Micro-fluidic chip and micro-droplet generation method

Abstract

The invention provides a micro-fluidic chip and a micro-droplet generation method. The micro-fluidic chip comprises a substrate, a first liquid storage tank, a second liquid storage tank and a micro-channel, wherein the first liquid storage tank, the second liquid storage tank and the micro-channel are arranged on the substrate; the method comprises the following steps: injecting a sample solution into the first liquid storage tank, and injecting a second liquid into the second liquid storage tank; first liquid is injected into the side, close to a pressure source, of the sample solution, and the first liquid and the sample solution are immiscible; driving pressure is applied to the first liquid storage pool and the second liquid storage pool based on a pressure source, so that the sample solution and the second liquid are pushed into the micro-channel, and micro-droplets are formed. The first liquid which is immiscible with the sample solution is injected above the sample solution as the isolation liquid to generate the micro-droplets, so that the possibility of pollution of a pressure source or other sample solutions caused by aerosol generated by evaporation of the sample solution is reduced, and the accuracy of a micro-droplet test result is improved.

Description

Technical Field

[0001] The present application relates to the field of microfluidics technology, and in particular to a microfluidics chip and a microdroplet generation method. Background Art

[0002] Polymerase Chain Reaction (PCR) is currently the main method for nucleic acid detection. The detection process of the droplet digital PCR system mainly involves three steps: droplet generation, PCR reaction and droplet detection.

[0003] Microdroplet generation usually relies on microfluidic chips. By applying pressure to the dispersed phase and the continuous phase surface, the liquid is determined to converge in the microfluidic chip to generate microdroplets. The dispersed phase is a sample solution to be tested, such as an aqueous phase, and the continuous phase is a solution in which the sample solution is dispersed, such as an oil phase. During the microdroplet generation process, the sample solution may evaporate to produce aerosols, contaminating the microfluidic chip or the test environment, thereby causing other sample solutions to be contaminated.

[0004] Therefore, there is an urgent need to provide a micro-droplet generation solution that reduces pollution. Summary of the invention

[0005] The present application provides a microfluidic chip and a microdroplet generation method, which reduces the possibility of aerosol generated by evaporation of the sample solution causing pressure source or other sample solution contamination by injecting a first liquid above the sample solution, thereby improving the accuracy of the microdroplet test results.

[0006] In a first aspect, the present application provides a method for generating microdroplets, the method being applied to a microfluidic chip, the microfluidic chip comprising a substrate and a first liquid reservoir, a second liquid reservoir and a microchannel arranged on the substrate; the method comprising:

[0007] A sample solution is injected into the first liquid reservoir, and a second liquid is injected into the second liquid reservoir; a first liquid is injected into a side of the sample solution close to a pressure source, wherein the first liquid is immiscible with the sample solution; and a driving pressure is applied to the first liquid reservoir and the second liquid reservoir respectively based on the pressure source to push the sample solution and the second liquid into the microchannel to form microdroplets.

[0008] Optionally, the method further includes:

[0009] The driving pressure of the pressure source of the first liquid reservoir is determined based on the surface tension coefficient of the first liquid and the sample solution and the density of the first liquid.

[0010] Optionally, the driving pressure P of the pressure source of the first liquid storage tank satisfies the following relationship:

[0011] PS≤σL-ρgV

[0012] Wherein, S is the surface area of ​​the first liquid in the first liquid storage tank; σ is the surface tension coefficient of the interface between the first liquid and the sample solution; L is the length of the interface between the first liquid and the sample solution; ρ is the density of the first liquid; V is the volume of the first liquid added to the first liquid storage tank; and g is the gravitational acceleration.

[0013] Optionally, the first liquid and the second liquid are the same liquid.

[0014] Optionally, the method further includes:

[0015] Obtaining the density of a plurality of candidate solutions and the surface tension coefficient of the interface between the candidate solutions and the sample solution;

[0016] The first liquid is determined from the plurality of candidate solutions based on the surface tension coefficient and the density of the candidate solutions.

[0017] Optionally, determining the first liquid from the plurality of candidate solutions based on the surface tension coefficient and the density of the candidate solution includes:

[0018] For each alternative solution, calculate the product of the surface tension coefficient of the interface corresponding to the alternative solution and the corresponding interface length to obtain the surface tension corresponding to the alternative solution; calculate the product of the density of the alternative solution, the gravitational acceleration and the preset volume to obtain the gravity of the alternative solution; determine that the alternative solution with the largest ratio of the difference between the surface tension and the gravity of the alternative solution and the surface area of ​​the first liquid in the first liquid storage tank is the first liquid.

[0019] Optionally, the surface tension coefficient of the interface between the first liquid and the sample solution is in the range of 1 mN / m to 100 mN / m.

[0020] In a second aspect, the present application provides a microfluidic chip, comprising a substrate and a first liquid reservoir, a second liquid reservoir, a microchannel and a droplet outlet arranged on the substrate; the first liquid reservoir and the second liquid reservoir are both connected to the microchannel to form microdroplets in the microchannel, and the microdroplets are discharged through the droplet outlet.

[0021] Optionally, the first liquid storage tank is used to cache the sample solution and the first liquid, the first liquid is immiscible with the sample solution, and in the first liquid storage tank the first liquid is located on the side of the sample solution close to the pressure source; the second liquid storage tank is used to cache the second liquid; under the action of the pressure source, the second liquid and the sample solution merge in the microchannel to form microdroplets.

