Sorting type micro-fluidic chip integrated with acousto-optic-electric method as well as preparation method and application of sorting type micro-fluidic chip

Abstract

The invention provides a sorting type micro-fluidic chip integrating an acousto-optic-electric method as well as a preparation method and application of the sorting type micro-fluidic chip, and belongs to the technical field of micro-fluidics. The micro-fluidic chip comprises a first substrate with at least one interdigital electrode, a second substrate with a micro-channel system and a charging electrode, at least one deflection electrode, at least one transmitting optical fiber and at least one receiving optical fiber. The interdigital electrode and the first substrate form an acoustic resonator. The micro-fluidic chip integrates the functions of flow focusing, acoustic focusing, light wave focusing and electrostatic induction sorting, all the functions cooperate with one another, and the micro-fluidic chip has excellent stability and sensitivity during cell sorting, shows high accuracy in liquid drop deflection and is suitable for large-scale cell enrichment or sorting.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic technology, and in particular relates to a sorting microfluidic chip integrating an acoustic-optical-electrical method, and a preparation method and application thereof. Background Art

[0002] The basic principle of microfluidics is to use immiscible multiphase fluids to generate controllable, uniform and continuous microdroplets under the action of shear force and surface tension. Such independent microdroplets provide a nearly perfect "compartment" space for biochemical reactions, which makes microfluidics a particularly effective method for single-cell analysis. Common methods for reaction analysis using microfluidic chips usually include three steps: the generation and collection process of droplets, the incubation process of droplets, and the sorting and collection process of droplets. However, in existing studies, these three steps are usually independent and require different microfluidic chips to complete. During the operation, manual operation by professionals is required, which is not conducive to the integration and automation of single-cell analysis experiments. In addition, during the droplet transfer process, the droplet quality may decrease, such as droplet fusion, which further affects the experimental results.

[0003] The Chinese patent application publication CN117586870A discloses an integrated droplet microfluidic chip and a nucleic acid extraction and detection device, which relates to an integrated droplet microfluidic chip and belongs to the field of microfluidic chips. The integrated droplet microfluidic chip includes: an injection channel, a continuous phase channel, a droplet generation mechanism, a heating mechanism, a storage amplification mechanism and a tail end connection port, wherein the injection channel and the continuous phase channel are both connected to the droplet generation mechanism, the droplet generation mechanism is connected to the storage amplification mechanism, the heating mechanism is provided on the microfluidic chip in the corresponding area of ​​the storage amplification mechanism, the heating mechanism includes heating columns arranged in an array, the heating columns all pass through the chip body from top to bottom, and the storage amplification mechanism is connected to the tail end connection port. CN116445246A discloses a microfluidic chip for living cell sorting, in which the sample channel of the microfluidic chip is connected to the liquid inlet end of the sorting channel, the inlet channel, the target channel and the non-target channel are connected to the liquid outlet end of the sorting channel, the target channel and the sorting channel have an angle of 100°-130°, and the non-target channel and the sorting channel have an angle of 100°-140°; the intersection of the axis of the inlet channel and the axis of the sorting channel is 0.02mm-0.05mm away from the intersection of the inlet channel, the target channel and the non-target channel.

[0004] The integrated droplet microfluidic chip in the above-mentioned prior art provides an idea of ​​integrating the entire experimental process, which can complete the automated process of nucleic acid extraction, detection, storage and amplification. However, the chip in CN117586870A only integrates the nucleic acid extraction, detection and amplification processes, and lacks a single cell sorting structure; the microfluidic chip for sorting living cells in CN116445246A provides a cell sorting solution based on gas and liquid phases, which cannot meet the needs of refined droplet manipulation.

[0005] Therefore, it is necessary to design a microfluidic sorting chip with high integration, high accuracy and high control precision to solve the problems of low integration and low degree of refinement of current microfluidic chip technology. Summary of the invention

[0006] Based on the above problems, the present invention provides a sorting microfluidic chip integrating acoustic-optical-electrical methods. The microfluidic chip integrates flow focusing, acoustic focusing, light wave focusing and electrostatic induction sorting functions, and each function cooperates with each other, has excellent stability and sensitivity in cell sorting, and shows high accuracy in droplet deflection, which is suitable for large-scale cell enrichment or sorting.

