CN104324769B - Generation method based on the drop of microchannel - Google Patents

Abstract

The present application provides a method for generating droplets based on micro-channels. First, a micro-channel with an opening at one end is provided, and the micro-channel is filled with a first liquid; an open container filled with a second liquid is provided; the micro-channel is located at the opening above the liquid level of the container; the first liquid and the second liquid are any two liquids that are immiscible or have two liquids that have interfacial reactions; then the microchannel moves downward from the liquid level of the open container, making the opening of the micro-channel contact and enter the second liquid, and the first liquid is located at the micro-channel opening; then the micro-channel opening into the second liquid is upwardly away from the open container The movement of the liquid surface separates the opening of the microchannel from the second liquid, and the first liquid at the opening of the microchannel separates from the microchannel to form droplets in the second liquid. The droplet generation method is simple, can form droplets with a controllable size, and improves the precision and efficiency of large-scale droplet generation.

Description

Droplet generation method based on microchannel

technical field

The present application relates to the technical field of measuring or distributing micro-volume liquids, in particular to a method for generating liquid droplets based on micro-channels.

Background technique

At the microscale, accurate manipulation of microliter to microliter level liquids is an important proposition in the research fields of modern engineering, physics, chemistry, materials science, microfabrication and bioengineering. In biochemical analysis, environmental monitoring It has a wide range of applications in medical and clinical testing and the synthesis and preparation of micro-nano materials. Traditional emulsion polymerization method (BoveyFA, etal.Emulsionpolymerization.NewYork:Intersciencepublishers1955,1-22), membrane emulsification method (NakashimaT, etal.Membraneemulsificationbymicroporousglass.KeyEngineeringMater1991,513:61-61) and spray emulsification method (LiuY, etal.Mixinginamulti- inletvortexmixer (MIVM) forfalshnano-precipitation.ChemicalEngineeringScience, 2008,63(11):2829-2842) can generate micro-droplets with relatively uniform particle size in large quantities, but the main application of these methods is to prepare microspheres and prepare drug carriers. There is a large error in the control accuracy of the droplet volume, and the droplet cannot be used as an accurate quantitative microreactor, and it is difficult to be applied in a complex biochemical reaction system. Microfluidics-based droplet generation technology has developed rapidly in recent years (TehSY, etal.Dropletmicrofluidics.LabonaChip2008,8(2):198-220), the generation of droplets is based on dispersed phase and continuous phase Interface destabilization at the junction in a microchannel. Through different microfluidic channel chip designs, droplets of uniform size can be generated, and operations such as fusion, reaction, and sorting can be performed. However, the generation of droplets on a microfluidic chip needs to meet certain conditions such as flow rate, oil-water interfacial tension, channel configuration, and channel surface modification, and the range of droplet volume adjustment is also restricted by the above factors. In addition, after the droplets are generated in the channel of the microfluidic chip, specific steps and devices are required to transfer them to the storage container. It is difficult to customize the conditions of a single droplet, and operations such as positioning, extraction, and analysis of the droplets are inconvenient.

Injecting or spraying a small amount of liquid through a microchannel such as a capillary, and injecting the liquid into a micropits or spotting a sample on a substrate is a simple droplet generation strategy in principle. However, in actual operation, when the droplet leaves the capillary, there is surface tension between the droplet and the continuous liquid in the tube, and the adhesion between the droplet and the surface of the nozzle, which affects the precise quantification of the droplet volume. Usually, the prior art adopts special injection or droplet excitation methods such as piezoelectric ceramics, thermal expansion, high-voltage electrospray, and ultrasound to increase the kinetic energy of the droplet leaving the outlet of the microchannel to overcome the influence of surface tension (TekinE, et al. Inkjetprintingasadepositionandpatterningtoolforpolymersandinorganicparticles.SoftMatter2008,4(4):703-713；MeachamJM,etal.Dropletformationandejectionfromamicromachinedultrasonicdropletgenerator:Visualizationandscaling.PhysicsofFluids2005,17(10):100605；FerraroP,etal.Dispensingnano-picodropletsandliquidpatterningbypyroelectrodynamicshooting.NatureNanotechnology2010,5:429-435)，通过微通道The special configuration of the outlet and silanization or coating treatment reduce the adhesion of droplets at the nozzle (TavanaH, et al.Nanolitreliquidpatterninginaqueousenvironmentsforspatiallydefinedreagentdeliverytomammaliancells.NatureMaterials2009,8(9):736-741).

However, the above-mentioned methods rely on relatively complex fluid drive devices, which are costly and difficult to precisely control the droplet volume.

Contents of the invention

In view of this, this application provides a method for generating droplets based on microchannels. The droplet generation method provided by this application is simple and easy, and can form droplets with controllable sizes, and improve the micro-reaction and mixing control of large batches of droplets. The accuracy and efficiency of these devices provide the basis for complex droplet-based biological and chemical applications.

The application provides a method for generating droplets based on microchannels, comprising the following steps:

a) providing a micro-channel with an opening at one end, the micro-channel is filled with the first liquid;

An open container containing a second liquid is provided; the microchannel is located above the liquid surface of the open container; the first liquid and the second liquid are any two liquids that are immiscible or have interfacial reactions;

b) In the step a), the micropipe moves downward from the liquid surface of the open container, so that the opening of the micropipe contacts and enters the second liquid, and the first liquid is located in the micropipe opening;

c) in the step b), the micropipe opening into the second liquid moves upwards away from the liquid surface of the open container, so that the opening of the micropipe is separated from the second liquid, and the micropipe located at the opening of the micropipe The first liquid exits the microchannel forming droplets in the second liquid.

Preferably, in the step a), the microchannel is a single single-core capillary, a single multi-core capillary, an array capillary, a microfluidic single channel or a microfluidic multichannel array.

Preferably, the opening size of the micro-channel is between 0.05 micron and 0.5 mm.

Preferably, the micropipe is a micropipe whose opening has been treated with low surface energy.

Preferably, in the step a), the other end of the micropipe is connected with a fluid driving device to generate the first liquid flow continuously or intermittently.

Preferably, the fluid-driven device comprises a peristaltic pump, a syringe pump, a pressure-driven pump, a pneumatic-driven pump or an electroosmotic-driven pump.

