CN112403538A

CN112403538A - Device and method for generating and fusing liquid drops

Info

Publication number: CN112403538A
Application number: CN201910784442.2A
Authority: CN
Inventors: 王立言; 段保峰; 郭肖杰
Original assignee: Wuxi Tmaxtree Biotechnology Co ltd
Current assignee: Wuxi Tmaxtree Biotechnology Co ltd
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2021-02-26

Abstract

The invention relates to a device for generating and fusing micro-droplets, which comprises: the device comprises a first pipeline, a second pipeline and a third pipeline, wherein two ends of the first pipeline respectively comprise a first connecting port and a second connecting port, one end of the second pipeline comprises a third connecting port, the other end of the second pipeline is communicated with the first pipeline, one end of the third pipeline comprises a fourth connecting port, the other end of the third pipeline is communicated with the first pipeline, the fourth pipeline comprises a pipeline with an upstream branch of 3 or more and a fifth connecting port, the other end of the fourth pipeline is communicated with the first pipeline through a common pipeline, the common pipeline comprises a continuously bent pipeline, a first communicating part of the second pipeline and the first pipeline is positioned at a second communicating part of the third pipeline and the first pipeline, and the middle of a third communicating part of the fourth pipeline and the first pipeline, and the distance between the first communicating part of the fourth pipeline and the third communicating part of the first pipeline is 500 mu m-2000 mu m; the first pipeline is communicated with a first power source through a first connecting port and a capillary pipeline, and is communicated with a first valve through a second connecting port and the capillary pipeline; the second pipeline is respectively communicated with a second power source and a second valve through a third connecting port and a capillary tube; the third pipeline is communicated with a third valve through a fourth connecting port and a capillary tube; and the fourth pipeline is respectively communicated with the power source connected with the branch and the corresponding valve thereof through a fifth connecting port and a capillary tube.

Description

Device and method for generating and fusing liquid drops

Technical Field

The invention belongs to the technical field of micro-fluidic, relates to a micro-droplet generation and fusion technology, and relates to a simple and practical device and a method for realizing multi-condition multi-factor micro-droplet generation and microorganism or single cell wrapping, in particular to a micro-droplet generation and fusion device and a method thereof.

Background

Conventional microbial strain screening and culture condition optimization are usually carried out in shake flasks, and the whole process is low in efficiency, high in cost, long in time and large in equipment and space. The micro-fluidic technology has extremely high efficiency, and as the structure is tiny, hundreds of microorganism culture units are easy to integrate on the chip at one time, so that a large amount of culture media can be saved; the integrated operation of software is adopted, the whole experiment process operation can be simulated on the chip, and the micro-fluidic chip is applied to culture and screening microorganisms, so that the problems can be effectively avoided.

Patent document 1 discloses in 2013 a pressurized cell culture system and method based on a microfluidic chip, including a microfluidic chip, a pressure driving device, a connecting pipeline and a pressure detection device, where the microfluidic chip includes a channel, the pressure driving device includes a container for holding a culture medium, the container is connected to the microfluidic chip through the connecting pipeline, the power driving device pushes the culture medium in the container to enter the microfluidic chip, and the pressure detection device is connected to the microfluidic chip through the connecting pipeline. The system can conveniently culture and research cells under the action of shearing force and pressure, is convenient to disassemble and assemble and carry, but cannot realize the functions of culturing and detecting microorganisms.

Patent document 2 discloses a high-flux detection system based on a droplet microfluidic chip in 2013, which mainly comprises a droplet microfluidic chip system (1), an optical path system (2) and a data acquisition and analysis system (3); the liquid drop micro-fluidic chip system (1) embeds microorganisms to be detected to form an independent single-liquid drop micro-reaction cell, laser-induced fluorescence detection signal transmission of microorganism samples in the single-liquid drop micro-reaction cell is carried out through the optical path system (2), and a data acquisition and analysis system (3) carries out detection and analysis on acquired signals through computer software. The invention is suitable for laser-induced fluorescence detection and analysis, can only realize the detection of micro-droplet unit samples, and cannot realize the functions of culture and sorting.

Patent document 3 discloses in 2016 a single-cell separation microfluidic chip, which includes a substrate and a microchannel formed on the substrate, wherein the microchannel includes a cell injection port, a single-cell collection pool, and a cell separation unit and a droplet output channel sequentially communicated between the cell injection port and the single-cell collection pool, a droplet generation and encapsulation unit is formed at a junction of the cell separation unit and the droplet output channel, the droplet generation and encapsulation unit is communicated with an oil phase delivery channel, and the cell separation unit is used for delivering cells arranged in a single row to the droplet generation and encapsulation unit. The invention adopts the spiral disk micro-channel to process the cell solution, so that the cells can be arranged in the pipeline in a single row, the single packaging of the cells is realized through the liquid drop packaging unit, the sorting of the same cell in different physiological states is also realized, but the online culture and the real-time monitoring of the microorganism can not be realized.

Although the above patent documents all relate to microfluidic chips, since the breeding of microorganisms is a special biological process, different culture factors may be changed for culturing, and then the target microorganisms are screened out after detection and evaluation. Therefore, how to realize the culture and detection of the microorganism with the micro-volume and the multi-factor and multi-condition by the micro-fluidic technology is one of the key problems of microorganism breeding.

Documents of the prior art

Patent document

Patent document 1: chinese patent application publication CN 104099247A;

patent document 2: chinese patent application publication CN 104007091A;

patent document 3: chinese patent application publication CN 105944775A.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a micro-droplet generation and fusion device and a method thereof on the basis of a microfluidic technology.

The purpose of the invention is realized by the following technical scheme.

1. A micro-droplet generation and fusion device, comprising:

a first pipeline, two ends of which respectively comprise a first connecting port and a second connecting port,

a second pipeline, one end of which comprises a third connecting port and the other end of which is communicated with the first pipeline,

one end of the third pipeline comprises a fourth connecting port, the other end of the third pipeline is communicated with the first pipeline,

a fourth pipeline, one end of which comprises a pipeline with 3 or more branches at the upstream and a fifth connecting port, the other end of which is communicated with the first pipeline through a section of common pipeline, the section of common pipeline comprises a continuous bending pipeline,

wherein the first communicating part of the second pipeline and the first pipeline is positioned at the second communicating part of the third pipeline and the first pipeline and in the middle of the third communicating part of the fourth pipeline and the first pipeline, the distance between the first communicating part and the second communicating part and the distance between the first communicating part and the third communicating part are respectively 500-2000 mu m, preferably 750-1800 mu m, and preferably 1000-1500 mu m,

the first pipeline is communicated with the first power source through the first connecting port and the capillary pipeline, the first pipeline is communicated with the first valve through the second connecting port and the capillary pipeline, the second pipeline is communicated with the second power source and the second valve through the third connecting port and the capillary pipeline respectively, the third pipeline is communicated with the third valve through the fourth connecting port and the capillary pipeline, and the fourth pipeline is communicated with the power source connected with the branch and the corresponding control valve through the fifth connecting port and the capillary pipeline respectively.