[0007] Specifically, the present invention adopts the following technical solutions to achieve the above objectives: A sorting microfluidic chip integrating an acoustic-optical-electrical method, the microfluidic chip comprising a first substrate, a second substrate combined with the first substrate, at least one deflection electrode, and at least one transmitting optical fiber and at least one receiving optical fiber; The material of the first substrate is a piezoelectric material, and at least one interdigital electrode is disposed on the first substrate; The second substrate is provided with a microfluidic system and a charging electrode; The microfluidic system includes a dispersed phase marrow fluid flow channel, a first dispersed phase sheath fluid flow channel, a second dispersed phase sheath fluid flow channel, a dispersed phase transport flow channel, a first continuous phase flow channel, a second continuous phase flow channel, a droplet transport flow channel, an optical fiber channel corresponding to the total number of the transmitting optical fiber and the receiving optical fiber, and a deflection electrode flow channel corresponding to the number of the deflection electrodes; the first dispersed phase sheath fluid flow channel and the second dispersed phase sheath fluid flow channel are respectively located on both sides of the dispersed phase marrow fluid flow channel and are both connected to the dispersed phase marrow fluid flow channel at a first intersection; the first intersection is connected to the entrance of the dispersed phase transport flow channel; the first continuous phase flow channel and the second continuous phase flow channel are respectively located on both sides of the dispersed phase transport flow channel and are both connected to the dispersed phase flow channel at a second intersection; the second intersection is connected to the entrance of the droplet transport flow channel; The interdigitated electrodes are located on one side or both sides of the dispersed phase transport channel; the deflection electrode is placed in the deflection electrode channel; the deflection electrode channel is located on one side or both sides of the dispersed phase transport channel; the transmitting optical fiber and the receiving optical fiber are respectively placed in the optical fiber channel; the optical fiber channel is located on one side or both sides of the dispersed phase transport channel; the charging electrode is located above the second intersection.

[0008] In a preferred embodiment, the first substrate is a lithium niobate substrate.

[0009] In a preferred embodiment, the material of the second substrate is PDMS.

[0010] In a preferred embodiment, the interdigitated electrodes are symmetrically arranged on both sides of the dispersed phase transport channel.

[0011] In a preferred embodiment, the deflection electrodes are symmetrically arranged on both sides of the droplet transport channel.

[0012] The present invention also provides a method for preparing the sorting microfluidic chip integrating the acoustic-optical-electrical method, comprising the following steps: S1, preparing the interdigital electrodes on the first substrate; preparing the microfluidic system and the charging electrode on the second substrate; preparing the deflection electrode; The preparation method of the microfluidic system comprises the following steps: preparing a positive mold of the microfluidic system on a single crystal silicon wafer; the positive mold of the microfluidic system comprises a dispersed phase marrow fluid flow channel, a first dispersed phase sheath fluid flow channel, a second dispersed phase sheath fluid flow channel, a dispersed phase transport flow channel, a first continuous phase flow channel, a second continuous phase flow channel, a droplet transport flow channel, at least one transmitting optical fiber channel, at least one receiving optical fiber channel, and at least one deflection electrode flow channel; performing a hydrophobic treatment on the positive mold of the microfluidic system; pouring the material of the second substrate on the positive mold of the microfluidic system to obtain the microfluidic system; placing the charging electrode above the second intersection, and pouring the material of the second substrate again on the surface of the microfluidic system; cutting and punching the obtained substrate to obtain a second substrate having a microfluidic system and a charging electrode; S2, combining the first substrate having the interdigital electrodes with the second substrate having the microfluidic system and the charging electrode; S3, placing the transmitting optical fiber in the transmitting optical fiber channel, and placing the receiving optical fiber in the receiving optical fiber channel; and obtaining the microfluidic chip.

[0013] In a preferred embodiment, step S1 includes a step of cleaning the first substrate.

[0014] In a preferred embodiment, step S1 includes a step of performing surface hydrophilic treatment on the first substrate.

[0015] In a further preferred embodiment, the method for cleaning and surface hydrophilic treatment of the first substrate includes the following steps: ultrasonically cleaning the first substrate with acetone, anhydrous ethanol and deionized water in sequence, taking it out and blowing it dry with a nitrogen gun, and then drying it to obtain a dry first substrate; and plasma cleaning the dried first substrate.

[0016] In a preferred embodiment, step S1 includes a step of cleaning the single crystal silicon wafer.

[0017] In a preferred embodiment, step S1 includes a step of performing surface hydrophilic treatment on the single crystal silicon wafer.

[0018] In a further preferred embodiment, the method for cleaning and surface hydrophilic treatment of the single crystal silicon wafer comprises the following steps: placing the single crystal silicon wafer in a piranha solution and heating it; after taking it out, ultrasonically cleaning it with acetone, anhydrous ethanol and deionized water in sequence; taking it out, blowing it dry with a nitrogen gun, and then drying it to obtain a cleaned single crystal silicon wafer; and plasma cleaning the cleaned single crystal silicon wafer.

[0019] In a preferred embodiment, the interdigital electrodes in step S1 are prepared by a Lift-Off process.

