WO2018113034A1 - 微流控装置、将物质导入细胞的系统及方法 - Google Patents

微流控装置、将物质导入细胞的系统及方法 Download PDF

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WO2018113034A1
WO2018113034A1 PCT/CN2017/000080 CN2017000080W WO2018113034A1 WO 2018113034 A1 WO2018113034 A1 WO 2018113034A1 CN 2017000080 W CN2017000080 W CN 2017000080W WO 2018113034 A1 WO2018113034 A1 WO 2018113034A1
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channel
flow field
cell
solution
microstructure
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PCT/CN2017/000080
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English (en)
French (fr)
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郑海荣
周伟
孟龙
牛丽丽
林争荣
王凯悦
黄小伟
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中国科学院深圳先进技术研究院
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Priority to US16/606,355 priority Critical patent/US11511277B2/en
Publication of WO2018113034A1 publication Critical patent/WO2018113034A1/zh

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    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • C12N15/895—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection using biolistic methods
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0436—Moving fluids with specific forces or mechanical means specific forces vibrational forces acoustic forces, e.g. surface acoustic waves [SAW]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0493—Specific techniques used
    • B01L2400/0496—Travelling waves, e.g. in combination with electrical or acoustic forces

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a microfluidic device, a system and method for introducing a substance into a cell.
  • Gene therapy refers to biology, physics and chemistry.
  • a normal gene or a therapeutic gene is introduced into a human target cell to correct a genetic defect or a functional disorder, thereby achieving the purpose of treating the disease. How to efficiently and safely transport foreign substances from outside the cells to the cells is the key to gene therapy.
  • methods for introducing substances into cells include viral gene introduction, injection method, gene gun method, electroporation method, and sonophoresis method.
  • sonic perforation is to add a large amount of cells and bubbles to a solution at the same time, and to combine the bubbles with Shake the cells evenly, so that the bubbles are evenly scattered between the cells.
  • the mixed solution is irradiated by ultrasound.
  • the bubbles will generate cavitation effects under the action of ultrasound, and generate physics such as expansion, implosion, micro-acoustic flow, micro-jet and shock wave. Phenomenon, these physical phenomena will form micropores on the cell surface, leading to changes in cell membrane permeability.
  • an object of embodiments of the present invention is to provide a microfluidic device, a system and method for introducing a substance into a cell, to solve the problem that the device for sono-perforation in the prior art cannot accurately control the cell and the bubble. The distance, thus causing the cells to suffer from the problem of unstable shear forces.
  • an embodiment of the present invention provides a microfluidic device, the device comprising:
  • a channel a channel, a bulk wave generating device, and a surface acoustic wave generating device
  • the inner wall of the channel is provided with a microstructure, and when the solution is injected into the channel, the solution forms bubbles at the microstructure;
  • the bulk wave generating device for generating a body wave to resonate the bubble to generate a flow field
  • the surface acoustic wave generating device is configured to generate a surface acoustic wave and control the position of the particles in the solution.
  • the microstructure is one or more;
  • the microstructure is a recess or hole that is concave on the inner wall of the channel.
  • the surface acoustic wave generating device controls the particles to be aligned in a line and to control the position of the particles in the flow field.
  • the channel is made of a transparent material, preferably a siloxane channel, more preferably a polydimethylsiloxane lumen;
  • the bulk wave generating device is a bulk wave transducer
  • the surface acoustic wave generating device is an interdigital transducer.
  • an embodiment of the present invention provides a system for introducing a substance into a cell, wherein the system comprises the microfluidic device provided by the first aspect;
  • An injection device coupled to the inlet of the chamber of the microfluidic device for injecting a solution comprising cells into a lumen of the microfluidic device, the solution being produced at a microstructure of the lumen bubble;
  • the microfluidic device for generating a body wave, resonating the bubble, causing vibration of a solution in the channel, generating a flow field in the channel, where each position in the flow field corresponds to a different shear Shear stress and control a position of the cell relative to the microstructure in the flow field, causing the cell to flow through a predetermined position when flowing in the flow field, the corresponding shear of the cell at the predetermined position Reversible perforation occurs under the action of stress, and the substance in the solution enters the cell through the reversible perforation.
  • the system further includes a signal generator and a power amplifier
  • the signal generator is configured to generate a sine wave signal and send the sine wave signal to the power amplifier;
  • the power amplifier is configured to amplify the sine wave signal and transmit the amplified sine wave signal to the bulk wave transducer and the interdigital transducer.