2. The micro-droplet generation and fusion device of claim 1, wherein the upstream branch of the fourth pipeline is 3, which are respectively a fourth pipeline a, a fourth pipeline b and a fourth pipeline c.

3. The micro-droplet generation and fusion device of claim 1, wherein the number of the upstream branches of the fourth pipeline is 4, and the four branches are respectively a fourth pipeline a, a fourth pipeline b, a fourth pipeline c and a fourth pipeline d.

4. The micro-droplet generation and fusion device of claim 1, wherein the number of the upstream branches of the fourth pipeline is 5, and the branches are respectively a fourth pipeline a, a fourth pipeline b, a fourth pipeline c, a fourth pipeline d and a fourth pipeline e.

5. The micro-droplet generation and fusion device of claim 1, wherein the continuous curved pipeline of which the upstream branch of the fourth pipeline is communicated with the first pipeline is composed of one or more of a P-type structure, an S-type structure, a U-type structure, a broken line type structure and a wave type structure.

6. The micro-droplet generation and fusion device according to any one of

claims

1 or 5, characterized in that the total volume of the liquid contained in the continuously bent pipeline is 4-100 μ L, preferably 4-50 μ L, preferably 4-20 μ L, preferably 6-12 μ L, and further preferably 8-10 μ L.

7. The droplet generation and fusion device of claim 1, wherein the first, second, third and fourth conduits are disposed on a substrate, and the substrate and the first, second, third and fourth conduits are formed from glass, Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS), preferably Polymethylmethacrylate (PMMA).

8. The droplet generation and fusion device of claim 1, wherein the first communication site is the same distance from the second communication site and the third communication site, respectively.

9. The micro-droplet generation and fusion device of any one of claims 11-8, wherein the cross-sectional area of the first, second, third and fourth conduits is in the range of 2.5 x 10^-3mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²It is further preferred that the cross-sectional areas of the first, second, third and fourth conduits are the same.

10. A method of microdroplet generation and fusion, comprising:

the first pipeline, the second pipeline, the third pipeline and the fourth pipeline are filled with oil phase,

respectively starting the power source of each upstream branch of the fourth pipeline to drive the connected liquid to form water-in-oil liquid drops a,

when the liquid drops a pass through the continuous bending pipeline of the fourth pipeline and reach the third communication position, the cutting of the liquid drops a is carried out, n liquid drops in parallel condition are formed and enter the first pipeline,

starting a first power source to drive n liquid drops under parallel conditions to move towards a second connecting port in a first pipeline, closing the first power source and simultaneously starting a second power source connected with a second pipeline and communicating a fusion electrode power source when the liquid drops sequentially reach the position under a first communicating part, wherein the second power source pushes bacterial liquid to enter the liquid drops under the parallel conditions,

wherein, two ends of the first pipeline respectively comprise a first connecting port and a second connecting port,

the first communication part of the second pipeline and the first pipeline is positioned between the second communication part of the third pipeline and the first pipeline and between the third communication part of the fourth pipeline and the first pipeline, and the distance between the first communication part and the second communication part and the distance between the first communication part and the third communication part are 500-2000 mu m, preferably 750-1800 mu m and preferably 1000-1500 mu m;

the first pipeline is communicated with a first power source through a first connecting port and a capillary pipeline, and is communicated with a first valve through a second connecting port and the capillary pipeline; the second pipeline is respectively communicated with a second power source and a second valve through a third connecting port and a capillary tube; the third pipeline is communicated with a third valve through a fourth connecting port and a capillary tube; and the fourth pipeline is respectively communicated with the power source connected with the branch and the corresponding control valve thereof through a fifth connecting port and a capillary tube.

11. The method for generating and fusing micro-droplets according to claim 10, wherein the cutting of the droplet a is performed by "pinching off the head and tail middle parts", the head and tail parts of the droplet a are cut off and flow out through the fourth connection port, and the droplets of the middle part are divided into droplets a1, droplets a2, … and droplets an on average.

12. The method for generation and fusion of micro-droplets according to claim 11, wherein the volumes of the droplets a1, a2, … and an are the same.

13. The method for generating and fusing microdroplets as claimed in claim 12, wherein the volume of the droplets a1, a2, … and an is one-half of the total volume of the droplets a, respectively, n + 2.

14. The micro-droplet generation and fusion device of claim 10, wherein the number of the upstream branches of the fourth pipeline is 3, and the branches are respectively a fourth pipeline a, a fourth pipeline b and a fourth pipeline c.

15. The method for droplet generation and fusion of claim 10, wherein the number of the fourth pipeline upstream branches into 4, namely a fourth pipeline a, a fourth pipeline b, a fourth pipeline c and a fourth pipeline d.

16. The method for droplet generation and fusion of claim 10, wherein the number of the upstream branches of the fourth pipeline is 5, and the branches are respectively a fourth pipeline a, a fourth pipeline b, a fourth pipeline c, a fourth pipeline d and a fourth pipeline e.

17. The method for droplet generation and fusion according to claim 10, wherein the upstream branch of the fourth pipeline is a continuous curved pipeline communicated with the first pipeline, and is preferably composed of one or more structures selected from P-type, S-type, U-type, fold line type and wave type.

18. The method for generation and fusion of micro-droplets according to claim 10, wherein the total volume of the liquid contained in the continuously bent pipeline is not less than the volume of the droplet a, and is 4-100 μ L, preferably 4-50 μ L, preferably 4-20 μ L, preferably 6-12 μ L, and further preferably 8-10 μ L.

19. The method for generation and fusion of micro-droplets according to claim 10, characterized in that the total volume of the droplets a is 4-100 μ L, preferably 4-50 μ L, preferably 4-20 μ L, preferably 6-12 μ L, and further preferably 8-10 μ L.

