Method and system for generating micro-droplets in situ

Abstract

The invention provides a method for in-situ generation of micro-droplets, and in the method, the micro-droplets are generated in situ and captured in situ; the micro-droplets are generated by a micro-droplet generating device, the micro-droplet generating device comprises one or more micro-droplet generation capture zones 101, the micro-droplet generation capture zones 101 are arranged in a flow channel 102, and the flow channel 102 is provided with a fluid inlet 103 and a fluid outlet 104. According to the invention, by controlling the size of the droplet generation capture area, the size of the formed micro-droplets can be controlled, and the flexibility and controllability are high; by increasing the number of the droplet generation and capture units, high-flux droplet generation and in-situ capture can be realized.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a method and a system for in-situ generation of micro-droplets. Background Art

[0002] Droplet microfluidics is an important branch of microfluidic chip technology. This technology achieves precise formation and control of tiny droplets by manipulating fluids in micron-scale channels. The basic principles of droplet microfluidics cover the microscopic scale effect of fluids, the role of surface tension, and the flow control of fluids. In microfluidic channels, liquids are divided into tiny droplets, and the formation of these droplets is closely related to the structure of the microchannels and the properties of the fluids.

[0003] Droplet microfluidics not only has the inherent advantages of traditional microfluidics, such as small size, low sample volume, fast speed, high throughput, etc., but also has the characteristics of uniform droplet size and internal stability. These characteristics make droplet microfluidics have broad application prospects in many fields. For example, droplet microfluidics in the field of biology can be used for the high integration of cell culture, experimental processing, imaging, detection and other steps; in the medical field, this technology is widely used in IVD testing, drug controlled release, antibody screening, biomarker detection, etc.; in addition, droplet microfluidics can also be used in the fields of particle material synthesis, droplet barcode sequencing, catalyst preparation and tissue engineering.

[0004] Droplet microfluidics technology usually involves operations such as droplet generation, splitting / fusion, capture, reaction, detection and sorting. For the generation of droplets, according to the different forces, it can be divided into passive droplet generation method and active droplet generation method. Among them, the passive droplet generation method is to separate the discrete phase fluid into uniform micro droplets in the continuous phase fluid through channel geometry (such as T-type / Y-type structure, flow focusing structure, coaxial flow focusing structure and step structure, etc.). This method has the advantages of uniform size, good monodispersity, and can effectively avoid external interference, but this type of droplet generation method has high requirements for the accuracy of channel geometry. The active droplet generation method uses external force fields such as batteries, magnetic fields, and sound fields to form droplets. In comparison, the active droplet generation method has better droplet control capabilities, but its droplet generation device is more complicated. For the capture of droplets, the currently commonly used droplet capture method is mainly to capture droplets through a special geometric "trap" structure, which has a more complex structure and is more difficult to process.

[0005] Usually, droplet generation and droplet capture need to be realized by two separate functional structures. When it is necessary to develop a droplet integration system, droplet generation, droplet capture and other required functional units are integrated together. On the one hand, this integrated structure is generally more complex and difficult to process. On the other hand, at the beginning of the startup of this system, due to factors such as equipment stability and changes in internal flow resistance of the chip, the size and shape of the droplets are usually not stable enough, and it takes a while to stably form droplets of controlled size. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for in-situ generation of micro-droplets, wherein the micro-droplets are generated in-situ and captured in-situ; the micro-droplets are generated by a micro-droplet generation device, the micro-droplet generation device comprises one or more micro-droplet generation and capture zones 101, the droplet generation and capture zones 101 are arranged in a flow channel 102, and the flow channel 102 is provided with a fluid inlet 103 and a fluid outlet 104;

[0007] When generating water-in-oil type micro-droplets, the surface of the area where the fluid flows through the flow channel 102 is a hydrophobic surface, and the contact angle between the hydrophobic surface and water is not less than 90°. The surface of the droplet generation and capture area 101 arranged in the flow channel 102 is a hydrophilic surface, and the contact angle between the hydrophilic surface and water is at least 40° smaller than the contact angle between the hydrophobic surface and water around it.

