A microfluidic chip, a preparation method and a separation method thereof

Abstract

The application provides a micro-fluidic chip and a preparation method and a separation method thereof. The micro-fluidic chip is formed by bonding a lithium niobate substrate and a PDMS substrate. The lithium niobate substrate is provided with a sound wave generator and a deflection electrode. The PDMS substrate is provided with a micro-fluid channel system and a charging electrode. The micro-fluid channel system comprises a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, a plurality of collection outlets, a dispersed phase conveying channel, a continuous phase conveying channel, a droplet generation channel, a fusion conveying channel and a plurality of sorting channels. The technical scheme of the application can solve the defect that the single cell screening and separation effect is not ideal in the prior art.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to a microfluidic chip and its fabrication and separation methods. Background Technology

[0002] Thanks to the continuous development of microfluidic chip technology, droplet microfluidics, as an important component of the microfluidics field, has developed rapidly from scratch. Droplet microfluidics aims to construct discrete microdroplets using incompatible multiphase fluids. The independent properties of these microdroplets ensure that biochemical reactions take place in a compartmentalized microfluidic environment. Therefore, droplet microfluidics is also known as "digital microfluidics," enabling digitization and programmability, and providing a platform for solving highly challenging research problems in biochemical medicine.

[0003] Given its advantages of low reagent consumption, high uniformity, large specific surface area, and independent controllability, microdroplet technology has become an important experimental platform in biological, chemical, medical, and materials preparation applications. Complex biochemical studies often involve a series of intricate processes such as droplet sample encapsulation, mixing, reaction, and measurement. Precise droplet manipulation technologies, such as droplet sorting, splitting, fusion, capture, and release, will make these complex processes more convenient and simpler. Among these, droplet sorting is the most fundamental technology based on droplet biochemical research.

[0004] Currently, high-throughput methods for generating single-cell droplets based on microdroplet microfluidics rely on precise pressure control of high-precision micropumps and high-precision MEMS-based chip fabrication. The generated single-cell droplets are still stored together in the same container, requiring each droplet to be detected individually through a microfluidic channel. This results in high equipment costs, complex systems, relatively complicated procedures, low automation, low experimental throughput, and long sample preparation times. Since current microdroplet microfluidics require the generation of single droplets before sorting, there is a drawback in the single-cell screening and separation effect, necessitating improvements. Summary of the Invention

[0005] The technical problem solved by the present invention is: how to overcome the defect of unsatisfactory single-cell screening and separation effect in the prior art.

[0006] To address the aforementioned technical problems, the present invention provides a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing. This chip is formed by bonding a lithium niobate substrate to a PDMS substrate. The lithium niobate substrate has an acoustic wave generator and deflection electrodes, while the PDMS substrate has a microfluidic system and charging electrodes. The microfluidic system includes: a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, multiple collection outlets, a dispersed phase transport channel, a continuous phase transport channel, a droplet generation channel, a fusion transport channel, and multiple sorting channels. The acoustic generator is adapted to focus the particles entering the dispersed phase inlet; The dispersed phase transport channel is connected to the dispersed phase inlet, and the continuous phase transport channel is connected to the first continuous phase inlet and the second continuous phase inlet, forming a cross with the dispersed phase transport channel. The droplet generation channel and the charging electrode are connected from the cross. The droplet generation channel is connected to the fusion transport channel, which has a converging end that radially divides into multiple separation channels. The multiple separation channels are respectively connected to the multiple collection outlets. The charging electrode is adapted to charge the droplets in the droplet generation channel; The deflection electrode is adapted to provide a deflection voltage to the fusion transport channel, causing the particles to separate into the separation channel at the converging end.

[0007] Optionally, the acoustic wave generator includes: a first interdigital electrode, a second interdigital electrode, a first reflective grid, and a second reflective grid disposed on the lithium niobate substrate; The first interdigital electrode and the second interdigital electrode each include several pairs of staggered linear electrodes and signal output terminals, and are symmetrically arranged with the central axis of the dispersed phase transport channel as the axis of symmetry; the first reflective grid and the second reflective grid are symmetrically arranged close to the signal input terminals of the first interdigital electrode and the second interdigital electrode, respectively.

[0008] Optionally, the interdigital width of the first interdigital electrode and the second interdigital electrode is 25 micrometers, and the metal strip width of the first reflective grid and the second reflective grid is 25 micrometers.

[0009] Optionally, the interdigitated electrodes are prepared using a Lift-Off process.

[0010] Optionally, the first interdigital electrode, the second interdigital electrode, the first reflective gate, and the second reflective gate are constructed by stacking silicon oxide-aluminum from the bottom layer to the top.

[0011] Optionally, the deflection electrode includes a first L-shaped electrode and a second L-shaped electrode disposed on the lithium niobate substrate, wherein the first L-shaped electrode and the second L-shaped electrode are symmetrically arranged with the central axis of the fusion transport channel as the axis of symmetry.

[0012] Optionally, the included angle between adjacent separation channels is 30°.

[0013] Optionally, the continuous phase transport channel is a rectangular channel with a width of 60 micrometers and a depth of 150 micrometers; The dispersed phase transport channel is a rectangular channel with a width of 100 micrometers and a depth of 150 micrometers. The droplet generation channel includes a first channel and a second channel. The first channel is a channel with unequal width, and the second channel is a channel with equal width. The inlet end of the first channel has a width of 70 micrometers, the outlet end has a width of 30 micrometers, a length of 30 micrometers, and a depth of 150 micrometers. The second channel has a width of 30 micrometers, a depth of 150 micrometers, and a length of 30 micrometers. The outlet end of the first channel is connected to the inlet end of the second channel. The fusion transport channel is a channel with unequal widths. The width of the inlet end of the fusion transport channel is 100 micrometers, the width of the outlet end is 300 micrometers, and the depth is 150 micrometers. The first, second, and third sorting channels are all equal-width channels. The width of the first and third sorting channels is 80 micrometers, the width of the second sorting channel is 120 micrometers, and the depth of each sorting channel is 150 micrometers.

[0014] Optionally, the microfluidic system further includes a deflection channel; the deflection channel is a first U-shaped channel and a second U-shaped channel symmetrically distributed on both sides of the converging end, the U-shaped channel having two channel ports, one channel port being a conductive solution inlet and the other channel port being an exhaust port; when the conductive solution is introduced into the conductive solution inlet, the deflection channel forms a spatial electric field region around the converging end, causing the charged particles to deflect.

[0015] Optionally, the first U-shaped flow channel and the second U-shaped flow channel are rectangular flow channels, with the bottom flow channel having a width of 400 micrometers and a depth of 150 micrometers, and the two side flow channels having a width of 100 micrometers and a depth of 150 micrometers.

[0016] Optionally, the microfluidic chip further includes: a first continuous phase inlet connector, a second continuous phase inlet connector, a dispersed phase inlet connector, and a plurality of collection outlet connectors disposed on the upper surface of the PDMS substrate; the first continuous phase inlet connector, the second continuous phase inlet connector, the dispersed phase inlet connector, and the plurality of collection outlet connectors are coaxially coupled and connected to the first continuous phase inlet, the second continuous phase inlet, the dispersed phase inlet, and the plurality of collection outlets in sequence.

[0017] Optionally, the charging electrode is a strip of conductive metal, with one end of the charging electrode disposed inside the PDMS substrate and the other end not enclosed by the PDMS substrate serving as the signal input terminal of the charging electrode.

[0018] Optionally, the charging electrode is arranged in a direction perpendicular to the transport direction axis of the droplet generation channel within the PDMS substrate, and is 300 micrometers away from the droplet generation channel in this perpendicular direction.

[0019] Optionally, the charging electrode is prepared by a secondary casting process.

[0020] To address the aforementioned technical problems, the present invention also provides a microfluidic chip for electrostatic sorting of droplets based on surface acoustic wave focusing, comprising: a PDMS substrate, wherein a microfluidic system and a charging electrode are disposed on the PDMS substrate, and the microfluidic system comprises: a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, multiple collection outlets, a dispersed phase transport channel, a continuous phase transport channel, a droplet generation channel, a fusion transport channel, and multiple sorting channels; The dispersed phase transport channel is connected to the dispersed phase inlet, and the continuous phase transport channel is connected to the first continuous phase inlet and the second continuous phase inlet, forming a cross with the dispersed phase transport channel. The droplet generation channel and the charging electrode are connected from the cross. The droplet generation channel is connected to the fusion transport channel, which has a converging end that radially divides into multiple separation channels. The multiple separation channels are respectively connected to the multiple collection outlets. The charging electrode is adapted to charge the droplets in the droplet generation channel.

[0021] Optionally, the included angle between adjacent separation channels is 30°.

