Dual-mode micro-droplet generation micro-fluidic chip and application thereof in generation of dual-emulsion droplets

Abstract

The invention discloses a dual-mode micro-droplet generation micro-fluidic chip and application thereof in generation of dual-emulsion droplets, and relates to the technical field of micro-fluidic chips. The method comprises the following steps: respectively injecting two different internal phase solutions from the left ends of respective iron tubes, guiding the internal phase solutions to a wedge-shaped tube orifice of a theta-shaped glass capillary tube through respective thin tubes, injecting an intermediate phase solution into a square glass tube through an intermediate phase inlet by using a No.1 needle, and injecting an external phase solution into the square glass tube through an external phase inlet by using a No.2 needle; the two internal phase solutions, the intermediate phase solution and the external phase solution converge at an inlet of the circular glass capillary tube, and after entering the circular glass capillary tube, water-in-oil-in-water double-emulsion droplets are obtained. The invention can obtain the dual-mode micro-droplet generation micro-fluidic chip and the application of the dual-mode micro-droplet generation micro-fluidic chip in generation of dual-emulsion droplets.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, and particularly relates to a dual-mode microdroplet generation microfluidic chip and application thereof in generating double emulsion droplets. BACKGROUND

[0002] Droplet microfluidic technology is a new technology for manipulating micro-volume liquid, and can make micro-nano reactions occur in droplets, and has become an effective means for observing and detecting biological and chemical reaction processes at microscale. Double emulsion droplets are the most widely used and most stable micro-reactors. Double emulsion droplets refer to a unique core-shell structure formed by one or more monodisperse droplets being wrapped in another mutually immiscible droplet. The outer droplet forms a layer of barrier around the inner droplet and between the inner droplets, which more effectively isolates the inner droplet from the external environment, and the reaction can be controlled by controlling the fusion sequence.

[0003] The traditional double emulsion droplet synthesis process is to perform twice emulsification by high-speed stirring. This process is accompanied by a violent stirring process, and the dispersed droplets are not uniform, and the wrapping efficiency is low and the raw material consumption rate is high. The new type of double emulsion droplet generation is realized by microfluidic chip. At present, the mainstream double emulsion droplet microfluidic chip mainly has two types, one is a polydimethylsiloxane (PDMS) microfluidic chip based on soft lithography, and the other is a microfluidic chip based on glass capillary flow focusing method. The PDMS chip has the advantage of being able to make three-dimensional structures, but the channel will swell under the action of organic solvents, affecting the experimental results, and it is not easy to perform local surface treatment, which affects the droplet generation and collection.

[0004] The glass capillary microfluidic chip can be divided into two types according to the different double emulsion droplets generated, one is a microfluidic chip made of θ-shaped glass capillary, which is used for generating double emulsion fusion droplets, and the other is a microfluidic chip in which two smaller outer diameter tubes are inserted into a tapered round glass capillary, which is used for generating double emulsion double core droplets. However, the former can only be used to generate double emulsion fusion droplets, and the latter can only be used to generate double emulsion double core droplets. Moreover, the manufacturing process and chip operation are complex, the cost is high, and the stability is poor during the generation process. SUMMARY

[0005] The present application aims to solve the above technical problems, and provides a dual-mode microdroplet generation microfluidic chip and application thereof in generating double emulsion droplets.

[0006] A dual-mode microdroplet generation microfluidic chip, comprising a glass substrate 14, a theta-shaped glass capillary 4, a square glass tube 7, a circular glass capillary 10, a collection tube 11 and a needle; the theta-shaped glass capillary 4, the square glass tube 7, the circular glass capillary 10 and the hose 11 are all fixed on the upper surface of the glass substrate 14;

[0007] One end of the theta-shaped glass capillary 4 is wedge-shaped, and the inner diameter of the wedge-shaped end is the smallest; a diaphragm 4-3 arranged in the axial direction of the theta-shaped glass capillary 4 divides the inside into two semicircular No. 1 inner phase channels 4-1 and No. 2 inner phase channels 4-2, and the entrances of the No. 1 inner phase channels 4-1 and the No. 2 inner phase channels 4-2 are used as the entrances of two kinds of inner phase solutions;