[0022] Optionally, the substrate includes a first carrier plate and a second carrier plate, the first carrier plate is provided with the first liquid reservoir, the second liquid reservoir and the microchannel, the second carrier plate is connected to the first carrier plate, and the droplet outlet is provided on the second carrier plate.

[0023] Optionally, the microfluidic channel includes a dispersed phase microfluidic channel, a continuous phase microfluidic channel and an emulsification microfluidic channel; the dispersed phase microfluidic channel is connected to the first liquid reservoir and the emulsification microfluidic channel respectively; the continuous phase microfluidic channel is connected to the second liquid reservoir and the emulsification microfluidic channel respectively; the depth of the emulsification microfluidic channel is less than the depth of the continuous phase microfluidic channel so that the sample solution is emulsified in a step-by-step manner; the sample solution after step-by-step emulsification flows out from the droplet outlet together with the second liquid in the continuous phase microfluidic channel to form microdroplets.

[0024] Optionally, the depth of the dispersed phase microfluidic channel is the same as the depth of the continuous phase microfluidic channel.

[0025] Optionally, the sample solution is an aqueous phase, and the first liquid and the second liquid are both oil phases; or, the sample solution is an oil phase, and the first liquid and the second liquid are both aqueous phases.

[0026] The microfluidic chip and microdroplet generation method provided by the present application are configured to reduce the contamination of other sample solutions and the laboratory environment by aerosols generated by the evaporation of the sample solution during the formation of microdroplets, so that a first liquid that is immiscible with the sample solution is injected on the side of the liquid reservoir of the microfluidic chip that is close to the pressure source for caching the sample solution. Since the first liquid is immiscible with the sample solution, when the pressure source drives the sample solution to flow, the first liquid forms an isolation barrier, thereby reducing the possibility of contamination of the sample solution. The scheme for reducing contamination is low-cost and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0028] Figure 1 A schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present application;

[0029] Figure 2 For this application Figure 1A schematic structural diagram of a first liquid storage tank provided in the illustrated embodiment;

[0030] Figure 3 A schematic diagram of the structure of a microfluidic chip provided in another embodiment of the present application;

[0031] Figure 4 A schematic diagram of the structure of a microfluidic chip provided in another embodiment of the present application;

[0032] Figure 5 A schematic diagram of the structure of a microfluidic chip provided in yet another embodiment of the present application;

[0033] Figure 6 A schematic diagram of the depths of a dispersed phase microchannel, a continuous phase microchannel, and an emulsified microchannel provided for one embodiment of the present application;

[0034] Figure 7 A schematic diagram of a process for generating micro-droplets provided in an embodiment of the present application;

[0035] Figure 8 A schematic diagram of the structure of a micro-droplet generation system provided in one embodiment of the present application.

[0036] Reference numerals:

[0037] 10-micro droplet generation system; 100-microfluidic chip; 200-control unit; 300-pressure source; 400-collection device;

[0038] 110-substrate; 111-first carrier plate; 112-second carrier plate;

[0039] 120-first liquid storage tank; 121-first connecting column;

[0040] 130 - second liquid storage tank; 131 - second connecting column;

[0041] 140-microfluidic channel; 141-dispersed phase microfluidic channel; 141a-main transport microfluidic channel; 141b-distribution microfluidic channel; 142-continuous phase microfluidic channel; 142a-continuous phase transport microfluidic channel; 142b-droplet transport microfluidic channel; 143-emulsification microfluidic channel;

[0042] 150 - droplet outlet; 151 - receiving tank.

[0043] The above drawings have shown clear embodiments of the present application, which will be described in more detail later. These drawings and text descriptions are not intended to limit the scope of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0044] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0045] First, the terms involved in this application are explained:

[0046] Continuous Phase: A liquid that flows continuously.

[0047] Dispersed Phase: Liquid that flows in droplets. The dispersed phase is immiscible with the continuous phase.

[0048] Microdroplet technology: A technology that applies a sufficiently large pressure to allow the dispersed phase to break through the interfacial tension and enter the continuous phase to form microdroplets. Under the action of the pressure source, the continuous phase and the dispersed phase enter different microchannels respectively. After meeting at the intersection of the microchannels, the droplets of the dispersed phase enter the continuous phase to form microdroplets.

[0049] The technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems are described in detail below with specific embodiments. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0050] Microfluidics is a technology that manipulates fluids at the micrometer scale. Droplet microfluidics, as an important branch of microfluidic chip research, has been developed in recent years based on the traditional microfluidic system. Droplet microfluidics technology can prepare picoliter (pL) droplets with good monodispersity. The droplets can encapsulate different samples, including chemical reagents, micro-nanoparticles, cells, DNA (Deoxyribonucleic Acid), and microorganisms. This enables the droplets to be used as independent reaction units for qualitative or quantitative detection. It has the advantages of low sample demand, portability, and high integration. It is currently widely used in the biomedical field.

[0051] A microfluidic chip is a micro-droplet generating device. Under the action of a pressure source, a sample solution (such as an aqueous phase) and a second liquid (such as an oil phase) flow into a microchannel respectively and merge to form micro-droplets.

[0052] Under the action of the pressure source, the sample solution may evaporate to produce aerosols that contaminate other sample solutions or the laboratory environment, thereby affecting the detection results of microdroplets.

[0053] In the related art, the solutions designed to reduce pollution include using disposable adapters, using non-contact pressurization methods, etc. These solutions are highly complex and have problems of being difficult to implement technically or having high costs.

[0054] In order to reduce the contamination of the sample solution, the embodiment of the present application provides a microfluidic chip, in which a first liquid that is immiscible with the sample solution is added to a reservoir for caching the sample solution in the microfluidic chip, and the first liquid is close to a pressure source; after the pressure source applies pressure, due to the surface tension between the added first liquid and the sample solution, the first liquid pushes the sample solution to flow in the microchannel until it merges with the second liquid to form microdroplets. The first liquid isolates the sample solution, thereby effectively reducing the possibility of aerosols generated by the evaporation of the sample solution contaminating other sample solutions and the sample test environment, and improving the cleanliness of the microdroplet test environment; at the same time, the pollution reduction scheme is easy to implement and low cost.

[0055] Figure 1 A schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present application is shown in FIG. Figure 1 As shown, the microfluidic chip 100 includes a substrate 110 and a first liquid reservoir 120, a second liquid reservoir 130, a microchannel 140 and a droplet outlet 150 disposed on the substrate 110. The first liquid reservoir 120 and the second liquid reservoir 130 are both connected to the microchannel 140 to form microdroplets in the microchannel 140, and the microdroplets are discharged through the droplet outlet 150.

[0056] The first liquid reservoir 120 is used to cache the sample solution and the first liquid. The first liquid and the sample solution are immiscible, and in the first liquid reservoir 120, the first liquid is located on the side of the sample solution close to the pressure source; the second liquid reservoir 130 is used to cache the second liquid; under the action of the pressure source, the second liquid and the sample solution merge in the microchannel 140 to form microdroplets. The microdroplets flow out through the droplet outlet 150, such as flowing to the collection device ( Figure 1 (not shown).

[0057] The material of the substrate 110 may be silicone rubber, glass or other materials.

[0058] The first liquid reservoir 120 and the second liquid reservoir 130 may be located on the same side of the microfluidic channel 140, or may be located on two opposite sides of the microfluidic channel 140. Figure 1 The example of being located on the same side.

[0059] The sample solution is the solution that needs to be tested, and the test content can be pathogen detection, gene mutation analysis, single-cell sequencing, etc.

[0060] If the sample solution is an aqueous phase, the first liquid and the second liquid are both oil phases. If the sample solution is an oil phase, the first liquid and the second liquid are both aqueous phases.

[0061] Exemplarily, the second liquid or the first liquid of the oil phase includes, but is not limited to, fluorinated oil, silicone oil, hydrocarbon oil, and the like.

[0062] The fluorinated oil can be any fluorinated organic compound. The fluorinated oil can be a perfluorocarbon, such as perfluorooctane or perfluorohexane; it can also be a partially fluorinated hydrocarbon, such as 1,1,1-trifluorooctane or 1,1,1,2,2-pentafluorodecane; it can also be a fluorinated ether, such as a mixture of 1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane, methyl nonafluorobutyl ether and perfluorobutyl methyl ether. The fluorinated oil can be linear, cyclic or heterocyclic. In addition to carbon and fluorine, the fluorinated organic compound can also contain atoms such as hydrogen, oxygen, nitrogen, sulfur, chlorine, bromine, etc.

[0063] In some embodiments, the second liquid or the first liquid may further include a surfactant, specifically a fluorinated surfactant (ie, including at least one fluorine atom).

[0064] Examples of surfactants include, but are not limited to, perfluoro-octanol, 1H,1H,2H,2H-perfluoro-1-octanol, perfluoro-decanol, 1H,1H,2H,2H-perfluoro-1-decanol, perfluoro-tetradecanoic acid, perfluoro-tetradecane oligoethylene glycol, perfluoropolyether diol, perfluoropolyether alcohol, perfluoropolyether-polyethylene glycol, perfluoropolyether-polyethylene glycol-perfluoropolyether, dodecafluoroheptanol, and mixtures thereof.

[0065] In an optional embodiment, in order to reduce the types of liquids injected into the microfluidic chip, the first liquid and the second liquid can be the same liquid, which reduces the cost of generating microdroplets and reduces the impact of the additionally injected first liquid on the detection of the sample solution.

[0066] Under the action of the pressure source, the first liquid pushes the sample solution to flow in the microchannel 140, and the second liquid also flows in the microchannel 140 under the action of the pressure. Figure 1 The sample solution (at the position indicated by the asterisk in the middle) merges with the second liquid to form oil-in-water or water-in-oil microdroplets.

[0067] Exemplarily, the pressure source is located above the first liquid reservoir 120 , and the first liquid is located above the sample solution. A through hole communicating with the microchannel 140 is provided on the lower surface or side wall of the first liquid reservoir 120 .

[0068] Figure 1The liquid reservoirs shown in the figure, including the first liquid reservoir 120 and the second liquid reservoir 130, and the shapes and positions of the microchannel 140 are only examples, and the present application does not limit them.

[0069] Exemplarily, the shape of the first liquid storage tank 120 may be cylindrical, cubic, or other shapes.

[0070] In some embodiments, the first liquid storage tank 120 and the second liquid storage tank 130 are truncated cone structures.

