A double-emulsion high-throughput microfluidic chip and its preparation method

Abstract

The present invention provides a double-emulsion high-throughput microfluidic chip and a preparation method thereof, wherein the chip comprises a substrate, an intermediate layer, and a cover plate. The present invention chooses not to process the substrate, but instead processes the main microchannels on both sides of the intermediate layer, and punches holes in the cover plate to facilitate the passage of fluid. A plurality of droplet generation units based on flow focusing technology are designed on one side of the intermediate layer to achieve the first generation of droplets, while a stepped droplet generation structure based on interfacial tension is designed on the other side to achieve the second generation of droplets. The structures on both sides of the chip are connected by through holes. The preparation method comprises performing photolithography to form a mold, using the mold to prepare an intermediate layer and forming the required microchannels on both sides of the intermediate layer, punching holes in the cover plate, and finally bonding the substrate, intermediate layer, and cover plate. The present invention can achieve rapid and large-scale preparation of double-emulsion droplets through flow channel and structural design, improves production efficiency, and is suitable for various applications such as drug delivery and biological research.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a preparation method and application of a double-emulsification high-throughput microfluidic chip. Background Art

[0002] Double emulsion technology, also known as multiple emulsion technology, is a technique for forming an emulsion within another emulsion. This technology is widely used in fields such as food, medicine, cosmetics, and materials science. Its core is the formation of complex emulsion systems, such as water-in-oil-in-water (w / o / w) and oil-in-water-in-oil (o / w / o). Double emulsion systems are achieved through two emulsification steps: a primary emulsion generates primary droplets, and a secondary emulsification encapsulates these primary droplets within secondary droplets, forming a multi-structured emulsion.

[0003] Traditional double emulsion methods rely primarily on high-speed stirring and phase inversion techniques. High-speed stirring uses high-shear equipment, such as a homogenizer or ultrasonic processor, to mix two immiscible liquids and form an emulsion. Phase inversion methods use a system with varying temperature or composition to cause a phase transition in an otherwise stable emulsion, thereby forming multiple emulsions. However, these methods often struggle to precisely control the size and distribution of droplets, and their production efficiency is low. Furthermore, the high-intensity mechanical action can damage sensitive substances, such as proteins or certain drug molecules.

[0004] In recent years, the rapid development of microfluidics has brought new opportunities for double emulsion technology. Microfluidics is a technology that manipulates and processes fluids at the micron scale. It can achieve precise generation of droplets and high-throughput production by designing precise flow channel structures and controlling fluid flow rates. In the development of microfluidics, double emulsion technology has shown great potential in applications such as droplet generation, drug delivery, and cell encapsulation. For example, in drug delivery, microfluidic double emulsion technology can prepare multilayer microcapsules with precise size and narrow distribution. These microcapsules can effectively encapsulate water-soluble and oil-soluble drugs. In the field of cell research, microfluidic double emulsion technology can be used to prepare artificial cells or tissues, providing new tools for tissue engineering and regenerative medicine. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation and application of a double-emulsion high-throughput microfluidic chip to solve the problems of stability and long-term stability, low production efficiency and high cost in the prior art.

[0006] In a first aspect, to solve the above technical problems, the present invention provides a double-emulsification high-throughput microfluidic chip, comprising: a substrate, an intermediate layer, and a cover plate;

[0007] The intermediate layer is installed between the substrate and the cover plate, and the intermediate layer is provided with a micro-channel for generating and transporting droplets;

[0008] A plurality of droplet generation units based on flow focusing technology are provided on one side of the intermediate layer for realizing the generation of the first droplets;

[0009] On the other side of the intermediate layer, a stepped droplet generation structure based on interfacial tension is provided to achieve a second droplet generation process, forming double-emulsion droplets. To minimize the impact of droplets generated by the stepped structure at the front end on droplets generated by the stepped structure at the rear end during the second droplet generation process, the entire stepped structure flow channel is divided into two parts, and two dispersed phase inlets and double-emulsion droplet outlets are provided. If too many dispersed phase inlets and double-emulsion droplet outlets are provided, more equipment for pumping liquids is required, increasing operational difficulty, experimental complexity, and the possibility of error.

[0010] Furthermore, the structures on both sides of the intermediate layer are connected by through holes, so that the droplets flow from one side to the other side for double emulsification processing;

[0011] The diameter of the through hole is slightly larger than the width of the flow channel to ensure that the droplets can pass through smoothly without breaking or merging. At the same time, the diameter should be kept within the width of the two flow channels to ensure that the droplets do not pass through in parallel.