[0022] Optionally, the continuous phase transport channel is a rectangular channel with a width of 60 micrometers and a depth of 150 micrometers; The dispersed phase transport channel is a rectangular channel with a width of 100 micrometers and a depth of 150 micrometers. The droplet generation channel includes a first channel and a second channel. The first channel is a channel with unequal width, and the second channel is a channel with equal width. The inlet end of the first channel has a width of 70 micrometers, the outlet end has a width of 30 micrometers, a length of 30 micrometers, and a depth of 150 micrometers. The second channel has a width of 30 micrometers, a depth of 150 micrometers, and a length of 30 micrometers. The outlet end of the first channel is connected to the inlet end of the second channel. The fusion transport channel is a channel with unequal widths. The width of the inlet end of the fusion transport channel is 100 micrometers, the width of the outlet end is 300 micrometers, and the depth is 150 micrometers. The first, second, and third sorting channels are all equal-width channels. The width of the first and third sorting channels is 80 micrometers, the width of the second sorting channel is 120 micrometers, and the depth of each sorting channel is 150 micrometers.

[0023] Optionally, the microfluidic system further includes a deflection channel; the deflection channel is a first U-shaped channel and a second U-shaped channel symmetrically distributed on both sides of the converging end, the U-shaped channel having two channel ports, one channel port being a conductive solution inlet and the other channel port being an exhaust port; when the conductive solution is introduced into the conductive solution inlet, the deflection channel forms a spatial electric field region around the converging end, causing the charged particles to deflect.

[0024] Optionally, the first U-shaped flow channel and the second U-shaped flow channel are rectangular flow channels, with the bottom flow channel having a width of 400 micrometers and a depth of 150 micrometers, and the two side flow channels having a width of 100 micrometers and a depth of 150 micrometers.

[0025] Optionally, the microfluidic chip further includes: a first continuous phase inlet connector, a second continuous phase inlet connector, a dispersed phase inlet connector, and a plurality of collection outlet connectors disposed on the upper surface of the PDMS substrate; the first continuous phase inlet connector, the second continuous phase inlet connector, the dispersed phase inlet connector, and the plurality of collection outlet connectors are coaxially coupled and connected to the first continuous phase inlet, the second continuous phase inlet, the dispersed phase inlet, and the plurality of collection outlets in sequence.

[0026] Optionally, the charging electrode is a strip of conductive metal, with one end of the charging electrode disposed inside the PDMS substrate and the other end not enclosed by the PDMS substrate serving as the signal input terminal of the charging electrode.

[0027] Optionally, the charging electrode is arranged in a direction perpendicular to the transport direction axis of the droplet generation channel within the PDMS substrate, and is 300 micrometers away from the droplet generation channel in this perpendicular direction.

[0028] Optionally, the charging electrode is prepared by a secondary casting process.

[0029] To address the aforementioned technical problems, the present invention also provides a method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, comprising: A lithium niobate substrate and a single-crystal silicon wafer were cleaned and subjected to surface hydrophilic treatment. Patterns of interdigitated electrodes and deflection electrodes are fabricated on the lithium niobate substrate; Based on the pattern, thin film metal electrodes of the interdigitated electrode and the deflection electrode are fabricated on the lithium niobate substrate to obtain the lithium niobate substrate; Based on the single-crystal silicon wafer, a chip positive mold is prepared to obtain a microfluidic system positive mold; The male mold of the microfluidic system is subjected to hydrophobic treatment; The prepared PDMS material was cast into the male mold of the microfluidic system to prepare the PDMS microfluidic system; A charging electrode is prepared and placed in the microfluidic system; The microfluidic system of the PDMS is cooled and the microfluidic system is cut and perforated to prepare a PDMS substrate; The lithium niobate substrate is bonded to the PDMS substrate.

[0030] Optionally, the lithium niobate substrate can be cleaned as follows: the lithium niobate substrate is ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 15 minutes respectively, removed and dried with a nitrogen gun, placed on a hot plate at room temperature, slowly heated to 120°C and heated for 30 minutes, and dried to obtain a dry lithium niobate substrate.

[0031] Optionally, the lithium niobate substrate may be subjected to surface hydrophilic treatment by placing it in a plasma cleaner for polishing.

[0032] Optionally, the pattern for fabricating the interdigitated electrodes and deflection electrodes on the lithium niobate substrate includes: Photoresist is injected into the center of the lithium niobate substrate; The lithium niobate substrate with photoresist is placed above the suction cup of the spin coater, and the photoresist is spin-coated using the spin coater to complete the pre-baking of the lithium niobate substrate. The pre-baked lithium niobate substrate is placed under a mask with the desired interdigitated electrode and deflection electrode pattern, and the lithium niobate substrate is photolithographically ... Post-baking is performed on the lithium niobate substrate after photolithography; After post-baking, the lithium niobate substrate is developed at room temperature until no white precipitate appears; Clean and dry the lithium niobate substrate.

[0033] Optionally, the preparation of the thin-film metal electrode of the interdigitated electrode and the deflection electrode on the lithium niobate substrate based on the pattern includes: placing the obtained lithium niobate substrate with the pattern in a magnetron sputtering machine, first sputtering a first layer of silicon oxide film, then sputtering an aluminum film, and finally taking out the coated lithium niobate substrate to obtain the electrode.

[0034] Optionally, the thickness of the first silicon oxide film is 150 nm, and the thickness of the aluminum film is 100 nm.

[0035] Optionally, the preparation method further includes: The lithium niobate substrate with the interdigitated electrode and deflection electrode is left to stand, rinsed and dried to prepare the lithium niobate substrate.

[0036] Optionally, the monocrystalline silicon wafer can be cleaned using the following steps: The single-crystal silicon wafer was placed in a piranha solution and heated; The heated monocrystalline silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 15 minutes each. It was then removed, dried with a nitrogen gun, and placed on a hot plate at 120°C for 30 minutes to ensure that the monocrystalline silicon wafer was completely dry.

[0037] Optionally, the monocrystalline silicon wafer can be hydrophilically treated by the following steps: place the cleaned monocrystalline silicon wafer with the surface facing up in a plasma cleaner to complete the cleaning process.

[0038] Optionally, the fabrication of the chip male model based on the single-crystal silicon wafer includes: Photoresist is injected into the center of a single-crystal silicon wafer, and the silicon wafer with photoresist is placed above the suction cup of a spin coater. The spin coater is then used to spin-coat the photoresist. The silicon wafer is placed on a heating stage for pre-baking, and a mask with a pre-designed flow channel system is placed on the photolithography machine mask holder. Photolithography is used to lithographically shape silicon wafers; After photolithography, the silicon wafer undergoes post-baking and development, is then dried with nitrogen, and finally placed on a heating stage for hard mold baking to obtain the chip positive mold.

[0039] Optionally, the thickness of the chip male mold is 150 μm.

[0040] Optionally, the hydrophobic treatment of the male mold of the microfluidic system includes: The obtained microfluidic system positive mold was placed in a volatilization cylinder. Three drops of perfluorodecyltriethoxysilane were added to the volatilization cylinder. After sealing, the volatilization cylinder was placed in an oven for heating and baking.

[0041] Optionally, the step of casting the prepared PDMS material onto the male mold of the microfluidic system to prepare the PDMS microfluidic system includes: The PDMS prepolymer and curing agent were mixed uniformly at a weight ratio of 10:1. After stirring evenly, the mixture was placed in a vacuum drying oven for degassing for 30 minutes. Then, a certain amount of PDMS was drawn up with a syringe and poured onto the anode template of the microfluidic system. The template was then placed in a vacuum drying oven for degassing for 30 minutes. Finally, the template was placed on a heating table for heating and curing. The PDMS microfluidic system was then prepared.

[0042] Optionally, the PDMS microfluidic system includes: a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, multiple collection outlets, a dispersed phase transport channel, a continuous phase transport channel, a droplet generation channel, a fusion transport channel, and multiple sorting channels; the dispersed phase transport channel is connected to the dispersed phase inlet, the continuous phase transport channel is connected to the first continuous phase inlet and the second continuous phase inlet, and forms a cross with the dispersed phase transport channel, from which the droplet generation channel and the charging electrode are connected; the droplet generation channel is connected to the fusion transport channel, the fusion transport channel has a converging end, the converging end is radially divided into the multiple separation channels, and the multiple separation channels are respectively connected to the multiple collection outlets.

[0043] Optionally, placing the charging electrode in the microfluidic system includes: The upper surface of the microfluidic system is cleaned by blowing it clean. The prepared charging electrode is placed on the upper surface of the microfluidic system, and the tip of the charging electrode is aligned with the cross intersection of the pre-set microfluidic channels under a microscope to obtain the final microfluidic system.

[0044] Optionally, the fabrication of the charging electrode includes: Stainless steel sheets with a thickness of 100μm to 300μm are used as the material for preparing the charging electrode. The stainless steel sheet is placed above a cutting table and cut into stainless steel sheets of a specified shape using a laser. The cut stainless steel sheets are then folded at a set angle along the length direction to obtain the charging electrode.

[0045] Optionally, the set angle is 90°.

[0046] Optionally, cooling the microfluidic system of the PDMS and cutting and punching the microfluidic system includes cutting and punching the microfluidic channels of the microfluidic system based on the corresponding inlets and outlets of the microfluidic channels.

[0047] Optionally, bonding the lithium niobate substrate to the PDMS substrate includes: The lithium niobate substrate and the PDMS substrate were ultrasonically cleaned sequentially using ethanol and deionized water. The lithium niobate substrate and PDMS substrate were placed in a plasma cleaner for oxygen plasma treatment. The lithium niobate substrate and the PDMS substrate are aligned, bonded, and heated to complete the bonding process.

[0048] Optionally, the deflection electrode has a conductive inlet, and the preparation method further includes injecting a conductive buffer solution into the conductive inlet.

[0049] To address the aforementioned technical problems, the present invention also provides a method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, comprising: A single-crystal silicon wafer is cleaned and its surface is treated with hydrophilicity. Based on the single-crystal silicon wafer, a chip positive mold is prepared to obtain a microfluidic system positive mold; The male mold of the microfluidic system is subjected to hydrophobic treatment; The prepared PDMS material was cast into the male mold of the microfluidic system to prepare the PDMS microfluidic system; A charging electrode is prepared and placed in the microfluidic system; The microfluidic system of the PDMS is cooled and then cut and perforated to prepare a PDMS substrate.

[0050] Optionally, the monocrystalline silicon wafer can be cleaned using the following steps: The single-crystal silicon wafer was placed in a piranha solution and heated; The heated monocrystalline silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 15 minutes each. It was then removed, dried with a nitrogen gun, and placed on a hot plate at 120°C for 30 minutes to ensure that the monocrystalline silicon wafer was completely dry.

[0051] Optionally, the monocrystalline silicon wafer can be hydrophilically treated by the following steps: place the cleaned monocrystalline silicon wafer with the surface facing up in a plasma cleaner to complete the cleaning process.

[0052] Optionally, the fabrication of the chip male model based on the single-crystal silicon wafer includes: Photoresist is injected into the center of a single-crystal silicon wafer, and the silicon wafer with photoresist is placed above the suction cup of a spin coater. The spin coater is then used to spin-coat the photoresist. The silicon wafer is placed on a heating stage for pre-baking, and a mask with a pre-designed flow channel system is placed on the photolithography machine mask holder. Photolithography is used to lithographically shape silicon wafers; After photolithography, the silicon wafer undergoes post-baking and development, is then dried with nitrogen, and finally placed on a heating stage for hard mold baking to obtain the chip positive mold.

[0053] Optionally, the thickness of the chip male mold is 150 μm.

[0054] Optionally, the hydrophobic treatment of the male mold of the microfluidic system includes: The obtained microfluidic system positive mold was placed in a volatilization cylinder. Three drops of perfluorodecyltriethoxysilane were added to the volatilization cylinder. After sealing, the volatilization cylinder was placed in an oven for heating and baking.

[0055] Optionally, the step of casting the prepared PDMS material onto the male mold of the microfluidic system to prepare the PDMS microfluidic system includes: The PDMS prepolymer and curing agent were mixed uniformly at a weight ratio of 10:1. After stirring evenly, the mixture was placed in a vacuum drying oven for degassing for 30 minutes. Then, a certain amount of PDMS was drawn up with a syringe and poured onto the anode template of the microfluidic system. The template was then placed in a vacuum drying oven for degassing for 30 minutes. Finally, the template was placed on a heating table for heating and curing. The PDMS microfluidic system was then prepared.

[0056] Optionally, the PDMS microfluidic system includes: a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, multiple collection outlets, a dispersed phase transport channel, a continuous phase transport channel, a droplet generation channel, a fusion transport channel, and multiple sorting channels; the dispersed phase transport channel is connected to the dispersed phase inlet, the continuous phase transport channel is connected to the first continuous phase inlet and the second continuous phase inlet, and forms a cross with the dispersed phase transport channel, from which the droplet generation channel and the charging electrode are connected; the droplet generation channel is connected to the fusion transport channel, the fusion transport channel has a converging end, the converging end is radially divided into the multiple separation channels, and the multiple separation channels are respectively connected to the multiple collection outlets.

[0057] Optionally, placing the charging electrode in the microfluidic system includes: The upper surface of the microfluidic system is cleaned by blowing it clean. The prepared charging electrode is placed on the upper surface of the microfluidic system, and the tip of the charging electrode is aligned with the cross intersection of the pre-set microfluidic channels under a microscope to obtain the final microfluidic system.

[0058] Optionally, the fabrication of the charging electrode includes: Stainless steel sheets with a thickness of 100μm to 300μm are used as the material for preparing the charging electrode. The stainless steel sheet is placed above a cutting table and cut into stainless steel sheets of a specified shape using a laser. The cut stainless steel sheets are then folded at a set angle along the length direction to obtain the charging electrode.

[0059] Optionally, the set angle is 90°.

[0060] Optionally, cooling the microfluidic system of the PDMS and cutting and punching the microfluidic system includes cutting and punching the microfluidic channels of the microfluidic system based on the corresponding inlets and outlets of the microfluidic channels.

[0061] Optionally, the preparation method further includes: The PDMS substrate is bonded to a lithium niobate substrate.

[0062] Optionally, bonding the PDMS substrate to a lithium niobate substrate includes: The lithium niobate substrate and the PDMS substrate were ultrasonically cleaned sequentially using ethanol and deionized water. The lithium niobate substrate and PDMS substrate were placed in a plasma cleaner for oxygen plasma treatment. The lithium niobate substrate and the PDMS substrate are aligned, bonded, and heated to complete the bonding process.

[0063] To address the aforementioned technical problems, the present invention also provides a separation method for a microfluidic chip based on surface acoustic wave focusing for droplet electrostatic sorting, comprising: An electrical signal of a preset frequency is input into the acoustic wave generator of the lithium niobate substrate to generate surface acoustic waves. The particles entering the dispersed phase inlet are focused based on the surface acoustic waves. Two incompatible continuously flowing fluids are introduced into the continuous phase inlet and the dispersed phase inlet, respectively, to compress and destabilize at the cross intersection to form droplets. The droplets in the droplet generation channel are charged based on the charging electrode; The deflection electrode is used to provide a deflection voltage to the fusion transport channel, causing the particles to separate into the separation channel at the converging end, thus completing the deflection and sorting of droplets.

[0064] The beneficial effects of the technical solution of the present invention include at least the following: The microfluidic chip of this invention features an acoustic wave generator and deflection electrode on a lithium niobate substrate, and a microchannel system and charging electrode on a PDMS substrate. The chip as a whole forms a surface acoustic wave focusing region, a droplet generation and charging region, and a droplet deflection region. Through the surface acoustic waves generated by the acoustic wave generator, the droplet microchannel structure, the charging electrode, and the deflection electrode, particles of different sizes are flexibly focused within the microchannels, generating droplets of different diameters to encapsulate the particles. The droplets encapsulating the particles are then charged, thereby controlling the droplet deflection and achieving effective sorting of cells encapsulated within the droplets. This overcomes the shortcomings of existing technologies in terms of unsatisfactory single-cell screening and separation.

[0065] The technical solution of this invention utilizes the non-contact nature of surface acoustic waves to solve the problem of poor separation effect in microfluidic chip technology, and has good biocompatibility. At the same time, it can achieve controllable, stable and continuous deflection for droplet charging, meeting the needs of droplet manipulation technology in fields including biochemical medicine.

[0066] The microfluidic chip prepared by the technical solution of this invention can realize the functions of cell focusing, cell detection, droplet generation, droplet charging, and effective screening and separation of target droplets under electric field control. Compared with the prior art, the technical solution of this invention solves the problem of low efficiency in single-cell sorting of traditional discrete droplet separation methods, thereby improving the screening and separation efficiency. The technical solution of this invention has a simple process flow, low preparation cost, and can be mass-produced. Attached Figure Description

[0067] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 A schematic diagram of a PDMS substrate structure for a droplet electrostatic sorting microfluidic chip based on surface acoustic wave focusing, provided for the technical solution of this invention; Figure 2 A schematic diagram of the structure of the connector on the upper surface of the PDMS substrate for the microfluidic chip provided by the present invention; Figure 3 A schematic diagram of the charging electrode structure provided on the microfluidic chip PDMS substrate for the technical solution of the present invention; Figure 4 A schematic diagram of the structure of a lithium niobate substrate and a PDMS substrate for a droplet electrostatic sorting microfluidic chip based on surface acoustic wave focusing, provided for the technical solution of this invention; Figure 5 A schematic diagram of the structure of the lithium niobate substrate for the microfluidic chip provided by the technical solution of the present invention; Figure 6 A schematic diagram illustrating the working principle of the microfluidic chip provided by the technical solution of this invention; Figure 7 A schematic flowchart of a droplet electrostatic separation microfluidic chip based on surface acoustic wave focusing is provided for the technical solution of this invention. Figure 8 A schematic flowchart of a method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, which is provided for the technical solution of this invention; Figure 9 A schematic diagram of another method for fabricating a microfluidic chip based on surface acoustic wave focusing for droplet electrostatic sorting provided by the present invention. Detailed Implementation

[0068] To better illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings.

[0069] Microfluidics refers to the technology of manipulating fluids at the micrometer scale, with functionality miniaturized to a chip of just a few square centimeters, hence the name "lab-on-a-chip." Microfluidic chips can manipulate fluid flow in tiny channels or structures ranging from tens to hundreds of micrometers. The rapid encapsulation and screening of specific cells within droplets, as well as the large-scale, low-cost, and rapid production of microfluidic chips, are pressing problems that need to be solved.

[0070] Microfluidic chips typically consist of a lithium niobate substrate and a PDMS substrate. The microfluidic chip of this invention achieves rapid liquid sieving by incorporating a pair of interdigitated electrodes and a pair of deflection electrodes on the lithium niobate substrate, and a microfluidic system and charging electrode on the PDMS substrate. This microfluidic chip offers low cost and rapid production capabilities. Furthermore, the microfluidic chip of this invention employs soft photolithography to obtain the microfluidic system, and includes a first continuous phase inlet connector, a second continuous phase inlet connector, a dispersed phase inlet connector, a first collection outlet connector, a second collection outlet connector, and a third collection outlet connector. The lithium niobate substrate and the PDMS substrate are bonded together with high strength through plasma oxidation treatment to obtain the complete chip.

[0071] The microfluidic chip region of this invention can be divided into a surface acoustic wave (SAW) focusing region, a droplet generation and charging region, and a droplet deflection region. This invention utilizes SAW generated by interdigitated electrodes, a droplet microfluidic structure, charging electrodes, and deflection electrodes to flexibly focus particles of different sizes within the microfluidic channel, generating droplets of different diameters to encapsulate the particles. The droplets encapsulating the particles are then charged, thereby controlling the droplet deflection and effectively sorting the cells encapsulated within the droplets. SAW offers non-contact operation and good biocompatibility, enabling controllable, stable, and continuous deflection of the droplets during charging, meeting the needs of droplet manipulation technology in fields including biomedicine.

[0072] Based on the above inventive concept, this embodiment is as follows: Figure 1 The embodiment shows a microfluidic chip 1 for electrostatic sorting of droplets based on surface acoustic wave focusing. In this embodiment, the microfluidic chip 1 can be composed of a PDMS substrate 100. The PDMS substrate 100 has a microchannel system 110 obtained by soft photolithography and a charging electrode for charging the droplets in the channels of the microchannel system 110. Figure 1 (Not shown in the image).

[0073] Specifically, the microfluidic system 110 is located on the lower surface of the substrate 100 and includes: First continuous phase transport channel 111, second continuous phase transport channel 112, dispersed phase transport channel 113, droplet generation channel 114, fusion transport channel 115, first sorting channel 116, second sorting channel 117, and third sorting channel 118.

[0074] The specific connection structure of the microfluidic system 110 is as follows: The first continuous phase transport channel 111 is coaxial with the second continuous phase transport channel 112. The dispersed phase transport channel 113 is perpendicular to both the first and second continuous phase transport channels 111 and 112, respectively. The ends of the dispersed phase transport channel 113, the first continuous phase transport channel 111, and the second continuous phase transport channel 112 intersect and connect. The dispersed phase transport channel 113 is coaxial with the droplet generation channel 114. The inlet end of the dispersed phase transport channel 113 is connected to the outlet end of the first continuous phase transport channel 111 and the second continuous phase transport channel 112, respectively. The outlet ends of the conveying channel 112 are connected; the droplet generating channel 114 and the fusion conveying channel 115 are coaxial, and the outlet end of the droplet generating channel 114 is connected to the inlet end of the fusion conveying channel 115; the outlet end of the fusion conveying channel 115 is connected to the inlet ends of the first sorting channel 116, the second sorting channel 117, and the third sorting channel 118, and the included angles between the first sorting channel 116 and the second sorting channel 117, and between the second sorting channel 117 and the third sorting channel 118 are 30° respectively.

[0075] More specifically: The first continuous phase transport channel 111 and the second continuous phase transport channel 112 are rectangular channels with a width of 60 micrometers and a depth of 150 micrometers. The dispersed phase transport channel 113 is a rectangular channel with a width of 100 micrometers and a depth of 150 micrometers. The droplet generation channel 114 includes a channel of unequal width and a channel of equal width. The unequal width channel has an inlet width of 70 micrometers, an outlet width of 30 micrometers, a length of 30 micrometers, and a depth of 150 micrometers. The equal width channel has a width of 30 micrometers, a depth of 150 micrometers, and a length of 30 micrometers. The outlet end of the unequal width channel is connected to the inlet end of the equal width channel. The fusion transport channel 115 is an unequal width channel with a narrow inlet end and a wide outlet end. The inlet end of the channel has a width of 100 micrometers, the outlet end has a width of 300 micrometers, and the channel depth is 150 micrometers. The first sorting channel 116, the second sorting channel 117, and the third sorting channel 118 are all equal-width channels. The width of the first sorting channel 116 and the third sorting channel 118 is 80 micrometers, the width of the second sorting channel 117 is 120 micrometers, and the depth of the sorting channels is 150 micrometers.

[0076] Continue to refer to Figure 1The microfluidic system 110 further includes: a first continuous phase inlet 121, a second continuous phase inlet 122, a dispersed phase inlet 123, a first collection outlet 124, a second collection outlet 125, and a third collection outlet 126. The specific connection structure is as follows: The inlet end of the first continuous phase conveyor 111 and the inlet end of the second continuous phase conveyor 112 are the first continuous phase inlet 121 and the second continuous phase inlet 122, respectively. The inlet end of the dispersed phase conveyor 113 is the dispersed phase inlet 123. The outlet ends of the first sorting channel 116, the second sorting channel 117 and the third sorting channel 118 are the first collection outlet 124, the second collection outlet 125 and the third collection outlet 126, respectively.

[0077] refer to Figure 1 It is known that the dispersed phase transport channel 113 is connected to the dispersed phase inlet 123, and the continuous phase transport channels (111, 112) are connected to the first continuous phase inlet 121 and the second continuous phase inlet 122. The continuous phase transport channels (111, 112) and the dispersed phase transport channel 113 form a cross, and the droplet generation channel 114 is connected from the cross. The droplet generation channel 114 is connected to the fusion transport channel 115, which has a converging end. The converging end is radially divided into a first separation channel to a third separation channel (116 to 118). The first separation channel to the third separation channel (116 to 118) are respectively connected to the first collection outlet to the third collection outlet (124 to 126).

[0078] The microfluidic system 110 also includes a deflection channel 131.

[0079] Specifically, the deflection channels 131 are a pair of U-shaped channels, symmetrically distributed at equal intervals on both sides of the fusion transport channel 115. The specific assembly structure of the deflection channels 131 is a pair of U-shaped channels open at both ends, with a wider bottom and narrower ends. Each U-shaped channel has two inlets located at the U-shaped ends. Either inlet can serve as an inlet for the conductive solution, while the other serves as an outlet. The deflection channels 131 function as follows: after the conductive solution is introduced into the deflection channels, a conductive electrode of the same height as the channel is formed. When the deflection channels are connected to the deflection electrode, the conductive solution in the deflection channels generates a region of strongest spatial electric field along the height of the channel, thereby effectively deflecting charged particles.

[0080] More specifically, the U-shaped bottom of the deflection channel 131 is a rectangular channel with a width of 400 micrometers and a depth of 150 micrometers, and the two side channels of the U-shape are rectangular channels with a width of 100 micrometers and a depth of 150 micrometers.

[0081] Combination Figure 2The upper surface of the substrate 100 is further provided with: a dispersed phase inlet connector 301, a first continuous phase inlet connector 302, a second continuous phase inlet connector 303, a first collection outlet connector 304, a second collection outlet connector 305, and a third collection outlet connector 306. The specific connection structure is as follows: The first continuous phase inlet connector 302 is coaxially fitted and connected to the first continuous phase inlet 121; the second continuous phase inlet connector 303 is coaxially fitted and connected to the second continuous phase inlet 122; the dispersed phase inlet connector 301 is coaxially fitted and connected to the dispersed phase inlet 123; the first collection outlet connector 304 is coaxially fitted and connected to the first collection outlet 124; the second collection outlet connector 305 is coaxially fitted and connected to the second collection outlet 125; and the third collection outlet connector 306 is coaxially fitted and connected to the third collection outlet 126.

[0082] Combination Figure 3 The PDMS substrate 100 is further provided with a charging electrode 140, which can charge the droplets in the droplet generation channel 114 while the droplets are being generated.