[0020] In a preferred embodiment, the deflection electrode is prepared by any one of the following methods: (1) The deflection electrode is prepared by a Lift-Off process; (2) The deflection electrode flow channel is filled with any one of a conductive solution, liquid metal, solid metal or conductive gel.

[0021] In a preferred embodiment, the method for preparing the microfluidic system in step S1 includes the following steps: uniformly mixing the PDMS prepolymer and the curing agent in a mass ratio of 10:1 to 15:1, and then drying and degassing to obtain PDMS; taking the PDMS and pouring it onto the positive mold of the microfluidic system, drying and degassing, and heating and curing to obtain the microfluidic system.

[0022] In a preferred embodiment, the method for hydrophobizing the positive mold of the microfluidic system in step S1 comprises the following steps: placing the positive mold of the microfluidic system in a volatile cylinder, dripping perfluorodecyltriethylsilane into the volatile cylinder, and after sealing, placing the volatile cylinder in an oven for heating and baking.

[0023] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) Different from the existing microfluidic chips, the sorting microfluidic chip provided by the present invention integrates the sorting functions of flow focusing, acoustic focusing, photodynamic focusing and electrostatic induction into the same chip, so that the various functions work together and the performance of the chip is more excellent.

[0024] (2) The sorting microfluidic chip of the present invention utilizes the electrostatic induction principle to enhance the charging phenomenon at the tip of the droplet for droplet charging and sorting, thereby reducing the damage to cells caused by direct mechanical or electrical manipulation, and ensuring a stable survival rate of more than 90% for the sorted cells.

[0025] (3) The sorting microfluidic chip of the present invention has excellent stability when performing cell sorting; it has high sensitivity in cell detection and high accuracy in droplet deflection. In the sorting experiment of large-sized H22 cells, it achieved a cell detection accuracy of 99.8% and a cell sorting accuracy of 99.3%.

[0026] (4) The sorting microfluidic chip provided by the present invention provides a robust solution for the high-purity enrichment of particulate matter with a small content in the solution. During the experiment, magnetic nanoparticles were used to simulate the particulate matter contained in the solution, and fluorescent dyes (such as FITC) were used to mark them before the experiment. Droplets containing particulate matter were generated by the sorting microfluidic chip, and the droplet diameter was controlled at 50-100 microns. The highest purity obtained in this enrichment experiment increased by 1029.9%.

[0027] (5) The preparation method of the sorting microfluidic chip provided by the present invention involves microchannel manufacturing, rapid prototyping of acoustic wave electrodes and deflection electrodes, integration of optical fibers, etc., providing a stable, efficient and simple preparation method for the preparation of highly integrated chips.

[0028] (6) The sorting microfluidic chip provided by the present invention can be widely used in situations such as cell counting, cell sorting or enrichment of single cells of various stains or different sizes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic structural diagram of a sorting microfluidic chip integrating an acoustic-optical-electrical method provided by the present invention.

[0030] In the figure: 101, interdigitated electrodes; 201, dispersed phase medullary fluid flow channel; 202, first dispersed phase sheath fluid flow channel; 203, second dispersed phase sheath fluid flow channel; 204, dispersed phase transport flow channel; 205, first continuous phase flow channel; 206, second continuous phase flow channel; 207, droplet transport flow channel; 208, optical fiber channel; 209, charging electrode; 301, deflection electrode; 401, first power supply; 402, second power supply; 501, charged droplets. DETAILED DESCRIPTION

[0031] The following content is combined with the embodiments to clearly and completely describe the technical solution of the present application so that those skilled in the art can fully understand the present application. Obviously, the described embodiments are only some preferred embodiments of the present application, rather than all embodiments. Any equivalent transformation or substitution made to the following implementation modes by those of ordinary skill in the art without creative work belongs to the protection scope of the present application.

[0032] Directional terms described in this application, such as "upper", "lower", "inner", "outer", "bottom", "upper surface", etc., indicate directions or positional relationships based on directions or positional relationships in the drawings of the specification, or directions or positional relationships in which the products of this application are usually placed when in use, and are only for the convenience of describing and understanding the product structure of this application. Therefore, directional terms cannot be understood as limiting this application. In this application, unless otherwise clearly defined, expressions such as "upper", "above", "above", and "upper surface" of a first feature on a second feature indicate that the first feature and the second feature may be in direct contact or indirect contact through an intermediate medium; the first feature may be directly above or obliquely above the second feature, or simply indicate that the first feature is higher in level than the second feature. Expressions such as "lower", "below", "below", and "lower surface" of a first feature on a second feature indicate that the first feature and the second feature may be in direct contact or indirect contact through an intermediate medium; the first feature may be directly below or obliquely below the second feature, or simply indicate that the first feature is lower in level than the second feature. Ordinal numbers used in this application, such as "first", "second", etc., are only used for descriptive purposes to distinguish similar objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. The methods not described in detail in the following embodiments are all conventional methods well known to those skilled in the art.