20. The method of droplet generation and fusion of claim 10, wherein the first, second, third and fourth conduits are disposed on a substrate, and the substrate and the first, second, third and fourth conduits are formed from glass, Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), Acrylonitrile Butadiene Styrene (ABS), preferably Polymethylmethacrylate (PMMA).

21. The method of droplet generation and fusion of claim 10, wherein the first communication site is the same distance from the second communication site and the third communication site, respectively.

22. The method of any one of claims 10 to 21, wherein the first, second, third and fourth conduits have cross-sectional areas in the range of 2.5 x 10^-3mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²It is further preferred that the cross-sectional areas of the first, second, third and fourth conduits are the same.

Drawings

Fig. 1 is a schematic structural diagram of a micro-droplet generation and fusion device according to the present invention.

FIG. 2 is a schematic diagram of a chip for droplet generation and fusion according to the present invention.

Description of the symbols:

1a first channel, 2 a second channel, 3 a third channel, 4a fourth channel (4a, 4b, 4c, 4d, 4e), 5a substrate, 7 holes, 8 holes, 9 a detection window I, 10 a detection window II, 11 a first connection port, 12 a second connection port, 21 a third connection port, 31 a fourth connection port, 41 '' '' a fifth connection port, 13 a first communication portion, 14 a second communication portion, 15 a third communication portion, 16 a fourth communication portion, 17 a fifth communication portion, 18 a sixth communication portion, 19 a seventh communication portion, 201 an electrode, 202 an electrode, 22 a first power source, 23 a second power source, 24 a third power source, 25 a fourth power source, 26 a fifth power source, 27 a sixth power source, 28 a seventh power source, 29 first valve, 30 second valve, 32 third valve, 33 fourth valve, 34 fifth valve, 35 sixth valve, 36 seventh valve, 37 eighth valve.

Detailed Description

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

To facilitate understanding of embodiments of the present invention, the following description will be made by way of example of several specific embodiments with reference to the accompanying drawings, each of which is not intended to limit the present invention.

The invention relates to a device for generating and fusing micro-droplets, as shown in fig. 1, comprising:

wherein the first communicating portion of the second duct and the first duct is located between the second communicating portion of the third duct and the first duct and the third communicating portion of the fourth duct and the first duct,

the first pipeline is communicated with a first power source through a first connecting port and a capillary pipeline, and is communicated with a first valve through a second connecting port and the capillary pipeline; the second pipeline is respectively communicated with a second power source and a second valve through a third connecting port and a capillary tube; the third pipeline is communicated with a third valve through a fourth connecting port and a capillary tube; and the fourth pipeline is respectively communicated with the power source connected with the branch and the corresponding control valve through a fifth connecting port and a capillary tube.

In a specific embodiment, the first, second, third and fourth channels are disposed on a substrate, the substrate is a microfluidic chip substrate formed of glass, polymethyl methacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS) and acrylonitrile-butadiene-styrene (ABS), and the first, second, third and fourth channels are formed inside the substrate. In the microfluidic chip of the present invention, the first, second, third, and fourth conduits may be formed inside the substrate by photolithography, hot pressing, engraving, injection molding, or may be independently formed conduits.

The material of the pipeline is selected from any one of glass, polymethyl methacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS) and acrylonitrile-butadiene-styrene copolymer (ABS), and the preferable forming material is polymethyl methacrylate (PMMA).

In the present embodimentThe cross-sectional shapes of the first, second, third and fourth pipes are not limited, and may be any shapes convenient for forming and circulating liquid drops, such as round, rectangular, oval, etc., and the cross-sectional area ranges from 2.5 × 10^-3mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²。

In a specific embodiment, the cross-sectional areas of the first, second, third and fourth conduits may be the same or different from each other. The first, second, third and fourth conduits may be tapered. In the present invention, as long as the cross-sectional area of the pipe satisfies the above-mentioned definition. The shape and cross-sectional area of the conduit can be designed by one skilled in the art depending on the size of the droplets contained in the conduit and the intended use of the droplets. In some embodiments, it is further preferable that the first conduit, the second conduit, the third conduit and the fourth conduit have the same shape and cross-sectional area, and under the same condition, the pressure of the liquid in the conduits can be ensured to be consistent, so as to facilitate the movement operation of the liquid drops.

In a specific embodiment, the distance between the first communicating part and the second communicating part and the distance between the first communicating part and the third communicating part are 500 to 2000 μm, preferably 750 to 1800 μm, and preferably 1000 to 1500 μm. Specifically, the distance may be 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm.

In one embodiment, the distances between the first communication portion and the second and third communication portions may be different or the same. In one embodiment, the first communication site is the same distance from the second communication site to the third communication site.

In an embodiment, the second duct, the third duct, and the fourth duct upstream branch duct are arranged in parallel with each other. The distance between the upstream branch pipelines of the fourth pipeline is 500-2000 mu m, preferably 750-1800 mu m, and preferably 1000-1500 mu m. Specifically, the upstream branch pipes of the fourth pipe are arranged in parallel with each other, and the distance between the upstream branch pipes is related to the size of liquid drops in the pipe and the cross-sectional area of the pipe. Specifically, the particles may be 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm. The spacing of the upstream branch conduits of the fourth conduit may be the same or different.

In a specific embodiment, the upstream branch of the fourth pipeline is composed of a nonlinear pipeline, preferably a continuously curved pipeline, and further preferably one or more of a P-type, an S-type, a U-type, a broken line type and a wave type, which is in communication with the first pipeline. That is, the structure may be a single P-type, S-type, U-type, zigzag-type, or wave-type repeating structure, or a combination of a plurality of structures in the shape, and may be regularly alternated or randomly combined.

No matter what shape is of the nonlinear pipeline or the bent pipeline, the volume of the nonlinear pipeline or the bent pipeline is not smaller than the total volume of liquid contained in the nonlinear pipeline, preferably 4-100 mu L, preferably 4-50 mu L, preferably 4-20 mu L, preferably 6-12 mu L, and further preferably 8-10 mu L. Further, the total volume of the liquid is 4-100 muL, preferably 4-50 muL, preferably 4-20 muL, preferably 6-12 muL, and further preferably 8-10 muL. Specifically, the volume is related to the size of the liquid drop in the pipeline, and the volume of the liquid drop can be changed according to the operation and the function of the liquid drop.

