CN101957383A - Micro-fluid control liquid drop generation system based on liquid drop sequence assembly technology and use method - Google Patents

Abstract

The field of the invention relates to the field of droplet analysis, in particular to a microfluidic droplet generation system based on the droplet sequential assembly technology. The microfluidic droplet generation system based on droplet sequential assembly technology of the present invention includes an automatic liquid presentation device, a liquid drive device, a capillary and a droplet array plate, and the automatic liquid presentation device is connected to the droplet array plate , the liquid driving device is connected with the capillary. The beneficial effects of the present invention are: (1) the system is simple and convenient to build, does not require complex micro-processing technology and expensive processing equipment, and is conducive to wide application in conventional laboratories; (2) the liquid in the droplet can be changed arbitrarily different types and mixing ratios to generate droplets with different compositions; (3) high-throughput automated generation of droplets with different compositions can be realized.

Description

Microfluidic droplet generation system and application method based on droplet sequential assembly technology

technical field

The field of the present invention is the field of droplet analysis, and more specifically, it relates to a microfluidic droplet generation system based on droplet sequential assembly technology and its application method.

Background technique

In recent years, microfluidic droplet analysis technology has received more and more attention. The droplet-based microfluidic analysis system uses immiscible liquid or gas phases as intervals between nanoliter to picoliter-sized droplets to form an independent reaction vessel, which has the advantages of eliminating diffusion, accelerating mixing, and automatic operation. With the advantages of fast analysis speed and low consumption of sample reagents, it is very suitable for high-throughput screening analysis, and has been successfully applied in the fields of chemistry, physics, biology and medicine.

However, so far, most droplet analysis systems use the instability of the oil-water two-phase interface to generate droplets at the mouth of T-shaped channels or cross-shaped channels. They only generate a large number of droplets with the same chemical composition, and there is still a lack of a The sample change method can conveniently change the type and composition of the sample in the droplet, so as to generate a droplet array with various components. At this stage, there are two main methods to change the droplet composition in the microchannel: one is the droplet fusion technology, which first generates two types of droplets with different compositions, and uses electricity, optical tweezers, microvalve, special channel configuration , or by methods such as partial modification of the surface to fuse the two droplets together. This technique needs to strictly control the generation frequency of each droplet and the interface properties of the droplets, which is not easy to be applied to droplet-based high-throughput screening systems. The second method is to use a droplet loader to pre-encapsulate droplets of different types or compositions, insert them into the microchannel of the chip during the experiment, and merge with the reagents to form a droplet reactor. This method truly applies droplet microfluidic technology to screening analysis, which is relatively easy to implement. However, this method requires manual preparation of the droplet loader, which is time-consuming and laborious, and requires the interface between the droplet loader and the chip to transfer the droplet into the chip. In addition, under the pressure drive, especially in the microfluidic system that uses bubbles to space the liquid section, and the mixing ratio of screening reagents and substrate reactants needs to be strictly controlled, which increases the difficulty of microfluidic manipulation. In addition, the above two methods are based on microfabricated chips, which require complex microfabrication technology and expensive processing equipment, which limits their wide application in conventional laboratories.

Contents of the invention

The purpose of the present invention is to solve the problems raised above, to provide a system that is simple and convenient to build, can arbitrarily change the type and mixing ratio of the liquid in the droplet, and can realize the high-throughput automatic generation of droplets of different compositions. A microfluidic droplet generation system for droplet sequential assembly technology, and a method of use thereof.

Technical scheme of the present invention is such:

A microfluidic droplet generation system based on droplet sequential assembly technology, including an automatic liquid delivery device, a liquid drive device, a capillary and a droplet array plate, the automatic liquid delivery device is connected to the droplet array plate, and the liquid The drive unit is connected to the capillary.

Preferably, the automatic liquid delivery device includes:

Multi-well plates and three-dimensional translation stages for holding liquids;

Or, an array of notched tubes holding liquid, a gear set controlled by a stepper motor.

Preferably, the material of the capillary can be quartz or glass or plastic or metal material, and the inner diameter of the capillary is between 0.5 micron and 5 mm.