[0008] When generating micro-droplets, the water phase fluid is driven to fill the micro-droplet generation capture zone 101 and the circulation channel 102 through the fluid inlet 103, and then the injection of the water phase fluid is stopped, and the oil phase fluid is driven to inject into the circulation channel 102 through the fluid inlet 103. In the process of the oil phase fluid covering the circulation channel 102, the droplet generation capture zone 101 is hydrophilic and will pull the water phase fluid in the droplet generation capture zone 101, while the water phase fluid not in the droplet generation capture zone 101 will be pushed toward the direction of the fluid outlet 104 along with the flow of the oil phase fluid; in this process, the water phase fluid can be sheared into micro-droplets of a certain size through the pulling / pushing away of the water phase fluid, and finally fixed at the location of the droplet generation capture zone 101;

[0009] Alternatively, when generating oil-in-water microdroplets, the surface of the area where the fluid flows through the flow channel 102 is a hydrophilic surface, and the contact angle between the hydrophilic surface and water is not greater than 90°, and the surface of the droplet generation and capture area 101 arranged in the flow channel 102 is a hydrophobic surface, and the contact angle between the hydrophobic surface and water is at least 40° greater than the contact angle between the hydrophilic surface and water around it;

[0010] When generating micro-droplets, the oil phase fluid is driven through the fluid inlet 103 to fill the micro-droplet generation capture area 101 and the flow channel 102, and then the injection of the oil phase fluid is stopped, and the water phase fluid is driven through the fluid inlet 103 to inject into the flow channel 102. In the process of the water phase fluid covering the flow channel 102, the droplet generation capture area 101 is hydrophobic and will pull the oil phase fluid in the droplet generation capture area 101, while the oil phase fluid not in the droplet generation capture area 101 will be pushed toward the direction of the fluid outlet 104 along with the flow of the water phase fluid; in this process, the oil phase fluid can be sheared into micro-droplets of a certain size through the pulling / pushing away of the oil phase fluid, and finally fixed at the location of the droplet generation capture area 101.

[0011] In one embodiment, when water-in-oil type microdroplets are generated, the contact angle between the hydrophobic surface and water is not less than 100°, and the contact angle between the hydrophilic surface and water is not greater than 60°.

[0012] In one embodiment, when water-in-oil type micro-droplets are generated, the flow channel 102 is entirely made of hydrophobic material, and only the droplet generation and capture area 101 is locally hydrophilic treated.

[0013] In one embodiment, the flow channel 102 is made of PDMS.

[0014] In one embodiment, the droplet generation and capture area 101 is locally hydrophilized by plasma treatment, chemical reagent modification, or chemical liquid deposition.

[0015] In one embodiment, the droplet generation capture zone 101 uses chemical liquid deposition to deposit poly (N-isopropylacrylamide) on the surface of the capture zone for local hydrophilic treatment.

[0016] In one embodiment, the droplet generation capture zone 101 is circular or polygonal.

[0017] In one embodiment, the size of the generated microdroplets is changed by changing the size of the droplet generation capture zone 101, and / or, when generating oil-in-water type microdroplets, the size of the generated microdroplets is changed by changing the flow rate of the oil phase fluid, and when generating oil-in-water type microdroplets, the size of the generated microdroplets is changed by changing the flow rate of the water phase fluid.

[0018] In one embodiment, a system for in situ generation of microdroplets for use in the above method is provided.

[0019] In one embodiment, the system includes a droplet generation and capture integrated chip, which includes a micro-droplet generation and capture unit 1, a first liquid inlet 2, a second liquid inlet 3, a liquid outlet 4, a first fluid inlet channel 5 connected to the first liquid inlet 2, a second fluid inlet channel 6 connected to the second liquid inlet 3, an inlet channel 7, a droplet channel 8, and an outlet channel 9. The micro-droplet generation and capture unit 1 is arranged in the droplet channel 8, and the micro-droplet generation and capture unit 1 includes at least one droplet generation and capture area 101; the first liquid inlet 2 and the second liquid inlet 3 are used to introduce water phase fluid and oil phase fluid, respectively; the first fluid enters the inlet channel 7 through the first fluid inlet channel 5, and the second fluid enters the inlet channel 7 through the second fluid inlet channel 6.