[0008] One end of the theta-shaped glass capillary 4 extends into the left end of the square glass tube 7; one end of the circular glass capillary 10 is in a tapered structure, and the inner diameter of the tapered structure end is 70-1000 μm; the tapered structure end of the circular glass capillary 10 extends into the right end of the square glass tube 7, and the straight-line distance between the wedge-shaped end of the theta-shaped glass capillary 4 and the tapered structure end of the circular glass capillary 10 is 50-500 μm; the left end of the square glass tube 7 is the middle phase entrance 6, and the right end is the outer phase entrance 8, and both the left end and the right end of the square glass tube 7 are sealed; the other end of the circular glass capillary 10 communicates with the collection tube 11.

[0009] The application of a dual-mode microdroplet generation microfluidic chip in generating double emulsion droplets, the application steps of the dual-mode microdroplet generation microfluidic chip in generating double emulsion droplets are as follows:

[0010] Two different inner phase solutions are respectively injected from the left end of the respective iron pipes 2, guided to the wedge-shaped pipe opening of the theta-shaped glass capillary 4 through the respective capillary tubes 3, and at the same time, the middle phase solution is injected into the square glass tube 7 through the middle phase entrance 6 using the No. 1 needle 5, and the outer phase solution is injected into the square glass tube 7 through the outer phase entrance 8 using the No. 2 needle 9; the two kinds of inner phase solutions, the middle phase solution and the outer phase solution converge at the entrance of the circular glass capillary 10, and after entering the circular glass capillary 10, the water-in-oil-in-water double emulsion droplets are obtained, the shell diameter of the water-in-oil-in-water double emulsion droplets is 100-1000 μm, and the core diameter is 70-900 μm; finally, the collection tube 11 is collected at the outlet.

[0011] The beneficial effects of the application are as follows:

[0012] (1) The microfluidic chip of the present application utilizes the interaction of shear force, viscous force and interfacial tension between fluids to generate droplets. During operation, two different inner phase solutions are guided from two iron pipe inlets to the wedge-shaped theta pipe orifice through a thin tube, and the inner phase fluid is sheared into two water-in-oil droplets by the extrusion of the intermediate phase fluid, and then the intermediate phase fluid is sheared by the outer phase fluid to wrap the inner phase droplets, forming oil-in-water-in-oil double emulsion droplets. By adjusting the operating parameters, double emulsion double core droplets (as shown in Figure 1 ) or double emulsion fusion droplets (as shown in Figure 2 ) can be generated. At the same time, by adjusting the flow rate ratio of the outer phase solution, the intermediate phase solution and the inner phase solution, the size of the overall water-in-oil-in-water droplets and the inner phase droplets can also be controlled, and the thickness of the oil shell wrapping the inner core droplets can also be adjusted.

[0013] (2) In the present application, the orifice of the theta-shaped glass capillary is wedge-shaped, which realizes the effect of generating double emulsion double core droplets and double emulsion fusion droplets in a single microfluidic chip. Without stopping the experiment or replacing the chip, by changing the operating parameters, double emulsion double core or double emulsion fusion droplets can be generated, and by adjusting the flow rate ratio of the outer phase solution, the intermediate phase solution and the inner phase solution, the size of the droplets and the thickness of the oil shell can be controlled, such as increasing the flow rate ratio of the intermediate phase solution while keeping the flow rate of the inner phase solution and the outer phase solution unchanged, which can realize smaller inner core and thicker oil shell of the generated droplets.

[0014] (3) The microfluidic chip of the present application is made of glass, which is overall colorless and transparent, and has good compatibility with various optical systems and detection systems. It can generate double emulsion fusion or double emulsion double core droplets, and the manufacturing process and operation are simple, stable, and the generated droplets are uniform, so it is very suitable for microreactor, drug powder screening, cosmetics, biomedical detection and other applications.

[0015] (4) By wedge-shaped processing of the orifice of the theta-shaped glass capillary, the present application realizes the generation of double emulsion double core and double emulsion fusion droplets without stopping the experiment and replacing the chip, and has low cost, simple manufacturing process and operation, and high stability.