Preferably, the liquid inlet of the capillary adopts a tapered tip structure, and the inner wall surface of the capillary and the outer wall surface of the tapered tip of the liquid inlet need to be treated with hydrophobic surface.

Preferably, the liquid driving device has two driving functions of forward push and reverse suction, and the flow rate of the liquid driven by the driving device ranges from 1 picoliter/minute to 100 microliter/minute.

As preferably, a plurality of small pits containing liquid droplets are processed on the droplet array plate, or a plurality of planar regions with a certain surface area that have affinity for droplets are processed; the volume range of a small pit is 1 picoliter to 100 microliters, the surface area of a flat area ranges from 1 square micron to 100 square millimeters.

The method of using the microfluidic droplet generation system based on the droplet sequential assembly technology, the steps are as follows:

1) Load the required different water phase liquids and oils immiscible with water in the automatic liquid delivery device, and the capillary is filled with oil;

2) Using an automatic liquid delivery device, the liquid it contains is in contact with the liquid inlet of the capillary, so that the liquid inlet of the capillary is inserted into the liquid, and a certain volume of water phase is sucked by using the reverse suction function of the liquid driving device Liquid enters the capillary;

3) Use the liquid delivery device to sequentially make the different aqueous phase liquids contained in it contact the capillary liquid inlet, and sequentially extract the required different aqueous phase liquids into the capillary;

4) The aqueous phase liquids introduced before and after are mixed in the capillary to generate droplets assembled from various liquids;

5) Use the liquid delivery device to make the contained oil contact with the liquid inlet of the capillary, extract a certain volume of oil into the capillary, and complete the assembly of a droplet;

6) cyclically perform the operations from step 2) to step 5), and generate a droplet array in the capillary;

7) Place the droplet array plate under the capillary, and use the forward push function of the liquid drive device to push out the droplets generated in the capillary from the liquid inlet of the capillary, and drop them onto the droplet array plate to generate two droplet array.

Preferably, the volume of the droplet and the mixing ratio of each liquid in the droplet are changed by adjusting the flow rate and time of absorbing the liquid; the composition of the droplet is changed by selecting the type of liquid to be absorbed.

Preferably, step 6) uses multiple capillaries to simultaneously generate droplets.

Preferably, said droplet array plate is immersed in oil.

The beneficial effects of the present invention are as follows:

(1) The system is simple and convenient to build, does not require complex micro-processing technology and expensive processing equipment, and is conducive to wide application in conventional laboratories;

(2) The type and mixing ratio of the liquid in the droplet can be changed arbitrarily to generate droplets with different compositions;

(3) High-throughput automatic generation of droplets with different compositions can be realized.

Description of drawings

Figure 1 is a schematic diagram of the operation of assembling droplets within a capillary;

2 is a schematic diagram of the device of the automatic droplet generation system based on the notched tube array in embodiment 1;

Fig. 3 is the schematic diagram of the device of the automatic droplet generation system based on the porous plate in embodiment 2;

4 is a schematic diagram of the operation of dropping droplets from a capillary onto a droplet array plate in Embodiment 1 and Embodiment 2;

Figure 5 is a top view of the two-dimensional droplet array generated by Embodiment 1 and Embodiment 2;

In the figure: 1 is capillary, 2 is oil, 3 is liquid inlet, 4 is liquid A, 5 is liquid B, 6 is liquid C, 7 is liquid droplet, 8 is droplet array plate, 9 is gear set, 10 11 is liquid D, 12 is liquid E, 13 is liquid F, 14 is liquid G, 15 is liquid H, 16 is a multi-well plate, and 17 is a three-dimensional translation stage.

Detailed ways

Embodiments of the present invention are described in further detail below in conjunction with accompanying drawings:

The microfluidic droplet generation system based on droplet sequential assembly technology includes an automatic liquid presentation device, a liquid drive device, a capillary 1 and a droplet array plate 8, the automatic liquid presentation device is connected to the droplet array plate 8, and the The liquid driving device described above is connected to the capillary 1.

The described automatic liquid delivery device comprises:

A porous plate 16 and a three-dimensional translation platform 17 for holding liquid;

Or, the gap tube 10 array containing the liquid, the gear set 9 controlled by the stepping motor.

