CN116408155A - A method for avoiding droplet fusion in a microfluidic system - Google Patents

Abstract

A method for avoiding droplet fusion in a microfluidic system, in which the water phase, the oil phase, and the gas phase are respectively fed into the three channels of the cross-shaped microchannel to generate "water-in-oil" droplets or "water Oil-packed droplets.

Description

A method for avoiding droplet fusion in a microfluidic system

technical field

The invention belongs to a droplet operation method of a microfluidic system, and in particular relates to a method for a microfluidic system to avoid droplet fusion.

Background technique

Droplet microfluidics is a scientific technology for generating and manipulating small-volume droplets. This technology can produce thousands of controllable micro-droplets per minute, and each micro-droplet is separated by an oil phase to become Compared with the traditional biochemical reaction system, a separate reactor has the advantages of less reagent consumption, high experimental efficiency, large throughput, and no cross-contamination. It can also use droplets as transport bodies or reactors to achieve real-time reactions and online observations, etc. Function. Although micro-droplets have such powerful application functions as above, the prerequisite is to form stable micro-droplets and maintain this state stably in the microsystem.

The manipulation technology of micro-droplets mainly includes droplet generation, directional displacement, fusion, splitting, mixing, culturing, incubation, sorting, etc. During these manipulations, there are many factors that affect the stability of micro-droplets, such as droplet formation During the process, due to the uneven size of the generated droplets, it is easy to cause the droplets to merge; the directional displacement speed of the droplets is unstable, and the droplets are close to each other under the action of external force. When the thickness of the liquid film between the droplets decreases to a critical value, resulting in liquid Droplet fusion; droplets are added with reagents for droplet fusion, and the input liquid affects the surface tension of the droplets, resulting in the fusion between the droplets; during the cultivation and incubation of the droplets, the metabolites generated affect the surface activity of the droplets, resulting in the fusion of the droplets etc. Micro-droplets are unstable and fused with each other, which can easily cause inaccurate droplet positioning and cross-contamination. Therefore, there is a need to provide a method to solve the above-mentioned droplet fusion problem.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, on the basis of the existing microfluidic T-shaped microchannels to generate "water-in-oil" droplets or "oil-in-water" droplets, to increase the intake channel and generate bubble intervals "Water-in-oil" droplets or "oil-in-water" droplets to avoid droplet fusion during manipulation.

The purpose of the present invention is achieved through the following technical solutions:

1. A method for avoiding droplet fusion in a microfluidic system, characterized in that: the water phase, the oil phase, and the gas phase are respectively introduced into the three pipelines of the cross-shaped microchannel, and the "water-in-oil" that generates bubble intervals in the fourth pipeline Liquid droplets or "oil-in-water" droplets.

2. A method for avoiding droplet fusion in a microfluidic system according to claim 1, characterized in that: the inner diameter of the cross-shaped microchannel is 0.1-10mm, preferably 1-3mm, more preferably 1mm.

3. A kind of microfluidic system according to claim 1 avoids the method for droplet fusion, it is characterized in that: the diameter of described bubble is greater than microchannel radius, and described " water-in-oil " droplet or " oil-in-water " "The diameter of the droplet is larger than the radius of the microchannel.

4. A method for avoiding droplet fusion in a microfluidic system according to claim 3, characterized in that: the length of the bubble is 0.05mm-1cm, and larger than the "water-in-oil" droplet or "oil-in-water" The diameter of the droplet.

5. A method for avoiding droplet fusion in a microfluidic system according to claim 3, characterized in that: the volume of the "water-in-oil" droplet or "oil-in-water" droplet is 0.6-10 μL , more preferably 0.7-7 μL, further preferably 0.8-6 μL, further preferably 1-3 μL, further preferably 2-3 μL.

6. A kind of microfluidic system according to claim 1 avoids the method for droplet fusion, it is characterized in that: the flow velocity of described criss-cross microchannel input water phase, oil phase is 0-1mL/min, gas phase input The air pressure in the pipeline is 0-100Kpa.

7. A method for avoiding droplet fusion in a microfluidic system according to claim 1, characterized in that: the gas input into the gas phase is a gas that does not react with the water phase and the oil phase.

8. A method for avoiding droplet fusion in a microfluidic system according to claim 7, characterized in that: the gas input into the gas phase is nitrogen, hydrogen or an inert gas.

9. A method for avoiding droplet fusion in a microfluidic system according to claim 8, characterized in that: the inert gas is any one of helium, argon, and neon.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention uses "water-in-oil" droplets or "oil-in-water" droplets that generate bubble intervals to solve the problems of droplet generation, directional displacement, fusion, splitting, mixing, cultivation, incubation, sorting, etc. Micro-droplet instability and its resulting inaccurate droplet positioning and cross-contamination occur in droplet manipulation techniques.

(2) The present invention controls the size of the liquid droplets and bubbles by controlling the inner diameter of the pipeline and the flow rate of the liquid inlet and air intake, so as to avoid deformation or drift of the liquid droplets and bubbles due to extrusion.

(3) The present invention uses gas that does not react with the water phase and the oil phase to generate bubbles, so as to avoid being completely absorbed and consumed by reacting with the pipeline water phase and oil phase.

Description of drawings

FIG. 1 is a schematic diagram of an embodiment of a method for avoiding droplet fusion in a microfluidic system according to the present invention.

Detailed ways

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific examples of structures employed by the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited by the examples set forth herein. Rather, these embodiments are provided for more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

In order to facilitate the understanding of the embodiments of the present invention, further explanations will be given below in conjunction with the accompanying drawings as examples, and the accompanying drawings do not constitute limitations on the method of the present invention.

As shown in Figure 1, the method of the present invention is implemented by using a cross-shaped microchannel, wherein the cross-shaped microchannel includes a first pipeline (1) for the oil inlet phase, a second pipeline (2) for the inlet phase, and a second pipeline (2) for the inlet phase. A third conduit (3) for the aqueous phase, and a fourth conduit (4) containing water-in-oil droplets or oil-in-water droplets that generate bubble spaces at the intersections. The power source (5) of the first pipeline (1), the power source (6) of the second pipeline (2), and the power source (7) of the third pipeline (3) are air pumps, peristaltic pumps, One of diaphragm pump, embolic pump, and syringe pump, preferably a syringe pump.

In one embodiment, the diameter of the cross-shaped microchannel is 0.1-10mm, and the diameter of the microchannel is the cross-sectional diameter of C-C shown in Figure 1, preferably 1-3mm, more preferably 1mm.

In one embodiment, the diameter of the air bubble is larger than the radius of the microchannel, and the diameter of the "water-in-oil" droplet or "oil-in-water" droplet is larger than the radius of the microchannel. Wherein, the diameter of the bubble is the inner diameter of the section A-A as shown in FIG. 1 , and the diameter of the droplet is the diameter of the section B-B as shown in FIG. 1 .

In one embodiment, the length of the bubbles is 0.05 mm-1 cm, and larger than the diameter of the "water-in-oil" droplet or the "oil-in-water" droplet, wherein the length of the bubbles is in the D-D direction shown in FIG. 1 .

In one embodiment, the intersection of the second pipeline (2) of the intake phase, the third pipeline (3) of the water phase, the first pipeline (1) and the fourth pipeline (4) is not necessarily perpendicular, that is, the intersection angle It does not have to be a right angle, and any angle cross connection can be formed as required.

In one embodiment, the cross-shaped microchannels, that is, the first pipe (1), the second pipe (2), the third pipe (3), and the fourth pipe (4) have a diameter of 0.1-10 mm, preferably 0.5-7 mm, preferably 1-3 mm, the inner diameters of the first pipe (1), the second pipe (2), the third pipe (3) and the fourth pipe (4) can be the same or different . When the inner diameters of the first pipeline (1), the second pipeline (2), the third pipeline (3) and the fourth pipeline (4) are completely the same, it is further preferably 1 mm.

In one embodiment, the volume of the "water-in-oil" droplet or "oil-in-water" droplet is 0.6-10 μL, more preferably 0.7-7 μL, further preferably 0.8-6 μL, further preferably 1-3 μL, further preferably 2-3 μL is preferred.

In one embodiment, the cross-shaped microchannels input the water phase and the oil phase at a flow rate of 0-1 mL/min, and the air pressure in the gas phase input pipeline is 0-100 Kpa. The gas input in the gas phase is any gas that does not react with the water phase and the oil phase, and is further a sterile gas, preferably nitrogen, hydrogen or an inert gas, and the inert gas is preferably helium, argon, neon of any kind.

In the present application, an optical detection platform is also provided on the fourth pipeline to identify and detect the generated droplets and air bubbles, and the optical detection platform is a high-speed imaging system and/or a droplet identifier, further specifically , the high-speed imaging system is a high-speed camera, and the droplet recognizer is a laser transmitter and a laser signal receiver arranged on both sides of the pipeline.

Example

In this implementation case, Bacillus subtilis is used, and its medium formula is: 20 g of glucose, 15 g of peptone, 0.5 g of beef extract, 5 g of sodium chloride, and 1 L of water.

Prepare the Bacillus subtilis seed liquid, pick the Bacillus subtilis colony from the plate, inoculate it into the above-mentioned medium, and culture it with shaking at 37 °C and 220 r/min for 1-2 h.

Using the above-mentioned criss-cross microchannel, wherein the internal diameters of the pipes are all 1 mm, the sample is injected.

Group a: mineral oil (containing surfactant span 80, 10 g/L) is fed into the first pipeline, nitrogen gas is fed into the second pipeline, and Bacillus subtilis culture solution for shaking culture for 1-2 h is fed into the third pipeline; the cross The flow rate of the input water phase and oil phase of the type microchannel is 30 uL/min, and the air pressure in the gas phase input pipeline is 80 Kpa. "Water-in-oil" droplets, air bubbles, "water-in-oil" droplets, air bubbles are sequentially generated at the cross intersections of the micro-pipes... that is, 100 "water-in-oil" droplets separated by bubbles are generated, and the volume of the droplets is It is about 2.5 μL, and the volume size of the bubble is about 5 μL.

Group b: Mineral oil (containing surfactant span 80, 10 g/L) is fed into the first pipe, the second pipe is filled with mineral oil to seal and prevent airflow and liquid flow from entering and leaving the second pipe, and the third pipe is fed into shaking culture 1-2 The bacillus subtilis culture fluid of h; The flow velocity of described cross-shaped microchannel input water phase, oil phase is 30 uL/min, generates 100 droplets of " water-in-oil " at the intersection and enters the fourth pipeline, wherein The volume size of the droplet is approximately 2.5 μL.

After the sample injection is completed, groups a and b are driven by the power source of the first pipeline and cultured back and forth at a flow rate of 100 uL/min. After culturing for half an hour, the droplets are recognized by the high-speed imaging system and the droplet recognizer And detection, the results show that there are 100 droplets in group a, and only 98 droplets in group b.

After the sample injection was completed, both groups a and b were cultured statically. After 48 hours, the droplets were identified and detected by a high-speed imaging system and a droplet recognizer. The number of droplets in group a remained at 100, and the number of droplets in group b There are only 95 of them.

It can be seen that the use of "water-in-oil" droplets separated by air bubbles can avoid the directional displacement of the droplets and the fusion of the droplets during the culture process.

Claims (9)

1. A method for avoiding droplet fusion in a microfluidic system, comprising the steps of: and respectively feeding water phase, oil phase and gas phase into three pipelines of the cross-shaped micro-channel to generate bubble-spaced water-in-oil droplets or oil-in-water droplets in a fourth pipeline.

2. A method of avoiding droplet fusion in a microfluidic system according to claim 1, wherein: the diameter of the cross-shaped micro-channels is 0.1-10mm, preferably 1-3mm, and more preferably 1mm.

3. A method of avoiding droplet fusion in a microfluidic system according to claim 1, wherein: the diameter of the bubbles is greater than the microchannel radius, and the diameter of the "water-in-oil" droplets or "oil-in-water" droplets is greater than the microchannel radius.

4. A method of avoiding droplet fusion in a microfluidic system according to claim 3, wherein: the bubbles have a length of 0.05mm-1cm and are greater than the diameter of the "water-in-oil" droplets or "oil-in-water" droplets.

5. A method of avoiding droplet fusion in a microfluidic system according to claim 3, wherein: the volume of the "water-in-oil" droplets or "oil-in-water" droplets is 0.6 to 10. Mu.L, more preferably 0.7 to 7. Mu.L, still more preferably 0.8 to 6. Mu.L, still more preferably 1 to 3. Mu.L, still more preferably 2 to 3. Mu.L.

6. A method of avoiding droplet fusion in a microfluidic system according to claim 1, wherein: the flow rate of the cross-shaped micro-channel input water phase and the oil phase is 0-1 mL/min, and the air pressure in the gas phase input pipeline is 0-100 Kpa.

7. A method of avoiding droplet fusion in a microfluidic system according to claim 1, wherein: the gas phase input gas is gas which does not react with the water phase and the oil phase.

8. The method for avoiding droplet fusion in a microfluidic system according to claim 7, wherein: the gas phase input gas is nitrogen, hydrogen or inert gas.

9. The method for avoiding droplet fusion in a microfluidic system according to claim 8, wherein: the inert gas is any one of helium, argon and neon.

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