[0071] The microfluidic chip provided in this embodiment is configured to reduce the contamination of other sample solutions and the laboratory environment by aerosols generated by evaporation of the sample solution during the formation of microdroplets, so that a first liquid that is immiscible with the sample solution is injected on the side of the liquid reservoir of the microfluidic chip that is close to the pressure source and caches the sample solution. Since the first liquid is immiscible with the sample solution, when the pressure source drives the sample solution to flow, the first liquid forms an isolation barrier, thereby reducing the possibility of contamination of the sample solution. The scheme for reducing contamination is low-cost and easy to implement.

[0072] Figure 2 For this application Figure 1 The schematic diagram of the structure of the first liquid storage tank provided in the embodiment shown is as follows: Figure 2 As shown, in the first liquid reservoir 120, the first liquid is located above the sample solution, and the length of the interface between the two is L. The driving pressure of the pressure source is applied from above the first liquid reservoir 120. Under the driving pressure, due to the surface tension on the interface between the first liquid and the sample solution, the first liquid is always located above the sample solution, and the first liquid pushes the sample solution into the microchannel 140. After all the sample solution in the first liquid reservoir 120 flows into the microchannel 140, the first liquid also enters the microchannel 140 under the action of the driving pressure, thereby pushing the first liquid remaining in the microchannel 140 into the collecting device (not shown in the figure). The collecting device is used to collect the formed micro droplets for detection.

[0073] There may be two pressure sources, one of which is used to apply pressure to the liquid in the first liquid storage tank 120 , and the other is used to apply pressure to the liquid in the second liquid storage tank 130 .

[0074] For example, Figure 3 A schematic diagram of the structure of a microfluidic chip provided in another embodiment of the present application is shown in FIG. Figure 1 and Figure 3The pressure source of the microfluidic chip 100 includes a first pressure source and a second pressure source. The first pressure source is used to apply pressure to the liquid in the first liquid reservoir 120 to push the first liquid and the sample solution into the microchannel to merge with the second liquid flowing into the microchannel; the second pressure source is used to apply pressure to the liquid in the second liquid reservoir 130 to push the second liquid into the microchannel; a droplet outlet 150 is also provided on the microfluidic chip 100, and the formed micro droplets flow from the microchannel (not shown in the figure) through the droplet outlet 150 into the collecting device.

[0075] Exemplarily, the collecting device may be an 8-tube connection.

[0076] In an optional embodiment, in order to prevent the first liquid and the sample solution from being emulsified or mixed under the action of a pressure source (such as the first pressure source), the driving pressure P of the pressure source of the first liquid reservoir 120 satisfies the following relationship:

[0077] PS≤σL-ρgV (1)

[0078] Wherein, S is the surface area of ​​the first liquid in the first liquid storage tank; σ is the surface tension coefficient of the interface between the first liquid and the sample solution; L is the length of the interface between the first liquid and the sample solution; ρ is the density of the first liquid; V is the volume of the first liquid added to the first liquid storage tank 120; and g is the gravitational acceleration.

[0079] L is a characteristic width of the first liquid reservoir 120 . The first liquid reservoir 120 may be cylindrical, and the length L of the interface between the first liquid and the sample solution may be the diameter of the first liquid reservoir 120 .

[0080] When the driving pressure P of the pressure source is constant, the size of the first liquid reservoir 120 can be adjusted based on the relationship (1), and parameters such as the volume of the first body fluid added to the first liquid reservoir 120 and the density of the first liquid can be adjusted.

[0081] When the specifications of the first liquid storage tank 120 and the first liquid are constant, the driving pressure P of the pressure source of the first liquid storage tank 120 can be determined based on the relational expression (1).

[0082] In some embodiments, at room temperature and atmospheric pressure, the surface tension of the first liquid and the sample solution at the interface may range from 1 mN.m-1 to 100 mN.m-1, preferably from 5 mN.m-1 to 20 mN.m-1.

[0083] The characteristic width of the first liquid storage tank 120, that is, the length L, may range from 1 mm to 10 mm, such as 2.5 mm, 4 mm, 6 mm, 8 mm, etc.

[0084] The water phase pores have a characteristic width L ranging from about 1 mm to about 10 mm, preferably from about 2.5 mm to about 8 mm, more preferably from about 4 mm to about 6 mm.

[0085] Figure 4 A schematic diagram of the structure of a microfluidic chip provided in another embodiment of the present application, combined with Figures 1 to 4 In this embodiment, the substrate 110 includes a first carrier plate 111 and a second carrier plate 112. The first carrier plate 111 is provided with a first liquid reservoir 120, a second liquid reservoir 130 and a microchannel 140. The second carrier plate 112 is connected to the first carrier plate 111, such as by bonding.

[0086] Continue to see Figure 4 In an optional embodiment, the first carrier plate 111 has a first through hole penetrating the first carrier plate 111, and the first through hole and the side surface of the second carrier plate 112 close to the first carrier plate 111 constitute the first liquid reservoir 120. The first carrier plate 111 has a second through hole penetrating the first carrier plate 111, and the second through hole and the side surface of the second carrier plate 112 close to the first carrier plate 111 constitute the second liquid reservoir 130.

[0087] In order to facilitate the connection between the chip and the pressure source, a hollow connecting column is provided above the first liquid reservoir 120 and the second liquid reservoir 130 to ensure reliable transmission of the pressure provided by the pressure source. Figure 4 As shown, the first liquid storage tank is connected to a first connecting column 121, and the second liquid storage tank is connected to a second connecting column 131. Both the first connecting column 121 and the second connecting column 131 are hollow structures and communicate with the liquid storage tanks.