Preferably, in the step a), the open container is a single reservoir, or a one-dimensional or two-dimensional array of reservoirs.

Preferably, in the step a), the first liquid is an aqueous solution, and the second liquid is an oily liquid immiscible with water;

Alternatively, the first liquid is an aqueous solution, and the second liquid is an aqueous liquid immiscible with water;

Alternatively, the first liquid is mineral oil, and the second liquid is perfluoroalkane oil immiscible with mineral oil;

Alternatively, the first liquid is an aqueous solution of sodium alginate, and the second liquid is an aqueous solution of calcium chloride, and there is an interfacial reaction between the two.

Preferably, the downward movement in the step b) and the upward liquid level movement away from the open container in the step c) are independently controlled by manual operation, manual translation stage operation or automatic moving stage operation Way.

Preferably, the frequency of the downward movement in the step b) and the upward movement away from the liquid surface in the step c) is between 0.0001 Hz and 1,000,000 Hz, and the amplitude is between 0.0001 Hz and 1,000,000 Hz relative to the liquid level. between 1 micron and 1 cm.

Compared with the prior art, the droplet generation method provided by the application is based on micro-channels (or called micro-channels), which are filled with the first liquid and have an opening at one end; the application also provides a second An open container of liquid, the microchannel is located above the liquid surface of the open container, and the first liquid and the second liquid are immiscible or have an interfacial reaction. In the present application, the micro-channel moves downward from the liquid surface of the open container, so that the opening of the micro-channel contacts and enters the second liquid, and the first liquid is located at the opening of the micro-channel place; the micro-channel that opens into the second liquid moves upward away from the liquid surface of the open container, so that the opening of the micro-channel is separated from the second liquid, and the first liquid at the opening of the micro-channel is separated from microchannels forming droplets in the second liquid. The present application uses the movement of the opening of the microchannel to overcome the surface tension and adhesion of the liquid at the outlet of the microchannel by utilizing the interfacial energy and fluid shear force of a small amount of liquid at the gas-liquid phase interface, so that the liquid flowing out of the nozzle of the microchannel Droplets can escape from microchannels smoothly and form droplets with controllable size. Therefore, the present application can quickly generate a large amount of liquid droplets with a fixed volume through continuous liquid flow and high frequency of reciprocating motion at the gas-liquid interface, thereby improving the accuracy and efficiency of liquid droplet generation such as biochemical samples. At the same time, the method for generating microdroplets provided by the present application can directly obtain droplets with controllable volume and quantity in the dispersed liquid phase, avoiding the evaporation effect of microdroplets, and simplifying the extraction and storage steps of droplets or microspheres; and The present application can use simple and low-cost microchannels to generate droplets, so the method for forming microdroplets using microchannels provided by the present application is simple and easy.

In addition, the diameter of the droplet generated by the present application can be controlled by the liquid flow rate in the micropipe, the liquid volume, the opening size of the micropipe, the movement frequency and amplitude of the micropipe, etc., and the control and adjustment of the droplet volume is more flexible; it is also possible By replacing the components of the microchannel or the first liquid flowing out of the microchannel, a plurality of droplets of different components and volumes are sequentially formed in the open container, which can be used to achieve high-throughput screening of large batches of microvolumes, as well as The multi-step ultra-trace biochemical reaction and detection can be realized, and has broad application prospects.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.

Fig. 1 is a schematic diagram of the droplet distribution generated by one embodiment of the present application;

Fig. 2 is a schematic diagram of the droplet distribution generated by one embodiment of the present application;

Fig. 3 is a schematic diagram of droplet distribution generated by another embodiment of the present application;

4 is a schematic diagram of the operation steps of the droplet generation method provided in Example 1 of the present application;

Fig. 5 is the curve graph of the flow velocity of pure water in the capillary in the embodiment 1 of the present application as a function of time;

Fig. 6 is the graph that the position of the capillary nozzle distance from the mineral oil liquid level varies with time in the embodiment 1 of the present application;

7 is a schematic diagram of the operation steps of the droplet generation method provided in Example 5 of the present application;

Fig. 8 is a graph showing the flow rate of pure water in the capillary as a function of time in Example 5 of the present application;

Fig. 9 is the graph that the position of the capillary nozzle distance from the mineral oil liquid level varies with time in the embodiment 5 of the present application;

Figure 10 is a schematic diagram of the droplet generation method provided in Example 7 of the present application;

Figure 11 is a schematic diagram of the droplet generation method provided in Example 8 of the present application;

Figure 12 is a schematic diagram of the droplet generation method provided in Example 9 of the present application;

Figure 13 is a schematic diagram of the droplet generation method provided in Example 10 of the present application;

Figure 14 is a schematic diagram of the droplet generation method provided in Example 11 of the present application;

Figure 15 is a schematic diagram of the droplet generation method provided in Example 12 of the present application;

Figure 16 is a microscopic view of the droplets generated at the bottom of the reservoir at a flow rate of 2.4 μl/min in Example 13 of the present application;

Figure 17 is a microscopic view of the droplets generated at the bottom of the reservoir at a flow rate of 4.8 microliters per minute in Example 14 of the present application;

Figure 18 is a microscopic view of the droplets generated at the bottom of the reservoir at a flow rate of 6 μl/min in Example 15 of the present application;

Figure 19 is a microscopic view of the droplets generated at the bottom of the reservoir at a flow rate of 12 microliters per minute in Example 16 of the present application;

Fig. 20 is a relationship diagram between droplets and flow velocity generated in Examples 13-21 of the present application;

Figure 21 is a schematic diagram of the process of the droplet generation method provided in Examples 22-26 of the present application;

Figure 22 is a schematic diagram of different solution ratios in the droplet generation method provided in Examples 22-26 of the present application;

Figure 23 is a microscopic image of the sodium alginate microspheres generated in Example 27 of the present application tiled on the bottom of the reservoir;

Figure 24 is a graph showing the diameter of the sodium alginate microspheres produced in Example 27 of the present application as the concentration of sodium alginate varies;

Figure 25 is a schematic diagram of the droplet generation method provided in Example 28 of the present application;

Figure 26 is a microscopic view of the droplets generated in Example 28 of the present application on the bottom of the reservoir;

Figure 27 is a size comparison diagram of the droplets generated in Example 28 of the present application and Comparative Examples 1-2;

28 is a schematic diagram of the droplet generation method provided in Comparative Example 1 of the present application;

Figure 29 is a microscopic view of the droplets generated in Comparative Example 1 of the present application on the bottom of the reservoir;

FIG. 30 is a schematic diagram of the droplet generation method provided in Comparative Example 2 of the present application;

Fig. 31 is a microscopic view of the liquid droplets generated in Comparative Example 2 of the present application laid flat on the bottom of the reservoir.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The application provides a method for generating droplets based on microchannels, comprising the following steps:

a) providing a micro-channel with an opening at one end, the micro-channel is filled with the first liquid;

An open container containing a second liquid is provided; the microchannel is located above the liquid surface of the open container; the first liquid and the second liquid are any two liquids that are immiscible or have interfacial reactions;

b) In the step a), the micropipe moves downward from the liquid surface of the open container, so that the opening of the micropipe contacts and enters the second liquid, and the first liquid is located in the micropipe opening;

c) in the step b), the micropipe opening into the second liquid moves upwards away from the liquid surface of the open container, so that the opening of the micropipe is separated from the second liquid, and the micropipe located at the opening of the micropipe The first liquid exits the microchannel forming droplets in the second liquid.

The present application provides a simple and flexible method for generating specific volume (micro) droplets by using microchannels, which has the advantages of high precision and high efficiency, and can be applied to the generation of biochemical sample droplets.

The main device involved in the embodiment of the present application includes a micropipe, which is open at one end; the micropipe is pre-filled with the first liquid, and the first liquid can be injected according to the set program during the movement of the micropipe; the embodiment of the present application also provides An open container containing the second liquid, the micro-channel is located above the open container, and the outlet of the channel (that is, the opening of the channel) is close to the gas-liquid interface of the second liquid in the open container.

The method provided in this application is based on a microchannel with openings, which can be a single single-core capillary, a single multi-core capillary, an array capillary, and a microfluidic single-channel or multi-channel array with one or more openings , preferably a single single-core capillary, a single multi-core capillary or an array capillary, which is simpler and less costly than a microfluidic droplet generation chip.

In the present application, the size of the opening at one end of the microchannel is preferably between 0.05 micron and 0.5 mm, more preferably between 5 micron and 0.3 mm. In order to make the generated droplets more uniform, the present application can adopt a micropipe outlet with a small outer diameter, preferably with an initial inner diameter of 100 microns and an outer diameter of 200 microns. In one embodiment of the present application, the tip of the capillary opening is in a tapered configuration, with an inner diameter of 30 microns and an outer diameter of 50 microns.

In order to make the generated droplets more uniform, the present application can carry out low surface energy treatment on the surface of the outlet of the micropipe, that is, the micropipe is preferably a micropipe with low surface energy treatment at the opening, so that the first liquid flowing out is easier Leaving the orifice, a droplet is formed in the second liquid. The low surface energy treatment can be low surface energy coating treatment, or silanization treatment, and the present application preferably adopts perfluorosilane to carry out silanization treatment on the outer wall of micropipes such as capillaries; the silanization treatment is well known to those skilled in the art technical means.

The microchannel of the present application is filled with the first liquid, and can inject the first liquid continuously or intermittently. In the embodiment of the present application, the liquid injected by the microchannel can be injected continuously with the movement of the microchannel mouth, or the first liquid can be injected according to the set flow rate and time starting when the microchannel moves to any specific position.

In the present application, preferably, the other end of the micropipe is connected with a fluid-driven device; the fluid-driven device is any fluid-driven device that may generate a continuous or intermittent flow of the first liquid. This application can use peristaltic pumps, syringe pumps, pressure-driven pumps, pneumatic-driven pumps or electroosmotic-driven pumps, etc., preferably micro-injection pumps, which have high precision and can set the injection volume to nanoliters. The present application has no special limitation on the way of connecting the micropipe and the fluid-driven device, for example, it can be connected through a Teflon thin tube, and the airtightness is guaranteed.

The application provides an open container, which is preloaded with a second liquid, and capable of receiving and storing droplets of the first liquid injected into the microchannel. The open container is any open container capable of storing microliter to milliliter volume of liquid, which can be called a liquid storage tank; each liquid storage tank can store more than one droplet of the first liquid, that is, the open container can be used as a single liquid. A separate storage container for droplets, which can also store large quantities of droplets. In the present application, the open container is preferably a single reservoir, a one-dimensional or two-dimensional array of reservoirs, more preferably a standard 96-well or 384-well microplate or PCR plate, as specific quantity, volume and Bulk storage cells for droplets of components, and array cells for mixing and reacting multiple droplet components. In the present application, the bottom of the open container may be a flat bottom, a round bottom or a conical bottom.

In the present application, the micro-channel is located above the liquid surface of the open container, and the open end of the micro-channel faces the liquid surface of the open container. Moreover, the first liquid in the microchannel of the present application is immiscible with the second liquid in the open container or has an interface reaction.

The first liquid and the second liquid can be any two immiscible liquids. In one embodiment of the present invention, the first liquid is an aqueous solution, and the second liquid is an oily liquid immiscible with water, Such as mineral oil (including n-tetradecane, etc.), vegetable oil, silicone oil and perfluoroalkane oil, etc., the generated droplets are aqueous solution droplets. Alternatively, the first liquid is mineral oil, such as organic phases such as tetradecane and n-hexane, and the second liquid is perfluoroalkane oil immiscible with mineral oil. The first liquid and the second liquid may be immiscible two-phase water, in another embodiment of the present invention, the first liquid is an aqueous solution, and the second liquid is an aqueous liquid immiscible with water, If the first liquid is dextran solution and the second liquid is polyethylene glycol (PEG) aqueous solution, the generated droplets are dextran solution droplets.

The first liquid and the second liquid can also be two liquids with interfacial reactions. In one embodiment of the present invention, the first liquid is an aqueous solution of sodium alginate, and the second liquid is an aqueous solution of calcium chloride , such as a calcium chloride aqueous solution with a mass concentration of 1%, there is an interfacial reaction between the two, and the generated droplets are calcium alginate gel microspheres. The present application can also form a plurality of droplets of different components and volumes in the open container by changing the components of the micropipe or the first liquid flowing out of the micropipe, which can be used to realize large-scale micro-volume high-pass Quantitative screening can also realize multi-step ultra-trace biochemical reactions and detection, which has broad application prospects.

In the embodiment of the present application, the gas-liquid interface movement of the immiscible second liquid loaded in the open container at the outlet of the micropipe is controlled to quickly generate micro-droplets of the first liquid. In the embodiment of the present application, the micropipe moves toward the open container, that is, moves downward from the liquid surface of the open container, so that the opening of the micropipe contacts the liquid surface of the second liquid in the open container, and Entering the second liquid; at this time, the first liquid is located at the opening of the micro-channel, and the first liquid injected by the micro-channel contacts and enters the second liquid, and can be wrapped by the second liquid.

In the present application, the control of the downward movement can be performed by manual operation, manual translation platform operation or automatic mobile platform operation. It should be noted that the control of the downward movement is the movement of the outlet of the microchannel solution relative to the liquid level of the open container, and has nothing to do with the movement of the channel as a whole. The downward movement path of the nozzle of the micropipe is gradually approaching the gas-liquid interface from a position far away from the liquid surface of the second liquid until the nozzle of the micropipe or the first liquid at the nozzle enters the second liquid solution. It should be noted that, during the movement process, the first liquid at the microchannel mouth touches the second liquid, not whether the microchannel penetrates below the liquid surface; down exercise.

In the present application, the frequency of the downward movement is preferably between 0.0001 Hz and 1,000,000 Hz, more preferably between 0.1 Hz and 1,000 Hz, most preferably 50 Hz; the distance amplitude of the movement relative to the liquid surface Preferably between 1 micron and 1 cm, more preferably between 5 microns and 0.5 mm.

After the downward movement, the micropipe described in the embodiment of the present application moves upward away from the liquid surface of the open container, and can return to the original position; the outlet of the pipe is separated from the second liquid in the open container, and at this time, the first liquid at the mouth of the micropipe Due to the influence of the main force of surface tension and shearing force, it leaves the opening of the micro-channel and is left in the second liquid, forming a micro-droplet in which the second liquid encloses the first liquid.

In the present application, the control of the liquid surface movement upward and away from the open container can be performed manually, manually with a translation platform or with an automatic mobile platform. In the present application, the frequency of the liquid surface moving upward away from the open container is preferably between 0.0001 Hz and 1,000,000 Hz, more preferably between 0.1 Hz and 1,000 Hz, most preferably 50 Hz; The magnitude of the distance relative to the liquid surface is preferably between 1 micron and 1 cm, more preferably between 5 microns and 0.5 mm.

In the embodiment of the present application, the process that the microchannel moves away from the open container causes the nozzle of the microchannel and the first liquid in the microchannel to separate from the droplets of the first liquid generated; this movement process can make the generated first liquid The droplets are collected in the second liquid in the open container. Preferably, the application moves upward vertically to the liquid surface of the second liquid.

In this application, the reciprocating movement of the opening of the microchannel on the gas-liquid interface of the second liquid enables the first liquid injected into the outlet of the microchannel to overcome the surface tension with the liquid flow in the tube and the adhesion between the droplet and the nozzle of the microchannel, so that the liquid The droplet breaks away from the microchannel to form a droplet. The droplet generation method of the present application is a process of generating one or more droplets. This process can be started, stopped and repeated times can be set according to program control to generate a specific number of droplets.

In the embodiment of the present application, droplets of the first liquid are generated. When the specific gravity of the first liquid is equal to that of the second liquid, the droplets are in a freely dispersed state in the open container, and the droplets may appear to be suspended in the open container. As shown in Figure 1, Figure 1 is a schematic diagram of the droplet distribution generated by one embodiment of the present application; when the specific gravity of the first liquid is smaller than that of the second liquid, the droplets float near the surface of the second liquid, as shown in Figure 2 Figure 2 is a schematic diagram of the distribution of droplets generated by an embodiment of the present application; when the specific gravity of the first liquid is greater than that of the second liquid, the droplets can sink to the bottom of the open container, as shown in Figure 3, Figure 3 Schematic diagram of the droplet distribution generated for another embodiment of the present application. When an open container with a conical bottom is used in the embodiment of the present application, and the specific gravity of the second liquid is smaller than that of the first liquid, the generated droplets can sink and collect at the tip of the conical shape.

The diameter of the droplet generated in this application can be controlled by the liquid flow rate in the micropipe, the volume of the liquid, the opening size of the micropipe, the movement frequency and amplitude of the micropipe, etc., and the control and adjustment of the droplet volume is relatively flexible. The adjustable range of the volume of the droplet generated in the embodiment of the present application can be from 20 picoliters to 10 nanoliters, reaching three orders of magnitude.

In summary, the droplet generation method provided by the present application is based on a microchannel (or called a microchannel), which is filled with a first liquid and has an opening at one end; the application also provides a microchannel filled with a second liquid. An open container, the microchannel is located above the liquid surface of the open container, and the first liquid and the second liquid are immiscible or have interfacial reactions. In the present application, the micro-channel moves downward from the liquid surface of the open container, so that the opening of the micro-channel contacts and enters the second liquid, and the first liquid is located at the opening of the micro-channel place; the micro-channel that opens into the second liquid moves upward away from the liquid surface of the open container, so that the opening of the micro-channel is separated from the second liquid, and the first liquid at the opening of the micro-channel is separated from microchannels forming droplets in the second liquid. The present application uses the movement of the opening of the microchannel to overcome the surface tension and adhesion of the liquid at the outlet of the microchannel by utilizing the interfacial energy and fluid shear force of a small amount of liquid at the gas-liquid phase interface, so that the liquid flowing out of the nozzle of the microchannel Droplets can escape from microchannels smoothly and form droplets with controllable size. Therefore, the present application can quickly generate a large amount of liquid droplets with a fixed volume through continuous liquid flow and high frequency of reciprocating motion at the gas-liquid interface, thereby improving the accuracy and efficiency of liquid droplets such as biochemical samples. At the same time, the method for generating microdroplets provided by the present application can directly obtain droplets with controllable volume and quantity in the dispersed liquid phase, avoiding the evaporation effect of microdroplets, and simplifying the extraction and storage steps of droplets or microspheres.

In order to further illustrate the present application, a method for generating droplets based on microchannels provided by the present application will be specifically described below in conjunction with examples, but they should not be construed as limiting the protection scope of the present application.

Example 1

FIG. 4 is a schematic diagram of the operation steps of the droplet generation method provided in Example 1 of the present application. In Fig. 4, 1 is a quartz capillary, 2 is a reservoir, 3 is mineral oil, 4 is a droplet of pure water, and 5 is pure water with an injection volume of 5 nanoliters. The inner diameter of the quartz capillary 1 is 25 microns, the outer diameter is 50 microns, and the length is 5 cm. Before use, the quartz capillary 1 is silanized with dichlorodimethylsilane to make the orifice hydrophobic. The upper end of the quartz capillary 1 is connected with a syringe pump (Harvard Apparatus, PicoElite, not shown in Figure 4) through a Teflon (Teflon) thin tube (300 microns in inner diameter and 600 microns in outer diameter), and the connected slit is covered with epoxy Filled with resin glue to ensure airtightness. The syringe pump was equipped with a syringe (not shown in Figure 4) with a volume of 10 microliters. Before use, fill the syringe, Teflon thin tube, and quartz capillary 1 with pure water, and check the liquid flow path for no leaks. The liquid reservoir 2 is a glass cuvette with a length of 1 cm, a width of 1 cm and a height of 5 cm, and the volume of the mineral oil 3 is 4 milliliters.

When the time is 0 seconds, the quartz capillary 1 is located at 5 mm above the liquid level of the mineral oil 3 to ensure that the quartz capillary 1 is filled with water and the liquid level of the water is flush with the nozzle. The quartz capillary moves at a speed of 5 mm per second, the vertical liquid surface moves downward, inserts 5 mm below the liquid surface of mineral oil at 2 seconds and remains stationary; start the syringe pump, the flow rate is 1 nanoliter/second, the mode is volume mode, and inject The volume is 5 nanoliters; after 5 seconds, the syringe pump is turned off. At this point (at 7 seconds), 5 mm below the surface of the mineral oil, a droplet with a diameter of 212 microns and a volume of 5 nanoliters was formed at the outlet of the capillary. Then, the capillary moves upwards at a speed of 5 millimeters per second, and the vertical liquid surface is lifted to 5 millimeters above the mineral oil liquid surface; , left in the mineral oil, forming pure water droplets 4 (at 9 seconds). Since the density of pure water is 1 kg/L and that of mineral oil is 0.85 kg/L, the droplet is heavier than the mineral oil, and the droplet sinks to the bottom of the reservoir. The present application can repeat the above operation steps to generate more pure water droplets with a volume of 5 nanoliters.

During the above process, the flow rate of pure water in the capillary varies with time as shown in Figure 5, which is a graph of the flow rate of pure water in the capillary as a function of time in Example 1 of the present application. During the movement, the position of the capillary nozzle from the mineral oil level varies with time as shown in Figure 6, and Figure 6 is a graph showing the position of the capillary nozzle from the mineral oil level over time in Example 1 of the present application .

Compared with direct capillary injection of droplets with a volume of 4 nanoliters or 5 nanoliters, this method relies on the contact and detachment of the liquid level of the liquid in the immiscible reservoir, such as changes in surface tension, to make the kinetic energy of capillary movement It is converted into surface tension energy, so that the outflowing solution can be smoothly detached from the capillary orifice, and there is no problem that the liquid cannot be detached due to adsorption on the capillary, thus ensuring the accuracy of the droplet volume. In addition, due to the use of mineral oil, the generated droplets can effectively avoid evaporation and can store microdroplets stably.

Example 2

According to the method of Example 1, a droplet with a volume of 2.5 nanoliters was obtained; the difference was that the flow rate of the syringe pump was 60 nanoliters/minute, and the injection time was changed to 2.5 seconds.

Example 3

According to the method of Example 1, a droplet with a volume of 2.5 nanoliters was obtained; the difference was that the flow rate of the syringe pump was changed to 30 nanoliters/minute, and the injection time was 5 seconds.

Example 4

According to the method of Example 1, a liquid droplet with a volume of 25 picoliters is obtained; the difference is that the outlet inner diameter of the pointed capillary is 8 microns, the outer diameter is 15 microns, the flow rate of the syringe pump is changed to 15 nanoliters/min, and the injection time for 0.1 seconds.

The above embodiments can accurately obtain volume-controllable pico- and nano-liter droplets according to the setting control of the syringe pump. Therefore, the present method belongs to a method for quantitatively measuring and manipulating liquids in picoliter and nanoliter volumes.

Example 5

FIG. 7 is a schematic diagram of the operation steps of the droplet generation method provided in Example 5 of the present application. In Fig. 7, 1 is a quartz capillary, 2 is a liquid reservoir, 3 is mineral oil, 4 is a pure water droplet, and 5 is injection pure water. The inner diameter of the quartz capillary 1 is 25 microns, the outer diameter is 50 microns, and the length is 5 cm. Before use, the quartz capillary 1 is silanized with dichlorodimethylsilane to make the orifice hydrophobic. The upper end of the quartz capillary 1 is connected with a syringe pump (Harvard Apparatus, PicoElite, not shown in Figure 7) through a Teflon (Teflon) thin tube (300 microns in inner diameter and 600 microns in outer diameter), and the connected slit is covered with epoxy Filled with resin glue to ensure airtightness. The syringe pump was equipped with a syringe (not shown in Figure 7) with a volume of 10 microliters. Before use, fill the syringe, Teflon thin tube, and quartz capillary 1 with pure water, and check the liquid flow path for no leaks. The liquid reservoir 2 is a glass cuvette with a length of 1 cm, a width of 1 cm, and a height of 5 cm, and the volume of the mineral oil 3 is 4 milliliters.