[0016] (5) Compared with the existing double emulsion double core droplet microfluidic chip, the microfluidic chip of the present application has lower manufacturing difficulty, simple operation, improved stability of the microfluidic chip in the droplet generation process and uniformity of the generated droplets.

[0017] The present application can obtain a double-mode microdroplet generation microfluidic chip and its application in generating double emulsion droplets. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1A diagram illustrating the generation process of the dual-emulsion, dual-nuclear droplets in this invention;

[0019] Figure 2 A diagram illustrating the formation process of the dual-emulsion fusion droplets in this invention;

[0020] Figure 3 This diagram illustrates the structure of a dual-mode microdroplet generation microfluidic chip according to the present invention. 1 represents the inner phase inlet, 2 represents the iron tube, 3 represents the thin tube, 4 represents the θ-type glass capillary, 5 represents needle No. 1, 6 represents the intermediate phase inlet, 7 represents the square glass tube, 8 represents the outer phase inlet, 9 represents needle No. 2, 10 represents the circular glass capillary, 11 represents the collection tube, and 14 represents the glass substrate.

[0021] Figure 4 The diagram shows the internal structure of a square glass tube. 4-1 represents the No. 1 internal phase channel, 4-2 represents the No. 2 internal phase channel, 4-3 represents the diaphragm, 4-4 represents the wedge-shaped opening, and 10-1 represents the collection port.

[0022] Figure 5 The diagram illustrates the generation process of the binuclear droplets of the present invention, where 12 represents the intermediate phase solution and 13 represents the external phase solution. Detailed Implementation

[0023] Specific Implementation Method 1: This implementation method is a dual-mode microdroplet generation microfluidic chip, including a glass substrate 14, a theta-shaped glass capillary 4, a square glass tube 7, a circular glass capillary 10, a collection tube 11, and a needle; the theta-shaped glass capillary 4, the square glass tube 7, the circular glass capillary 10, and the tube 11 are all fixed on the upper surface of the glass substrate 14.

[0024] The θ-type glass capillary 4 has a wedge-shaped opening at one end, with the wedge-shaped opening at the end having the smallest inner diameter. The diaphragm 4-3 arranged along the axial direction inside the θ-type glass capillary 4 divides its interior into two semi-circular inner phase channels 1-1 and 2-2. The inlets of inner phase channels 1-1 and 2-2 serve as the inlets for the two inner phase solutions, respectively.

[0025] One end of the θ-shaped glass capillary 4 extends into the square glass tube 7 from the left end; one end of the circular glass capillary 10 has a conical structure with an inner diameter of 70~1000 μm; the conical end of the circular glass capillary 10 extends into the square glass tube 7 from the right end, and the straight-line distance between the wedge-shaped end of the θ-shaped glass capillary 4 and the conical end of the circular glass capillary 10 is 50~500 μm; the left end of the square glass tube 7 is the intermediate phase inlet 6, and the right end is the outer phase inlet 8, and both the left and right ends of the square glass tube 7 are sealed; the other end of the circular glass capillary 10 is connected to the collecting tube 11.

[0026] In this embodiment, the square glass tube 7 is fixed in the middle of the glass substrate 14, serving as the chamber for generating droplets in the microfluidic chip. The θ-shaped glass capillary 4, as a key component of the microfluidic chip, has diaphragms 4-3 that ensure the two encapsulated internal phase solutions do not contact each other before encapsulation, ensuring the accuracy of the reaction structure. Simultaneously, it serves as the inflow channel for the internal phase fluid required for the synthesis of the dual emulsion droplets. One end of the θ-shaped glass capillary 4 is wedge-shaped, acting as the generation port for the microfluidic chip, allowing the two internal phase solutions to flow out from the oblique side, thus generating dual-emulsion dual-core or dual-emulsion fused droplets. One end of the circular glass capillary 10 is tapered, serving as a collection channel for the fluids of each phase. A flexible tube 11 is attached to the rear end as a collection channel, leading the generated dual emulsion droplets out of the microfluidic chip.