The invention can realize high-precision and smooth liquid transmission without pulsation by using a power source. In a specific embodiment of the present invention, the first power source, the second power source, and the fourth pipe-connected power source are each independently selected from any one of 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, and the fourth pipe-connected power source are syringe pumps. In the present invention, the range of the power source is not limited, and a person skilled in the art can appropriately select a syringe pump, a pressure pump, a peristaltic pump, a diaphragm pump and/or a plunger pump with an appropriate range according to the amount of the sample to be injected.

In the present invention, the flow direction of the liquid droplets in each pipe is controlled by the change of the pressure by using a valve and changing the pressure in each closed pipe by opening and closing the valve. In the present invention, the control valves connected to the first valve, the second valve, the third valve, and the fifth connection port of the fourth line branch are each independently selected from any one of a solenoid valve, a rotary valve, a rocker valve, and a pinch-off valve. Preferably, the control valves connected to the first valve, the second valve, the third valve, and the fifth connecting port of the fourth pipe branch are rotary valves.

In some embodiments, it will be understood by those skilled in the art that the valve may be replaced by other mechanisms or components, such as a syringe pump used as a power source to serve as the valve, as long as the pressure in the closed pipeline can be changed by opening and closing.

In the present invention, the first power source and the second power source are only used to indicate power sources that perform different functions, and are not intended to limit the number of power sources.

In the present invention, the first valve, the second valve and the third valve are only used to indicate valves that perform different functions, and are not intended to limit the number of valves, and 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.

In a particular embodiment of the invention, the capillary channel has a cross-sectional area in the range of 2.5 x 10^-3mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²。

The pipeline connecting port is connected with the capillary, the connecting position is sealed, and then the pipeline connecting port is communicated with the power source and/or the valve through the capillary. In order to ensure the stable conduction of the power source pressure, the capillary tube is a hard tube, and the pipeline has no flexible change. Further preferably, the capillary tube is any one of a polytetrafluoroethylene tube (PTFE tube), a copolymer tube of perfluoropropyl perfluorovinyl ether and polytetrafluoroethylene (PFA tube), a polyetheretherketone tube (PEEK tube), a polycarbonate tube (PC tube), and a polystyrene tube (PS tube).

In an embodiment of the present invention, the first channel, the second channel, the third channel and the fourth channel are disposed on a substrate to form a microfluidic chip, i.e. a chip for generating and fusing micro-droplets, the substrate is a microfluidic chip substrate, the substrate has 2

holes

7 and 8 on the substrate, the

holes

7 and 8 are located near the first communication portion 13 of the first channel 1 and the second channel 2, and their positions are not fixed, and can be located on both sides of the second channel 2 or on both sides of the first channel 1, the distance between the

holes

7 and 8 and the first communication portion 13 of the first channel 1 and the second channel 2 is 0.1mm to 1cm, preferably 0.3mm to 5mm, and further preferably 0.5mm to 2mm, the

holes

7 and 8 are used for accommodating the positive and

negative electrodes

201 and 202 of the additionally disposed fusion electrode, and those skilled in the art can arrange the

holes

7 and 8 in the above range according to the chip design requirements, And 8 position. The frequency of the voltage loaded on the fusion electrode is 0-20000Hz, preferably 1000-10000Hz, and the voltage of the electrode is 1-5000V, preferably 500-1000V. When the fusion electrode is connected with a power supply, an electric field is generated to act on the liquid drops at the first communication part, and the purpose is to promote the liquid drop fusion. The electric field can be any of an alternating electric field or a constant electric field, and the voltage applied by the electrodes is 1-5000V, preferably 500-1000V.

Of course, in the present invention, the specific shape of the

holes

7 and 8 and the size of the holes are not limited as long as they can be used to place the fusion electrodes. Wherein the fusion electrode may refer to the fusion electrode described above for the device of the present invention.

In a specific embodiment, the chip substrate further includes a first detection window 9 and a second detection window 10 formed on the first pipeline, the first detection window 9 and the second detection window 10 are only required to be used for monitoring and detecting micro-droplets in the pipeline, and there is no limitation on the specific shape and form thereof, if the basic material of the chip is formed by a transparent material, the first detection window 9 and the second detection window 10 are two detection points of the first pipeline, and if the chip substrate and the pipeline material are opaque, the first detection window 9 and the second detection window 10 are located on the first pipeline itself to form two transparent portions, that is, both the pipeline and the substrate material are transparent here. The position of the second detection window 10 is not limited; the first detection window 9 is arranged on the first pipeline and located between the third communication part and the first connection port, and the distance between the first detection window 9 and the third communication part is 1mm-1cm, preferably 1.5-3 mm, so that the micro-droplets moving in the pipeline can be monitored and detected more conveniently and more accurately.

In one embodiment of the invention, the first conduit contains droplets to be fused entering from the fourth conduit. The first power source is started, the first valve is opened, the fused liquid drops are pushed to stay under the first communication part under the driving of the first power source, the first power source is closed, the driving is stopped, the second power source is started to provide driving force for the pipeline through the third connecting port, and therefore the liquid of the third connecting port is pushed to enter the liquid drops quantitatively, and the liquid drop fusion is completed. During the liquid drop fusion process, the fusion electrode is connected with a power supply, and an electric field is generated to act on the first connection part to promote the liquid drop fusion.

Further, a plurality of droplets to be fused may exist in the chip, and the droplet fusion is sequentially performed by the above method.

In the present invention, one end of the fourth pipe includes a pipe whose upstream branch is 3 or more parallel to each other and a fifth connection port. The upstream branch pipeline can realize automatic preparation of culture solution with different factors and different concentrations.

Further, a microfluidic chip comprises a substrate, and a first pipeline, a second pipeline, a third pipeline and a fourth pipeline which are formed in the substrate. Wherein, first pipeline both ends include first connector and second connector respectively. One end of the second pipeline comprises a third connecting port, and the other end of the second pipeline is communicated with the first pipeline; one end of the third pipeline comprises a fourth connecting port, the other end of the third pipeline is communicated with the first pipeline, the upstream branch of the fourth pipeline is 3, 4, 5 or more, the quantity of the upstream branch of the fourth pipeline can be designed according to the requirement, one end of the upstream branch of the fourth pipeline is respectively connected with a fifth connecting port, and the fifth connecting ports are respectively connected with different power sources and valves for realizing oil phase and water phase sample introduction.

