used in sample injection system II. In a specific embodiment, the oil phase bottle can be shared. In this example, as shown in Figure 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, and the three sample injection buffer bottles 61 respectively contain the aqueous bacterial solution, culture medium, and chemical factors for addition. Six syringe pumps 63 are the power sources for the sample injection systems. Each of the six syringe pumps 63 is connected to an oil bottle 60. Three of the syringe pumps push the oil-phase medium from the oil bottle 60 into the microfluidic chip system 46, forming the three sample injection systems I shown in FIG. 8. The other three syringe pumps push the oil-phase medium from the oil bottle 60 into three sample injection buffer bottles 61, respectively. The liquid pressure of the oil-phase medium pushes the aqueous liquid in the sample injection buffer bottles 61 into the microfluidic chip system 46, forming the three sample injection systems II shown in FIG. 8. The syringe pumps 63, oil bottles 60, sample injection buffer bottles 61, rotary valve 65, waste bottle 62, and microfluidic chip system 46 are connected via capillary channels (not shown) according to the connection scheme of Example 2. With the cooperation of the above-mentioned sample injection system and valve control, the bacterial liquid to be cultured was divided and fused within the microfluidic chip system 46 to form multiple water-in-oil microbial droplets, which were then cultured by moving back and forth within the channels of the microfluidic chip system 46 under the action of the syringe pump 63.

[0113] The temperature control system includes a temperature-raising element, a temperature-reducing element, and a temperature-control element. As shown in FIG. 2, the temperature-raising element includes an air heater 54 and a metal bath 53. A sample injection buffer bottle 61 is placed in the metal bath 53. The air heater 54 heats the microbial culture environment within the device box via a heating fan. The temperature-reducing element includes two fans 64, one for lowering the temperature of the device's operating environment and the other for lowering the temperature of the microbial culture environment. A temperature probe 67 measures the temperature of the microbial culture environment within the box and provides feedback to the device's control system, which then controls the operation of the temperature-raising element and the temperature-reducing element to heat and regulate the temperature of the droplets within the microfluidic chip. In this example, the temperature control curve is shown in FIG. 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 illuminates a first detection window of the microfluidic chip system 46, identifies aqueous phase sample droplets passing through the first detection window, and transmits droplet position information back to the apparatus control device via 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 detects the spectral signal of the aqueous phase sample droplet passing through the second detection window of the microfluidic chip system via the spectrometer 58.

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

[0117] The steps in microbial culture are as follows: 1) The equipment was sterilized with the ultraviolet lamp turned on for 30 minutes; 2) The device lines were evacuated, 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, which was then placed in a vacuum degassing box for 30 minutes for degassing treatment. Fresh culture medium (LB medium) was added to one sample injection buffer bottle, and the chemical factor to be added, i.e., ascorbic acid solution, was added to the other sample injection buffer bottle; 4) The chip was attached to the bracket and connected to the inlets of the sample injection buffer bottle and oil bottle; 5) Open the instrument software, select the "growth curve" measurement mode, and set the basic parameters as follows: detection cycle 0.5 h, droplet number 100, detection wavelength 620 nm; 6) Clicked the Run button; 7) After 46 hours of running, data was acquired and the results are shown in Figure 10.

[0118] The present invention provides accurate temperature control, increases the reliability of results, allows for smooth growth of microorganisms, and allows for continuous cultivation for up to 90 days. Industrial Applicability

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

[0120] Although the embodiments of the present invention have been described in combination with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are merely illustrative and instructive, and are not limiting. Under the teachings of this specification, a person skilled in the art can make many modifications without departing from the scope of protection of the claims of the present invention, and all of them are within the scope of protection of the present invention.