[0083] Specifically, the charging electrode 140 is a strip of conductive metal, which is horizontally placed inside the PDMS substrate. Figure 3 In the horizontal direction shown, the axis of the charging electrode 140 is perpendicular to the second segment axis of the droplet generation channel 114, and the right side of the charging electrode 140 is flush with the outlet end of the droplet generation channel 114. Figure 3 In the vertical direction shown, the lower part of the charging electrode 140 is 300 micrometers away from the top of the droplet generation channel 114. One end of the charging electrode 140 is not wrapped by the PDMS substrate, and thus serves as the signal input terminal. The charging electrode 140 can be fabricated by a secondary casting process.

[0084] This embodiment also provides a method such as Figure 4 The microfluidic chip 2 shown is a droplet electrostatic sorting microfluidic chip based on surface acoustic wave focusing. The microfluidic chip 2 can be formed by bonding a lithium niobate substrate 200 and a PDMS substrate 100 using a high-strength bonding method, such as plasma oxidation activation bonding. The lithium niobate substrate 200 is equipped with an acoustic wave generator and deflection electrodes, while the PDMS substrate 100 is equipped with a microfluidic system and charging electrodes. The specific structure of the microfluidic system and charging electrodes on the PDMS substrate can be found in [reference needed]. Figures 1 to 3 As shown. The acoustic wave generator and deflection electrode structure provided on the lithium niobate substrate 200 can be combined with... Figure 4 and Figure 5 As shown.

[0085] Specifically, the lithium niobate substrate 200 has interdigitated electrodes and a matching reflective grid assembly, forming an acoustic wave generator through the structure of the interdigitated electrodes and the reflective grid assembly. The acoustic wave generator can focus the particles entering the dispersed phase inlet 123 of the microfluidic system.

[0086] More specifically, combined Figure 5 The acoustic wave generator of the lithium niobate substrate 200 includes: a first interdigital electrode 201, a second interdigital electrode 202, a first reflective grid 203, and a second reflective grid 204 disposed on the lithium niobate substrate 205. The specific connection structure is as follows: The first interdigital electrode 201 is composed of a first long finger 2011, a second long finger 2012, a first bus bar 2013, and a second bus bar 2014. The first bus bar 2013 and the second bus bar 2014 both extend vertically and are arranged opposite each other. The first long finger 2011 and the second long finger 2012 both extend horizontally and are located between the first bus bar 2013 and the second bus bar 2014. The first long finger 2011 is arranged alternately vertically and is connected to the first bus bar 2013. The second long finger 2012 is arranged alternately vertically and is connected to the second bus bar 2014. The first long finger 2011 and the second long finger 2012 have a gap of 25 μm.

[0087] The second interdigitated electrode is composed of a third long finger 2021, a fourth long finger 2022, a third bus bar 2023, and a fourth bus bar 2024. The third bus bar 2023 and the fourth bus bar 2024 both extend vertically and are arranged opposite each other. The third long finger 2021 and the fourth long finger 2022 both extend horizontally and are located between the third bus bar 2023 and the fourth bus bar 2024. The third long finger 2021 is arranged alternately vertically and is connected to the third bus bar 2023. The fourth long finger 2022 is arranged alternately vertically and is connected to the fourth bus bar 2024. The third long finger 2021 and the fourth long finger 2022 have a 25µm gap.

[0088] Both the first interdigital electrode 201 and the second interdigital electrode 202 have two signal input terminals. The first reflective grid 203 is located near the signal input side of the first interdigital electrode 201, and the second reflective grid 204 is located near the signal input side of the second interdigital electrode 202. The interdigital electrodes and reflective grids are positioned above the lithium niobate substrate. In the horizontal direction, the left side of the interdigital electrode is flush with the inlet end of the dispersed phase transport channel. In the vertical direction, the center of symmetry of the first interdigital electrode 201 and the second interdigital electrode 202 coincides with the center of the dispersed phase transport channel. The first interdigital electrode 201 and the second interdigital electrode 202 are symmetrically arranged, each including 20 pairs of interdigitates with a width of 25 micrometers. The first reflective grid 203 and the second reflective grid 204 are each composed of 10 metal strips with a width of 25 micrometers.

[0089] Continue to refer to Figure 4 and Figure 5 The lithium niobate substrate 205 has deflection electrodes 206 on its surface. The specific connection structure of the deflection electrodes 206 is as follows: the deflection electrodes 206 are a pair of L-shaped electrodes, arranged with their bottoms facing each other, and signal input terminals are provided at the top of the two electrodes; Figure 4 In the horizontal direction, the right side of the deflection electrode is aligned with the outlet end of the fusion transport channel 115; in the vertical direction, the center of symmetry of the deflection electrode 206 coincides with the center of symmetry of the fusion transport channel 115, and the bottom of the deflection electrode is 150 micrometers away from the edge of the transport channel. The deflection electrode is placed on the surface of the lithium niobate substrate 205. The deflection channel and the deflection electrode have a structural relationship; the deflection channel is located on the PDMS substrate, and the deflection electrode is located on the lithium niobate substrate. In the vertical direction, the deflection channel partially overlaps with the deflection electrode.

[0090] More specifically, the aforementioned interdigitated electrodes can be fabricated using a lift-off process. The lithium niobate substrate 205 is specifically made of single-sided polished 128° Y-type lithium niobate. The first interdigitated electrode 201, the second interdigitated electrode 202, the first reflective gate 203, and the second reflective gate 204 can be constructed by stacking silicon oxide-aluminum from the bottom layer upwards.

[0091] Combination Figure 6 , Figure 6 This is a schematic diagram illustrating the working principle of the microfluidic chip 2. The lithium niobate substrate 200 uses a piezoelectric substrate, and the interdigitated electrode structures 21 and 22 are interdigitated electrodes and reflective gate arrays placed opposite each other on the lithium niobate substrate to generate surface acoustic waves. The focused fluid 25 enters from the dispersed phase inlet of the microfluidic system 110, while the continuous phase fluid 27 enters from the two continuous phase inlets of the microfluidic system 110.

[0092] When a specific frequency electrical signal is input into an interdigital transducer printed on a lithium niobate substrate 100, the lithium niobate substrate 100 can generate surface acoustic waves propagating along the surface. The superposition of two surface waves can form a standing wave field. When the flow channel is placed perpendicular to the direction of standing wave propagation, minimum and maximum pressure regions are formed in the fluid domain under the action of the standing wave. Since the pressure in different regions is different, the cells in the fluid will be pushed to the region of minimum pressure, thereby completing the focusing of the fluid cells 25. In the cross-shaped flow channel of the microfluidic system 110, the fluid 25 enters from the middle dispersed phase transport channel, and the continuous phase fluid 27 enters from the continuous phase transport channels on both sides. The fluid 25 and the continuous phase fluid 27 are incompatible continuously flowing fluids. The fluid in the middle channel will be squeezed and destabilized under the action of the fluid shear force in the two side channels to form droplets. The size and frequency of droplet formation can be controlled by controlling the fluid velocity and velocity ratio. Conductive fluid is introduced into the flow channel at the cross intersection through the charging electrode 26, and a certain voltage of positive or negative charge is applied to the conductive fluid. When the fluid approaches the electrode 26 above the cross intersection flow channel, the electrode 26 has a polarity opposite to that of the fluid. Under the action of electrostatic induction, the charge in the fluid below the electrode 26 will be redistributed and accumulate, that is, the fluid below the electrode becomes charged. When the fluid passes through the cross intersection, the droplets 28 generated in the droplet generation channel will carry a certain amount of charge.

[0093] A pair of oppositely placed electrodes are arranged at both ends of the sorting channel of the microfluidic system 110. Figure 6 The diagram shows a pair of deflection electrodes 29. These electrodes are connected to electrodes of opposite polarity, and an electric field can be formed between the two electrodes that runs through the sorting channel. When a charged droplet 28 passes through the channel, the charge on the droplet 28 will generate an electrostatic force acting on the droplet under the action of the electric field, thereby causing the droplet 28 to move along the direction of the electrostatic force, thus completing the deflection and sorting of the droplet 28.

[0094] based on Figure 6 The working principle of the chip, combined with Figure 7 This embodiment also provides a separation method for droplet electrostatic sorting microfluidic chips based on surface acoustic wave focusing, which includes the following steps: S100, an electrical signal of a preset frequency is input into the acoustic wave generator of the lithium niobate substrate to generate surface acoustic waves; S101, Focusing the particles entering the dispersed phase inlet based on the surface acoustic wave; S102, two incompatible continuously flowing fluids are introduced into the continuous phase inlet and the dispersed phase inlet respectively, so as to compress and destabilize at the cross intersection to form droplets; S103, the droplet in the droplet generation channel is charged based on the charging electrode; S104, the deflection electrode is used to provide a deflection voltage to the fusion transport channel, so that the particles are separated into the separation channel at the converging end, thus completing the deflection and sorting of droplets.