In one embodiment, the upstream of the fourth pipeline is branched into 3, which are respectively a fourth pipeline a, a fourth pipeline b and a fourth pipeline c, and one end of each of the four pipelines is connected to the

fifth connection ports

41, 41' and 41 ″. For example, the fifth connection port 41 is connected to the third power source and the fourth valve through a capillary tube, the fifth connection port 41' is connected to the fourth power source and the fifth valve through a capillary tube, and the fifth connection port 41 ″ is connected to the fifth power source and the sixth valve through a capillary tube. In the invention, the upstream branches of the fourth pipeline are 3, and the preparation of the culture solution with single factor and different concentrations is realized by controlling the sample injection of the oil phase and the water phase.

In one embodiment, the upstream of the fourth pipeline is branched into 4, which are respectively a fourth pipeline a, a fourth pipeline b, a fourth pipeline c and a fourth pipeline d, and one end of each of the four pipelines is connected to the

fifth connection ports

41, 41 ', 41 ″ and 41 ″', respectively. For example, the fifth connection port 41 is communicated with the third power source and the fourth valve through a capillary tube, the fifth connection port 41' is communicated with the fourth power source and the fifth valve through a capillary tube, the fifth connection port 41 ″ is communicated with the fifth power source and the sixth valve through a capillary tube, and the fifth connection port 41 ″ is communicated with the sixth power source and the seventh valve through a capillary tube. In the invention, the upstream branches of the fourth pipeline are 4, and the preparation of culture solution with two factors and different concentrations is realized by controlling the sample injection of oil phase and water phase. In one embodiment, the upstream of the fourth pipeline is branched into 5, which are respectively a fourth pipeline a, a fourth pipeline b, a fourth pipeline c, a fourth pipeline d and a fourth pipeline e, and one end of each of the four pipelines is connected with the

fifth connection ports

41, 41 ', 41 "' and 41" ', respectively. Further, the first pipeline, the second pipeline, the third pipeline and the fourth pipeline are arranged on the substrate to form the microfluidic chip, as shown in fig. 2. Specifically, the fifth connection ports 41, 41 '' '' are connected with different power sources and valves respectively, and are used for realizing oil phase and water phase sample injection. For example, the fifth connection port 41 is communicated with the third power source and the fourth valve through a capillary tube, the fifth connection port 41 ' is communicated with the fourth power source and the fifth valve through a capillary tube, the fifth connection port 41 ″ is communicated with the fifth power source and the sixth valve through a capillary tube, the fifth connection port 41 ″ ' is communicated with the sixth power source and the seventh valve through a capillary tube, and the fifth connection port 41 ″ ' is communicated with the seventh power source and the eighth valve through a capillary tube. In the invention, the upstream branches of the fourth pipeline are 5, and the preparation of the three-factor culture solution with different concentrations is realized by controlling the sample injection of the oil phase and the water phase.

By utilizing the device for generating and fusing the micro-droplets, the method for generating and fusing the micro-droplets comprises the following steps:

the first step is as follows: the first pipeline, the second pipeline, the third pipeline and the fourth pipeline are filled with oil phase,

the second step is as follows: respectively starting the power source of each upstream branch of the fourth pipeline to drive the connected liquid to form water-in-oil liquid drops a,

the third step: when the liquid drops a pass through the continuous bending pipeline of the fourth pipeline and reach the third communication position, the cutting of the liquid drops a is carried out, n liquid drops in parallel condition are formed and enter the first pipeline,

the fourth step: and starting the first power source to drive the n liquid drops under the parallel condition to move towards the second connector in the first pipeline, and when the liquid drops sequentially reach the position under the first communication part, closing the first power source, starting the second power source connected with the second pipeline and communicating the fusion electrode power source, wherein the second power source pushes the bacterial liquid to enter the liquid drops under the parallel condition.

Wherein, the two ends of the first pipeline respectively comprise a first connecting port and a second connecting port, and the second pipeline, one end of the third pipeline comprises a fourth connecting port, the other end of the third pipeline is communicated with the first pipeline, the fourth pipeline, one end of the first pipeline is provided with a pipeline with 3 or more branches at the upstream and a fifth connecting port, the other end of the first pipeline is communicated with the first pipeline through a common pipeline, the common section of tubing comprises a continuously curved tubing, the first communication of the second tubing with the first tubing being intermediate the second communication of the third tubing with the first tubing, and the third communication of the fourth tubing with the first tubing, the distance between the first communication part and the second communication part and the distance between the first communication part and the third communication part are 500-2000 mu m, preferably 750-1800 mu m, and preferably 1000-1500 mu m; the first pipeline is communicated with a first power source through a first connecting port and a capillary pipeline, and is communicated with a first valve through a second connecting port and the capillary pipeline; the second pipeline is respectively communicated with a second power source and a second valve through a third connecting port and a capillary tube; the third pipeline is communicated with a third valve through a fourth connecting port and a capillary tube; and the fourth pipeline is respectively communicated with the power source connected with the branch and the corresponding control valve thereof through a fifth connecting port and a capillary tube.

In the method, the upstream branch of the fourth pipeline is 3, 4 or 5, that is, the fourth pipeline a, the fourth pipeline b or the fourth pipeline c, or the fourth pipeline a, the fourth pipeline b, the fourth pipeline c or the fourth pipeline d, or the fourth pipeline a, the fourth pipeline b, the fourth pipeline c, the fourth pipeline d or the fourth pipeline e.

In the method, the upstream branch of the fourth pipeline is a continuous bent pipeline communicated with the first pipeline, and preferably consists of one or more structures of P type, S type, U type, broken line type and wave type.

In the method, the total volume of the liquid contained in the continuous bent pipeline is not less than the volume of the liquid drop a, and is 4-100 muL, preferably 4-50 muL, preferably 4-20 muL, preferably 6-12 muL, and further preferably 8-10 muL. Further, the total volume of the liquid drops a is 4-100 muL, preferably 4-50 muL, preferably 4-20 muL, preferably 6-12 muL, and further preferably 8-10 muL.

The first, second, third and fourth pipes are disposed on a substrate, and the substrate and the first, second, third and fourth pipes are formed of glass, Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS), preferably Polymethylmethacrylate (PMMA).

Preferably, the first communicating portion is the same distance as the second communicating portion and the third communicating portion, respectively.