[0121] (1) a sample injection system for injecting aqueous and oil phase samples into a microdroplet processing device, the sample injection system 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 conduit 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 a fiber optic spectrometer and a halogen light source; and Control system for controlling each system in a micro liquid processing device A microdroplet processing device comprising: (2) The droplet identification system identifies sample droplets of the aqueous phase passing through a first detection window of the microfluidic chip system using a photoelectric sensor and a laser light source; the droplet detection system detects a spectral signal of the aqueous phase sample droplet passing through a second detection window of the microfluidic chip system using a fiber optic spectrometer and a halogen light source; (1) The device described in (1). (3) The sample injection systems each include at least two sample injection systems I and one sample injection system II; The device according to (1) or (2). (4) The sample injection system includes three sample injection systems I and three sample injection systems II; (3) The device described in (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 liquid that is incompatible with the sample solution to be injected and is incompressible; The power source drives the liquid through an import line into a buffer bottle; The buffer bottle contains the sample solution, and when driven by a power source, the liquid enters the buffer bottle through an inlet pipe, thereby forcing the sample solution into the microfluidic chip system through an outlet pipe, and the liquid container, the power source, and the buffer bottle are in communication with each other through a capillary pipe. The device according to (3) or (4). (6) The microfluidic chip system substrate; and a first conduit, a second conduit, and a third conduit formed in the substrate; a first detection window and a second detection window, which are transparent regions formed on the first conduit; and a capillary passage in sealed communication with the first passage, the second passage, and the third passage; Including, the first conduit includes a first connection port and a second connection port at both ends thereof, the second conduit includes a third connection port at one end thereof and communicates with the first conduit at the other end thereof; the third conduit includes a fourth connection port at one end thereof and communicates with the first conduit at the other end thereof; The first communication part between the first conduit and the second conduit is located upstream of the second communication part between the third conduit and the first conduit in the droplet movement direction, 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, and preferably 1000 μm to 1500 μm. The device according to any one of (1) to (5). (7) the first pipe branched into a first pipe a, a first pipe b, a first pipe c, and a first pipe d upstream of the first communication portion; the first detection window and the second detection window are formed on the first conduit a; the first conduit a, the first conduit b, the first conduit c, and the first conduit d are sealedly connected to the capillary channel through the first connection port and the second connection port thereon; the second conduit and the third conduit are sealedly connected to the capillary conduit via the third connection port and the fourth connection port; (6) The device described in (6). (8) the substrate, the first conduit, the second conduit, and the third conduit are made of one or more materials selected from the group consisting of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and acrylonitrile-butadiene-styrene copolymer (ABS), and are preferably made of polymethyl methacrylate (PMMA); The capillary channel is a rigid tube, and more preferably, the capillary channel is any one selected from the group consisting of a polytetrafluoroethylene tube (PTFE tube), a perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene copolymer tube (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 area of ​​the first conduit, the second conduit, the third conduit, and the capillary conduit is less than 2.5×10 -3 mm 2 ~4mm 2 , preferably 0.01 to 3 mm 2 , more preferably 0.1 to 2.5 mm 2 , and more preferably 0.25 to 1 mm 2 That is, The device according to (6) or (7). (9) The device according to any one of (1) to (8), wherein the sample injection system and the microfluidic chip system are connected to the capillary channel via a connection port to form a sealed closed-loop control structure. (10) The sample injection system, microfluidic chip system, droplet identification system, and 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 a range of 10°C to 50°C, and the temperature fluctuation range is controlled within a range of ±0.5°C. (1) The device described in (1). (11) The apparatus of (10), wherein the box further comprises a sterilizer, preferably an ultraviolet lamp. (12) the control system includes a controller and a PC display control system; The controller is connected to the sample injection system, the temperature control system, the droplet identification system, and the droplet detection system, respectively, and controls them through a digital circuit of the control system; The PC display control system displays, stores, and analyzes information from 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 (1) to (11).

Claims

[Claim 1] a sample injection system for injecting aqueous and oil phase samples into a microdroplet processing device, the sample injection system 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; Microfluidic chip systems; 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 a fiber optic spectrometer and a halogen light source; and Control system for controlling each system in a micro liquid processing device Including, The microfluidic chip system includes: a substrate; and a first conduit, a second conduit, and a third conduit formed in the substrate; a first detection window and a second detection window, which are transparent regions formed on the first conduit; and a capillary passage in sealing communication with the first conduit, the second conduit, and the third conduit; the first conduit includes a first connection port and a second connection port at both ends thereof, the second conduit includes a third connection port at one end thereof and communicates with the first conduit at the other end thereof; the third conduit includes a fourth connection port at one end thereof and communicates with the first conduit at the other end thereof; a first communication portion between the first conduit and the second conduit is located upstream of a second communication portion between the third conduit and the first conduit in a droplet movement direction, and a distance between the first communication portion and the second communication portion is 1000 μm to 1500 μm; The cross-sectional areas of the first, second and third conduits are 0.25 to 1 mm ２ and The droplet volumes of the droplet a to be split and the droplet b to be merged contained in the microfluidic chip system are 0.9 to 5 μl; the first conduit is controlled to accommodate a droplet a to be split and a droplet b to be fused before cutting and fusing, and when cutting and fusing, the first conduit is controlled to accommodate a second portion of droplet a2 and a new droplet c; The second conduit is controlled to receive the droplet a1 of the cut first portion, the droplet a1 of the first portion and the droplet b to be merged are controlled to merge in the first communicating portion, the third conduit is controlled to receive the droplet a2 of the cut second portion; The microdroplet processing device, wherein the length of the first detection window is 200 μm to 1 mm.

Images