[0095] Based on microfluidic chip 1, this embodiment also provides a method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, such as... Figure 8 As shown, it includes: Step S200: Clean a single-crystal silicon wafer and perform a surface hydrophilic treatment.

[0096] Cleaning a single-crystal silicon wafer involves placing an appropriate-sized single-crystal silicon wafer in a beaker, pouring in an appropriate amount of piranha solution, and heating the beaker on a 120° hot plate for two hours. After removing it, ultrasonically clean it for 15 minutes each with acetone, anhydrous ethanol, and deionized water. After removing it, dry it with a nitrogen gun and heat it on a 120° hot plate for 30 minutes to ensure the silicon wafer is completely dry.

[0097] The surface hydrophilic treatment of the monocrystalline silicon wafer specifically includes: placing the cleaned monocrystalline silicon wafer (the surface of the silicon wafer used to prepare the positive mold pattern) with its surface facing upwards in a plasma cleaner to complete the cleaning process. The cleaning parameters of the oxygen plasma cleaner are: oxygen flow rate: 50 sccm, chamber pressure: 750 mTorr, power: 150 W, and cleaning time: 10 min.

[0098] Step S201: Prepare a chip positive mold based on the single crystal silicon wafer to obtain a microfluidic system positive mold.

[0099] The fabrication of a chip positive mold based on a single-crystal silicon wafer to obtain a microfluidic system positive mold can specifically include the following process: SU8-2100 photoresist is applied to the center of the silicon wafer. The chip with photoresist is placed above the spin coater chuck, and the photoresist is spin-coated using the spin coater. Then, the wafer is placed on a heating stage for pre-baking. The designed mask (fluidic system) is placed on the photolithography machine mask holder, and the chuck is activated for fixation. The pre-baked silicon wafer is placed below the mask, and the distance between the mask and the silicon wafer is adjusted to ensure close contact. Photolithography is performed using a photolithography machine, setting the energy value (220 mJ·cm²) and the exposure time to 25 s. After photolithography, post-baking is performed. After post-baking, development is carried out at room temperature using a solution of propylene glycol methyl ether acetate and isopropanol. Finally, the wafer is dried with nitrogen and placed on the heating stage again for hard mold baking, resulting in a positive mold with a thickness of 150 μm. In the above process, the spin coating parameters can be: Level I - 750 rpm - 30 s; Level II - 3000 rpm - 50 s. The pre-baking heating program is: 45℃ → 10 min; 45-65℃ → 5 min; 65℃ → 240 min; 65℃ → room temperature. The photolithography parameters are: set the energy value (220 mJ·cm²), the exposure time is 25 s, and after photolithography, perform post-baking. The post-baking program is: 65℃ → 5 min; 65-95℃ → 5 min; 95℃ → 10 min; 95℃ → room temperature. The development parameters are: immerse in propylene glycol methyl ether acetate solution for 15 s, remove, immerse in isopropanol solution for 15 s, repeat this cycle until no white flocculent matter precipitates in the isopropanol solution. The mold baking parameters are: 110℃ → 30 min for mold baking, resulting in a positive mold thickness of 150 μm.

[0100] Step S202: Perform hydrophobic treatment on the male mold of the microfluidic system.

[0101] The hydrophobication process for the microfluidic system positive mold includes: placing the obtained microfluidic system positive mold in a volatilization cylinder, adding three drops of perfluorodecyltriethoxysilane to the volatilization cylinder, sealing it, and then placing the volatilization cylinder in an oven for heating and baking. The baking program is as follows: 25℃→125℃-30min; 125℃→125℃-180min; 125℃→room temperature.

[0102] Step S203: The prepared PDMS material is poured into the male mold of the microfluidic system to prepare the PDMS microfluidic system.

[0103] In step S203, the process of preparing the PDMS microfluidic system is as follows: PDMS prepolymer and curing agent are uniformly mixed at a weight ratio of 10:1. After stirring evenly, the mixture is placed in a vacuum drying oven for degassing for 30 minutes. Then, a certain amount of PDMS is drawn up using a syringe and poured onto the SU-8 anode template of the microfluidic system. The template is then placed in a vacuum drying oven for degassing for 30 minutes, followed by heating on a heating platform for curing. The heating program is as follows: 65℃→5min; 65-95℃→5min; 95℃→5min; 95℃-65℃→5min; 65℃→room temperature.

[0104] Step S204: Prepare a charging electrode and place the charging electrode in the microfluidic system.

[0105] The specific process for preparing the charging electrode includes: using stainless steel with a thickness of 100μm to 300μm as the material for preparing the charging electrode, placing the stainless steel sheet above the cutting stage, using a laser to cut the stainless steel into a stainless steel sheet of a specified shape, and folding the cut stainless steel sheet 90° along the length direction to obtain an L-shaped charging electrode.

[0106] The specific process of filling and placing the charging electrode into the microfluidic system includes: cleaning the upper surface of the microfluidic system, placing the prepared charging electrode on the upper surface of the microfluidic system, aligning the tip of the charging electrode with the cross intersection of the microfluidic channels under a microscope, and repeating the steps to prepare the microfluidic system.

[0107] Step S205: Cool the microfluidic system of the PDMS and cut and punch holes in the microfluidic system to prepare a PDMS substrate.

[0108] The process of cooling, cutting, and drilling the microfluidic system specifically includes: after cooling, slowly peeling it off the template, and then cutting and drilling to obtain a PDMS microfluidic system with charging electrodes. The holes involved in the cutting and drilling include: a first continuous phase inlet, a second continuous phase inlet, a dispersed phase inlet, a first collection outlet, a second collection outlet, a third collection outlet, a first deflection electrode inlet, a first deflection electrode outlet, a second deflection electrode inlet, and a first deflection electrode outlet in the microfluidic chip 1.

[0109] Based on microfluidic chip 2, this embodiment also provides a method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, such as... Figure 9 As shown, it includes: Step S300: Clean a lithium niobate substrate and a single-crystal silicon wafer and perform surface hydrophilic treatment.

[0110] The cleaning process for the lithium niobate substrate may specifically include: placing the Y128° lithium niobate substrate in a beaker, pouring in an appropriate amount of piranha solution, and heating the beaker on a 120°C hot plate. After removal, it is ultrasonically cleaned for 15 minutes each with acetone, anhydrous ethanol, and deionized water. It is then dried with a nitrogen gun and placed on a room-temperature hot plate, slowly heated to 120°C for 30 minutes, and finally dried to obtain a dry lithium niobate substrate. The heating process can specifically be: 25°C → 120°C for 30 minutes, 120°C → 120°C for 120 minutes, 120°C → room temperature.

[0111] The specific process for hydrophilic treatment of the lithium niobate substrate surface includes: placing the obtained silicon wafer in a plasma cleaner with the polished surface of the lithium niobate substrate facing upwards. The cleaning parameters of the oxygen plasma cleaner are: oxygen flow rate: 50 sccm, chamber pressure: 750 mTorr, power: 150 W, and cleaning time: 10 min.

[0112] The process of cleaning and hydrophilic surface treatment of monocrystalline silicon wafers can be referred to in step S200, and will not be repeated here.

[0113] Step S301: Patterns of interdigitated electrodes and deflection electrodes are fabricated on the lithium niobate substrate.

[0114] The process of fabricating interdigitated electrodes and deflection electrodes on a lithium niobate substrate includes the following steps: NR7-1500py negative photoresist is injected into the center of the substrate. The chip with the photoresist is placed above the spin coater chuck, and the photoresist is spin-coated using the spin coater. Then, the designed mask (interdigitated electrode and deflection electrode patterns) is placed on the photolithography mask holder under a heated stage. The chuck is activated for fixation. The pre-baked silicon wafer is placed below the mask, and the distance between the mask and the substrate is adjusted to ensure close contact. Photolithography is performed using a photolithography machine. After photolithography, post-baking is performed. After post-baking, RD6 is developed at room temperature until no white precipitates appear, and the wafer is immediately rinsed in deionized water. After rinsing, it is quickly dried with a nitrogen gun. The spin-coating parameters can be: Level I - 500 rpm - 30 s; Level II - 3000 rpm - 50 s. The pre-baking heating program is: 25-150℃ → 5 min, 150℃ → room temperature. The photolithography parameters were: energy set at 220 mJ·cm², and exposure time at 10 s. The post-baking program was: 25-95℃ → 5 min; 95℃ → room temperature.

[0115] Step S302: Based on the pattern, thin film metal electrodes of the interdigitated electrode and the deflection electrode are prepared on the lithium niobate substrate to obtain the lithium niobate substrate.