Cross-sections of the first, second, third and fourth conduitsThe product range is 2.5 × 10^- ³mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²It is further preferred that the cross-sectional areas of the first, second, third and fourth conduits are the same.

In the second step, when the upstream of each fourth pipeline branches into 3 mutually parallel fourth pipelines a, b and c, the corresponding fifth connection ports are the

fifth connection ports

41, 41' and 41 ″ which are respectively connected with different power sources and valves for realizing oil phase and water phase sampling. For example, the fifth connection port 41 is connected to the third power source and the fourth valve through a capillary tube, the fifth connection port 41' is connected to the fourth power source and the fifth valve through a capillary tube, and the fifth connection port 41 ″ is connected to the fifth power source and the sixth valve through a capillary tube. Before use, all the pipes are filled with oil phase, preferably mineral oil as filling medium, so that the pipes connected to the

fifth connection ports

41, 41', 41 ″ are filled with mineral oil. In use, the fifth connection ports 41', 41 ″ are connected to fresh basic culture solution, basic culture solution containing chemical factors, substrate reaction solution, etc. through corresponding connection capillaries and power sources, and can be prepared according to the experimental requirements. For example, the fifth connection port 41' is connected to a fresh basic culture solution, and the fifth connection port 41 ″ is connected to a basic culture solution containing the culture factor x. The specific implementation operation mode is as follows: opening the first valve, and respectively starting a fourth power source and a fifth power source to fill corresponding culture solution in the fourth pipeline b and the fourth pipeline c; starting a fourth power source, opening a first valve at the same time, closing the fourth power source when the basal culture solution in the fourth pipeline b quantitatively enters the main pipeline of the fourth pipeline through a fourth communication part, starting the third power source, and forming a liquid drop p of the water-in-oil basal culture solution at the fourth communication part; the liquid drop p further reaches the position right below the fifth communication part under the drive of the third power source, the third power source is closed, the fifth power source is started to inject the basic culture solution containing the culture factor x into the liquid drop p, and a culture medium liquid drop p' containing the culture factor x is formed. The culture factor x is used as a single factor or a single factor, the flow velocity and the flow of the injected liquid are controlled by a corresponding power source, namely a fifth power source, the entering amount of the culture factor is changed, so that the concentration of the culture factor x is controlled, different concentration gradients are formed, and the formation of the culture solution with the single factor and different concentrations is realized.

In the second step, when the upstream of each of the fourth pipes is branched into 4 parallel fourth pipes a, b, c, d, one end of each of the fourth pipes is connected to the

fifth connection ports

41, 41 ', 41 "', respectively. In addition to the structure in which the fourth channel is branched at 3 upstream sides, a fourth channel d and a fifth connection port 41 '' 'thereof are added, the fifth connection port 41' '' is connected to a sixth power source and a seventh valve through a capillary tube, the fifth connection port 41 '' 'is connected to a base culture solution containing a culture factor y through a capillary tube, and when a culture medium droplet p' containing the culture factor x reaches a sixth connection portion, the sixth power source connected to the fifth connection port 41 '' 'is driven, and the base culture solution containing the culture factor y is quantitatively controlled to enter the culture medium droplet p' to form a culture solution droplet p '' containing the culture factor x and the culture factor y. In this embodiment, the control of the culture medium with different concentration by a single factor may be realized by individually controlling the amount of the culture factor x or the culture factor y, or the formation of the culture medium with different concentration by two factors may be realized by separately controlling the amount of the culture factor x and the culture factor y.

In the second step, when the upstream of each of the fourth pipes branches into 5 fourth pipes a, b, c, d, and d, which are parallel to each other, one end of each of the fourth pipes is connected to the

fifth connection ports

41, 41 ', 41 "', and 41" ', respectively. Specifically, the fifth connection ports 41, 41 '' '' are connected to different power sources and valves, respectively, and further, on the basis of the above-mentioned power source and valve structures branched at 4 upstream, the fifth connection port 41 '' '' is connected to a seventh power source and an eighth valve through a capillary tube, respectively, for realizing the introduction of the oil phase and the water phase. For example, in the case of the formation of a culture solution having a concentration different from the single factor and the formation of a culture solution having a concentration different from the two factors, an oil phase sample injection system is connected to the connection port of the tube immediately before the chip is used, and the tube is filled with an oil phase medium, and the fifth connection ports 41 ', 41 "', and 41" ' can be used to receive a fresh culture solution, a chemical factor, a substrate reaction solution, and the like. Further, for example, a fresh medium is connected to the fifth connection port 41 ', a water-in-oil culture medium droplet p is formed at the fourth communication site, and the fifth connection ports 41 ' ', 41 ' ' ', and 41 ' ' ' ' are connected to a culture factor x, a culture factor y, and a culture factor z, respectively, which are fused with the water-in-oil culture medium droplet p in the pipe at the fifth communication site, the sixth communication site, and the seventh communication site, respectively, to finally form a new culture medium droplet p ' ' ' or a droplet a containing a culture factor of a different culture factor and/or a culture factor of a different concentration. Different culture factors or culture factors with different concentrations are realized by controlling the entering amount of the corresponding power source. The culture medium drops p ' ' ' or drops a containing different culture factors and/or culture factors with different concentrations are arranged at the continuous bent pipeline, and effectively promote the buffering and mixing of the different culture factors. In the present embodiment, the automatic preparation of the micro-system of the three-factor multi-level microbial culture solution is realized by the power source connected to the fifth connection ports 41, 41 '' ''. Of course, those skilled in the art can realize the preparation of the multi-factor and multi-level microorganism culture solution according to more branches and connectors of the fourth pipeline, thereby realizing the culture of the multi-factor and multi-level microorganisms.

In the third step, the liquid drops move to the continuous bending pipeline through the driving power supply, and the buffering and mixing of different culture factors can be effectively promoted through the special shape and structure of the bend.

In the third step, the water-in-oil droplets a are cut into n droplets with parallel conditions, i.e. the volume size and the content of the droplets are completely the same. In one embodiment, droplet a1, droplets a2, …, and droplet an are "n + 2" times the total volume of droplet a. In the microorganism culture experiment, 3 or 5 microorganisms with the same conditions are often used for parallel culture, so that the experimental error is reduced, and therefore n is preferably 3 or 5.

In the invention, after the preparation of culture media with different factors and different levels is finished, the formed liquid drops a directly enter the first pipeline under the driving of the third power source, or a plurality of parallel liquid drops enter the first pipeline through the cutting of the liquid drops at the third communication part.