[0012] Furthermore, the cover plate is provided with a plurality of holes for introducing different fluids, thereby realizing the multifunctional application of the microfluidic chip;

[0013] The positions of these holes correspond to the positions where the liquid flows into the middle layer flow channel. The hole diameter is larger than the width of the flow channel to ensure that the fluid can enter the microchannel evenly without affecting the overall structural strength of the chip.

[0014] Furthermore, the substrate, the middle layer and the cover plate are assembled through a bonding process to form a complete double-emulsion high-throughput microfluidic chip.

[0015] The bonding process may be plasma activated bonding, thermal bonding or UV light curing. The selected bonding method needs to ensure close bonding between the various parts of the chip while not affecting the structure and function of the microchannel.

[0016] Furthermore, the substrate is not processed, and all microchannels are processed on both sides of the middle layer to reduce errors during chip bonding.

[0017] This design approach can simplify the manufacturing process, improve chip consistency and reliability, and also facilitate subsequent quality control and mass production.

[0018] Furthermore, the intermediate layer adopts specific materials and processing technology to ensure stability and efficiency during the droplet generation process.

[0019] The specific material may be a material having good optical transparency, chemical stability and biocompatibility, such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA) or epoxy resin.

[0020] Fabrication techniques can include soft lithography, micromolding, or 3D printing, which can accurately replicate the complex structures of microfluidic channels.

[0021] Preferably, the double-emulsion high-throughput microfluidic chip can rapidly and massively prepare double-emulsion droplets, such as o / w / o, w / o / w, etc., through the flow channel and structural design, thereby greatly improving production efficiency.

[0022] The flow channel design utilizes capillary phenomena, shear force, interfacial tension, etc. to achieve stable droplet generation and control.

[0023] Preferably, the double-emulsion high-throughput microfluidic chip is suitable for various applications such as drug delivery and biological research.

[0024] In drug delivery, double emulsion droplets can be used as drug carriers to improve drug stability and bioavailability. In biological research, they can be used in single-cell analysis, protein crystallization, and enzymology research.

[0025] In a second aspect, based on the same inventive concept, the present invention also provides a method for preparing a double-emulsification high-throughput microfluidic chip, comprising the following steps:

[0026] The first step is to perform photolithography to form a mold. The photolithography process includes steps such as mask design, photoresist coating, exposure and development. The parameters of each step need to be precisely controlled to ensure the accuracy of the mold. Two molds need to be prepared, corresponding to the flow channels on both sides of the middle layer.

[0027] The second step is to use a mold to prepare an intermediate layer and form the required microfluidic channels on both sides of the intermediate layer. The preparation process requires aligning the two molds and placing them in a suitable container. Liquid PDMS is then poured into the molds. After degassing and thermal curing, an intermediate layer with a microfluidic channel structure is obtained.

[0028] The third step is to punch holes in the cover plate to allow fluid to flow in and out. The punching process can use methods such as laser cutting or mechanical drilling, and the position and size of the holes need to be controlled to ensure precise docking with the microchannel.

[0029] The fourth step is to bond the substrate, the middle layer, and the cover plate to obtain the final double-emulsion high-throughput microfluidic chip. The bonding process needs to be carried out in a clean environment to prevent dust and other impurities from affecting the performance of the chip.

[0030] Compared with the prior art, the present invention has the following beneficial effects: through the flow focusing structure design of the first emulsification and the stepped structure design of the second emulsification, the generation of a majority of double-emulsion droplets can be achieved; this intermediate layer double-sided design flow channel, compared with multi-layer flow channel bonding, simplifies the production process and reduces the error caused by chip bonding; through this structural design, the chip area is smaller, and more flow channels can be integrated on the same area, making the production efficiency higher, realizing the rapid preparation of a large number of double-emulsion droplets, greatly improving production efficiency, and being suitable for a variety of application fields.