When the time is 0 seconds, the quartz capillary 1 is placed vertically, and the initial position is 5 mm above the liquid surface of the mineral oil 3 . The quartz capillary moves at a speed of 2 mm/s, the vertical liquid surface moves downward, inserts the mineral oil 5 mm below the liquid surface and immediately changes the movement direction of the capillary, moves vertically upwards, and returns to the initial position. At this time, immediately change the direction of motion to be vertically downward, and enter the next motion cycle. During the reciprocating movement, the capillary is connected to the syringe pump to ensure that the capillary is filled with pure water. The syringe pump continuously injects pure water at a flow rate of 1 nanoliter/second. In the above continuous injection mode, the cycle time is 5 seconds, and the capillary is co-injected. The volume is 5 nanoliters. During the movement, when the capillary breaks away from the mineral oil surface, due to effects such as surface tension, the droplets at the capillary mouth leave the capillary mouth and stay in the mineral oil to form pure water droplets 4 (at 9 seconds). Since pure water has a higher specific gravity than mineral oil, the droplets sink to the bottom of the reservoir. After the first cycle, the subsequent cycles can stably obtain pure water droplets with a volume of 5 nanoliters.

During the operation, the flow rate of pure water in the capillary varies with time as shown in FIG. 8 . FIG. 8 is a graph showing the change of flow rate of pure water in the capillary with time in Example 5 of the present application. During the movement, the position of the capillary nozzle from the mineral oil level varies with time as shown in Figure 9, and Figure 9 is a graph showing the position of the capillary nozzle from the mineral oil level over time in Example 5 of the present application .

In the above steps, the syringe pump does not need to be paused and restarted, and the movement of the capillary continues without pausing. Therefore, the efficiency of generating droplets in this embodiment is higher than that in Embodiment 1, and the operation steps are more simplified. Through the cyclic operation steps, the present application can obtain a large amount of pure water droplets with a volume of 5 nanoliters. Due to its higher specific gravity than mineral oil, the resulting droplets sink to the bottom of the reservoir.

Example 6

According to the method of Example 5, pure water droplets with a volume of 2.5 nanoliters were obtained. The difference is that the flow rate of the syringe pump is changed to 0.5 nanoliter/second; 5% (v/v) Span80 (Span 80) is pre-added to the mineral oil to avoid fusion of droplets when a large number of droplets are generated.

Example 7

FIG. 10 is a schematic diagram of the droplet generation method provided in Example 7 of the present application. In Fig. 10, 1 is three parallel capillaries, and 2 is three droplets generated synchronously.

The parameters of the capillary in this embodiment and the operation steps of droplet generation are the same as those in Embodiment 4, the difference is that in this embodiment, three parallel capillaries are used to generate three droplets synchronously, wherein the distance between the capillaries is 2 mm.

The array capillary design of the present application can generate droplets in large quantities by continuously increasing the number of capillaries.

Example 8

FIG. 11 is a schematic diagram of the droplet generation method provided in Example 8 of the present application. In Fig. 11, 1 is a microfluidic chip containing 5 parallel droplet generation channels, and 2 is 5 droplets generated simultaneously.

The operation steps of droplet generation in this embodiment are the same as those in Embodiment 7, the difference is that a microfluidic chip is used instead of a capillary, which also achieves the purpose of increasing the flux of droplet generation. Among them, the microfluidic chip is made of plastic, and the channel is 30 microns deep, 100 microns wide, and 1 cm long. The channel pitch is 1.5 mm, and the chip is 2 mm thick, 8.5 mm wide, and 2 cm long. The outlet of the channel adopts a pointed design to prevent the generated droplets from being adsorbed on the chip and cannot fall.

The method can simply generate liquid droplets in batches through arrayed microchannels, and the efficiency is high.

Example 9

FIG. 12 is a schematic diagram of the droplet generation method provided in Example 9 of the present application. In Fig. 12, 1 is a double-core capillary, that is, there is a partition inside a capillary to separate it into two channels that are not connected to each other, I is the first channel, and II is the second channel; 2 is the generated two-component Mixed droplets.

The operation steps for generating droplets in this embodiment are the same as in Example 1, the difference is that this embodiment uses a double-core capillary to generate a two-component mixed droplet; wherein, the size of the double-core capillary is 300 microns in outer diameter and 200 microns in inner diameter , The thickness of the intermediate partition is 50 microns; the fluorescein solution of 1 mole/liter is passed into the first channel I of the double-core capillary, and the pure aqueous solution is passed into the second channel II; the two-component mixed droplet generated is Diluted droplets of fluorescein from these two solutions are mixed, and the concentration of fluorescein in the droplet is determined by the ratio of the flow rates of the two channels.

The method can also use a multi-core capillary with more than two partitions to generate multi-component mixed droplets mixed with more than two solutions.

Example 10

FIG. 13 is a schematic diagram of the droplet generation method provided in Embodiment 10 of the present application. In Fig. 13, 1 is three single-channel capillaries arranged closely together, I is the first capillary, II is the second capillary, III is the third capillary; 2 is the generated three-component mixed droplet.

The parameters of a single single-channel capillary in this embodiment and the operation steps for generating droplets are the same as in Example 1, the difference is that this embodiment uses three single-channel capillaries that are closely arranged side by side to generate three-component mixed droplets; wherein, Three kinds of colorless solutions of potassium iodide, potassium iodate and dilute sulfuric acid are respectively injected into the first capillary I, the second capillary II and the third capillary III, and the three-component mixed droplets of the generation are mixed with these three kinds of solutions. Droplets, due to the oxidation-reduction reaction to generate elemental iodine, the droplets are yellow-brown.

Example 11

FIG. 14 is a schematic diagram of the droplet generation method provided in Example 11 of the present application. In Fig. 14, 1 is a four-channel bus chip, I is the first channel, II is the second channel, III is the third channel, and IV is the fourth channel; 2 is the generated four-component mixed droplet.

The operation steps for generating droplets in this embodiment are the same as in Example 1, the difference is that this embodiment uses a four-channel confluence chip to generate four-component mixed droplets; wherein, four channels in the four-channel confluence chip are finally merged into one Straight channel, the material of the chip is polydimethoxysilane (PDMS), the size is 1.5 cm wide, 2.5 cm high, and 0.5 cm thick. Pigment, yellow pigment, green pigment, and blue pigment are four kinds of solutions, which merge in the rear straight channel; the generated four-component mixed droplet is a dark droplet mixed with these four kinds of pigment solutions.