[0027] Two iron tubes 2 serve as inlets for the two internal phase solutions to enter the microfluidic chip, and are fixed at the very front of the chip. Two thin tubes 3 extend one end into the iron tubes 2 and the other end into the two channels of the θ-shaped glass capillary 4, acting as guides to direct the liquid entering the iron tubes 2 into the θ-shaped glass capillary 4. Two needles are placed and fixed at both ends of a square glass tube 7, serving as inlets for the intermediate phase solution and the external phase solution to enter the chip, respectively.

[0028] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that the dual-mode microdroplet generation microfluidic chip further includes two thin tubes 3. The right end of one thin tube 3 extends into the inlet of the No. 1 inner phase channel 4-1, and the right end of the other thin tube 3 extends into the inlet of the No. 2 inner phase channel 4-2. The inner diameter of the two thin tubes 3 is 70~300 μm, and the outer diameter is 100~500 μm.

[0029] The other steps are the same as in Specific Implementation Method 1.

[0030] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 or 2 is that the dual-mode microdroplet generation microfluidic chip also includes two iron tubes 2. The left end of one thin tube 3 extends into the right end of one iron tube 2, and the left end of the other thin tube 3 extends into the right end of the other iron tube 2. The right ends of both iron tubes 2 are sealed. The left ends of the two iron tubes 2 serve as inlets for the two internal phase solutions, respectively.

[0031] The other steps are the same as in Specific Implementation Method 1 or 2.

[0032] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Methods One to Three is that the dual-mode microdroplet generation microfluidic chip further includes needles, including needle No. 1 5 and needle No. 2 9; needle No. 1 5 is provided in the intermediate phase inlet 6, and needle No. 2 9 is provided in the outer phase inlet 8.

[0033] The other steps are the same as those in Specific Implementation Methods One to Three.

[0034] Specific Implementation Method 5: The difference between this implementation method and Specific Implementation Methods 1 to 4 is that the inner diameter of the θ-type glass capillary 4 is 0.9~1.17 mm, the outer diameter is 1~1.5 mm, and the thickness of the diaphragm 4-3 is 140~210 μm; the outer surface of the θ-type glass capillary 4 is subjected to silanization hydrophobic treatment before use, and the specific steps are as follows: xylene and octadecyltrichlorosilane are thoroughly mixed to obtain a silanized OTS solution, wherein the mass ratio of xylene to octadecyltrichlorosilane is (95~99):1; the θ-type glass capillary 4 is immersed in the silanized OTS solution for 90~120 s and then removed, dried with nitrogen, and then the θ-type glass capillary 4 is placed on a drying rack at 165~180℃ for 45~60 min.

[0035] The other steps are the same as those in Specific Implementation Methods One through Four.

[0036] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Methods One to Five is that the inner diameter of the circular glass capillary tube 10 is 580~840 μm and the outer diameter is 1~1.5 mm.

[0037] The other steps are the same as those in Specific Implementation Methods 1 to 5.

[0038] Specific Implementation Method Seven: The difference between this implementation method and Specific Implementation Methods One to Six is ​​that the collection tube 11 is a Teflon tube with an inner diameter of 0.96~1.5 mm and an outer diameter of 1.26~1.8 mm.

[0039] The other steps are the same as those in Specific Implementation Methods 1 to 6.

[0040] Specific Implementation Method Eight: This implementation method describes the application of a dual-mode microdroplet generation microfluidic chip in the generation of dual emulsion droplets. The steps for applying the dual-mode microdroplet generation microfluidic chip in the generation of dual emulsion droplets are as follows:

[0041] Two different internal phase solutions are injected from the left end of their respective iron tubes 2 and guided through their respective thin tubes 3 to the wedge-shaped opening of the θ-type glass capillary 4. At the same time, the intermediate phase solution is injected into the square glass tube 7 through the intermediate phase inlet 6 using needle No. 1 5, and the external phase solution is injected into the square glass tube 7 through the external phase inlet 8 using needle No. 2 9. The two internal phase solutions, the intermediate phase solution, and the external phase solution converge at the inlet of the circular glass capillary 10. After entering the circular glass capillary 10, a water-in-oil-in-water double emulsion droplet is obtained. The outer shell diameter of the water-in-oil-in-water double emulsion droplet is 100~1000 μm, and the inner core diameter is 70~900 μm. Finally, it is collected at the outlet of the collection tube 11.