[0031] In addition, the double-emulsion high-throughput microfluidic chip of the present invention also has the following advantages: High integration: the generation, processing and collection of double-emulsion droplets are realized on one chip, which reduces sample transfer and operation steps and reduces experimental errors. Strong controllability: by adjusting the flow rate of the fluid and the geometric parameters of the flow channel, the size, monodispersity and composition of the generated droplets can be precisely controlled. Saving reagents: compared with traditional batch preparation methods, microfluidic technology can significantly reduce the consumption of reagents and is particularly suitable for the processing of precious or expensive samples. High throughput: the parallel operation of multiple droplet generation units enables the chip to generate a large number of double-emulsion droplets in a short time, meeting the needs of high-throughput screening. Flexibility: by changing the type and proportion of the injected fluid, double-emulsion droplets of different types and compositions can be easily prepared to meet diverse experimental needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0033] Figure 1 A schematic diagram of the three-dimensional structure of a double-emulsification high-throughput microfluidic chip provided in an embodiment of the present invention;

[0034] Figure 2 for Figure 1 The double-emulsification high-throughput microfluidic chip is shown as a top 45° exploded view;

[0035] Figure 3 for Figure 1 The double-emulsification high-throughput microfluidic chip is shown in a 45° exploded view from above;

[0036] Figure 4 for Figure 1 The front view of the middle layer of the double-emulsification high-throughput microfluidic chip shown;

[0037] Figure 5 for Figure 4 A partial enlarged view of the interfacial tension-based stepwise generation of droplet structures in the middle layer of the double-emulsification high-throughput microfluidic chip shown;

[0038] Figure 6 for Figure 1 The rear view of the middle layer of the double-emulsification high-throughput microfluidic chip is shown;

[0039] Figure 7 for Figure 6 A partial enlarged view of the structure of the droplet generation unit based on flow focusing technology in the middle layer of the double-emulsification high-throughput microfluidic chip shown;

[0040] Figure 8 for Figure 1 The side view of the middle layer of the double-emulsification high-throughput microfluidic chip is shown;

[0041] Figure 9 A schematic diagram of the three-dimensional structure of the flow channel of a double-emulsification high-throughput microfluidic chip provided by an embodiment of the present invention;

[0042] Figure 10 for Figure 1 The preparation flow chart of mold 1 for the middle layer of the double-emulsification high-throughput microfluidic chip is shown;

[0043] Figure 11 for Figure 1 The preparation flow chart of mold 2 for the middle layer of the double-emulsification high-throughput microfluidic chip is shown;

[0044] Figure 12 for Figure 1 The flowchart for preparing the middle layer of the double-emulsification high-throughput microfluidic chip is shown.

[0045] Figure 1: 1-cover plate; 101-cover plate outlet 1; 102-cover plate inlet 1; 103-cover plate inlet 2; 104-cover plate inlet 3; 105-cover plate inlet 4; 106-cover plate outlet 2; 2-middle layer; 201-middle layer outlet 1; 202-middle layer inlet 1; 203-middle layer inlet 2; 204-middle layer inlet 3; 205-middle layer inlet 4; 206-middle layer outlet 2; 3-substrate; 4-stepped droplet generation structure based on interfacial tension; 401-inlet for the second generation of droplets; 402-dispersed phase flow of the second generation of droplets Channel; 403-trumpet-mouth structure; 404-continuous phase flow channel for the second generation of droplets; 5-droplet generation unit structure based on flow focusing technology; 501-dispersed phase flow channel for the first generation of droplets; 502-continuous phase flow channel for the first generation of droplets; 503-flow focusing structure; 504-outlet for the first generation of droplets; 6-vertical connection flow channel; 7-silicon wafer; 8-photoresist; 9-X-ray; 10-mask plate one; 11-copper; 12-mask plate two; 13-mold one; 14-mask plate three; 15-mask plate four; 16-mold two; 17-PDMS. DETAILED DESCRIPTION

[0046] The technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0047] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0048] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0049] Example 1

[0050] like Figure 1-9 As shown, a double-emulsion high-throughput microfluidic chip provided in this embodiment includes: a cover plate 1, an intermediate layer 2 and a substrate 3; the intermediate layer 2 is installed between the substrate 3 and the cover plate 1, and the intermediate layer 2 is provided with a microchannel for generating and transporting droplets.

[0051] On the basis of the above characteristics, it is further preferred that a plurality of droplet generation units 5 based on flow focusing technology are provided on one side of the intermediate layer 2 to realize the generation of the first droplets; and a stepped droplet generation structure 4 based on interfacial tension is provided on the other side of the intermediate layer 2 to realize the generation of the second droplets to form double emulsion droplets.