[0020] In the method of the present invention, microdroplets are generated and captured in situ, and the generation of microdroplets and the capture of microdroplets are integrated into a structure with a simple structure and high system integration, which is convenient for controlling the process of microdroplet generation and for detecting microdroplets. In the method of the present invention, flexible and controllable generation and in situ capture of microdroplets are achieved through local hydrophilic / hydrophobic treatment.

[0021] In the method of the present invention, after the droplets are generated and captured, the continued flow of the oil phase as the continuous phase will not only not push the generated droplets away from the droplet generation and capture area, but will also drive the liquid flow inside the aqueous phase droplets, that is, the internal circulation phenomenon of the aqueous phase droplets, which can further improve the mass transfer efficiency within the droplets.

[0022] In the method of the present invention, the pump valve stability and flow resistance change at the beginning of the system startup have little effect on droplet generation, the system has high stability, can stably and reliably generate micro droplets, and avoid instability in the micro droplet production process. In the present invention, by controlling the size of the droplet generation capture area, the size of the formed micro droplets can be controlled, and the flexibility and controllability are high; in the present invention, by increasing the number of droplet generation capture units, high-throughput droplet generation and in-situ capture can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 is a schematic diagram of an embodiment of a micro-droplet generation capture zone of the present invention;

[0025] Figure 2It is a picture showing the actual micro-droplet generation and capture process of the micro-droplet generation and capture zone of the present invention;

[0026] Figure 3 is a diagram showing the simulation results of the fluid flow state of the trapped droplet when the continuous phase continues to flow;

[0027] Figure 4 It is a schematic diagram of an embodiment of the droplet generation and capture integrated chip of the present invention and a diagram of experimental results thereof;

[0028] Figure 5 The present invention shows the capture effect diagram of droplet generation and capture areas of different sizes (the diameters of the droplet generation and capture areas from left to right are 0.5 mm, 0.4 mm, and 0.3 mm, respectively);

[0029] Figure 6 The present invention shows the result data graph of droplet diameters generated and captured in droplet generation and capture zones of different sizes at different flow rates. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0031] Example 1 Method for in-situ generation of microdroplets of the present invention

[0032] The present invention provides a method for in-situ generation and in-situ capture of microdroplets, such as Figure 1 As shown, in the micro-droplet generation device of the present invention, it includes a micro-droplet generation and capture area 101, a flow channel 102, an inlet 103 and an outlet 104. The droplet generation and capture area 101 can be in various shapes such as a circle and a polygon, and the outer dimensions (such as length, width, and diameter) are 0.1 mm or more, and the channel width of the flow channel 102 is 0.1 mm or more, and the channel depth is 0.05 mm or more.

[0033] For the capture of water in oil (W / O) droplets, the entire flow channel is made of hydrophobic material (such as PDMS material) (the contact angle of the hydrophobic material is greater than 90°, preferably greater than 100°), and only local hydrophilic treatment is performed in the droplet generation and capture area 101 (for example, plasma treatment, chemical reagent modification or chemical liquid deposition, the contact angle of the hydrophilic area must be less than 60°), and the contact angles of the hydrophobic material and the hydrophilic material must have a difference of 40° or more.

[0034] During operation, the water phase fluid first fills the droplet generation capture area 101 and the circulation channel 102 through the inlet 103 under the action of the fluid drive pump, and then stops injecting the water phase fluid, and instead drives the oil phase fluid to be injected into the circulation channel 102 through the inlet 103. In this way, in the process of the oil phase fluid covering the circulation channel 102, the droplet generation capture area 101, due to the hydrophilic coating, will pull the water phase fluid near the hydrophilic coating, while the water phase fluid not in the droplet generation capture area 101 will be pushed toward the outlet 104 with the flow of the oil phase. In this process, the water phase fluid can be sheared into droplets of a certain size through the pulling / pushing away of the water phase fluid, and finally fixed at the location of the droplet generation capture area 101 (see the specific experimental results). Figure 2 After the droplets are generated and captured, the continued flow of the oil phase as the continuous phase will not only not push the generated droplets away from the droplet generation and capture zone 101, but will also drive the liquid flow inside the water phase droplets (see Figure 3 Simulation results), that is, the internal circulation phenomenon of water phase droplets, which can further improve the mass transfer efficiency within the droplets.