[0116] The specific process for fabricating thin-film metal electrodes on lithium niobate substrates includes: placing the patterned lithium niobate substrate in a magnetron sputtering machine for silicon oxide-aluminum coating; firstly, sputtering a first layer of silicon oxide film, then sputtering an aluminum film; and finally removing the coated substrate. The thickness of the first layer of silicon oxide film can be 150 nm, and the thickness of the aluminum film is 100 nm. The silicon oxide coating conditions are: target material: silicon oxide; target position: radio frequency target; vacuum degree: 3.0 x 10e-4 Pa; argon vacuum degree: 0.005 Pa; argon flow rate: 40 SCCM; sputtering power: 150 W; sputtering time: 150 min. The conditions for aluminum metal coating are as follows: target material: high purity aluminum (99.999%), target position: DC target position, vacuum degree: 3.0x10e-4Pa, argon vacuum degree: 0.005Pa, argon flow rate: 40SCCM, sputtering power: 150W, sputtering time: 15min.

[0117] The obtained lithium niobate substrate can be placed in acetone solution and left to stand for 5 hours. After that, it can be taken out, rinsed with anhydrous ethanol for 5 minutes, rinsed with deionized water, and the surface of the substrate can be dried with a nitrogen gun. The substrate can be placed on a hot plate at room temperature and heated slowly to 120°C for 30 minutes. After drying, a dry lithium niobate substrate with interdigitated electrodes and deflection electrodes can be obtained.

[0118] Step S303: Based on the single-crystal silicon wafer, a chip positive mold is prepared to obtain a microfluidic system positive mold.

[0119] In this step, the process of preparing the chip positive mold can be referred to step S201, and will not be repeated here.

[0120] Step S304: Perform hydrophobic treatment on the male mold of the microfluidic system.

[0121] In this step, the process of hydrophobizing the male mold of the microfluidic system can be referred to step S202, and will not be repeated here.

[0122] Step S305: The prepared PDMS material is poured into the male mold of the microfluidic system to prepare the PDMS microfluidic system.

[0123] In this step, the process of casting the prepared PDMS material into the male mold of the microfluidic system can be referred to step S203, and will not be repeated here.

[0124] Step S306: Prepare a charging electrode and place the charging electrode in the microfluidic system.

[0125] In this step, the specific process of preparing the charging electrode and placing the charging electrode in the microfluidic system can be referred to step S204, and will not be repeated here.

[0126] Step S307: Cool the microfluidic system of the PDMS and cut and punch holes in the microfluidic system to prepare a PDMS substrate.

[0127] In this step, the specific process of cooling the microfluidic system of the PDMS and cutting and drilling the microfluidic system can be referred to step S205, and will not be repeated here.

[0128] Step S308: Bond the lithium niobate substrate to the PDMS substrate.

[0129] The obtained microfluidic system with charging electrodes is bonded to a lithium niobate substrate with interdigitated electrodes and deflection electrodes. The bonding process may specifically include: ultrasonically cleaning the obtained microfluidic system with charging electrodes and the obtained lithium niobate substrate with interdigitated electrodes and deflection electrodes with ethanol and deionized water for 5 min in sequence; placing the microfluidic system with the lower surface facing up and the lithium niobate substrate with the upper surface facing up in a plasma cleaner for oxygen plasma treatment for 2 min; aligning and bonding the two parts together under a microscope; and heating them on a 110℃ heating stage for 1 h to complete the bonding.

[0130] Finally, a conductive buffer solution can be injected into the deflection electrode inlet of the prepared chip (after bonding) to obtain the chip (i.e., microfluidic chip 2).

[0131] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing, comprising a lithium niobate substrate bonded to a PDMS substrate, characterized in that, An acoustic wave generator and a deflection electrode are provided on the lithium niobate substrate, and a microfluidic system and a charging electrode are provided on the PDMS substrate. The microfluidic system includes: a dispersed phase inlet, a first continuous phase inlet, a second continuous phase inlet, multiple collection outlets, a dispersed phase transport channel, a continuous phase transport channel, a droplet generation channel, a fusion transport channel, and multiple sorting channels. The acoustic generator is adapted to focus the particles entering the dispersed phase inlet; The dispersed phase transport channel is connected to the dispersed phase inlet, and the continuous phase transport channel is connected to the first continuous phase inlet and the second continuous phase inlet, forming a cross with the dispersed phase transport channel. The droplet generation channel and the charging electrode are connected from the cross intersection. The droplet generation channel is connected to the fusion transport channel, which has a converging end that radially divides into multiple sorting channels. The multiple sorting channels are respectively connected to the multiple collection outlets. The charging electrode is adapted to charge the droplets in the droplet generation channel; The deflection electrode is adapted to provide a deflection voltage to the fusion transport channel, causing the particles to separate into the sorting channel at the converging end.

2. The microfluidic chip as described in claim 1, characterized in that, The acoustic wave generator includes: a first interdigital electrode, a second interdigital electrode, a first reflective grid, and a second reflective grid disposed on the lithium niobate substrate; The first interdigitated electrode and the second interdigitated electrode are arranged symmetrically with the central axis of the dispersed phase transport channel as the axis of symmetry; the first reflective grid and the second reflective grid are arranged symmetrically near the signal input ends of the first interdigitated electrode and the second interdigitated electrode, respectively. The first interdigitated electrode is composed of a first long finger, a second long finger, a first busbar, and a second busbar; the first busbar and the second busbar both extend vertically and are arranged opposite each other; the first long finger and the second long finger both extend horizontally and are located between the first busbar and the second busbar; the first long finger is arranged alternately vertically and is connected to the first busbar; the second long finger is arranged alternately vertically and is connected to the second busbar; the first long finger and the second long finger have a certain gap. The second interdigitated electrode is composed of a third long finger, a fourth long finger, a third bus bar, and a fourth bus bar; the third and fourth bus bars extend vertically and are arranged opposite each other; the third and fourth long fingers extend horizontally and are located between the third and fourth bus bars; the third long fingers are arranged alternately vertically and are all connected to the third bus bar; the fourth long fingers are arranged alternately vertically and are all connected to the fourth bus bar; the third and fourth long fingers have a certain gap.

3. The microfluidic chip as described in claim 2, characterized in that, The width of the first, second, third, and fourth long fingers is 25 micrometers, and the width of the metal strips of the first and second reflective gratings is 25 micrometers.

4. The microfluidic chip as described in claim 2, characterized in that, The first interdigital electrode and the second interdigital electrode are prepared by the Lift-Off process.

5. The microfluidic chip as described in claim 2, characterized in that, The first interdigital electrode, the second interdigital electrode, the first reflective grid, and the second reflective grid are constructed by stacking silicon oxide-aluminum oxide from the bottom layer to the top.

6. The microfluidic chip as described in claim 1, characterized in that, The deflection electrode includes a first L-shaped electrode and a second L-shaped electrode disposed on the lithium niobate substrate, wherein the first L-shaped electrode and the second L-shaped electrode are symmetrically arranged with the central axis of the fusion transport channel as the axis of symmetry.

7. The microfluidic chip as described in claim 1, characterized in that, The angle between adjacent sorting channels is 30°.

8. The microfluidic chip as described in claim 1, characterized in that, The continuous phase transport channel is a rectangular channel with a width of 60 micrometers and a depth of 150 micrometers; The dispersed phase transport channel is a rectangular channel with a width of 100 micrometers and a depth of 150 micrometers. The droplet generation channel includes a first channel and a second channel. The first channel is a channel with unequal width, and the second channel is a channel with equal width. The inlet end of the first channel has a width of 70 micrometers, the outlet end has a width of 30 micrometers, a length of 30 micrometers, and a depth of 150 micrometers. The second channel has a width of 30 micrometers, a depth of 150 micrometers, and a length of 30 micrometers. The outlet end of the first channel is connected to the inlet end of the second channel. The fusion transport channel is a channel with unequal widths. The width of the inlet end of the fusion transport channel is 100 micrometers, the width of the outlet end is 300 micrometers, and the depth is 150 micrometers. The microchannel system includes: a first sorting channel, a second sorting channel, and a third sorting channel; the first sorting channel, the second sorting channel, and the third sorting channel are all equal-width channels, the width of the first sorting channel and the third sorting channel is 80 micrometers, the width of the second sorting channel is 120 micrometers, and the depth of the sorting channels is 150 micrometers.

9. The microfluidic chip as described in claim 1, characterized in that, The microfluidic system further includes a deflection channel; the deflection channel is a first U-shaped channel and a second U-shaped channel symmetrically distributed on both sides of the converging end. The U-shaped channel has two channels, one channel port is a conductive solution inlet and the other channel port is an exhaust port; when the conductive solution is introduced into the conductive solution inlet, the deflection channel forms a spatial electric field region around the converging end, causing the charged particles to deflect.

10. The microfluidic chip as described in claim 9, characterized in that, The first and second U-shaped flow channels are rectangular flow channels. The bottom flow channel has a width of 400 micrometers and a depth of 150 micrometers, while the two side flow channels have a width of 100 micrometers and a depth of 150 micrometers.