[0052] Among them, the structures on both sides of the intermediate layer 2 are connected by a vertical connecting channel 6, which is used for the droplets to flow from the first droplet generation outlet 504 to the second droplet generation inlet 401 for double emulsification treatment; the diameter of the vertical connecting channel 6 is slightly larger than the width of the channel to ensure that the droplets can pass smoothly without breaking or merging, and at the same time the diameter should be kept within the width of the two channels to ensure that the droplets do not pass in parallel.

[0053] More preferably, the cover plate 1 is provided with multiple holes, including cover plate outlet 1 101, cover plate inlet 1 102, cover plate inlet 2 103, cover plate inlet 3 104, cover plate inlet 4 105, and cover plate outlet 2 106, for the passage and discharge of different fluids, realizing the multifunctional application of the microfluidic chip. The location of these holes corresponds to the position of the liquid inflow into the middle layer flow channel. The hole diameter is larger than the width of the flow channel to ensure that the fluid can enter and exit the microfluidic channel evenly without affecting the overall structural strength of the chip.

[0054] Cover plate inlet 102 introduces the dispersed phase liquid of the first droplet generation, i.e., the inner phase liquid of the double emulsion droplets; cover plate inlet 4 105 introduces the continuous phase liquid of the first droplet generation, i.e., the middle phase liquid of the double emulsion droplets, also known as the isolation layer liquid; cover plate inlet 2 103 and cover plate inlet 3 104 introduce the continuous phase liquid of the second droplet generation, i.e., the outer phase liquid of the double emulsion droplets; and cover plate outlet 101 and cover plate outlet 2 106 discharge the double emulsion droplets. Cover plate inlet 2 103 can interchangeably function with cover plate outlet 101, and cover plate inlet 3 104 can interchangeably function with cover plate outlet 2 106. Four different usage modes are possible depending on the permutation and combination.

[0055] The droplet generation unit 5 based on flow focusing technology includes: a dispersed phase flow channel 501 for the first generation of droplets, a continuous phase flow channel 502 for the first generation of droplets, a flow focusing structure 503, and an outlet 504 for the first generation of droplets. The step-by-step droplet generation structure 4 based on interfacial tension includes: an inlet 401 for the second generation of droplets, a dispersed phase flow channel 402 for the second generation of droplets, a bell-mouth structure 403, and a continuous phase flow channel 404 for the second generation of droplets.

[0056] When the chip is in use, the dispersed phase and continuous phase liquids of the first droplet generation enter the middle layer inlet 1 202 and the middle layer inlet 205 through the cover plate inlet 1 102 and the cover plate inlet 4 105 respectively, and then flow into the dispersed phase flow channel 501 and the continuous phase flow channel 502 for the first droplet generation, forming the first droplet at the flow focusing structure 503, and flowing out from 504, entering the inlet 401 for the second droplet generation through the vertical connecting flow channel 6, and then flowing into the dispersed phase flow channel 402 for the second droplet generation, and meeting the continuous phase liquid flowing in from the continuous phase flow channel 404 for the second droplet generation at the trumpet structure 403 to form the second droplet, realizing the double emulsification process.

[0057] Furthermore, the substrate 3, the intermediate layer 2, and the cover plate 1 are assembled through a specific bonding process to form a complete double-emulsion high-throughput microfluidic chip. The bonding process can be plasma activated bonding, thermal bonding, or UV light curing. The selected bonding method should ensure a tight bond between the various components of the chip while not affecting the structure and function of the microfluidic channels.

[0058] It is worth noting that the substrate 3 is not machined, and all microfluidic channels are machined on both sides of the intermediate layer 2 to reduce errors during chip bonding. This design method can simplify the manufacturing process, improve the consistency and reliability of the chip, and also facilitate subsequent quality control and mass production.

[0059] In this embodiment, the intermediate layer 2 is made of polydimethylsiloxane (PDMS) and processed using soft lithography. PDMS has excellent optical transparency, chemical stability, and biocompatibility, making it suitable for the fabrication of microfluidic chips. Soft lithography can accurately replicate the complex structure of microchannels, ensuring stability and efficiency during droplet generation.

[0060] The double-emulsion high-throughput microfluidic chip of this embodiment, through its flow channel and structural design, can rapidly and massively produce double-emulsion droplets, such as o / w / o and w / o / w, significantly improving production efficiency. The flow channel design utilizes capillary phenomena, shear forces, and interfacial tension to achieve stable droplet generation and control.