[0035] For the generation and capture of oil-in-water (O / W) droplets, the droplet generation and capture area 101 is hydrophobic, and other positions are hydrophilic. The specific generation and capture mechanism of oil-in-water droplets is similar to that of oil-in-water droplets, which will not be repeated here.

[0036] Example 2 System for in-situ generation of microdroplets of the present invention

[0037] The system for in-situ generation of micro-droplets of the present invention comprises a droplet generation and capture integrated chip. Figure 4 In one embodiment shown, the droplet generation and capture integrated chip includes a micro-droplet generation and capture unit 1, a first liquid inlet 2, a second liquid inlet 3, a liquid outlet 4, a first fluid inlet channel 5 connected to the first liquid inlet 2, a second fluid inlet channel 6 connected to the second liquid inlet 2, a liquid inlet channel 7, a droplet channel 8, and a liquid outlet channel 9. The micro-droplet generation and capture unit 1 includes at least one droplet generation and capture area 101, and the micro-droplet generation and capture unit 1 is arranged in the droplet channel 8. The first liquid inlet 2 and the second liquid inlet 3 are used to introduce water phase and oil phase fluids respectively.

[0038] For the generation and capture of water-in-oil microdroplets, the droplet generation and capture integrated chip is made of hydrophobic materials (such as PDMS) as a whole, and only the droplet generation capture area 101 of the microdroplet generation capture unit 1 is locally hydrophilic (for example, using a plasma treatment + chemical reagent modification method or a chemical liquid phase deposition method). During operation, the aqueous phase fluid first flows into the chip through the first liquid inlet 2 under the action of the fluid drive pump, and passes through and fills the first fluid inlet channel 5, the liquid inlet channel 7, the droplet channel 8, and the droplet generation capture area 101 in sequence, and then flows out of the chip through the liquid outlet channel 9 and the liquid outlet 4. Thereafter, the aqueous phase stops injecting liquid, and the oil phase fluid flows into the microfluidic chip through the second liquid inlet 3, and fills the second fluid inlet channel 6, the liquid inlet channel 7, the droplet channel 8 in sequence, and then flows out of the chip through the liquid outlet channel 9 and the liquid outlet 4. In the process of the oil phase fluid covering the droplet channel 8, the droplet generation and capture area 101, due to the hydrophilic coating, will pull the water phase fluid near the hydrophilic coating, while the water phase fluid not in the droplet generation and capture area 101 will be pushed toward the liquid outlet 4 with the flow of the oil phase. In this process, the pulling / pushing away of the water phase fluid near the droplet generation and capture area 101 will shear the water phase fluid into micro-droplets of a certain size. After the micro-droplets are generated, they will be fixed at the location of the droplet generation and capture area 101, thereby achieving the generation and in-situ capture of micro-droplets.

[0039] Example 3 Generation of microdroplets of the present invention

[0040] like Figure 4 In the embodiment shown, the droplet generation and capture integrated chip has 8 identical circular droplet generation and capture areas 101 with a diameter of 0.3 mm, the channel width of the flow channel 102 is 1 mm, the depth of the entire chip is 0.1 mm, the entire chip is made of PDMS material with a contact angle of about 110°, and a layer of poly N-isopropylacrylamide (PNIPAAm) hydrophilic coating is formed on the surface of the droplet generation and capture area by chemical liquid deposition, and the contact angle is about 50°. The specific operation is as follows:

[0041] (1) The aqueous fluid is injected into the chip from the first liquid inlet 2 at a flow rate of 0.3 μL / min, passes through and fills the first fluid inlet channel 5, the liquid inlet channel 7, the droplet channel 8, and the droplet generation capture area 101 in sequence, and then flows out of the chip through the liquid outlet channel 9 and the liquid outlet 4.