[0042] Specific Implementation Method Nine: The difference between this implementation method and Specific Implementation Method Eight is that the two internal phase solutions are a navy blue pigment solution and a lemon yellow pigment solution, each with a concentration of 1%. The navy blue pigment solution is prepared by mixing navy blue compound coloring agent and deionized water, and the lemon yellow pigment solution is prepared by mixing lemon yellow compound coloring agent and deionized water.

[0043] The preparation steps of the intermediate phase solution are as follows: polymethylsiloxane and silicone oil are mixed at a volume ratio of 3:7 and stirred with a magnetic stirrer for 12-18 h.

[0044] The external phase solution is a 5% polyvinyl alcohol solution by mass. The specific preparation steps are as follows: polyvinyl alcohol powder is added to deionized water and heated and stirred at 65~75℃ for 12~20 h with a magnetic stirrer.

[0045] The other steps are the same as in Specific Implementation Method 8.

[0046] Specific Implementation Method 10: The difference between this implementation method and Specific Implementation Method 8 or 9 is that the water-in-oil-in-water double emulsion droplet is a double emulsion double core droplet or a double emulsion fusion droplet.

[0047] The other steps are the same as in specific implementation method eight or nine.

[0048] The beneficial effects of the present invention are verified using the following embodiments:

[0049] Example 1: A dual-mode microdroplet generation microfluidic chip (e.g.) Figure 3 As shown, it includes a glass substrate 14, a θ-shaped glass capillary 4, a square glass tube 7, a circular glass capillary 10, a collection tube 11, and a needle; the θ-shaped glass capillary 4, the square glass tube 7, the circular glass capillary 10, and the flexible tube 11 are all fixed to the upper surface of the glass substrate 14.

[0050] like Figure 4 As shown, one end of the θ-type glass capillary 4 is wedge-shaped, and the inner diameter of the wedge-shaped opening at the end of the capillary is the smallest; the diaphragm 4-3 arranged along the axial direction inside the θ-type glass capillary 4 divides its interior into two semi-circular inner phase channels 1-1 and 2-2, and the inlets of inner phase channels 1-1 and 2-2 serve as the inlets of the two inner phase solutions, respectively.

[0051] One end of the θ-shaped glass capillary 4 extends into the square glass tube 7 from the left end; one end of the circular glass capillary 10 has a conical structure with an inner diameter of 100~500 μm; the conical end of the circular glass capillary 10 extends into the square glass tube 7 from the right end, and the straight-line distance between the wedge-shaped end of the θ-shaped glass capillary 4 and the conical end of the circular glass capillary 10 is 150~300 μm; the left end of the square glass tube 7 is the intermediate phase inlet 6, and the right end is the outer phase inlet 8, and both the left and right ends of the square glass tube 7 are sealed; the other end of the circular glass capillary 10 is connected to the collecting tube 11.

[0052] The dual-mode microdroplet generation microfluidic chip also includes two thin tubes 3. The right end of one thin tube 3 extends into the inlet of the No. 1 inner phase channel 4-1, and the right end of the other thin tube 3 extends into the inlet of the No. 2 inner phase channel 4-2. The inner diameter of the two thin tubes 3 is 100 μm and the outer diameter is 170 μm.

[0053] The dual-mode microdroplet generation microfluidic chip also includes two iron tubes 2. The left end of one thin tube 3 extends into the right end of one iron tube 2, and the left end of the other thin tube 3 extends into the right end of the other iron tube 2. The right ends of both iron tubes 2 are sealed. The left ends of the two iron tubes 2 serve as inlets for the two internal phase solutions, respectively.

[0054] The dual-mode microdroplet generation microfluidic chip also includes needles, namely needle 1 (5) and needle 2 (9); needle 1 (5) is provided in the intermediate phase inlet 6, and needle 2 (9) is provided in the outer phase inlet 8.

[0055] The glass capillaries are tapered using a theta-shaped glass capillary tube drawing instrument and a needle breaker: the drawing instrument heats and stretches the desired portion of the glass capillary tube and the theta-shaped glass capillary tube into a cone shape; the needle breaker cuts an opening at the top of the tapered theta-shaped glass capillary tube, and then processes the opening into a wedge shape. After tapering the circular glass capillary tube, the cut edge is polished with 2000-grit sandpaper to make the cross-section of the tube opening smooth and flat, and then nitrogen is used to blow away the polishing debris.