[0061] Furthermore, the double-emulsion high-throughput microfluidic chip of this embodiment is suitable for a variety of applications, including drug delivery and biological research. In drug delivery, double-emulsion droplets can serve as drug carriers, improving drug stability and bioavailability. In biological research, it can be used in fields such as single-cell analysis, protein crystallization, and enzymology research.

[0062] Example 2

[0063] An embodiment of the present invention provides a method for preparing a double-emulsion high-throughput microfluidic chip, which mainly includes chip mold preparation and chip preparation.

[0064] The steps for preparing chip mold 1 are as follows:

[0065] Step 1: Clean the silicon wafer 7 with ethanol, deionized water, etc. and dry it. Figure 10 As shown in a;

[0066] Step 2: Place the silicon wafer on the coating machine and apply photoresist 8. Since the thickness of the spin-coated photoresist is large, it can be spin-coated in multiple steps. After each spin coating, it is necessary to obtain Figure 10 b;

[0067] Step 3: Use mask 10 to align the silicon wafer 7 coated with photoresist 8 and expose it with X-rays, as shown in the following example: Figure 10 As shown in c;

[0068] Step 4: placing the exposed silicon wafer 7 on a heating table for post-baking to further cure the photoresist;

[0069] Step 5: Immerse the silicon wafer 7 in a developer for development. The area irradiated by the X-rays 9 undergoes a chemical reaction and is more easily dissolved in the developer. The pattern after development is as follows: Figure 10 As shown in d;

[0070] Step 6: Add copper 11 as a seed layer, such as Figure 10 As shown in e;

[0071] Step 7: Electroplating is performed on the seed layer to fill the gaps in the photoresist with copper 11 to form the desired metal structure, such as Figure 10 f shown;

[0072] Step 8: After the electroplating in step 7 is completed, spin-coat the appropriate photoresist 8 and perform pre-baking to solidify the photoresist and prepare for the second layer structure processing, such as Figure 10 As shown in g;

[0073] Step 9: Replace mask plate 10 with mask plate 2 12 and repeat steps 3-7. Figure 10 As shown in hk;

[0074] Step 10: Remove excess photoresist and seed layer to finally obtain mold 13, as shown in FIG. Figure 10 l shown.

[0075] The steps for preparing chip mold 2 are similar to those for preparing chip mold 1. Mask plate 3 14 and mask plate 4 15 are required to replace mask plate 10 and mask plate 2 12, and the thickness of the photoresist is changed according to the depth of the flow channel. The steps are as follows: Figure 11 As shown in al, the mold 2 16 is finally obtained.

[0076] The steps for preparing the chip are as follows:

[0077] Step 1: Align mold 13 and mold 2 16, as shown in the following example: Figure 12 As shown in a, place in a suitable container;

[0078] Step 2: Mix PDMS and curing agent in a ratio of 1:10, and pour the mixed PDMS17 into the space between the two molds several times. Each time you pour PDMS17, you need to place it in a vacuum drying oven to remove bubbles, such as Figure 12 As shown in b;

[0079] Step 3: Place the mold filled with PDMS17 and after removing bubbles on a heating table and bake at 80°C for 3-4 hours to solidify the PDMS17.

[0080] Step 4: Separate the mold 13, mold 2 16 and the cured PDMS 17, as shown in FIG. Figure 12 As shown in c;

[0081] The double-emulsion high-throughput microfluidic chip can be prepared by the above steps.

[0082] This embodiment provides a method for performing enzymology research using the double-emulsification high-throughput microfluidic chip, comprising the following steps:

[0083] (1) Preparation of enzyme and substrate solutions

[0084] Select an appropriate enzyme and corresponding substrate and prepare them into aqueous solutions of appropriate concentrations. For example, consider β-galactosidase as the enzyme and luciferin di-β-D-galactoside as the substrate, preparing solutions at 1 mg / mL and 0.5 mM, respectively. These solutions will serve as the internal aqueous phase.

[0085] (2) Prepare the oil phase

[0086] Select an oil phase with good biocompatibility, such as perfluoropolyether (HFE-7500), which will serve as the isolation layer.

[0087] (3) Prepare the external aqueous phase

[0088] Prepare an aqueous solution containing a surfactant, such as a 0.5% (w / v) aqueous solution of polyoxyethylene (20) sorbitan monooleate (Tween 80). This will serve as the external aqueous phase.