Example 12

FIG. 15 is a schematic diagram of the droplet generation method provided in Example 12 of the present application. In Fig. 15, 1 is the droplet generation flow chip, and 2 is the generated double emulsion phase droplet.

The operation steps for generating droplets in this embodiment are the same as those in Embodiment 1, the difference is that this embodiment uses a droplet generation flow chip to generate droplets of the double emulsion phase; wherein, the droplet generation flow chip has two inlets, upper and lower , inject mineral oil with surfactant added into the upper inlet, divide it into two channels, pinch off the water phase injected into the lower inlet, and form water-in-oil droplets in the rear channel; the material of the chip is Polydimethoxysilane (PDMS), the size is 2 cm wide, 3 cm long, and 5 mm thick. The end of the chip is cut into a tapered design with a blade; the double emulsion phase droplets generated in the reservoir are oil-in-water Droplets of the double emulsion phase of water.

In the method, by changing the flow velocity and vibration frequency of the chip, one complex emulsion droplet can wrap different numbers of droplets.

Examples 13-16

Following the method of Example 1, droplets were generated by adjusting the flow rate. The difference is that in Examples 13-16, the first liquid is deionized water, and the second liquid contains 4% AbilEM90 (cetyl polyethylene glycol/polypropylene glycol-10/1 dimethiconol, sourced from Tetradecane from Germany/Evonik Goschmidt Co., Ltd., the flow rates of the syringe pumps were 2.4 microliters/minute, 4.8 microliters/minute, 6 microliters/minute, and 12 microliters/minute.

Figure 16 is a microscopic view of the droplets generated at the bottom of the reservoir at a flow rate of 2.4 microliters/minute in Example 13 of the present application; Figure 17 is a flow rate of 4.8 microliters/minute in Example 14 of the present application A microscopic view of the generated droplets at the bottom of the reservoir; FIG. 18 is a microscopic view of the droplets generated at the bottom of the reservoir in Example 15 of the present application at a flow rate of 6 μl/min; FIG. 19 is A microscopic view of the liquid droplets generated at the flow rate of 12 μl/min in Example 16 of the present application on the bottom of the reservoir.

The present application calculates the volumes of droplets generated at different flow rates by measuring the radii of the droplets. Refer to FIG. 20 for the results. FIG. 20 is a graph showing the relationship between the droplets generated in Examples 13-21 of the present application and the flow rate.

Examples 17-21

Following the method of Example 13, droplets were generated by adjusting the flow rate. The difference is that the flow rates of the syringe pumps in Examples 17-21 are 0.06 microliter/minute, 0.30 microliter/minute, 0.60 microliter/minute, 1.2 microliter/minute, and 18 microliter/minute respectively.

It can be seen from FIG. 20 that the volume of droplets generated by this method at different flow rates has a linear relationship with the corresponding flow rates.

Examples 22-26

FIG. 21 is a schematic diagram of the process of the droplet generation method provided in Examples 22-26 of the present application. In Fig. 21, 1 is a 96-well plate with a conical bottom, I is a nanoliter sample droplet, II is a nanoliter reaction reagent solution, III is mineral oil, and I+II is a generated multi-component droplet.

Embodiments 22 to 26 Using the method in Example 1, first generate a sample droplet I with a nanoliter sample volume in each well of the 96-well plate 1, and then generate another droplet containing the reaction reagent in each well II, the specific gravity of these two droplets is larger than that of the oil phase, so they sink to the bottom of the tube; because the bottom of the tube is V-shaped, the two droplets will converge at the bottom tip; when there is no or very small amount of surfactant in the oil phase When , the two droplets merge into a droplet I+II, which mixes the sample I and the reaction reagent solution II.

Different volumes of different solutions are generated in different tubes, and the droplets formed by fusion are mixed with different proportions of different solutions. Referring to FIG. 22 , FIG. 22 is a schematic diagram of different solution ratios in the droplet generation method provided in Examples 22-26 of the present application. In Examples 22 to 26, using the method of Example 1, in five tubes, the samples were first used to generate 10 nanoliters, 20 nanoliters, 30 nanoliters, 40 nanoliters, and 50 nanoliters of droplets, and then In these five tubes, droplets of 50 nanoliters, 40 nanoliters, 30 nanoliters, 20 nanoliters, and 10 nanoliters were generated with the reaction reagent solution; in each tube, two droplets sank to the bottom, and Fusion occurs at the tip of the base.

The volume of fused droplets formed in each tube is equal, 60 nanoliters; but they are mixed with different ratios of sample and reagent solution, 1:5, 1:2, 1:1, 2, respectively. :1, 5:1.

The method can realize the control of the mixing volume and ratio of multi-component droplets in large batches, and can be applied to high-throughput screening experiments.

Example 27

According to the method of Example 5, solidified sodium alginate microspheres were obtained by changing the components of the first liquid and the second liquid. This embodiment specifically includes:

The first liquid flowing out of the capillary is replaced with micro-sodium alginate aqueous solution, and the second liquid in the reservoir is replaced with calcium chloride aqueous solution with a mass concentration of 0.5%.

When in use, the capillary is placed vertically, the initial position is 5 mm above the liquid surface of the calcium chloride aqueous solution, the capillary moves vertically downward at a speed of 2 mm/s, and is inserted 5 mm below the calcium chloride aqueous solution, and the capillary changes immediately. The direction of movement is to move vertically upwards and return to the place 5 mm above the liquid level of the calcium chloride aqueous solution. At this time, immediately change the direction of motion to be vertically downward, and enter the next motion cycle. The time of each motion cycle is 0.02 seconds. During the reciprocating movement, the capillary is connected to the syringe pump to ensure that the capillary is filled with sodium alginate aqueous solution, and the syringe pump continuously injects the liquid at a flow rate of 0.12 μl/min. During exercise, when the sodium alginate aqueous solution enters the calcium chloride aqueous solution through the capillary, due to the action of calcium ions, the sodium alginate is cross-linked and solidified, and the viscosity increases. Since it takes a certain amount of time for the sodium alginate aqueous solution to solidify in the calcium chloride aqueous solution, the sodium alginate aqueous solution can still flow out in a liquid state during a cycle of the capillary. Due to surface tension and other effects, the aqueous solution of sodium alginate forms droplets that leave the capillary orifice and enter the reservoir.