[0056] The θ-type glass capillary 4 has an inner diameter of 1.02 mm, an outer diameter of 1.5 mm, and a diaphragm 4-3 thickness of 200 μm. Before use, the θ-type glass capillary 4 undergoes a silanization hydrophobic treatment, the specific steps of which are as follows: 14.85 g of xylene and 0.15 g of octadecyltrichlorosilane are thoroughly mixed to obtain a silanized OTS solution; the θ-type glass capillary 4 is immersed in the silanized OTS solution for 90 s, then removed and dried with nitrogen gas, and then the θ-type glass capillary 4 is heat-treated on a baking rack at 165℃ for 45 min.

[0057] A glass slide measuring 11 cm long, 3 cm wide, and 1.5 mm thick was used as the chip substrate. A 1.5 mm × 1.5 mm square tube was glued to the center of the slide as a channel, and the slide was exposed to ultraviolet light for 10 seconds for curing. The prepared θ-shaped glass capillary and a circular glass capillary were fixed inside the square tube with AB glue, ensuring that the axes of the two capillaries were on the same horizontal line. The slide was left to stand for 30 minutes to allow the AB glue to fully cure. The end of the circular glass capillary was connected to a 15 L Teflon tube with AB glue, and the end of the θ-shaped glass capillary was connected to a thin steel tube with an outer diameter of 1.08 mm with AB glue for the injection of the inner phase solution.

[0058] The packaged microfluidic chip needs to be left to stand for 12 hours before it can be used for the synthesis of dual emulsion droplets.

[0059] The circular glass capillary 10 has an inner diameter of 840 μm and an outer diameter of 1.5 mm.

[0060] The collection tube 11 is a Teflon tube with an inner diameter of 1.5 mm and an outer diameter of 1.8 mm.

[0061] An application of a dual-mode microdroplet generation microfluidic chip in the generation of dual emulsion droplets, the steps of which are as follows:

[0062] Two different internal phase solutions are injected from the left end of their respective iron tubes 2 and guided through their respective thin tubes 3 to the wedge-shaped opening of the θ-type glass capillary 4. At the same time, the intermediate phase solution is injected into the square glass tube 7 through the intermediate phase inlet 6 using needle No. 1 5, and the external phase solution is injected into the square glass tube 7 through the external phase inlet 8 using needle No. 2 9. The two internal phase solutions, the intermediate phase solution, and the external phase solution converge at the inlet of the circular glass capillary 10. After entering the circular glass capillary 10, a water-in-oil-in-water double emulsion droplet is obtained. The outer shell diameter of the water-in-oil-in-water double emulsion droplet is 300~600 μm, and the inner core diameter is 100~300 μm. Finally, it is collected at the outlet of the collection tube 11.

[0063] The water-in-oil-in-water biemulsion droplets are biemulsion binuclear droplets or biemulsion fusion droplets.

[0064] The two internal phase solutions are a 1% solution of navy blue pigment and a 1% solution of lemon yellow pigment. The navy blue pigment solution is prepared by mixing navy blue compound colorant and deionized water, and the lemon yellow pigment solution is prepared by mixing lemon yellow compound colorant and deionized water.

[0065] The preparation steps of the intermediate phase solution are as follows: polymethylsiloxane and silicone oil are mixed at a volume ratio of 3:7 and stirred with a magnetic stirrer for 12-18 h.

[0066] The external phase solution is a 5% polyvinyl alcohol solution by mass. The specific preparation steps are as follows: polyvinyl alcohol powder is added to deionized water and heated and stirred at 65~75℃ for 12~20 h with a magnetic stirrer.