  • the system further comprises a cell recovery container
  • the cell recovery vessel is connected to an outlet of the channel.
  • an embodiment of the present invention provides a method of introducing a substance into a cell, wherein the method comprises:
  • Controlling by the surface acoustic wave generating device in the microfluidic device, a position of the cell relative to the microstructure in the first flow field, causing the cell to flow while flowing in the first flow field Over a predetermined position, the cell produces a reversible perforation under the corresponding shear stress at the predetermined position, and the substance in the solution enters the cell through the reversible perforation.
  • the corresponding shear stress at each location in the first flow field is determined by:
  • the solution containing the tracer particles is injected into the PDMS channel, and the injected solution containing the tracer particles generates arc-shaped bubbles at the microstructure in the PDMS channel;
  • a corresponding shear stress at each of the second flow fields is determined as a corresponding shear stress at each of the first flow fields.
  • the method further includes:
  • Amplifying the sine wave signal by the power amplifier and transmitting the amplified sine wave signal to the bulk wave transducer and the interdigital transducer;
  • the position of the cell relative to the microstructure in the first flow field is adjusted by adjusting the phase of the sine wave signal produced by the signal generator.
  • the surface acoustic wave generating device can accurately control the position of the particles in the solution, thereby realizing precise control of the particles in the solution. Shear stress.
  • Embodiment 1 is a schematic structural view of a microfluidic device provided by Embodiment 1 of the present invention.
  • FIG. 2 is a schematic structural view of a cavity in a microfluidic device according to Embodiment 1 of the present invention
  • FIG. 3 is a schematic structural view of an interdigital transducer in a microfluidic device according to Embodiment 1 of the present invention.
  • FIG. 4 is a schematic diagram showing a specific structure of a microfluidic device provided by Embodiment 1 of the present invention.
  • FIG. 5 is a schematic structural view showing a system for introducing a substance into a cell according to Embodiment 2 of the present invention.
  • FIG. 6 is a schematic view showing a second structure of a system for introducing a substance into a cell according to Embodiment 2 of the present invention.
  • Fig. 7 is a flow chart showing a method of introducing a substance into a cell according to Embodiment 3 of the present invention.
  • microinjection pump 650
  • tubing 660
  • cell recovery container 670
  • power amplifier 680
  • embodiments of the present invention provide a microfluidic device, a system and method for introducing a substance into a cell, which are described below by way of embodiments.
  • the invention relates to a microfluidic device, comprising:
  • a channel having a microstructure disposed on an inner wall thereof, the microstructure being configured to form a bubble at the microstructure when the solution is injected into the channel;
  • a bulk wave generating device for generating a body wave resonates the bubble to generate a flow field.
  • the microfluidic device further includes a surface acoustic wave generating device for generating surface acoustic waves and controlling the position of the particles in the solution.
  • An embodiment of the present invention provides a microfluidic device, as shown in FIG. 1, the device includes a cavity 110, a bulk wave generating device 120, and a surface acoustic wave generating device 130;
  • a microstructure is disposed on the interior of the channel 110, and when the solution is injected into the channel 110, the solution forms bubbles at the microstructure;
  • a body wave generating device 120 for generating a body wave to resonate the bubble and generate a flow field
  • the surface acoustic wave generating device 130 is for generating surface acoustic waves and controlling the position of the particles in the above solution.
  • the channel 110 may be made of a transparent material, and the channel 110 may be a siloxane channel or a PDMS (polydimethylsiloxane) channel.
  • the bulk wave generating device 120 can be a bulk wave transducer.
  • the surface acoustic wave generating device 130 may be an interdigital transducer.
  • the microstructures in the above described channels 110 are one or more.
  • the microstructure is a recess or hole that is concave on the inner wall of the channel 110.
  • FIG. 2 a schematic structural view of one of the possible channels 110 is shown.
  • the solution When the solution is injected into the channel through the inlet 111 of the channel 110, the solution does not flow into the microstructures 112 of the channel 110, but instead forms a bubble at the microstructure 112 and the bubbles formed are arc-shaped bubbles.
  • 113 is the outlet of the channel 110, and particles or solutions in the solution exit the channel 110 from the outlet 113.
  • FIG 2 shows only the case where the five microstructures 112 are included in the cavity 110, but the embodiment of the present invention is not limited thereto.
  • the specific number of the microstructures 112 can be set according to specific application scenarios.