[0033] Example 1 Reference Figure 1, a sorting microfluidic chip integrating an acoustic-optical method, comprising a first substrate, a second substrate combined with the first substrate, at least one deflection electrode 301, and at least one transmitting optical fiber and at least one receiving optical fiber. The material of the first substrate is a piezoelectric material. At least one interdigitated electrode 101 is arranged on the first substrate. A microfluidic system and a charging electrode 209 are arranged on the second substrate. The microfluidic system comprises a dispersed phase medullary fluid flow channel 201, a first dispersed phase sheath fluid flow channel 202, a second dispersed phase sheath fluid flow channel 203, a dispersed phase transport flow channel 204, a first continuous phase flow channel 205, a second continuous phase flow channel 206, a droplet transport flow channel 207, an optical fiber channel 208 and a deflection electrode flow channel. The first dispersed phase sheath fluid flow channel 202 and the second dispersed phase sheath fluid flow channel 203 are respectively located on both sides of the dispersed phase medullary fluid flow channel 201, and are both connected to the dispersed phase medullary fluid flow channel 201 at the first intersection. The first intersection is the outlet of the dispersed phase medullary fluid flow channel 201, the first dispersed phase sheath fluid flow channel 202, and the second dispersed phase sheath fluid flow channel 203. The first intersection is connected to the inlet of the dispersed phase transport flow channel 204. The first continuous phase flow channel 205 and the second continuous phase flow channel 206 are respectively located on both sides of the dispersed phase transport flow channel 204, and are both connected to the dispersed phase transport flow channel 204 at the second intersection. The second intersection is the outlet of the dispersed phase transport flow channel 204, the first continuous phase flow channel 205, and the second continuous phase flow channel 206. The second intersection is connected to the inlet of the droplet transport flow channel 207. The outlet of the droplet transport flow channel 207 is bifurcated into multiple branch flow channels for transporting different droplets. The number of optical fiber channels 208 corresponds to the total number of transmitting optical fibers and receiving optical fibers. The transmitting optical fiber is placed in a part of the optical fiber channel 208, and the receiving optical fiber is placed in the remaining optical fiber channel 208. The optical fiber channels 208 may all be located on any side of the dispersed phase delivery channel 204, or may be located on both sides of the dispersed phase delivery channel 204. When located on both sides of the dispersed phase delivery channel 204, the number of optical fiber channels 208 on both sides may be the same or different. The charging electrode 209 is located above the second intersection.

[0034] The interdigital electrodes 101 are located on one side or both sides of the dispersed phase transport channel 204. When the number of the interdigital electrodes 101 is one, the interdigital electrodes 101 are located on any side of the dispersed phase transport channel 204. When the number of the interdigital electrodes 101 is multiple, the interdigital electrodes 101 can be located on any side of the dispersed phase transport channel 204 or on both sides of the dispersed phase transport channel 204. When the interdigital electrodes 101 are located on both sides of the dispersed phase transport channel 204, the number of the interdigital electrodes 101 on both sides can be the same (for example, the interdigital electrodes 101 are symmetrically arranged on both sides of the dispersed phase transport channel 204) or different.

[0035] The number of deflection electrode channels corresponds to the number of deflection electrodes 301. The deflection electrode 301 is arranged in the deflection electrode channel. When the number of the deflection electrode 301 is one, the deflection electrode 301 is located on any side of the droplet transport channel 207. When the number of the deflection electrodes 301 is multiple, the deflection electrode 301 can be located on any side of the droplet transport channel 207 or on both sides of the droplet transport channel 207. When the deflection electrodes 301 are located on both sides of the droplet transport channel 207, the number of the two sides can be the same (for example, the deflection electrodes 301 are symmetrically arranged on both sides of the dispersed phase transport channel 204) or different.

[0036] When the above-mentioned microfluidic chip is used to sort cells, the dispersed phase cerebrospinal fluid enters the dispersed phase cerebrospinal fluid flow channel 201 from the inlet of the dispersed phase cerebrospinal fluid flow channel 201 ( Figure 1 The direction indicated by the arrow above is the direction of liquid flow). The first dispersed phase sheath liquid enters the first dispersed phase sheath liquid flow channel 202 from the inlet of the first dispersed phase sheath liquid flow channel 202, and the second dispersed phase sheath liquid enters the second dispersed phase sheath liquid flow channel 203 from the inlet of the second dispersed phase sheath liquid flow channel 203. The dispersed phase spinal fluid, the first dispersed phase sheath liquid and the second dispersed phase sheath liquid (the three are collectively referred to as dispersed phase liquids) merge at the first intersection and flow into the dispersed phase delivery flow channel 204. The first continuous phase enters the first continuous phase flow channel 205 from the inlet ( Figure 1 The direction indicated by the upper arrow is the direction of liquid flow), and the second continuous phase enters the second continuous phase flow channel 206 from the entrance of the second continuous phase flow channel 206. The first continuous phase and the second continuous phase are collectively referred to as continuous phase liquid. When the dispersed phase liquid flows from the dispersed phase delivery channel 204 through the second intersection, it is sheared by the shear force of the continuous phase liquid to generate micro-droplets. The micro-droplets enter the droplet delivery channel 207, and under the action of the charging electrode 209 (the first power supply 401 provides voltage to the charging electrode 209), charged droplets 501 are formed. Under the action of the deflection electrode 301 (the second power supply 402 provides voltage to the deflection electrode 301), the charged droplets 501 are pushed to one side of the droplet delivery channel 207, and then enter the corresponding branch channel and are successfully collected.

[0037] Example 2 A method for preparing a sorting microfluidic chip integrating an acoustic-optical-electrical method comprises the following steps: S1. Prepare interdigital electrodes and deflection electrodes on a first substrate; the material of the first substrate is piezoelectric material.

[0038] A microfluidic system and a charging electrode are prepared on a second substrate. First, a positive mold of a microfluidic system is prepared on a single crystal silicon wafer. The positive mold of the microfluidic system includes a dispersed phase medullary fluid flow channel, a first dispersed phase sheath fluid flow channel, a second dispersed phase sheath fluid flow channel, a dispersed phase transport flow channel, a first continuous phase flow channel, a second continuous phase flow channel, a droplet transport flow channel, at least one transmitting optical fiber channel, at least one receiving optical fiber channel, and at least one deflection electrode flow channel. The positive mold of the microfluidic system is subjected to a hydrophobic treatment. The material of the second substrate is cast on the positive mold of the microfluidic system to prepare a second substrate having a microfluidic system. Then, the charging electrode is placed at the intersection where the dispersed phase transport flow channel is connected to the first continuous phase flow channel and the second continuous phase flow channel, and the material of the second substrate is cast again on the surface of the microfluidic system to fix the charging electrode; the obtained substrate is cut and punched to obtain a second substrate having a microfluidic system and a charging electrode.

[0039] S2. Combining the first substrate with the interdigital electrodes with the second substrate with the microfluidic system and the charging electrodes by bonding technology.

[0040] S3. Integrate the optical fiber in the optical fiber channel. The specific method is as follows: Use an optical fiber cutter to cut the end face of the optical fiber with a diameter of 100 μm flat, push the optical fiber along the optical fiber channel until the end face of the optical fiber reaches the end of the optical fiber channel, and use ultraviolet light curing glue to fix the optical fiber at the entrance of the optical fiber channel.

[0041] As an example, the material of the first substrate is lithium niobate.

[0042] As an example, step S1 includes the steps of cleaning the first substrate and performing surface hydrophilic treatment.

[0043] Furthermore, as an example, the method for cleaning a lithium niobate substrate includes the following steps: ultrasonically cleaning the lithium niobate substrate with acetone, anhydrous ethanol and deionized water for 15 minutes each, taking it out and blowing it dry with a nitrogen gun, placing it on a hot plate at room temperature, slowly heating it to 120°C and maintaining it for 30 minutes, and drying it to obtain a dry lithium niobate substrate.

[0044] Further, as an example, a method for treating the surface hydrophilicity of a lithium niobate substrate includes the following steps: placing the cleaned lithium niobate substrate in an oxygen plasma cleaning machine for surface activation (generally cleaning for 2 minutes).

[0045] As an example, step S1 includes preparing patterns of interdigital electrodes and deflection electrodes on a first substrate. A specific method for preparing the pattern includes the following steps: injecting photoresist AZ5214 at the center of a lithium niobate substrate. Fix the lithium niobate substrate with AZ5214 photoresist above the suction cup of a coating machine, and use the coating machine to spin coat the lithium niobate substrate. The coating parameters are: acceleration of 500 rpm, accelerated to 3000 rpm and maintained for 40 seconds. After the coating is completed, dry at 95°C for 2 minutes to complete the pre-baking of the lithium niobate substrate. Place the pre-baked lithium niobate substrate under a mask designed with a pattern of interdigital electrodes and a pattern of deflection electrodes, and use a photolithography machine to perform photolithography on the lithium niobate substrate. Dry at 95°C for 3 minutes to complete the post-baking of the lithium niobate substrate after photolithography. After the post-baking is completed, develop the lithium niobate substrate at room temperature until no white precipitate appears.