[0089] (4) Chip operation

[0090] Connect the prepared inner aqueous phase, oil phase, and outer aqueous phase to the corresponding inlets of the chip. Adjust the flow rate ratio of each phase to keep it within the appropriate range.

[0091] (5) Generation of double emulsion droplets

[0092] The syringe pump is started, and w / o single emulsion droplets are first formed through the droplet generation unit based on flow focusing technology in the chip. Then, they pass through the through-holes in the chip and enter the other side, where they form the final w / o / w double emulsion droplets through a stepped droplet generation structure based on interfacial tension.

[0093] (6) Collection and observation

[0094] The generated double emulsion droplets were collected into a pre-prepared microplate. The droplets were observed using a fluorescence microscope, and the changes in fluorescence intensity over time were recorded.

[0095] (7) Data Analysis

[0096] By analyzing the fluorescence intensity versus time curve, we can calculate enzyme kinetic parameters, such as the Michaelis constant (Km) and maximum reaction rate (Vmax). We can also compare the changes in kinetic parameters under different conditions (such as pH, temperature, and the presence of inhibitors) to further understand the catalytic mechanism of the enzyme.

[0097] This method leverages the advantages of double-emulsion high-throughput microfluidic chips to enable enzymology studies at the level of a single microreactor. Each double-emulsion droplet can be considered an independent microreactor. The enzyme and substrate in the inner aqueous phase are isolated by the oil phase, preventing mutual interference. The outer aqueous phase facilitates droplet manipulation and detection. This method offers advantages such as low sample consumption, controllable reaction conditions, and high throughput. It enables rapid and efficient enzymology studies, providing a powerful tool for fields such as enzyme engineering and drug screening.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-emulsion high-throughput microfluidic chip, characterized by: The invention comprises a substrate, an intermediate layer and a cover plate, wherein the intermediate layer is installed between the substrate and the cover plate, and the intermediate layer is provided with a micro-channel for generating and transporting droplets; a plurality of droplet generating units based on flow focusing technology are provided on one side of the intermediate layer for generating the first droplets; a stepped droplet generating structure based on interfacial tension is provided on the other side of the intermediate layer for generating the second droplets to form double emulsion droplets; all the stepped structure flow channels are divided into two parts, and two dispersed phase inlets and double emulsion droplet outlets are provided; the structures on both sides of the intermediate layer are connected by through holes , used for droplets to flow from one side to the other side for double emulsification treatment; the substrate is not processed, and all microchannels are processed on both sides of the middle layer; the diameter of the through hole is slightly larger than the width of the channel, and the diameter should be kept within the width of two channels; a plurality of holes are provided on the cover plate for introducing different fluids to realize the multifunctional application of the microfluidic chip; the positions of these holes correspond to the positions of the liquid flowing into the middle layer channel, and the hole diameter is larger than the width of the channel; the substrate, middle layer and cover plate are assembled by a bonding process, and the bonding process is plasma activated bonding, thermal bonding or UV light curing.

2. The double-emulsion high-throughput microfluidic chip according to claim 1, characterized in that: The middle layer is made of polydimethylsiloxane, polymethyl methacrylate or epoxy resin; the processing technology includes soft lithography technology, micro molding or 3D printing.

3. A method for preparing a double-emulsion high-throughput microfluidic chip, characterized in that: The process includes the following steps: first, performing photolithography to form a mold. The photolithography process includes four steps: mask design, photoresist coating, exposure, and development. The parameters of each step need to be precisely controlled to ensure the accuracy of the mold. Two molds need to be prepared, one for each flow channel on both sides of the middle layer; The second step is to use a mold to prepare an intermediate layer and form the desired microfluidic channels on both sides of the intermediate layer. The preparation process requires aligning the two molds and placing them in a suitable container. Liquid polydimethylsiloxane is then poured into the molds. After degassing and thermal curing, the intermediate layer with the microfluidic channel structure is obtained. The third step is to punch holes in the cover plate to allow fluid to flow in and out. The punching process uses laser cutting or mechanical drilling, and the position and size of the holes need to be controlled to ensure precise docking with the microchannel. In the fourth step, the substrate, the middle layer and the cover plate are bonded to obtain the final double-emulsification high-throughput microfluidic chip. The bonding process is carried out in a clean environment to prevent impurities from affecting the performance of the chip.

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