[0088] The droplet outlet 150 may be disposed on the first carrier plate 111 or the second carrier plate 112 to allow the micro droplets to flow out. The droplet outlet 150 may be a through hole penetrating the first carrier plate 111 or the second carrier plate 112 .

[0089] Figure 4 In the example, the droplet outlet 150 is located on the second carrier plate 112, and the second carrier plate 112 is provided with the droplet outlet 150. The first carrier plate 111 is provided with a receiving groove 151, which is connected to the microfluidic channel 140 and is used to buffer the microdroplets formed in the microfluidic channel 140 and discharge them through the droplet outlet 150. The receiving groove 151 can be omitted.

[0090] When the first carrier plate 111 and the second carrier plate 112 are bonded, the droplet outlet 150 and the receiving groove 151 need to be aligned so that the micro droplets flow out through the receiving groove 151 and the droplet outlet 150. The droplet outlet 150 is provided with a through hole on the side facing the first carrier plate 111. The droplet outlet 150 and the receiving groove 151 are specifically aligned so that the orthographic projection of the receiving groove 151 on the second carrier plate 112 coincides with the through hole of the droplet outlet 150 or is located in the through hole.

[0091] By arranging all the microchannels 140 on the first carrier 111, the alignment between the microchannels is not involved in the bonding process between the first carrier 111 and the second carrier 112, which reduces the difficulty of alignment, thereby reducing the manufacturing difficulty of the microfluidic chip 100 and improving the yield.

[0092] See also Figure 4 and Figure 5 In this embodiment, the microchannel 140 includes a dispersed phase microchannel 141 , a continuous phase microchannel 142 and an emulsified microchannel 143 .

[0093] The dispersed phase microchannel 141 is connected to the first liquid reservoir 120 and the emulsification microchannel 143 respectively; the continuous phase microchannel 142 is connected to the second liquid reservoir 130 and the emulsification microchannel 143 respectively; the depth h of the emulsification microchannel 143 is less than the depth H2 of the continuous phase microchannel 142, so that the sample solution is emulsified in a step-by-step manner; the sample solution after step-by-step emulsification flows out from the droplet outlet 150 together with the second liquid in the continuous phase microchannel 142 to form microdroplets, which flow into the collection device for detection.

[0094] Figure 5 The shapes, lengths, positional relationships, etc. of the dispersed phase microchannel 141, the continuous phase microchannel 142, and the emulsified microchannel 143 shown in the figure are only examples, and the present application does not limit them.

[0095] The principle of step softening is that in a microchannel with a depth difference (such as a step-shaped difference), the sample solution is subjected to shear forces of different gradients at the depth difference, thereby generating microdroplets.

[0096] In one embodiment, the depth difference between the continuous phase microchannel 142 and the emulsification microchannel 143 should be greater than or equal to the diameter of the generated microdroplets.

[0097] Exemplarily, the depth difference may be between 15 μm and 1000 μm, such as 15 μm, 25 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1000 μm.

[0098] In an optional embodiment, the depth H2 of the continuous phase microchannel 142 may be between 20 μm and 1050 μm, and the depth h of the emulsification microchannel 143 may be between 5 μm and 50 μm.

[0099] Exemplarily, the depth H2 of the continuous phase microchannel 142 may be 20 μm, 50 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1050 μm. The depth h of the emulsification microchannel 143 may be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.

[0100] Preferably, the depth H2 of the continuous phase microchannel 142 may be between 100 μm and 500 μm, such as between 150 μm and 250 μm; the depth h of the emulsification microchannel 143 may be between 10 μm and 30 μm, such as between 15 and 25 μm.

[0101] The depth H1 of the dispersed phase microchannel 141 is greater than the depth h of the emulsification microchannel 143 .

[0102] In some embodiments, the depth H1 of the dispersed phase microchannel 141 can be the same as the depth H2 of the continuous phase microchannel 142 , so that the microchannel 140 only includes two different depths, which reduces the difficulty of the manufacturing process and injection molding, thereby reducing the manufacturing cost of the microchannel 140 .

[0103] Figure 6 A schematic diagram of the depths of the dispersed phase microchannel, the continuous phase microchannel and the emulsified microchannel provided in one embodiment of the present application is shown in FIG. Figure 6 As shown, the depth H1 of the dispersed phase microchannel 141 is the same as the depth H2 of the continuous phase microchannel 142 , and both are greater than the depth h of the emulsified microchannel 143 .

[0104] In the above-mentioned microfluidic chip 100, the sample solution (dispersed phase) enters the dispersed phase microchannel 141 from the first liquid reservoir 120 under the impetus of the first liquid, and then enters the emulsification microchannel 143; the second liquid (continuous phase) enters the continuous phase microchannel 142 from the second liquid reservoir 130, and flows along the continuous phase microchannel to the droplet outlet 150; the sample solution flows in the emulsification microchannel 143 to the intersection of the emulsification microchannel 143 and the continuous phase microchannel 142, and step emulsification occurs to form droplets, and the droplets then flow out from the droplet outlet 150 along with the second liquid flowing in the continuous phase microchannel 142.

[0105] By limiting the depth of the emulsification microchannel 143 to be smaller than the depth of the continuous phase microchannel 142, the dispersed phase liquid (sample solution) is step-emulsified at the intersection of the emulsification microchannel 143 and the continuous phase microchannel 142 where there is a depth difference, so as to generate droplets with high size consistency, thereby improving the accuracy of the detection results.