The material of the capillary 1 can be quartz or glass or plastic or metal material, and the inner diameter of the capillary 1 is between 0.5 micron and 5 mm.

The liquid inlet 3 of the capillary 1 adopts a tapered tip structure, and the inner wall surface of the capillary 1 and the outer wall surface of the tapered tip of the liquid inlet 3 need to be treated with hydrophobic surface.

The liquid driving device has two driving functions of forward push and reverse suction, and the flow rate of the liquid driven by the driving device ranges from 1 picoliter/minute to 100 microliter/minute.

A plurality of small pits containing liquid droplets 7 are processed on the droplet array plate 8, or a plurality of planar regions with a certain surface area having affinity for the liquid droplets 7 are processed; the volume range of a small pit is 1 picoliter to 100 microliters, the surface area of a flat area ranges from 1 square micron to 100 square millimeters.

Fig. 1 is a schematic diagram of the operation principle of the microfluidic droplet generation system based on the droplet sequential assembly technology established in the present invention to assemble droplets in a capillary. The liquid inlet of the capillary 1 is sharpened into the shape of the tapered tip 3, and the other end of the capillary 1 is connected with the liquid driving device. Before the droplets are generated, the inner wall of the capillary 1 and the outer wall of the capillary liquid inlet 3 are subjected to hydrophobic treatment. The capillary 1 is filled with oil 2 . Using the liquid presenting device, the liquid A4 contained in it is contacted with the liquid inlet 3 of the capillary 1, so that the liquid inlet 3 of the capillary is inserted into the liquid A4, and a certain volume of liquid A4 is sucked into the capillary 1. Then insert the liquid inlet 3 of the capillary into the liquid B5, and draw a certain volume of the liquid B5 into the capillary 1. Then insert the liquid inlet 3 of the capillary into the liquid C6, and draw a certain volume of the liquid C6 into the capillary 1. The liquid inlet 3 of the capillary is inserted into the oil 2, and a certain volume of oil 2 is sucked into the capillary 1. Liquid A4, liquid B5, and liquid C6 introduced in sequence are mixed within the capillary to generate a droplet 7 assembled from various liquids. The liquid droplets generated in the capillary 1 are pushed out from the liquid inlet 3 of the capillary by using the positive push function of the liquid driving device, and drop onto the droplet array plate 8 of the immersion oil 2 .

Example 1

Fig. 2 is a schematic diagram of the device of the automatic droplet generation system based on the notched tube array in Example 1. The system consists of three parts: a sample presentation device based on a notched tube array, a quartz capillary, and a microsyringe pump. The sample providing device based on the notched tube array is composed of a gear set 9 composed of two gears, one big and one small, and the small gear is controlled by a stepping motor to drive the big gear to rotate. On the large gear, 6 notched tubes 10 are equidistantly arranged; on the small gear, 3 notched tubes 10 are fixed according to an isosceles triangle. Liquid A4, liquid B5, liquid C6, liquid D11, liquid E12, liquid F13, liquid G14, liquid H15 and oil 2 required for generating droplet arrays are contained in the notched tubes respectively. When generating liquid droplets, the gear set 9 is rotated so that the required liquid in the notched tube 10 contacts the liquid inlet 3 of the capillary 1 in turn, absorbs a certain volume of liquid into the capillary 1, and assembles to form a liquid droplet 7 . Embodiment 1 device can generate successively by liquid A4, liquid B5 and liquid C6, liquid A4, liquid B5 and liquid D11, liquid A4, liquid B5 and liquid E12, liquid A4, liquid B5 and liquid F13, liquid A4, liquid B5 and liquid Liquid G14, liquid A4, liquid B5 and liquid H15 assembled into droplets of different composition 7 . In this embodiment: the inner diameter of the capillary 1 is 5 mm, and the flow rate of the liquid driven by the driving device is 100 microliters/minute.