11. The microfluidic chip as described in claim 1, characterized in that, Also includes: A first continuous phase inlet connector, a second continuous phase inlet connector, a dispersed phase inlet connector, and multiple collection outlet connectors are arranged on the upper surface of the PDMS substrate; The first continuous phase inlet connector, the second continuous phase inlet connector, the dispersed phase inlet connector, and the multiple collection outlet connectors are coaxially fitted and connected to the first continuous phase inlet, the second continuous phase inlet, the dispersed phase inlet, and the multiple collection outlets in sequence.

12. The microfluidic chip as described in claim 1, characterized in that, The charging electrode is a strip of conductive metal. One end of the charging electrode is disposed inside the PDMS substrate, and the other end, which is not wrapped by the PDMS substrate, is the signal input terminal of the charging electrode.

13. The microfluidic chip as described in claim 12, characterized in that, The charging electrode is arranged in a direction perpendicular to the transport direction axis of the droplet generation channel inside the PDMS substrate, and is 300 micrometers away from the droplet generation channel in this perpendicular direction.

14. The microfluidic chip as described in claim 1, 12, or 13, characterized in that, The charging electrode is prepared by a secondary casting process.

15. A method for fabricating a microfluidic chip for droplet electrostatic sorting based on surface acoustic wave focusing as described in claim 1, characterized in that, include: A lithium niobate substrate and a single-crystal silicon wafer were cleaned and subjected to surface hydrophilic treatment. Patterns of interdigitated electrodes and deflection electrodes are fabricated on the lithium niobate substrate; Based on the pattern, thin film metal electrodes of the interdigitated electrode and the deflection electrode are prepared on the lithium niobate substrate to obtain the lithium niobate substrate; Based on the single-crystal silicon wafer, a chip positive mold is prepared to obtain a microfluidic system positive mold; The male mold of the microfluidic system is subjected to hydrophobic treatment; The prepared PDMS material was cast into the male mold of the microfluidic system to prepare the PDMS microfluidic system; A charging electrode is prepared and placed in the microfluidic system; The microfluidic system of the PDMS is cooled and the microfluidic system is cut and perforated to prepare a PDMS substrate; The lithium niobate substrate is bonded to the PDMS substrate.

16. The preparation method according to claim 15, characterized in that, The lithium niobate substrate was cleaned as follows: the lithium niobate substrate was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 15 minutes respectively, removed and dried with a nitrogen gun, placed on a hot plate at room temperature, slowly heated to 120°C and heated for 30 minutes, and dried to obtain a dry lithium niobate substrate.

17. The preparation method according to claim 15, characterized in that, The lithium niobate substrate is subjected to surface hydrophilic treatment as follows: the lithium niobate substrate is placed in a plasma cleaner for polishing.

18. The preparation method according to claim 15, characterized in that, The patterns for fabricating interdigitated electrodes and deflection electrodes on the lithium niobate substrate include: Photoresist is injected into the center of the lithium niobate substrate; The lithium niobate substrate with photoresist is placed above the suction cup of the spin coater, and the photoresist is spin-coated using the spin coater to complete the pre-baking of the lithium niobate substrate. The pre-baked lithium niobate substrate is placed under a mask with the desired interdigitated electrode and deflection electrode pattern, and the lithium niobate substrate is photolithographically ... Post-baking is performed on the lithium niobate substrate after photolithography; After post-baking, the lithium niobate substrate is developed at room temperature until no white precipitate appears; Clean and dry the lithium niobate substrate.

19. The preparation method according to claim 15, characterized in that, The preparation of the interdigitated electrode and deflection electrode on the lithium niobate substrate based on the pattern includes: placing the obtained lithium niobate substrate with the pattern in a magnetron sputtering machine, first sputtering a first layer of silicon oxide film, then sputtering an aluminum film, and finally taking out the coated lithium niobate substrate to obtain the electrode.

20. The preparation method according to claim 19, characterized in that, The first silicon oxide film has a thickness of 150 nm, and the aluminum film has a thickness of 100 nm.

21. The preparation method according to claim 15, characterized in that, Also includes: The lithium niobate substrate with the interdigitated electrode and deflection electrode is left to stand, rinsed and dried to prepare the lithium niobate substrate.

22. The preparation method according to claim 15, characterized in that, Clean the monocrystalline silicon wafer using the following steps: The single-crystal silicon wafer was placed in a piranha solution and heated; The heated monocrystalline silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 15 minutes each. It was then removed, dried with a nitrogen gun, and placed on a hot plate at 120°C for 30 minutes to ensure that the monocrystalline silicon wafer was completely dry.

23. The preparation method according to claim 15, characterized in that, Perform surface hydrophilic treatment on monocrystalline silicon wafers as follows: Place the cleaned monocrystalline silicon wafers with the surface facing up in a plasma cleaner to complete the cleaning process.

24. The preparation method according to claim 15, characterized in that, The preparation of the chip positive mold based on the single-crystal silicon wafer includes: Photoresist is injected into the center of a single-crystal silicon wafer, and the silicon wafer with photoresist is placed above the suction cup of a spin coater. The spin coater is then used to spin-coat the photoresist. The silicon wafer is placed on a heating stage for pre-baking, and a mask with a pre-designed flow channel system is placed on the photolithography machine mask holder. Photolithography is used to lithographically shape silicon wafers; After photolithography, the silicon wafer undergoes post-baking and development, is then dried with nitrogen, and finally placed on a heating stage for hard mold baking to obtain the chip positive mold.

25. The preparation method according to claim 24, characterized in that, The thickness of the chip male mold is 150 μm.

26. The preparation method according to claim 15, characterized in that, The hydrophobication treatment of the male mold of the microfluidic system includes: The obtained microfluidic system positive mold was placed in a volatilization cylinder. Three drops of perfluorodecyltriethoxysilane were added to the volatilization cylinder. After sealing, the volatilization cylinder was placed in an oven for heating and baking.

27. The preparation method according to claim 15, characterized in that, The step of casting the prepared PDMS material onto the male mold of the microfluidic system to prepare the PDMS microfluidic system includes: The PDMS prepolymer and curing agent were mixed uniformly at a weight ratio of 10:

1. After stirring evenly, the mixture was placed in a vacuum drying oven for degassing for 30 minutes. Then, a certain amount of PDMS was drawn up with a syringe and poured onto the anode template of the microfluidic system. The template was then placed in a vacuum drying oven for degassing for 30 minutes. Finally, the template was placed on a heating table for heating and curing. The PDMS microfluidic system was then prepared.

28. The preparation method according to claim 15, characterized in that, The step of placing the charging electrode in the microfluidic system includes: The upper surface of the microfluidic system is cleaned by blowing it clean. The prepared charging electrode is placed on the upper surface of the microfluidic system, and the tip of the charging electrode is aligned with the cross intersection of the pre-set microfluidic channels under a microscope to obtain the final microfluidic system.

29. The preparation method according to claim 15, characterized in that, The preparation of the charging electrode includes: Stainless steel sheets with a thickness of 100μm to 300μm are used as the material for preparing the charging electrode. The stainless steel sheet is placed above a cutting table and cut into stainless steel sheets of a specified shape using a laser. The cut stainless steel sheets are then folded at a set angle along the length direction to obtain the charging electrode.

30. The preparation method according to claim 29, characterized in that, The set angle is 90°.

31. The preparation method according to claim 15, characterized in that, The cooling of the microfluidic system of the PDMS and the cutting and perforation of the microfluidic system include: cutting and perforating the microfluidic channels of the microfluidic system based on the corresponding inlet and outlet of the microfluidic channels.

32. The preparation method according to claim 15, characterized in that, The bonding of the lithium niobate substrate to the PDMS substrate includes: The lithium niobate substrate and the PDMS substrate were ultrasonically cleaned sequentially using ethanol and deionized water. The lithium niobate substrate and PDMS substrate were placed in a plasma cleaner for oxygen plasma treatment. The lithium niobate substrate and the bonding surfaces of the PDMS substrate are aligned, bonded, and heated to complete the bonding.

33. The preparation method according to claim 15, characterized in that, The deflection electrode has a conductive inlet, and the preparation method further includes injecting a conductive buffer solution into the conductive inlet.

34. A separation method for droplet electrostatic sorting microfluidic chips based on surface acoustic wave focusing, wherein the microfluidic chip is based on the microfluidic chip as described in claims 1 to 14, characterized in that, include: An electrical signal of a preset frequency is input into the acoustic wave generator of the lithium niobate substrate to generate surface acoustic waves. The particles entering the dispersed phase inlet are focused based on the surface acoustic waves. Two incompatible continuously flowing fluids are introduced into the continuous phase inlet and the dispersed phase inlet, respectively, to compress and destabilize at the cross intersection to form droplets. The droplets in the droplet generation channel are charged based on the charging electrode; The deflection electrode provides a deflection voltage to the fusion transport channel, causing the particles to separate at the converging end into the sorting channel, thus completing the deflection and sorting of the droplets.

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