[0106] The first liquid reservoir 120 is the inlet of the sample solution, so that the sample solution flows into the dispersed phase microchannel 141 through the first liquid reservoir 120. The second liquid reservoir 130 is the inlet of the second liquid, so that the second liquid flows into the continuous phase microchannel 142 through the second liquid reservoir 130, and flows to the outlet along the continuous phase microchannel 142. The sample solution generates droplets through step emulsification at the intersection of the emulsification microchannel 143 and the continuous phase microchannel 142, and the droplets flow out from the droplet outlet 150 together with the flowing second liquid. Such a setting reduces the risk of droplet fusion caused by accumulation of droplets inside the microfluidic chip 100, improves the fluidity of the droplets, and thus improves the consistency of the particle size of the microfluidic liquid.

[0107] The receiving groove 151 may be located at one end of the continuous phase microfluidic channel 142 away from the second liquid reservoir 130 and communicate with the continuous phase microfluidic channel 142. The depth of the receiving groove 151 may be the same as the depth of the continuous phase microfluidic channel 142.

[0108] Continue to see Figure 5 The dispersed phase microchannel 141 includes a main transmission microchannel 141a and a distribution microchannel 141b that are interconnected. The end of the main transmission microchannel 141a away from the distribution microchannel 141b is connected to the first liquid reservoir 120, and is used to transmit the sample solution and the first liquid injected from the first liquid reservoir 120 to the distribution microchannel 141b. The end of the emulsification microchannel 143 away from the continuous phase microchannel 142 is connected to the side wall of the distribution microchannel 141b. Figure 5 Taking the multiple emulsification microchannels 143 as an example, the multiple emulsification microchannels 143 can generate droplets at the same time, thereby improving the generation efficiency of the liquid.

[0109] The main transmission microfluidic channel 141a can be connected to the distribution microfluidic channel 141b at the middle position of the distribution microfluidic channel 141b, dividing the distribution microfluidic channel 141b into two branches, such as a first branch and a second branch. The cross-sectional area of ​​the main transmission microfluidic channel 141a should be greater than or equal to the cross-sectional area of ​​the first branch and the second branch, so that the sample solution can flow from the main transmission microfluidic channel 141a into the two branches continuously and stably.

[0110] The continuous phase microfluidic channel 142 includes a continuous phase transport microfluidic channel 142a and a droplet transport microfluidic channel 142b that are interconnected. The end of the continuous phase transport microfluidic channel 142a away from the droplet transport microfluidic channel 142b is connected to the second liquid reservoir 130, and is used to transport the second liquid injected from the second liquid reservoir 130 to the droplet transport microfluidic channel 142b. Figure 5 The middle dotted line is the dividing line between the continuous phase transmission microfluidic channel 142a and the droplet transmission microfluidic channel 142b. The end of the emulsification microfluidic channel 143 away from the dispersed phase microfluidic channel 141 is connected to the side wall of the droplet transmission microfluidic channel 142b, and droplets are formed at the intersection of the emulsification microfluidic channel 143 and the droplet transmission microfluidic channel 142b. The end of the droplet transmission microfluidic channel 142b away from the continuous phase transmission microfluidic channel 142a is connected to the receiving tank 151, so that the formed micro droplets flow into the droplet outlet 150 through the receiving tank 151, and then flow out of the microfluidic chip 100. The droplet outlet 150 is externally connected to a collection device to collect micro droplets for sample detection.

[0111] In an optional embodiment, the cross-sectional area of ​​the continuous phase transport microchannel 142a is larger than the cross-sectional area of ​​the droplet transport microchannel 142b, thereby facilitating the transport of the second liquid and the formation of droplets at the intersection of the emulsification microchannel 143 and the droplet transport microchannel 142b.

[0112] Furthermore, the depth of the droplet transport microchannel 142b is greater than or equal to 1.5 standard droplet particle sizes, where the standard droplet particle size is the desired particle size of the formed droplets, such as 20 μm. The droplets being formed are spaced apart from the newly generated droplets in the depth direction of the droplet transport microchannel 142b to reduce the risk of droplet fusion due to accumulation of droplets in the droplet transport microchannel 142b, thereby improving the uniformity of the droplet particle size and facilitating the smooth outflow of the droplets. It should be noted that the droplet particle size mentioned in this application refers to the diameter of the droplets.

[0113] The sample solution enters the main transmission microchannel 141a from the first liquid reservoir 120, and then enters the distribution microchannel 141b and multiple emulsification microchannels 143 in sequence; the second liquid enters the continuous phase transmission microchannel 142a from the second liquid reservoir 130, and then enters the droplet transmission microchannel 142b; at the intersection of the emulsification microchannel 143 and the droplet transmission microchannel 142b, the sample solution is subjected to different gradient shear forces to generate microdroplets, which are transmitted to the holding tank 151 along with the flowing second liquid, and are transmitted to the outside of the microfluidic chip 100 through the droplet outlet and collected by the collection device.

[0114] In some embodiments, the microfluidic chip 100 also includes a control unit, which is used to control the flow rate of the sample solution and / or the second liquid entering the corresponding microchannel (dispersed phase microchannel 141 or continuous phase microchannel 142), thereby controlling the speed of microdroplet generation and outflow from the microfluidic chip 100, improving the controllability of the microdroplet generation process, and further reducing the risk of microdroplet accumulation inside the microfluidic chip 100 by controlling the microdroplet generation and outflow speed.

[0115] The flow rate of the second liquid is greater than the flow rate of the sample solution, or the sample solution may be pressurized after the second liquid has been pressurized for a period of time.

[0116] Figure 7 This is a schematic flow chart of a method for generating micro-droplets provided in an embodiment of the present application. The method is applied to the microfluidic chip provided in any embodiment of the present application, such as the microfluidic chip 100.

[0117] like Figure 7 As shown, the micro-droplet generation method provided in this embodiment includes the following steps:

[0118] Step S701: injecting a sample solution into the first liquid storage tank, and injecting a second liquid into the second liquid storage tank.

[0119] Step S702: injecting a first liquid into a side of the sample solution close to a pressure source, wherein the first liquid is immiscible with the sample solution.

[0120] The method of injecting liquid (including sample solution, first liquid and second liquid) includes but is not limited to a syringe, a syringe pump, etc. This embodiment does not limit the method of liquid injection.

[0121] Exemplarily, taking the sample solution as an example, the syringe pump is connected to the first liquid reservoir 120 in the microfluidic chip 100 through a capillary to inject the sample solution into the first liquid reservoir 120 .

[0122] Step S703: applying driving pressure to the first liquid reservoir and the second liquid reservoir respectively based on a pressure source to push the sample solution and the second liquid into the microchannel to form microdroplets.

[0123] The micro-droplet generation method provided in this embodiment is to inject a first liquid that is immiscible with the sample solution into the first liquid reservoir for caching the sample solution before applying pressure, so that after applying pressure, under the action of the surface tension of the interface between the first liquid and the sample solution, the first liquid pushes the sample solution and the second liquid to merge in the microchannel to form micro-droplets. Since the first liquid plays the role of isolation and barrier, it greatly hinders the spread of aerosols generated by the evaporation of the sample solution under the action of pressure, thereby reducing the contamination of the sample solution to other sample solutions or the environment, improving the cleanliness of the micro-droplet formation and the test environment, effectively avoiding the phenomenon of incorrect test results due to sample contamination, and improving the accuracy of sample detection.

[0124] After the first liquid and the sample solution are injected or after the first liquid corresponding to the sample solution is determined, the driving pressure (which may be simply referred to as pressure) applied by the first liquid reservoir 120 needs to be determined.

[0125] Optionally, the method further includes:

[0126] The driving pressure of the pressure source of the first liquid reservoir 120 is determined based on the surface tension coefficient of the first liquid and the sample solution and the density of the first liquid.

[0127] Optionally, the driving pressure P of the first liquid storage tank pressure source satisfies the aforementioned relationship (1).

[0128] Specifically, the surface tension coefficient and the density of the first liquid may be input into equation (1) to obtain the maximum value of the driving pressure, and a pressure less than or equal to the maximum value is selected as the driving pressure of the first liquid storage tank 120 .

[0129] Optionally, the first liquid and the second liquid are the same liquid.

[0130] Optionally, the method further includes:

[0131] The densities of a plurality of candidate solutions and the surface tension coefficients of the interfaces between the candidate solutions and the sample solution are obtained; and the first liquid is determined from the plurality of candidate solutions based on the surface tension coefficients and the densities of the candidate solutions.

[0132] The alternative solution is a first liquid that is optional for the sample solution and is immiscible with the sample solution.

[0133] The candidate solution may be determined based on the sample solution. Specifically, multiple candidate solutions may be determined based on the type of the sample solution.

[0134] The density of the candidate solution and the surface tension coefficient of the interface between the candidate solution and the sample solution can be obtained from the stored candidate solution parameter table by looking up the table based on the solution identification of the candidate solution. The candidate solution parameter table is used to store the identification of each candidate solution, the density of the candidate solution, and the surface tension coefficient of the interface between the candidate solution and the sample solution.

[0135] In order to select a suitable candidate solution from a plurality of candidate solutions as the first liquid to improve the effect of reducing contamination, a candidate solution with a small density and a large surface tension coefficient at the interface with the sample solution may be selected as the first liquid.

[0136] Optionally, determining the first liquid from the plurality of candidate solutions based on the surface tension coefficient and the density of the candidate solution includes:

[0137] For each alternative solution, calculate the product of the surface tension coefficient of the interface corresponding to the alternative solution and the corresponding interface length, i.e., σL, to obtain the surface tension corresponding to the alternative solution; calculate the product of the density of the alternative solution, the gravitational acceleration and the preset volume, i.e., ρgV, to obtain the liquid pressure of the alternative solution; determine that the alternative solution with the largest ratio of the difference between the surface tension and the liquid pressure (i.e., σL-ρgV) and the surface area of ​​the first liquid in the first liquid storage tank is the first liquid.

[0138] Figure 8 A schematic diagram of the structure of a micro-droplet generation system provided in one embodiment of the present application is shown in FIG. Figure 8 As shown, the micro-droplet generation system 10 includes a microfluidic chip 100 , a control unit 200 , a pressure source 300 and a collection device 400 .

[0139] The pressure source 300 includes a first pressure source 310 and a second pressure source 320. The first pressure source 310 is used to apply pressure to the first liquid reservoir 120 in the microfluidic chip 100 to drive the first liquid to push the sample solution into the microchannel and flow to the droplet outlet 150 in the microchannel; the second pressure source 320 is used to apply pressure to the second liquid reservoir 130 to push the second liquid into the microchannel and flow to the droplet outlet 150 in the microchannel. The microdroplets flowing out of the droplet outlet are collected by the collection device 400 for subsequent detection, such as pathogen detection, gene mutation analysis, single cell sequencing, etc.