[0046] Further, as an example, the method for preparing the interdigital electrodes and the deflection electrodes in step S1 adopts the Lift-Off process, which specifically includes the following steps: placing the lithium niobate substrate with the interdigital electrodes and the deflection electrode pattern in a magnetron sputtering machine, first sputtering the first layer of silicon oxide film, then sputtering the aluminum film, stripping the photoresist and various films, and obtaining the first substrate with the interdigital electrodes and the deflection electrodes. The thickness of the first layer of silicon oxide film is 100~200nm, for example, 100nm, 130nm, 150nm, 180nm, 200nm, but not limited thereto; preferably 150nm. The thickness of the aluminum film is 60~140nm, for example, 60nm, 80nm, 100nm, 120nm, 140nm, but not limited thereto; preferably 100nm.

[0047] As an example, the material of the second substrate in step S1 is PDMS (polydimethylsiloxane).

[0048] As an example, step S1 includes the steps of cleaning the single crystal silicon wafer and performing surface hydrophilic treatment.

[0049] Further, as an example, the steps for cleaning a single crystal silicon wafer are as follows: place the single crystal silicon wafer in a piranha solution and heat it to 50°C for 5 hours. After taking it out, ultrasonically clean it with acetone, anhydrous ethanol and deionized water for 15 minutes each, take it out and blow dry it with a nitrogen gun, place it on a hot plate at 120° and heat it for 30 minutes to obtain a cleaned single crystal silicon wafer.

[0050] Further, as an example, a method for treating the surface hydrophilicity of a single crystal silicon wafer includes the following steps: placing the cleaned single crystal silicon wafer with its surface facing upward in an oxygen plasma cleaning machine for surface activation (generally cleaning for 2 minutes).

[0051] As an example, the method for preparing a positive mold of a microfluidic system in step S1 includes the following steps: injecting SU8 photoresist into the center of a single crystal silicon wafer, placing the single crystal silicon wafer with SU8 photoresist on top of a coating machine suction cup, and using a coating machine to spin coat the single crystal silicon wafer. The coating parameters are: acceleration of 500 rpm, accelerating to 1000 rpm and maintaining for 62 seconds. After the coating is completed, the single crystal silicon wafer is placed on a heating table and dried at 45°C for 5 minutes to complete the pre-baking. The mask with a microfluidic system pattern is placed on a mask holder of a photolithography machine, and the single crystal silicon wafer is photolithographically processed. Dry at 65°C for 5 minutes to complete the post-baking of the single crystal silicon wafer after photolithography. Then the single crystal silicon wafer is developed. Finally, it is dried with nitrogen and placed on the heating table again for hard mold baking (for example, kept at 75°C for 120 minutes) to obtain a positive mold of a microfluidic system.

[0052] As an example, the method for hydrophobizing the positive mold of the microfluidic system includes the following steps: placing the obtained positive mold of the microfluidic system in a volatile cylinder, dropping three drops of perfluorodecyltriethylsilane into the volatile cylinder, and after sealing, placing the volatile cylinder in an oven for heating and baking (for example, 110°C for 60 minutes).

[0053] As an example, the method for preparing the second substrate with a microfluidic system in step S2 includes the following steps: PDMS prepolymer (Sylgard 184, Dow Corning) and dimethylsiloxane (DMS) are mixed evenly at a mass ratio of 10:1 to 15:1 (for example, the mass ratio of PDMS prepolymer to curing agent is 10:1, 12:1 or 15:1, preferably 10:1), and placed in a vacuum drying oven for degassing at 25°C for 30 minutes. Then, PDMS is drawn with a syringe and poured onto the positive mold of the microfluidic system, and placed in a vacuum drying oven for degassing at 25°C for 30 minutes, and then placed on a heating table for heating and curing at 105°C.

[0054] It is understandable that the deflection electrode can also be prepared by other methods besides the Lift-Off process. For example, a conductive solution or liquid metal or conductive gel can be filled into the deflection electrode microchannel to serve as the deflection electrode; solid metal can also be heated to liquid state and then filled into the deflection electrode microchannel, and then cooled to solid state to obtain solid metal as the deflection electrode.

[0055] Example 3 This example uses the microfluidic chip in Example 1 to sort H22 cells (using magnetic beads for enrichment), and the specific process is as follows: R1 and H22 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2. The cell concentration was adjusted to 1×10 6 / mL and labeled with fluorescent dye (Hoechst 33342 or FITC) to form a sample solution.