[0067] The microfluidic chip in this embodiment utilizes the interaction of shear force, viscosity, and interfacial tension between fluids to generate droplets. During the generation of the biemulsion binuclear droplet, two inner-phase solutions flow into the θ-shaped glass capillary 4 and flow out obliquely to both sides at the wedge-shaped opening. The shear stress of the intermediate phase solution along the oblique direction of the wedge-shaped opening overcomes the surface tension of the inner-phase solution, causing the inner-phase solution to form two droplets. The two inner-phase droplets continue to flow forward under the encapsulation of the intermediate phase. At this time, the outer-phase solution generates a vertical shear stress on the intermediate phase solution and overcomes the surface tension of the intermediate phase solution, forming a biemulsion binuclear droplet encapsulating two droplets.

[0068] Preparation of biemulsion fusion droplets: Based on the above operation process for generating biemulsion binuclear droplets, while keeping the flow rates of the outer phase solution and the intermediate phase solution constant, the flow rate of the inner phase solution is increased, so that the inner phase solution, which is squeezed by the intermediate phase solution, breaks at the front end of the wedge-shaped opening to form droplets, and then the outer phase solution shears the intermediate phase solution to form biemulsion fusion droplets (as shown in Figure 2).

[0069] The dual-emulsion fusion droplets prepared in this embodiment can be applied to the detection of tomato yellow leaf curl virus. Based on the high throughput of microfluidics, each dual-emulsion droplet can be generated as a miniature solution reactor. By statistically analyzing the proportion of positive droplets, absolute quantification independent of a standard curve can be achieved, greatly improving quantitative accuracy and detection sensitivity, theoretically reaching the single-molecule level. Furthermore, this detection and statistical method can effectively prevent aerosol contamination of amplification products, significantly reducing the cost of expensive reagents.

[0070] Enzyme-catalyzed reactions are an important method for compound synthesis and biomarker detection, mimicking synthetic strategies in biological organisms. High-precision detection requires analysis of large-sample microreactions, which conventional microreaction platforms struggle to meet. This paper presents a glucase-catalyzed reaction experiment using a dual-emulsion, dual-nuclear droplet as a microreactor, based on Trinder's reaction, ultimately achieving large-sample analysis. Therefore, the dual-emulsion, dual-nuclear droplets and dual-emulsion fusion droplets prepared in this embodiment have been successfully applied in their respective fields, overcoming long-standing technical challenges and significantly improving the efficiency and accuracy of detection and analysis.

Claims

1. A dual-mode microdroplet generation microfluidic chip, characterized in that, The dual-mode microdroplet generation microfluidic chip includes a glass substrate (14), a theta-shaped glass capillary (4), a square glass tube (7), a circular glass capillary (10), a collection tube (11), and a needle; the theta-shaped glass capillary (4), the square glass tube (7), the circular glass capillary (10), and the flexible tube (11) are all fixed on the upper surface of the glass substrate (14); The θ-type glass capillary (4) has a wedge-shaped opening at one end, and the wedge-shaped opening at the end of the capillary has the smallest inner diameter. The diaphragm (4-3) arranged along the axial direction inside the θ-type glass capillary (4) divides its interior into two semi-circular inner phase channels 1 (4-1) and 2 (4-2). The inlets of inner phase channels 1 (4-1) and 2 (4-2) serve as the inlets of the two inner phase solutions, respectively. One end of the θ-type glass capillary (4) extends into the left end of the square glass tube (7); one end of the circular glass capillary (10) is a conical structure with an inner diameter of 70~1000 μm; the conical end of the circular glass capillary (10) extends into the right end of the square glass tube (7), and the straight distance between the wedge-shaped end of the θ-type glass capillary (4) and the conical end of the circular glass capillary (10) is 50~500 μm; the left end of the square glass tube (7) is the intermediate phase inlet (6), and the right end is the outer phase inlet (8), and both the left and right ends of the square glass tube (7) are sealed; the other end of the circular glass capillary (10) is connected to the collection tube (11).

2. The dual-mode microdroplet generation microfluidic chip according to claim 1, characterized in that, The dual-mode microdroplet generation microfluidic chip also includes two thin tubes (3). The right end of one thin tube (3) extends into the inlet of the No. 1 inner phase channel (4-1), and the right end of the other thin tube (3) extends into the inlet of the No. 2 inner phase channel (4-2). The inner diameter of the two thin tubes (3) is 70~300 μm and the outer diameter is 100~500 μm.