Example 2

Fig. 3 is a schematic diagram of the device of the automatic droplet generation system based on a porous plate in Example 2. The system consists of three parts: a sample presentation device based on a porous plate 16, a quartz capillary, and a micro-injection pump. The sample presentation device based on the porous plate 16 is composed of the porous plate 16 for containing various liquids and a three-dimensional translation stage 17 . When generating liquid droplets, move the three-dimensional translation stage 17, so that the different liquids contained in the porous plate 16 contact the liquid inlet 3 of the capillary 1 in sequence, absorb a certain volume of liquid into the capillary 1, and assemble and generate liquid droplets 7. In the example: the inner diameter of the capillary 1 is 0.5 micron, and the flow rate of the liquid driven by the driving device is 1 picoliter/minute.

Fig. 4 is a schematic diagram of the operation of dropping droplets from the capillary onto the droplet array plate in Embodiment 1 and Embodiment 2. Place the droplet array plate 8 under the vertically placed capillary 1, adjust the position of the oil-immersed droplet array plate 8 manually, or fix the oil-immersed droplet array plate on a three-dimensional translation platform, and control the three-dimensional position by a computer program. The movement of the translation stage adjusts the position of the oil-immersed droplet array plate 8 . The droplets 7 in the capillary are sequentially dropped onto the oil-immersed droplet array plate 8 under the push of the syringe pump to form a two-dimensional droplet array.

FIG. 5 is a top view of the two-dimensional droplet array generated by Embodiment 1 and Embodiment 2. The oil-immersed droplet array plate 8 is made of glass, and the small pits containing the liquid droplets 7 on the droplet array plate 8 are processed by photolithography and chemical etching. The volumes of the small pits are 100 microliters and 1 picoliter respectively. Lift.

Example 3

The oil-immersed droplet array plate 8 is made of glass, and the small pit containing the liquid droplet 7 on the droplet array plate 8 is processed by photolithography and chemical etching. The volume of the small pit is 20 nanoliters, and the capillary 1 The inner diameter is 150 microns, the flow rate of the liquid driven by the driving device is 10 nanoliters/minute, and the rest is the same as in Embodiment 2.

The volume of the wells can also be 1 nanoliter or 1 microliter, ranging from 1 picoliter to 100 microliters.

Example 4

The oil-immersed droplet array plate 8 is processed with a plurality of planar regions with a certain surface area that have affinity for the droplets 7, and the surface area of one planar region is 1 square micron, and the rest are the same as in Example 2.

The surface area of the planar region may be 500 square micrometers or 1 square millimeter or 100 square millimeters, ranging from 1 square micrometer to 100 square millimeters.

The method of using the microfluidic droplet generation system based on the droplet sequential assembly technology, the steps are as follows:

1) Load the required different water phase liquids and oil 2 immiscible with the water phase in the automatic liquid delivery device, and the capillary 1 is filled with oil 2;

2) Adopt an automatic liquid delivery device, contact the liquid contained in it with the liquid inlet 3 of the capillary 1, insert the liquid inlet 3 of the capillary 1 into the liquid, and use the reverse suction function of the liquid driving device to absorb a certain amount of liquid. A volume of aqueous phase liquid enters the capillary 1;

3) Use the liquid delivery device to sequentially make the different aqueous phase liquids contained in it contact the liquid inlet 3 of the capillary 1, and sequentially extract the required different aqueous phase liquids into the capillary 1;

4) The aqueous phase liquids introduced before and after are mixed in the capillary 1 to generate droplets 7 assembled from various liquids;

5) Using the liquid delivery device to make the contained oil 2 contact the liquid inlet 3 of the capillary 1, extract a certain volume of oil 2 into the capillary 1, and complete the assembly of a droplet 7;

6) cyclically perform the operations from step 2) to step 5), and generate an array of droplets 7 in the capillary 1;

7) Place the droplet array plate 8 under the capillary 1, and use the forward push function of the liquid drive device to push the generated droplets 7 in the capillary 1 from the liquid inlet 3 of the capillary 1, and drop them onto the droplet On the array plate 8, a two-dimensional array of droplets 7 is generated.

The volume of the droplet 7 and the mixing ratio of each liquid in the droplet 7 can be changed by adjusting the flow rate and time of absorbing the liquid; the composition of the droplet 7 can be changed by selecting the type of liquid to be absorbed.

Step 6) Using a plurality of capillaries 1, the operation of generating droplets 7 is performed simultaneously.