[0056] R2. Use a syringe pump to pass the first dispersed phase sheath liquid (1×PBS, pH=7.4), the second dispersed phase sheath liquid (1×PBS, pH=7.4), and the sample liquid into the first dispersed phase sheath liquid flow channel 202, the second dispersed phase sheath liquid flow channel 203, and the dispersed phase spinal fluid flow channel 201 of the microfluidic chip. Pass the first continuous phase (mineral oil) and the second continuous phase (mineral oil) into the first continuous phase flow channel 205 and the second continuous phase flow channel 206 of the microfluidic chip respectively. Control the flow rates of the first dispersed phase sheath liquid and the second dispersed phase sheath liquid to be 6μL / min, the flow rate of the sample liquid to be 3μL / min, and the flow rates of the first continuous phase and the second continuous phase to be 15μL / min. At the same time, a 19.8MHz, 0dB RF sinusoidal signal amplified by a power amplifier is applied to the interdigital electrode 101, and the cells will be focused into a straight line and flow to the detection optical fiber (including the transmitting optical fiber and the receiving optical fiber) located in the optical fiber channel 208. After the first dispersed phase sheath liquid, the second dispersed phase sheath liquid and the sample liquid merge at the first intersection, they enter the dispersed phase delivery channel 204 and then are sheared by the first continuous phase and the second continuous phase at the second intersection. Under this condition, charged droplets 501 encapsulating cells with a particle size of 80-100 μm are stably generated and enter the droplet delivery channel 207.

[0057] R3. Use a data acquisition card with a photomultiplier tube to collect signals at a frequency of 1kHz, and set the threshold of the fluorescence pulse detected to 200 photon units. According to the detection signal, output the pulse to the voltage amplifier (the voltage amplifier multiple is ×100), and set the pulse signal to a rectangular wave pulse with a delay of 20ms, a pulse width of 1ms, and a pulse voltage of 5V. The voltage amplifier is connected to the first power supply 401 as a signal source. The first power supply 401 provides power for the charging electrode 209. The cell detection accuracy under this condition reaches 99.8%.

[0058] R4, the second power supply 402 sets the positive and negative voltages to ±1kV, provides a deflection voltage to the deflection electrode 301, and deflects the charged droplets to one side of the droplet transport channel 207 to achieve cell enrichment. The outlet of the droplet transport channel 207 is connected to three branch channels. Use three collection bottles to collect droplets at the outlet of each branch channel. The components are analyzed by flow cytometry, and the concentration of cells or magnetic beads is calculated, and the cell sorting accuracy is 99.3%. Before enrichment, the concentration of magnetic nanoparticles measured by the flow cytometer was 7.7%. After enrichment by the microfluidic chip, the concentration of magnetic nanoparticles rose to 87.0%. R5. Perform cell viability test on the successfully sorted H22 cells, and use 0.4wt% trypan blue dye to stain the cells in the sorted droplets. The staining time is 5 minutes to ensure that the cells are fully exposed to the dye. Use a cell counting plate (such as a Neubauer counting plate) to count cells. In the large squares of the counting plate, count the number of living cells that are not stained with trypan blue and the number of dead cells that are stained. The volume of each large square is 0.1mm³, and ensure that at least 4 large squares are counted to improve statistical accuracy. Calculate the proportion of living cells (unstained cells) to all cells (living cells + dead cells) in each large square. Use the formula: survival rate = (number of living cells / total number of cells) × 100%. Perform at least three independent experiments to ensure the reliability of the results. The average value of the statistical results of each experiment is used as the final cell survival rate, and the calculated cell survival rate is 94.3%.

[0059] The above-described embodiments are only preferred embodiments of the present application and are not intended to limit the scope of protection of the present application. For any person skilled in the art, the present application may have various changes and modifications. Any simple equivalent changes and modifications made based on the scope of protection of the present application and the contents of the specification should be included in the scope of protection of the present application.

Claims

1. A sorting microfluidic chip integrating acoustic-optical-electrical methods, characterized in that: The microfluidic chip includes a first substrate, a second substrate combined with the first substrate, at least one deflection electrode, and at least one transmitting optical fiber and at least one receiving optical fiber; The material of the first substrate is a piezoelectric material, and at least one interdigital electrode is disposed on the first substrate; The second substrate is provided with a microfluidic system and a charging electrode; The microfluidic system includes a dispersed phase marrow fluid flow channel, a first dispersed phase sheath fluid flow channel, a second dispersed phase sheath fluid flow channel, a dispersed phase transport flow channel, a first continuous phase flow channel, a second continuous phase flow channel, a droplet transport flow channel, an optical fiber channel corresponding to the total number of the transmitting optical fiber and the receiving optical fiber, and a deflection electrode flow channel corresponding to the number of the deflection electrodes; the first dispersed phase sheath fluid flow channel and the second dispersed phase sheath fluid flow channel are respectively located on both sides of the dispersed phase marrow fluid flow channel and are both connected to the dispersed phase marrow fluid flow channel at a first intersection; the first intersection is connected to the entrance of the dispersed phase transport flow channel; the first continuous phase flow channel and the second continuous phase flow channel are respectively located on both sides of the dispersed phase transport flow channel and are both connected to the dispersed phase flow channel at a second intersection; the second intersection is connected to the entrance of the droplet transport flow channel; The interdigitated electrodes are located on one side or both sides of the dispersed phase transport channel; the deflection electrode is placed in the deflection electrode channel; the deflection electrode channel is located on one side or both sides of the dispersed phase transport channel; the transmitting optical fiber and the receiving optical fiber are respectively placed in the optical fiber channel; the optical fiber channel is located on one side or both sides of the dispersed phase transport channel; the charging electrode is located above the second intersection.

2. The sorting microfluidic chip integrating the acoustic-optical-electrical method according to claim 1, characterized in that: The first substrate is a lithium niobate substrate; or / and the material of the second substrate is PDMS.

3. The sorting microfluidic chip integrating the acoustic-optical-electrical method according to claim 1, characterized in that: The interdigitated electrodes are symmetrically arranged on both sides of the dispersed phase transport channel; or / and the deflection electrodes are symmetrically arranged on both sides of the droplet transport channel.

4. The method for preparing a sorting microfluidic chip integrating an acoustic-optical-electrical method according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, preparing the interdigital electrodes on the first substrate; preparing the microfluidic system and the charging electrode on the second substrate; preparing the deflection electrode; The preparation method of the microfluidic system comprises the following steps: preparing a positive mold of the microfluidic system on a single crystal silicon wafer; the positive mold of the microfluidic system comprises a dispersed phase marrow fluid flow channel, a first dispersed phase sheath fluid flow channel, a second dispersed phase sheath fluid flow channel, a dispersed phase transport flow channel, a first continuous phase flow channel, a second continuous phase flow channel, a droplet transport flow channel, at least one transmitting optical fiber channel, at least one receiving optical fiber channel, and at least one deflection electrode flow channel; performing a hydrophobic treatment on the positive mold of the microfluidic system; pouring the material of the second substrate on the positive mold of the microfluidic system to obtain the microfluidic system; placing the charging electrode above the second intersection, and pouring the material of the second substrate again on the surface of the microfluidic system; cutting and punching the obtained substrate to obtain a second substrate having a microfluidic system and a charging electrode; S2, combining the first substrate having the interdigital electrodes with the second substrate having the microfluidic system and the charging electrode; S3, placing the transmitting optical fiber in the transmitting optical fiber channel, and placing the receiving optical fiber in the receiving optical fiber channel; and obtaining the microfluidic chip.

5. The preparation method according to claim 4, characterized in that: Step S1 includes the steps of cleaning the first substrate and treating its surface for hydrophilicity; or / and Step S1 includes the steps of cleaning the single crystal silicon wafer and treating its surface for hydrophilicity.

6. The preparation method according to claim 5, characterized in that: The method for cleaning and treating the surface hydrophilicity of the first substrate comprises the following steps: ultrasonically cleaning the first substrate with acetone, anhydrous ethanol and deionized water in sequence, taking it out and blowing it dry with a nitrogen gun, and then drying it to obtain a dried first substrate; plasma cleaning the dried first substrate; or / and the method for cleaning and treating the surface hydrophilicity of the single crystal silicon wafer comprises the following steps: placing the single crystal silicon wafer in a piranha solution and heating it; taking it out and ultrasonically cleaning it with acetone, anhydrous ethanol and deionized water in sequence; taking it out, blowing it dry with a nitrogen gun, and then drying it to obtain a cleaned single crystal silicon wafer; plasma cleaning the cleaned single crystal silicon wafer.

7. The preparation method according to claim 4, characterized in that: The interdigital electrodes in step S1 are prepared by a Lift-Off process; Or / and, the preparation method of the deflection electrode is selected from any one of the following methods: (1) The deflection electrode is prepared by a Lift-Off process; (2) The deflection electrode flow channel is filled with any one of a conductive solution, liquid metal, solid metal or conductive gel.

8. The preparation method according to claim 4, characterized in that: The method for preparing the microfluidic system in step S1 includes the following steps: uniformly mixing the PDMS prepolymer and the curing agent in a mass ratio of 10:1 to 15:1, and then drying and degassing to obtain PDMS; taking the PDMS and pouring it onto the positive mold of the microfluidic system, drying and degassing, and heating and curing to obtain the microfluidic system.

9. The preparation method according to claim 4, characterized in that: The method for hydrophobizing the positive mold of the microfluidic system in step S1 comprises the following steps: placing the positive mold of the microfluidic system in a volatilization cylinder, dripping perfluorodecyltriethylsilane into the volatilization cylinder, sealing the cylinder, and placing the volatilization cylinder in an oven for heating and baking.

10. Use of the sorting microfluidic chip integrating the acoustic-optical-electrical method according to any one of claims 1 to 3 in at least one of cell counting, cell sorting and cell enrichment.

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