In the present invention, the water-in-oil droplet a is not limited to a single droplet, and may be a plurality of droplets having different levels of different factors, and further, for example, if there are m different culture conditions, i.e., different levels of different factors, the droplet a is the droplet a₁Droplet a₂Droplet a₃…, droplet a_mFormed by cutting (droplets a)₁1. Liquid droplet a₁2.… droplet a₁n), (droplet a)₂1. Liquid droplet a₂2.… droplet a₂n), …, (droplet a)_m1. Liquid droplet a_m2.… droplet a_mn）。

In order to ensure the consistency of the droplet volumes and reduce operation errors, in the third step, droplet division is preferably performed in a mode of 'pinching off the head and tail middle parts', namely, the head and tail parts of the droplet a are cut off and flow out through the fourth connecting port, and the middle part droplet is divided into the droplet a1, the droplet a2, … and the droplet an on average.

The invention discloses a specific implementation mode of removing the middle part of the tail by nipping the head, which comprises the following steps:

pinching: and (3) starting the power source connected with the fourth pipeline a, opening the first valve, enabling the liquid drop a to reach a third communication part through the continuous bent pipeline of the fourth pipeline, when the head liquid a0 of the liquid drop a enters the first pipeline, closing the power source and the first valve connected with the fourth pipeline a, opening the third valve and starting the first power source, driving the liquid a0 to move towards the fourth connecting port to finish the cutting of the liquid drop a0, and further enabling the liquid drop a0 to flow out of the fourth connecting port under the driving of the first power source.

The middle part is as follows: starting a first power source to suck reversely, opening a control valve connected with a fourth pipeline a, enabling liquid drops a to be quantified to enter a first pipeline, enabling the liquid to move towards a first connecting port, closing the control valve connected with the fourth pipeline a, opening the first valve, completing the cutting of the liquid drops and forming liquid drops a1 moving towards the first connecting port, completing the cutting of the liquid drops forming parallel conditions according to a cutting step of the liquid drops a1, forming a1, liquid drops a2, … and liquid drops an, and keeping the liquid drops an on the left side of a third connecting part of the first pipeline

During the cutting operation of the middle part of the droplet, the first detection window 9 performs operation control thereon, for example, when the droplet a1 reaches the first detection window 9, the cutting operation of the droplet a2 is performed, when the droplet a2 reaches the first detection window 9, the cutting operation of the droplet a3 is performed, and so on.

Removing tails: and after the cutting operation of the liquid drops with the same volume of n volumes is finished, closing the first power source and the first valve, starting the power source connected with the fourth pipeline a, and opening the third valve to enable the residual liquid after the cutting of the liquid drops a to flow out through the fourth connecting port.

Before the operation of 'pinching the head and removing the tail and the middle part', the volume of the liquid drop a and the number and the volume of the liquid drops to be formed by cutting need to be calculated in advance, and the total volume of the liquid drops 'pinching the head and removing the tail' is usually 0.5-2 liquid drops an, which are discharged through the third connecting port. The 'pinching and removing' operation is beneficial to ensuring that the size and the volume of the liquid drops under a plurality of parallel conditions are consistent.

In one embodiment, the volume of the droplets a1, a2, … and an is the same, preferably, the volume is one half of the total volume of the droplets a, i.e., "n + 2", and the total volume of the droplets cutting the head and the tail of the droplets a accounts for two parts of the total volume of the droplets a, i.e., "n + 2".

In the fourth step, when all the liquid drops enter the first pipeline, the liquid drops are to be fused and sequentially reach the position right below the first communication part under the driving of the first power source, the first power source is closed, and the second power source connected with the second pipeline drives the bacterial liquid connected with the second power source to sequentially enter the liquid drops a1, a2, … and the liquid drops an respectively and complete the fusion of the liquid drops. In the process of liquid drop fusion, the fusion electrode is communicated with a power supply to generate an electric field effect to promote fusion. In the operation process, the first detection window 9 identifies the liquid drops, and fusion operation control is facilitated. In one embodiment, the microorganism culture solution, whether single-factor multi-level, two-factor multi-level, three-factor multi-level, or multi-factor multi-level, reaches the third communication position through the continuous bending pipeline, and is cut into a plurality of droplets under parallel conditions.

Examples

The first pipeline, the second pipeline, the third pipeline and the fourth pipeline are pipelines formed in the chip substrate, the size of the chip substrate is 3cm x 5cm x 4mm (length x width x thickness), the material used by the chip substrate is PMMA, the first pipeline, the second pipeline, the third pipeline and the fourth pipeline formed in the chip substrate are pipelines with square cross sections, and the cross sections of the first pipeline, the second pipeline, the third pipeline and the fourth pipeline are pipelines with 1mm cross sections²(the side length is 1mm), the first pipeline is respectively communicated with the second pipeline, the third pipeline and the fourth pipeline, the first communication part of the second pipeline and the first pipeline is positioned at the second communication part of the third pipeline and the first pipeline, and the distance between the first communication part of the fourth pipeline and the third communication part of the first pipeline is 1.5 mm. The pipeline is filled with oily medium. The upstream branches of the fourth pipelines are 5, namely a fourth pipeline a, a fourth pipeline b, a fourth pipeline c, a fourth pipeline d and a fourth pipeline e which are arranged in parallel, and the distance between the fourth pipeline a, the fourth pipeline b, the fourth pipeline c, the fourth pipeline d and the fourth pipeline e is 1.5 mm. The 5 branches of fourth pipeline upper reaches link to each other with power supply and valve through the pipeline respectively, and the fourth pipeline other end is through one section S type pipeline and first pipeline intercommunication.

A first connecting port of the first pipeline is connected with an injection pump A, and a second connecting port is connected with a rotary first valve; the third connecting port of the second pipeline is connected with a syringe pump B and a rotary second valve, the fourth connecting port of the third pipeline is connected with a rotary third valve, and the fourth pipeline a, the fourth pipeline B, the fourth pipeline C, the fourth pipeline D and the fourth pipeline E are respectively connected with a syringe pump C, a rotary fourth valve, a syringe pump D, a rotary fifth valve, a syringe pump E, a rotary sixth valve, a syringe pump F, a rotary seventh valve, a syringe pump G and a rotary eighth valve. The pipeline is connected with the injection pump and the rotary valve through a capillary, the inner diameter of the capillary is 1.0 mm, and the material is polytetrafluoroethylene.