Under the above continuous injection mode, sodium alginate microspheres with a volume of 0.0042 nanoliters to 4.2 nanoliters can be stably obtained due to the difference in the inner diameter of the capillary used, as shown in Figure 23, which is generated in Example 27 of the present application The microscopic image of sodium alginate microspheres tiled on the bottom of the reservoir.

In order to ensure the mechanical properties of the sodium alginate microspheres, after the formation of the microspheres, 400 microliters of calcium chloride aqueous solution with a mass concentration of 5% was added to the reservoir to further solidify the sodium alginate microspheres.

The microsphere volume of the method is mainly determined by the inner diameter of the capillary outlet, and sodium alginate microspheres with diameters ranging from 20 microns to 200 microns can be obtained by changing the inner diameter of the capillary outlet.

The properties of the liquid droplets in this method can be adjusted by changing the concentration of the sodium alginate aqueous solution. At the same time, when the concentration of the sodium alginate aqueous solution changes, the diameter of the microspheres will change accordingly due to changes in its viscosity and solidification rate, as shown in Figure 24, which is the diameter of the sodium alginate microspheres generated in Example 27 of the present application Graph as a function of sodium alginate concentration.

Example 28

FIG. 25 is a schematic diagram of the droplet generation method provided in Example 28 of the present application. In Fig. 25, 1 is a capillary, 2 is a reservoir, 3 is tetradecane containing 3% EM90, and 4 is pure water droplets. Among them, the model of capillary 1 has an inner diameter of 30 microns and an outer diameter of 50 microns, and the outer wall of the capillary is silanized and hydrophobically treated with dichlorodimethylsilane.

According to the method of Example 1, droplets were generated. Referring to FIG. 26 , FIG. 26 is a microscopic view of the droplets generated in Example 28 of the present application spreading on the bottom of the reservoir.

The diameters of the droplets were calculated, as shown in FIG. 27 , which is a comparison diagram of the sizes of the droplets generated in Example 28 and Comparative Examples 1-2 of the present application.

Comparative example 1

Droplets were generated according to the method of Example 28, except that the orifice of the capillary reciprocated under the tetradecane liquid surface.

Referring to FIG. 28 , FIG. 28 is a schematic diagram of the droplet generation method provided in Comparative Example 1 of the present application. Referring to FIG. 29 , FIG. 29 is a microscopic view of the droplets generated in Comparative Example 1 of the present application spreading flat on the bottom of the reservoir. It can be seen from Figure 29 that the generated droplets are relatively inhomogeneous. Referring to FIG. 27 , it can be seen that the diameter of the droplets generated in this comparative example fluctuates more than that in Example 28.

Comparative example 2

Droplets were generated according to the method of Example 28, except that the orifice of the capillary reciprocated on the surface of the tetradecane liquid.

Referring to FIG. 30 , FIG. 30 is a schematic diagram of the droplet generation method provided in Comparative Example 2 of the present application. Referring to FIG. 31 , FIG. 31 is a microscopic view of the droplets generated in Comparative Example 2 of the present application spreading flat on the bottom of the reservoir. As can be seen from Figure 31, the resulting droplet size is too large. Referring to FIG. 27 , it can be seen that the diameter of the droplets generated in this comparative example fluctuates more than that in Example 28.

It can be known from the above examples that the present application is based on the movement of the outlet of the micropipe at the gas-liquid interface, and the injection generates droplets with precise and controllable sizes. The droplet generation method provided by the present application has the advantages of simplicity, high precision and efficiency, etc., and has broad application prospects.

Claims (10)

1., based on a generation method for the drop of microchannel, comprise the following steps:

A) provide one end to have the microchannel of opening, in described microchannel, be full of first liquid；

The open containers filling second liquid is provided；Described microchannel is positioned at the ullage of open containers；Described first liquid and second liquid are arbitrarily immiscible two kinds of liquid or two kinds of liquid with interfacial reaction；

B) in described step a), microchannel moves downward from the ullage of described open containers, makes the openings contact of described microchannel and enters described second liquid, and described first liquid is positioned at described microchannel opening part；

C) microchannel of the described second liquid of described step b) split shed entrance is upwardly away from the motion of melt surface of described open containers, the opening making described microchannel departs from second liquid, and described in be positioned at microchannel opening part first liquid depart from microchannel, in described second liquid formed drop。

2. generation method according to claim 1, it is characterised in that in described step a), described microchannel is single single capillary tube, single multicore capillary tube, array capillary, micro-fluidic single channel or micro-fluidic multichannel array。

3. generation method according to claim 2, it is characterised in that the openings of sizes of described microchannel is between 0.05 micron to 0.5 millimeter。

4. generation method according to claim 1, it is characterised in that described microchannel is the microchannel that opening part processes through low-surface-energy。

5. generation method according to claim 1, it is characterised in that in described step a), the other end of described microchannel is connected to fluid drives equipment, continuously or intermittently produces first liquid liquid stream。

6. generation method according to claim 5, it is characterised in that described fluid drives equipment includes peristaltic pump, syringe pump, pressure-driven pump, air pressure driving pump or driven by electroosmosis pump。

7. generation method according to claim 1, it is characterised in that in described step a), described open containers is the liquid storage tank array of single liquid storage tank, one-dimensional or two-dimensional arrangements。

8. generation method according to claim 1, it is characterised in that in described step a), described first liquid is aqueous solution, and described second liquid is oil-based liquid immiscible with water；

Or, described first liquid is aqueous solution, and described second liquid is waterborne liquid immiscible with water；

Or, described first liquid is mineral oil, and described second liquid is perfluorine oil immiscible with mineral oil；

Or, described first liquid is sodium alginate aqueous solution, and described second liquid is calcium chloride water, both Presence of an interface reactions。

9. generation method according to claim 1, it is characterized in that, move downward described in described step b) and adopt manual operation, the operation of manual translation platform independently with the motion of melt surface being upwardly away from described open containers described in described step c) or automatically move the control mode of platform operation。

10. generation method according to claim 1, it is characterized in that, move downward and be upwardly away from described in described step c) frequency of motion of melt surface of described open containers described in described step b) between 0.0001 hertz to 1000000 hertz, amplitude relative to liquid level between 1 micron to 1 centimetre。