The droplet array plate 8 is immersed in the oil 2 to prevent the droplet 7 from volatilizing or being polluted in the air.

What has been described above is only a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the core technical features of the present invention. Improvements and retouches should also be considered within the protection scope of the present invention.

Claims (10)

1. The microfluidic droplet generating system based on droplet sequential assembly technology is characterized in that it includes an automatic liquid delivery device, a liquid drive device, a capillary (1) and a droplet array plate (8), and the automatic liquid delivery The device is connected with the droplet array plate (8), and the liquid driving device is connected with the capillary (1). 2. The microfluidic droplet generating system based on droplet sequential assembly technology according to claim 1, wherein the automatic liquid delivery device comprises: A porous plate (16) and a three-dimensional translation platform (17) for holding liquid; Or, the notch tube (10) array containing the liquid, the gear set (9) controlled by the stepper motor. 3. the microfluidic droplet generation system based on droplet sequential assembly technology according to claim 1, is characterized in that, the material of described capillary (1) can be quartz or glass or plastics or metal material, capillary ( 1) The inner diameter is between 0.5 microns and 5 mm. 4. The microfluidic droplet generation system based on droplet sequential assembly technology according to claim 1 or 3, characterized in that, the liquid inlet (3) of the capillary (1) adopts a tapered tip structure, The surface of the inner wall of the capillary (1) and the surface of the outer wall of the tapered tip of the liquid inlet (3) need to be treated with hydrophobic surface. 5. The microfluidic droplet generation system based on droplet sequential assembly technology according to claim 1, wherein the liquid drive device has both forward push and reverse suction drive functions, The drive unit drives the liquid at a flow rate ranging from 1 picoliter/minute to 100 microliters/minute. 6. The microfluidic droplet generation system based on droplet sequential assembly technology according to claim 1, characterized in that, on the droplet array plate (8), a plurality of small microfluidic droplets (7) containing liquid droplets (7) are processed. Pit, or process a plurality of planar regions with a certain surface area that have affinity for droplets (7); the volume of a small pit ranges from 1 picoliter to 100 microliters, and the surface area of a planar region ranges from 1 square micron to 100 square millimeters. 7. The method for using the microfluidic droplet generation system based on droplet sequential assembly technology according to claim 1, wherein the steps are as follows: 1) Load the required different water phase liquids and water-immiscible oils (2) in the automatic liquid delivery device, and the capillary (1) is filled with oil (2); 2) Using an automatic liquid delivery device, the liquid contained in it is brought into contact with the liquid inlet (3) of the capillary (1), so that the liquid inlet (3) of the capillary (1) is inserted into the liquid, and the Reverse suction function, absorbing a certain volume of aqueous phase liquid into the capillary (1); 3) Using the liquid delivery device to sequentially make the different aqueous phase liquids contained in it contact the liquid inlet (3) of the capillary (1), and sequentially extract the required different aqueous phase liquids into the capillary (1); 4) The aqueous phase liquids introduced before and after are mixed in the capillary (1) to generate droplets (7) assembled from various liquids; 5) Use the liquid delivery device to make the contained oil (2) contact with the liquid inlet (3) of the capillary (1), extract a certain volume of oil (2) into the capillary (1), and complete a droplet (7 ) assembly; 6) cyclically perform the operations from step 2) to step 5), and generate an array of droplets (7) in the capillary (1); 7) Place the droplet array plate (8) under the capillary (1), and use the forward push function of the liquid drive device to feed the generated droplets (7) into the capillary (1) from the capillary (1) The mouth (3) is pushed out, and the droplets fall onto the droplet array plate (8), generating a two-dimensional droplet (7) array. 8. The method of use according to claim 7, characterized in that, the volume of the droplet (7) and the mixing ratio of each liquid in the droplet (7) are changed by adjusting the flow velocity and the suction time of the suction liquid; by selecting The type of liquid absorbed changes the composition of the droplet (7). 9. The method according to claim 7, characterized in that, in step 6), a plurality of capillary tubes (1) are used to simultaneously generate the droplets (7). 10. The use method according to claim 7, characterized in that, the droplet array plate (8) is immersed in oil (2).

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