In this embodiment, the syringe pumps a, B, C, D, E, F and G used are all industrial syringe pumps, and the valve heads of the syringe pumps are three-way valves, and are commercially available from MSP1-C2 of north Heiben Baoding Lange constant flow pump, Inc.

The first valve, the second valve, the third valve, the fourth valve, the fifth valve, the sixth valve, the seventh valve and the eighth valve are all Mrv-01 high-pressure two-way valves purchased from Nanjing wetting fluid control devices, Inc.

Before the chip is used, an oil phase is filled in a pipeline, then a fifth connecting port is respectively connected with the oil phase, a culture medium and various culture factor solutions, and corresponding liquids are filled in a branch pipeline at the upstream of a fourth pipeline, wherein the medium oil phase is mineral oil, an injection pump C is used for driving the oil phase, an injection pump D is used for driving a culture solution (called initial culture solution for short) filled with 10G/L tryptone and 10G/L yeast extract, and an injection pump E, an injection pump F and an injection pump G are respectively used for driving the initial culture solution of a culture factor X, a culture factor Y and a culture factor Z; in this example, the culture factor X, the culture factor Y and the culture factor Z were 50g/L NaCl, 25mol/L NaOH and 250mg/L ampicillin, respectively.

Starting the injection pump C, simultaneously opening a first valve, driving an oil phase in a fourth pipeline a to enter a pipeline, closing the injection pump C, simultaneously starting the injection pump D, keeping the opening state of the first valve unchanged, immediately closing the injection pump D when the ' initial culture solution ' in the fourth pipeline b is injected to a position below a fourth communication position, starting the injection pump C, and forming a liquid drop p of the culture solution in oil bag below the fourth communication position by the 4 muL ' of the ' initial culture solution '; under the driving of the injection pump C, the liquid drop p moves to reach the position right below the fifth communication part, the injection pump C is closed, the injection pump E is started at the same time, 2 muL of 50g/L NaCl 'initial culture solution in the fourth pipeline C is driven to enter the liquid drop p in the pipeline, the injection pump E is closed, the injection pump C is started, and the liquid drop p' is formed at the moment; under the driving of the injection pump C, the liquid drop p 'moves to reach the position right below the sixth communication part, the injection pump C is closed, the injection pump F is started at the same time, 2 muL of 25mol/LNaOH initial culture solution in the fourth pipeline d is driven to enter the liquid drop p' in the pipeline, the injection pump F is immediately closed, the injection pump C is started, and the liquid drop p '' is formed at the moment; under the driving of the injection pump C, the liquid drop p ' ' moves to reach the position right below the seventh communication position, the injection pump C is closed, the injection pump G is started, 2 muL of 250mg/L ampicillin ' initial culture solution in the fourth pipeline e is driven to enter the liquid drop p ' ' in the pipeline, the injection pump G is immediately closed, the injection pump C is started, and at the moment, the liquid drop p ' ' or called liquid drop a is formed. At this time, the culture solution containing 10g/L of tryptone, 10g/L of yeast extract, 10g/L of NaCl, 5mol/L of LNaOH and 50mg/L of ampicillin was 10. mu.L of p ' ' ' or a droplet a in the droplet.

And further pushing the liquid drops a to reach a third communication position through a continuous bent pipeline by an injection pump C, and cutting the liquid drops by 'pinching and removing the tail and the middle part', wherein 3 liquid drops a1, a2 and a3 with the volume of 2 mu L are formed into middle parallel liquid drops, and the liquid drops with the pinched heads and the removed tails are about 2 mu L and are discharged through a third pipeline. In the formation of the droplet a1, the droplet a2 and the droplet a3, the first detection window 9 is located on the first pipe between the third communication portion and the first connection port, the first detection window 9 is operated and controlled at a distance of 2mm from the third communication portion, the cutting operation of the droplet a2 is performed when the droplet a1 reaches the first detection window 9, and the cutting operation of the droplet a3 is performed when the droplet a2 reaches the first detection window 9.

The first rotary valve is opened, the syringe pump A is driven continuously, and the liquid drops a1, a2 and a3 are pushed to move towards the second connecting port. When the liquid drop a1 reaches the position right below the first communication part, the injection pump A stops driving, the injection pump B is started to provide driving force, so that the bacterium liquid B connected with the second pipeline is pushed to enter the liquid drop a1, the fusion electrode is communicated with a 500V power supply while the injection pump B provides the driving force, and an electric field is generated to promote the fusion of the liquid drops. In the examples, the bacterial suspension b is Escherichia coli. The steps are circularly repeated, and the fusion operation of the liquid drop a2 and the liquid drop a3 is completed, so that the inoculation culture of 3 colibacillus with parallel concentration is realized. The first detection window 9 serves as a droplet identification point, identifying the position of the droplet; and the second detection window 10 is used for detecting the liquid drop detection point and the bacteria concentration OD.

Industrial applicability

The micro-fluidic chip for culturing and detecting microorganisms can be manufactured and used in the field of micro-fluidic chips. The invention is suitable for satisfying the growth condition of microorganism culture by multiple factors and levels, and is suitable for adaptive evolution, drug resistance mechanism research, culture optimization and microorganism detection.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.

The present application is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, the application is not intended to be limited to the particular forms disclosed. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the application, which is defined by the appended claims and their legal equivalents.

The numerical ranges recited in the present invention each include data for both endpoints of the numerical range, and also include each of the specific values in the numerical range, and the numerical values can be combined with the endpoints at will to form a new subrange.

Claims

1. A micro-droplet generation and fusion device, comprising:

6. The micro-droplet generation and fusion device according to any one of claims 1 or 5, characterized in that the total volume of the liquid contained in the continuously bent pipeline is 4-100 μ L, preferably 4-50 μ L, preferably 4-20 μ L, preferably 6-12 μ L, and further preferably 8-10 μ L.

9. The micro-droplet generation and fusion device of any one of claims 1-8, wherein the cross-sectional area of the first, second, third and fourth conduits is in the range of 2.5 x 10^-3mm²~4mm²Preferably 0.01 to 3mm in thickness²More preferably 0.1 to 2.5mm²More preferably 0.25 to 1mm in thickness²It is further preferred that the cross-sectional areas of the first, second, third and fourth conduits are the same.

10. A method of microdroplet generation and fusion, comprising: