High-throughput microfluidic strain screening device

Abstract

The invention discloses a high-flux microfluidic strain screening device, and relates to the technical field of microfluidics, the high-flux microfluidic strain screening device comprises a microdroplet generation unit and a microdroplet collection unit, the microdroplet generation unit comprises an inner water phase pipe, an oil phase pipe, an outer water phase pipe and a liquid outlet pipe which are coaxially arranged; the micro-droplet collecting unit comprises a collecting plate, a micro-droplet liquid inlet, a micro-droplet collecting cavity and a waste liquid discharging opening are formed in the collecting plate, a plurality of positioning grooves for capturing micro-droplets are formed in the bottom of the micro-droplet collecting cavity, and an extraction channel penetrating to the bottom surface of the collecting plate is formed in the bottom of each positioning groove; one end of the microdroplet liquid outlet of the liquid outlet pipe is inserted into the microdroplet liquid inlet; in order to solve the problems of low flux, easy cross contamination and false positive screening in single strain separation and culture in the prior art, the invention provides a high-flux microfluidic strain screening device capable of quickly separating, culturing and screening a single strain collected in an environment.

Description

Technical Field

[0001] The invention relates to the field of microfluidic technology, and in particular to a high-throughput microfluidic strain screening device. Background Art

[0002] With the rapid development of environmental microbiology and biotechnology, screening strains with specific functions from natural environments has become an important task for scientific research and industrial applications. These functional strains have broad application potential in the fields of pollutant degradation, biopharmaceutical production, and agricultural adjuvant development. However, microorganisms in environmental samples usually exist in the form of mixed communities, and the isolation and purification of single strains is a complex and time-consuming task.

[0003] Traditional isolation methods, such as plate streaking and dilution coating, rely on repeated manual operations and cultivation. Although these methods have played an important role in the early research of microbiology, they are inefficient when faced with a large number of environmental samples. On the one hand, the long cultivation and screening cycles limit the separation speed; on the other hand, the number of samples that can be processed in each experiment is limited, which makes it difficult to meet the needs of large-scale strain screening. In addition, operations in an open environment are prone to cross-contamination, affecting the purity and reliability of the separation.

[0004] It is particularly noteworthy that traditional methods are prone to cross-contamination between strains during the separation process, and this cross-contamination may lead to inhibition between different strains. Metabolites secreted by some bacteria may inhibit the growth of other strains, resulting in the inability to effectively separate certain target strains. In addition, cross-contamination may also cause false positive results in screening, that is, during the screening process, a certain strain is mistakenly believed to have a specific function, which is actually caused by interference from contaminating strains. This phenomenon not only wastes time and resources, but may also lead to misjudgment of microbial functions, affecting the accuracy and effectiveness of research.

[0005] Another challenge is that there are a large number of unknown bacterial communities in environmental samples, and many strains cannot grow under single culture conditions, resulting in the neglect of potentially valuable microbial resources. These uncultured strains may have important metabolic functions or new biological activities, and traditional methods are difficult to effectively separate them for in-depth research. Therefore, it is particularly urgent to develop an efficient, controllable and high-throughput strain separation technology.

[0006] Microfluidics technology provides a new means to solve these problems. Microfluidics devices can accurately control fluid flow in tiny channels and quickly generate closed emulsion droplets. Each droplet can be used as an independent microreactor for the isolation and cultivation of single strains. However, existing microfluidics devices still have shortcomings in practical applications, such as single function, difficulty in introducing multiple reagents at the same time, and difficulties in efficient collection and sorting of microdroplets. Summary of the invention

[0007] In order to solve the problems of low throughput, easy cross contamination and false positive screening in the isolation and cultivation of single strains in the prior art, the present invention proposes a high-throughput microfluidic strain screening device that can quickly separate, culture and screen strains collected from the environment.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: A high-throughput microfluidic strain screening device comprises a droplet generation unit and a droplet collection unit. The droplet generation unit comprises an inner water phase tube, an oil phase tube, an outer water phase tube and a liquid outlet tube, all of which are coaxially arranged; one end of the inner water phase tube is an inner water phase outlet, and the other end is provided with an inner water phase inlet interface; The oil phase tube is sleeved on the outside of the inner water phase tube, one end of the oil phase tube is the oil phase water outlet, the other end of the port is blocked with the outer wall of the inner water phase tube, and the oil phase tube is provided with an oil phase water inlet interface; one end of the liquid outlet tube is arranged opposite to the oil phase water outlet of the oil phase tube, and the other end is a droplet liquid outlet; one end of the outer water phase tube is sleeved on the outside of the liquid outlet tube and the port is blocked with the outer wall of the liquid outlet tube, the other end is blocked with the outer wall of the oil phase tube, and the outer water phase tube is provided with an outer water phase water inlet interface; The droplet collection unit comprises a collection plate, in which a droplet inlet, a droplet collection cavity and a waste liquid discharge port are arranged, wherein the droplet collection cavity and the waste liquid discharge port are respectively located on both sides of the droplet collection cavity and are both connected to the droplet collection cavity; a plurality of positioning grooves for capturing droplets are arranged at the bottom of the droplet collection cavity, and an extraction channel which passes through to the bottom surface of the collection plate is arranged at the bottom of each positioning groove; one end of the droplet outlet of the liquid outlet pipe is plugged into the droplet inlet.

[0009] Preferably, the inner water phase outlet of the inner water phase tube is a tapered outlet with a gradually decreasing diameter, and the oil phase outlet of the oil phase tube is also a tapered outlet with a gradually decreasing diameter, the oil phase outlet is located in front of the inner water phase outlet and the diameter of the oil phase outlet is larger than the diameter of the inner water phase outlet; the diameter of the liquid outlet tube toward one end of the oil phase tube is larger than the diameter of the oil phase outlet.

[0010] Preferably, the positioning groove is a hemispherical groove.

[0011] Preferably, a micro-liquid limiting plate is provided at the upper opening of each positioning groove in the micro-liquid collecting chamber. The micro-liquid limiting plate is arranged in a U shape and its opening is toward the direction of the droplet inlet. A water pressure balance port for the passage of reagents is provided on the rear side of the micro-liquid limiting plate.

[0012] Preferably, a culture medium filling port connected to the micro-liquid collecting chamber is provided on the collecting plate, and the connecting port between the culture medium filling port and the micro-liquid collecting chamber is located near the micro-droplet inlet.

[0013] Preferably, the collecting plate is formed by bonding a bottom plate and a cover plate, and the micro-droplet inlet, micro-liquid collecting chamber and waste liquid discharge port are all arranged on the bonding surface between the bottom plate and the cover plate.

[0014] Preferably, the inner water phase tube is a three-channel tube, the cross section of the inner water phase tube is a three-pointed star structure, the three channels are evenly distributed at 120°, and each channel is provided with a liquid injection port.

[0015] The present invention has the following beneficial effects: 1. Each droplet, as an independent reactor, can effectively separate and culture a single strain, avoid cross contamination between strains, and reduce inhibition and false positive results; 2. A droplet collection unit is provided, which can efficiently collect, sort and recover the target strain. The device is combined with a microscope observation system or a fluorescence detection system to achieve real-time monitoring of the growth status and function of the strain, and is suitable for multiple fields such as environmental microbial screening, drug sensitivity testing and bacterial biofilm research; 3. The inner water phase tube can be set as a three-channel tube to achieve the simultaneous introduction of strains, culture media and reagents, thereby improving the flexibility and efficiency of screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural diagram of embodiment 1.

[0017] Figure 2 It is a structural schematic diagram of embodiment 1 from another angle.

[0018] Figure 3 It is a perspective view of the first embodiment.

[0019] Figure 4 It is an exploded view of the first embodiment.

[0020] Figure 5 4 is a cross-sectional view of the droplet generation unit in the first embodiment.

[0021] Figure 6 yes Figure 5 Enlarged view of point a in the middle.

[0022] Figure 7 It is a schematic diagram of the structure of the micro-droplet inlet, the micro-liquid collection chamber and the waste liquid discharge port in the first embodiment.

[0023] Figure 8 yes Figure 7 Enlarged view of point b in the middle.

[0024] Fig. 9 It is a cross-sectional view of the inner water phase tube in the second embodiment.

[0025] Fig.10 Schematic diagram of droplet generation under a microscope.

[0026] 1: inner water phase tube; 101: inner water phase outlet; 102: inner water phase inlet interface; 2: oil phase tube; 201: oil phase outlet; 202: oil phase inlet interface; 3: outer water phase tube; 301: outer water phase inlet interface; 4: liquid outlet tube; 401: droplet outlet; 5: collection plate; 501: bottom plate; 502: cover plate; 6: droplet inlet; 7: micro-liquid collection chamber; 701: positioning groove; 702: extraction channel; 703: micro-liquid limiting plate; 704: water pressure balance port; 8: waste liquid discharge port; 9: culture medium filling port. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] Example 1: See Figures 1 to 8 A high-throughput microfluidic strain screening device comprises a microdroplet generating unit and a microdroplet collecting unit.

[0029] The droplet generation unit comprises an inner water phase tube 1, an oil phase tube 2, an outer water phase tube 3 and a liquid outlet tube 4 which are all coaxially arranged.

[0030] The inner water phase tube 1 is a glass tube with an inner diameter of 5 to 50 microns and an outer diameter of 80 to 150 microns. One end of the inner water phase tube 1 is an inner water phase outlet 101. The inner water phase outlet 101 of the inner water phase tube 1 is a tapered mouth with a gradually decreasing diameter. The other end is provided with an inner water phase inlet interface 102. The inner water phase tube 1 is used to guide the inner water phase fluid.

[0031] The oil phase tube 2 is a glass tube with an inner diameter of 100 to 200 microns and an outer diameter of 150 to 300 microns. It is sleeved on the outside of the inner water phase tube 1. One end of the oil phase tube 2 is an oil phase outlet 201. The oil phase outlet 201 of the oil phase tube 2 is also a tapered port with a gradually decreasing diameter. The oil phase outlet 201 is located in front of the inner water phase outlet 101 and the diameter of the oil phase outlet 201 is larger than the diameter of the inner water phase outlet 101. The port at the other end of the oil phase tube 2 is blocked from the outer wall of the inner water phase tube 1. An oil phase water inlet interface 202 is provided on the oil phase tube 2. The oil phase tube 2 is used to guide the oil phase fluid.

[0032] One end of the liquid outlet pipe 4 is arranged opposite to the oil phase water outlet 201 of the oil phase pipe 2 , and the other end is a droplet outlet 401 ; the diameter of the end of the liquid outlet pipe 4 facing the oil phase pipe 2 is larger than the diameter of the oil phase water outlet 201 .

[0033] One end of the external water phase pipe 3 is sleeved on the outside of the liquid outlet pipe 4 and the port is sealed with the outer wall of the liquid outlet pipe 4, and the other end is sealed with the outer wall of the oil phase pipe 2. The external water phase pipe 3 is provided with an external water phase water inlet interface 301, and the external water phase pipe 3 is used to guide the external water phase fluid.

[0034] The droplet collection unit includes a collection plate 5, which is formed by bonding a bottom plate 501 and a cover plate 502. A droplet inlet 6, a micro-liquid collection cavity 7 and a waste liquid discharge port 8 are provided in the collection plate 5. The droplet inlet 6, the micro-liquid collection cavity 7 and the waste liquid discharge port 8 are all arranged on the bonding surface between the bottom plate 501 and the cover plate 502. The micro-liquid collecting chamber 7 and the waste liquid discharge port 8 are respectively located on both sides of the micro-liquid collecting chamber 7 and are both connected to the micro-liquid collecting chamber 7; a plurality of positioning grooves 701 for capturing droplets are provided at the bottom of the micro-liquid collecting chamber 7, the positioning grooves 701 are hemispherical grooves, the diameter of the positioning grooves 701 is 50 to 100 microns, and an extraction channel 702 is provided at the bottom of each positioning groove 701 that passes through the bottom surface of the collecting plate 5, the aperture of the extraction channel 702 is 10 to 20 microns, and is used for capillary suction and transfer of emulsion droplets, and a micro-liquid limiting plate 703 is provided at the upper opening of each positioning groove 701, the micro-liquid limiting plate 703 is arranged in a U shape and the opening is facing the direction of the droplet inlet 6, and a water pressure balance port 704 for the passage of reagents is provided on the rear side of the micro-liquid limiting plate 703.

[0035] The collecting plate 5 is provided with a culture medium filling port 9 connected to the micro-liquid collecting chamber 7 , and the connecting port between the culture medium filling port 9 and the micro-liquid collecting chamber 7 is located near the micro-droplet inlet 6 .

[0036] One end of the micro-droplet liquid outlet 401 of the liquid outlet tube 4 is plugged into the micro-droplet liquid inlet 6 .

[0037] When in use, the inner water phase (strain suspension) is introduced into the inner water phase tube 1 through the inner water phase outlet 101; the oil phase (which may be silicone oil or fluorinated oil) is introduced into the oil phase tube 2 through the oil phase outlet 201; the outer water phase (2% to 10% polyvinyl alcohol aqueous solution) is introduced into the outer water phase tube 3 through the outer water phase inlet interface 301; the three streams of fluid are focused and merged at the inner water phase outlet 101 and the oil phase outlet 201, the inner water phase is squeezed by the oil phase and the outer water phase, and droplets are generated when the inner water phase flows out from the inner water phase outlet 101, and are wrapped by the oil phase flowing out of the oil phase outlet 201, and are dispersed in the outer water phase to form monodisperse double emulsion droplets, and finally triple emulsion droplets of water-oil-water (w / o / w) fluid flow mode are formed, and the diameter of the emulsion droplets is between 50 and 300 microns, such as Fig.10 Furthermore, the size and structure of the droplets can be precisely controlled by adjusting the flow rate ratio of the inner water phase, the oil phase, and the outer water phase.

[0038] Then, the generated emulsion droplets pass through the droplet outlet 401 and the droplet inlet 6 in sequence and then enter the micro-liquid collection chamber 7 of the collection plate 5. When the emulsion droplets flow in the micro-liquid collection chamber 7, when they fall into the positioning groove 701 or hit the U-shaped micro-liquid limiting plate 703, they will be restricted in the corresponding positioning groove 701 to prevent the droplets from moving or cross-contamination. Excess reagents are discharged through the waste liquid discharge port 8.

[0039] Finally, the strains on the detection collection plate 5 can be observed through a microscope observation system. When the target strain is observed and detected, a negative pressure pump is used to connect the capillary glass tube to absorb the target droplets from the extraction channel 702 at the bottom of the positioning groove 701 for subsequent culture or analysis. In addition, a culture medium filling port 9 is provided on the collection plate 5, which can transport culture medium or other reagents into the microfluid collection chamber 7 to maintain strain growth or perform further screening.

[0040] Example 2: See Fig. 9 The remaining structures of this embodiment are consistent with those of the first embodiment, except that: the inner water phase tube 1 is a three-channel tube, the cross section of the inner water phase tube 1 is a three-pointed star structure, the three channels are evenly distributed at 120°, and each channel is provided with a liquid injection port.

[0041] The three channels can be a strain channel, a culture medium channel, and an antibiotic or enzyme substrate channel. The strain channel is used to introduce the strain suspension to be screened, the culture medium channel is used to introduce the culture medium suitable for strain growth, and the antibiotic or enzyme substrate channel is used to introduce the reagents required for screening, such as antibiotics or enzyme substrates. Through the three-channel design, multiple components can be introduced simultaneously when generating droplets to provide support for functional screening.

Claims

1. A high-throughput microfluidic strain screening device, characterized in that: comprising a droplet generating unit and a droplet collecting unit, The droplet generation unit comprises an inner water phase tube (1), an oil phase tube (2), an outer water phase tube (3) and a liquid outlet tube (4) which are all coaxially arranged; one end of the inner water phase tube (1) is an inner water phase outlet (101), and the other end is provided with an inner water phase inlet interface (102); the oil phase tube (2) is sleeved on the outer side of the inner water phase tube (1), one end of the oil phase tube (2) is an oil phase outlet (201), and the port at the other end is sealed with the outer wall of the inner water phase tube (1). , an oil phase water inlet interface (202) is provided on the oil phase tube (2); one end of the liquid outlet tube (4) is arranged opposite to the oil phase water outlet (201) of the oil phase tube (2), and the other end is a droplet liquid outlet (401); one end of the external water phase tube (3) is sleeved on the outside of the liquid outlet tube (4) and the port is blocked between the external wall of the liquid outlet tube (4), and the other end is blocked between the external wall of the oil phase tube (2), and the external water phase tube (3) is provided with an external water phase water inlet interface (301); The micro-droplet collection unit comprises a collection plate (5), wherein a micro-droplet liquid inlet (6), a micro-liquid collection chamber (7) and a waste liquid discharge port (8) are provided in the collection plate (5), wherein the micro-liquid collection chamber (7) and the waste liquid discharge port (8) are respectively located on both sides of the micro-liquid collection chamber (7) and are both connected to the micro-liquid collection chamber (7); a plurality of positioning grooves (701) for capturing micro-droplets are provided at the bottom of the micro-liquid collection chamber (7), and an extraction channel (702) is provided at the bottom of each positioning groove (701) and is connected to the bottom surface of the collection plate (5); and one end of the micro-droplet liquid outlet (401) of the liquid outlet pipe (4) is plugged into the micro-droplet liquid inlet (6).

2. A high-throughput microfluidic strain screening device according to claim 1, characterized in that: The inner water phase outlet (101) of the inner water phase tube (1) is a tapered outlet with a gradually decreasing diameter, and the oil phase outlet (201) of the oil phase tube (2) is also a tapered outlet with a gradually decreasing diameter. The oil phase outlet (201) is located in front of the inner water phase outlet (101) and has a diameter greater than that of the inner water phase outlet (101); the diameter of the end of the liquid outlet tube (4) facing the oil phase tube (2) is greater than the diameter of the oil phase outlet (201).

3. A high-throughput microfluidic strain screening device according to claim 1, characterized in that: The positioning groove (701) is a hemispherical groove.

4. A high-throughput microfluidic strain screening device according to claim 3, characterized in that: A micro-liquid limiting plate (703) is provided at the upper opening of each positioning groove (701) in the micro-liquid collecting chamber (7); the micro-liquid limiting plate (703) is arranged in a U-shape and its opening faces the direction of the micro-droplet inlet (6); a water pressure balancing port (704) for the passage of reagents is provided at the rear side of the micro-liquid limiting plate (703).

5. The high-throughput microfluidic strain screening device according to claim 1, characterized in that: The collection plate (5) is provided with a culture medium filling port (9) connected to the microfluid collection chamber (7); the communication port between the culture medium filling port (9) and the microfluid collection chamber (7) is located close to the microdroplet inlet (6).

6. A high-throughput microfluidic strain screening device according to claim 1, characterized in that: The collecting plate (5) is formed by bonding a bottom plate (501) and a cover plate (502), and the micro-droplet inlet (6), the micro-liquid collecting chamber (7) and the waste liquid discharge port (8) are all arranged on the bonding surface between the bottom plate (501) and the cover plate (502).

7. A high-throughput microfluidic strain screening device according to any one of claims 1 to 6, characterized in that: The inner water phase tube (1) is a three-channel tube, the cross section of the inner water phase tube (1) is a three-pointed star structure, the three channels are evenly distributed at 120 degrees, and each channel is provided with a liquid injection port.

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