[0140] The control unit 200 is used to control the driving pressure P of the pressure source 300 , including controlling the magnitude of the driving pressure P and the application time.

[0141] The control unit 200 can determine the driving pressure P of the pressure source 300 based on the relationship (1) and parameters related to the first liquid and the first liquid storage tank input by the user, such as the type of the first liquid, the specifications of the first liquid storage tank 120, and the volume or height of the first liquid added to the first liquid storage tank 120.

[0142] The control unit 200 may store a plurality of densities of first liquids and surface tension coefficients of interfaces between the first liquids and the sample solution, so as to search for the density of the first liquid of the corresponding type and the surface tension coefficient of the interface between the first liquid and the sample solution based on the type of the first liquid input by the user.

[0143] After obtaining various required parameters, including the density of the first liquid, the volume of the first liquid added to the first liquid reservoir, the surface tension coefficient of the interface between the first liquid and the sample solution, and the characteristic width of the first liquid reservoir 120, these parameters are substituted into the aforementioned relationship (1) to obtain the maximum value of the driving pressure P, and a driving pressure less than or equal to the maximum value is selected to drive the first liquid reservoir 120.

[0144] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the invention disclosed herein. The present application is intended to cover any modification, use or adaptation of the present application, which follows the general principles of the present application and includes common knowledge or customary techniques in the art that are not disclosed in the present application. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present application are indicated by the following claims.

[0145] It should be understood that the present application is not limited to the precise structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. A method for generating microdroplets, characterized in that: The method is applied to a microfluidic chip, which includes a substrate and a first liquid reservoir, a second liquid reservoir and a microchannel arranged on the substrate; the method includes: Injecting a sample solution into the first liquid reservoir, and injecting a second liquid into the second liquid reservoir; Injecting a first liquid into a side of the sample solution close to a pressure source, wherein the first liquid is immiscible with the sample solution; A driving pressure is applied to the first liquid reservoir and the second liquid reservoir based on a pressure source, so as to push the sample solution and the second liquid into the microfluidic channel to form micro droplets.

2. The method according to claim 1, characterized in that The method further comprises: The driving pressure of the pressure source of the first liquid reservoir is determined based on the surface tension coefficient of the first liquid and the sample solution and the density of the first liquid.

3. The method according to claim 2, characterized in that The driving pressure P of the first liquid storage tank pressure source satisfies the following relationship: PS≤σL-ρgV Wherein, S is the surface area of ​​the first liquid in the first liquid storage tank; σ is the surface tension coefficient of the interface between the first liquid and the sample solution; L is the length of the interface between the first liquid and the sample solution; ρ is the density of the first liquid; V is the volume of the first liquid added to the first liquid storage tank; and g is the gravitational acceleration.

4. The method according to any one of claims 1 to 3, characterized in that: The first liquid and the second liquid are the same liquid.

5. The method according to claim 1, characterized in that The method further comprises: Obtaining the density of a plurality of candidate solutions and the surface tension coefficient of the interface between the candidate solutions and the sample solution; The first liquid is determined from the plurality of candidate solutions based on the surface tension coefficient and the density of the candidate solutions.

6. The method according to claim 5, characterized in that The step of determining the first liquid from the plurality of candidate solutions based on the surface tension coefficient and the density of the candidate solution comprises: For each candidate solution, calculate the product of the surface tension coefficient of the interface corresponding to the candidate solution and the corresponding interface length to obtain the surface tension corresponding to the candidate solution; Calculating the product of the density of the candidate solution, the gravitational acceleration, and the preset volume to obtain the gravity of the candidate solution; The candidate solution having the largest ratio of the difference between the surface tension and the gravity of the candidate solution to the surface area of ​​the first liquid in the first liquid reservoir is determined to be the first liquid.

7. A microfluidic chip, characterized in that: It includes a substrate, a first liquid reservoir, a second liquid reservoir, a microchannel and a droplet outlet arranged on the substrate; the first liquid reservoir and the second liquid reservoir are both connected to the microchannel to form microdroplets in the microchannel, and the microdroplets are discharged through the droplet outlet.

8. The microfluidic chip according to claim 7, characterized in that: The substrate comprises a first carrier plate and a second carrier plate, the first carrier plate is provided with the first liquid reservoir, the second liquid reservoir and the microfluidic channel, the second carrier plate is connected to the first carrier plate; the droplet outlet is provided on the second carrier plate.

9. The microfluidic chip according to claim 8, characterized in that: The microfluidic channel comprises a dispersed phase microfluidic channel, a continuous phase microfluidic channel and an emulsified microfluidic channel; The dispersed phase microchannel is communicated with the first liquid storage tank and the emulsification microchannel respectively; The continuous phase microchannel is respectively connected to the second liquid reservoir and the emulsification microchannel; The depth of the emulsification microchannel is smaller than the depth of the continuous phase microchannel, so as to achieve step-by-step emulsification of the sample solution; The sample solution after step emulsification flows out from the droplet outlet together with the second liquid in the continuous phase microfluidic channel to form micro droplets.

10. The microfluidic chip according to claim 7, characterized in that: The depth of the dispersed phase microfluidic channel is the same as the depth of the continuous phase microfluidic channel.

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