[0042] (2) Set the aqueous phase fluid flow rate to 0 μL / min.

[0043] (3) The oil phase fluid is injected into the chip from the second liquid inlet 3 at a flow rate of 0.3 μL / min, and fills the second fluid inlet channel 6, the liquid inlet channel 7, and the droplet channel 8 in sequence, and then flows out of the chip through the liquid outlet channel 9 and the liquid outlet 4. In the process of the oil phase fluid covering the droplet channel 8, the eight circular droplet generation and capture areas 101 generate droplets in sequence and fix the droplets on the droplet generation and capture areas 101. The specific experimental results are shown in Figure 4 , the size of the generated droplets is very uniform.

[0044] Example 4 Experiment on factors affecting the formation of microdroplets of the present invention

[0045] like Figure 5 , 6 In the embodiment shown, the droplet generation and capture integrated chip has six circular droplet generation and capture areas 101 with diameters of 0.5 mm, 0.5 mm, 0.4 mm, 0.4 mm, 0.3 mm, and 0.3 mm, respectively, the channel width of the flow channel 102 is 1.3 mm, and the depth of the entire chip is 0.1 mm. The entire chip is made of PDMS material with a contact angle of about 110°, and a layer of poly N-isopropylacrylamide (PNIPAAm) hydrophilic coating is formed on the surface of the droplet generation and capture area by chemical liquid deposition, and the contact angle is about 50°. The specific operation is as follows:

[0046] (1) The aqueous fluid is injected into the chip from the first liquid inlet 2 at a flow rate of 0.3 μL / min, passes through and fills the first fluid inlet channel 5, the liquid inlet channel 7, the droplet channel 8, and the droplet generation capture area 101 in sequence, and then flows out of the chip through the liquid outlet channel 9 and the liquid outlet 4.

[0047] (2) Set the aqueous phase fluid flow rate to 0 μL / min.

[0048] (3) The oil phase fluid is injected into the chip from the second inlet 3 at a flow rate of 0.2 to 0.8 μL / min, and sequentially fills the second fluid inlet channel 6, the inlet channel 7, and the droplet channel 8, and then flows out of the chip through the outlet channel 9 and the outlet 4. In the process of the oil phase fluid covering the droplet channel 8, the circular droplet generation capture area 101 generates droplets in turn and fixes the droplets on the droplet generation capture area 101. Figure 5 and 6 It can be seen that ① the size of the droplet generation capture area is proportional to the size of the generated droplets; ② the greater the flow rate, the smaller the size of the generated and captured droplets. When the flow rate exceeds 0.8μL / min, droplets will not be generated in the droplet generation capture area 101.

[0049] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for in-situ generation of microdroplets, characterized in that: In the method, the microdroplets are generated in situ and captured in situ; the microdroplets are generated by a microdroplet generation device, the microdroplet generation device comprises one or more microdroplet generation and capture zones (101), the droplet generation and capture zones (101) are arranged in a circulation channel (102), and the circulation channel (102) is provided with a fluid inlet (103) and a fluid outlet (104); When generating water-in-oil type micro-droplets, the surface of the area where the fluid flows through the circulation channel (102) is a hydrophobic surface, and the contact angle between the hydrophobic surface and water is not less than 90°; the surface of the droplet generation and capture area (101) arranged in the circulation channel (102) is a hydrophilic surface, and the contact angle between the hydrophilic surface and water is at least 40° smaller than the contact angle between the hydrophobic surface and water around it; When generating micro-droplets, the water phase fluid is driven through the fluid inlet (103) to fill the micro-droplet generation capture zone (101) and the circulation channel (102), and then the injection of the water phase fluid is stopped, and the oil phase fluid is driven through the fluid inlet (103) to inject into the circulation channel (102). In the process of the oil phase fluid covering the circulation channel (102), the droplet generation capture zone (101) is hydrophilic and will pull the water phase fluid in the droplet generation capture zone (101), while the water phase fluid not in the droplet generation capture zone (101) will be pushed toward the direction of the fluid outlet (104) along with the flow of the oil phase fluid. In this process, the water phase fluid can be sheared into micro-droplets of a certain size through the pulling / pushing away of the water phase fluid, and finally fixed at the location of the droplet generation capture zone (101); Alternatively, when generating oil-in-water microdroplets, the surface of the area where the fluid flows through the circulation channel (102) is a hydrophilic surface, and the contact angle between the hydrophilic surface and water is not greater than 90°, and the surface of the droplet generation and capture area (101) arranged in the circulation channel (102) is a hydrophobic surface, and the contact angle between the hydrophobic surface and water is at least 40° greater than the contact angle between the hydrophilic surface and water around it; When generating micro-droplets, the oil phase fluid is driven through the fluid inlet (103) to fill the micro-droplet generation capture zone (101) and the circulation channel (102), and then the injection of the oil phase fluid is stopped, and the water phase fluid is driven through the fluid inlet (103) to inject into the circulation channel (102). In the process of the water phase fluid covering the circulation channel (102), the droplet generation capture zone (101) is hydrophobic and will pull the oil phase fluid in the droplet generation capture zone (101), while the oil phase fluid not in the droplet generation capture zone (101) will be pushed toward the direction of the fluid outlet (104) along with the flow of the water phase fluid. In this process, the oil phase fluid can be sheared into micro-droplets of a certain size through the pulling / pushing away of the oil phase fluid, and finally fixed at the location of the droplet generation capture zone (101).

2. The method according to claim 1, characterized in that When water-in-oil type microdroplets are generated, the contact angle between the hydrophobic surface and water is not less than 100°, and the contact angle between the hydrophilic surface and water is not more than 60°.

3. The method according to claim 1, characterized in that When water-in-oil type micro-droplets are generated, the circulation channel (102) is entirely made of hydrophobic material, and only the droplet generation and capture area (101) is locally hydrophilic.

4. The method according to claim 3, characterized in that The circulation channel (102) is made of PDMS.

5. The method according to claim 3, characterized in that: The droplet generation and capture area (101) is locally hydrophilized by plasma treatment, chemical reagent modification or chemical liquid phase deposition.

6. The method according to claim 5, characterized in that The droplet generation capture area (101) uses chemical liquid deposition to deposit poly N-isopropylacrylamide on the surface of the capture area for local hydrophilic treatment.

7. The method according to claim 1, characterized in that The droplet generation and capture area (101) is circular or polygonal.

8. The method according to claim 1, characterized in that The size of the generated microdroplets is changed by changing the size of the droplet generation capture zone (101), and / or, when generating oil-in-water type microdroplets, the size of the generated microdroplets is changed by changing the flow rate of the oil phase fluid, and when generating oil-in-water type microdroplets, the size of the generated microdroplets is changed by changing the flow rate of the water phase fluid.

9. A system for in situ generation of microdroplets for use in the method of any one of claims 1 to 8.

10. The system according to claim 9, characterized in that The system comprises a droplet generation and capture integrated chip, the droplet generation and capture integrated chip comprising a micro-droplet generation and capture unit (1), a first liquid inlet (2), a second liquid inlet (3), a liquid outlet (4), a first fluid inlet channel (5) connected to the first liquid inlet (2), a second fluid inlet channel (6) connected to the second liquid inlet (3), a liquid inlet channel (7), a droplet channel (8), and a liquid outlet channel (9); the micro-droplet generation and capture unit (1) is arranged in the droplet channel (8); the micro-droplet generation and capture unit (1) comprises at least one droplet generation and capture area (101); the first liquid inlet (2) and the second liquid inlet (3) are respectively used for introducing water phase fluid and oil phase fluid; the first fluid enters the liquid inlet channel (7) through the first fluid inlet channel (5), and the second fluid enters the liquid inlet channel (7) through the second fluid inlet channel (6).

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