3. A dual-mode microdroplet generation microfluidic chip according to claim 1 or 2, characterized in that, The dual-mode microdroplet generation microfluidic chip also includes two iron tubes (2). The left end of one thin tube (3) extends into the right end of one iron tube (2), and the left end of the other thin tube (3) extends into the right end of the other iron tube (2). The right ends of both iron tubes (2) are sealed. The left ends of the two iron tubes (2) serve as inlets for the two internal phase solutions, respectively.

4. A dual-mode microdroplet generation microfluidic chip according to claim 1, characterized in that, The dual-mode microdroplet generation microfluidic chip also includes needles, which include needle 1 (5) and needle 2 (9); needle 1 (5) is provided in the intermediate phase inlet (6), and needle 2 (9) is provided in the outer phase inlet (8).

5. A dual-mode microdroplet generation microfluidic chip according to claim 1, characterized in that, The inner diameter of the θ-type glass capillary (4) is 0.9~1.17 mm, the outer diameter is 1~1.5 mm, and the thickness of the diaphragm (4-3) is 140~210 μm. The outer surface of the θ-type glass capillary (4) is subjected to silanization hydrophobic treatment before use. The specific steps are as follows: xylene and octadecyltrichlorosilane are thoroughly mixed to obtain a silanized OTS solution. The mass ratio of xylene to octadecyltrichlorosilane is (95~99):

1. The θ-type glass capillary (4) is immersed in the silanized OTS solution for 90~120 s and then taken out and dried with nitrogen. Then the θ-type glass capillary (4) is placed on a drying rack at 165~180℃ for 45~60 min.

6. A dual-mode microdroplet generation microfluidic chip according to claim 1, characterized in that, The inner diameter of the circular glass capillary (10) is 580~840 μm and the outer diameter is 1~1.5 mm.

7. A dual-mode microdroplet generation microfluidic chip according to claim 1, characterized in that, The collection tube (11) is a Teflon tube with an inner diameter of 0.96~1.5 mm and an outer diameter of 1.26~1.8 mm.

8. The application of a dual-mode microdroplet generation microfluidic chip as described in any one of claims 1-7 in the generation of dual emulsion droplets, characterized in that, The application steps of the dual-mode microdroplet generation microfluidic chip in generating dual emulsion droplets are as follows: Two different internal phase solutions are injected from the left end of their respective iron tubes (2) and guided through their respective thin tubes (3) to the wedge-shaped opening of the θ-type glass capillary (4). At the same time, the intermediate phase solution is injected into the square glass tube (7) through the intermediate phase inlet (6) using needle No. 1 (5), and the external phase solution is injected into the square glass tube (7) through the external phase inlet (8) using needle No. 2 (9). The two internal phase solutions, the intermediate phase solution and the external phase solution converge at the inlet of the circular glass capillary (10). After entering the circular glass capillary (10), a water-in-oil-in-water double emulsion droplet is obtained. The outer shell diameter of the water-in-oil-in-water double emulsion droplet is 100~1000 μm and the inner core diameter is 70~900 μm. Finally, it is collected at the outlet of the collection tube (11).

9. The application of a dual-mode microdroplet generation microfluidic chip according to claim 8 in the generation of dual emulsion droplets, characterized in that, The two internal phase solutions are a 1% solution of navy blue pigment and a 1% solution of lemon yellow pigment. The navy blue pigment solution is prepared by mixing navy blue compound colorant and deionized water, and the lemon yellow pigment solution is prepared by mixing lemon yellow compound colorant and deionized water. The preparation steps of the intermediate phase solution are as follows: polymethylsiloxane and silicone oil are mixed at a volume ratio of 3:7 and stirred with a magnetic stirrer for 12-18 h. The external phase solution is a 5% polyvinyl alcohol solution by mass. The specific preparation steps are as follows: polyvinyl alcohol powder is added to deionized water and heated and stirred at 65~75℃ for 12~20 h with a magnetic stirrer.

10. The application of a dual-mode microdroplet generation microfluidic chip according to claim 8 in the generation of dual emulsion droplets, characterized in that, The water-in-oil-in-water biemulsion droplets are biemulsion binuclear droplets or biemulsion fusion droplets.

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