  • the body wave generating device 120 When the body wave generating device 120 is in operation, the body wave is emitted outward, and the frequency of the body wave emitted is the resonance frequency of the bubble generated at the microstructure. Therefore, the generated body wave causes resonance of the bubble, and the bubble resonance drives the cavity.
  • the flow of solution in channel 110 produces a flow field in channel 110.
  • the above resonance frequency can be determined according to the radius of the circle where the arc of the arc-shaped bubble is located, and specifically includes:
  • the above resonance frequency is determined by the following formula
  • f is the resonant frequency of the bubble
  • ⁇ is the surface tension of the fluid in the flow field
  • p is the fluid pressure in the flow field
  • ⁇ is the multi-index of the gas inside the bubble
  • ⁇ is the density of the fluid in the flow field
  • a is the radius of the circle in which the bubble is located.
  • the surface acoustic wave generating device 130 described above controls the particles in the solution in the channel to be aligned in a straight line and to control the position of the particles in the flow field.
  • the surface acoustic wave generating device 130 controls the position of the particles in the flow field relative to the microstructure, and the surface acoustic wave generating device 130 can control the position of the particles in the vertical direction in the flow field when the channel is horizontally placed.
  • the phase of the input signal of the surface acoustic wave generating device 130 can be adjusted, thereby adjusting the position of the particle relative to the microstructure in the flow field, that is, in the flow field.
  • the position in the vertical direction specifically includes:
  • a one-dimensional standing wave field is formed in the cavity 110, and under the action of the ultrasonic radiation force generated by the surface acoustic wave generating device 130, in the cavity 110
  • the particles are arranged at the position of the standing wave node, and the frequency of the input signal of the surface acoustic wave generating device 130 is adjusted such that the surface acoustic wave generated by the surface acoustic wave generating device 130 is moved up or down, thereby causing another
  • the standing wave node superposed by the surface acoustic wave generated by the surface wave generating device 130 is moved up or down. Since the particles are locked at the position of the nodes, the particles in the channel 110 move up and down as the nodes move up and down, so that the position of the particles in the vertical direction in the flow field in the channel can be adjusted.
  • the distance in which the particles move in the vertical direction and the phase of the input signal of the surface acoustic wave generating device 130 satisfy the following relationship:
  • ⁇ x is the displacement of the particle movement
  • ⁇ is the wavelength of the surface acoustic wave generated by the surface acoustic wave generating device 130
  • the position of the particles in the vertical direction of the flow field does not change, and therefore, the position of the particles in the vertical direction in the flow field can be precisely controlled.
  • the surface acoustic wave generating device 130 may include a pair of interdigital transducers, that is, a pair of interdigital electrodes are plated on the piezoelectric substrate, and a schematic structural view is shown in FIG. 3, and one of the surface acoustic wave generating devices 130 is used. A possible structural schematic is shown in Figure 3.
  • the surface acoustic wave generating device 130 includes a piezoelectric substrate 310 and a pair of interdigital electrodes 320 formed by plating a pair of interdigital electrodes 320 on the piezoelectric substrate 310.
  • the interdigital transducer in the embodiment of the present invention can select 128° YX double-sided polished lithium niobate as the piezoelectric substrate.
  • the interdigital transducer includes a piezoelectric substrate and an interdigital electrode, that is, formed by plating an interdigital electrode on the piezoelectric substrate.
  • the interdigital transducer can be fabricated by a process such as gluing, photolithography, coating, and stripping.
  • the piezoelectric substrate was glued.
  • the front side A34620 was spin-coated at 5000 rpm for 30 s, and the chip was placed on a 120° hot plate for 3 minutes, and the light was rubbed with a step meter.
  • the thickness of the glue is tested, and the test result is that the thickness of the photoresist is about 5 ⁇ m;
  • the coated piezoelectric substrate is exposed and developed, and the prepared film sheet is overlaid on the coated piezoelectric substrate for exposure, and the light is transmitted through the place where the light is transmitted, and the AZ400 is used for development.
  • the above cured portion will not be dissolved, the non-cured portion will be dissolved, and then baked on a hot plate at 150 ° C for 10 minutes after development;
  • the substrate of the above-mentioned aluminum film on which the metal layer was grown was placed in an acetone solution, and the photoresist was peeled off by ultrasonic vibration of an ultrasonic cleaner to obtain an interdigital transducer.
  • the channel 110 in the embodiment of the present invention may be a PDMS channel, and the PDMS channel is fabricated by a process such as pretreatment, gluing and prebaking, exposure and development, pouring PDMS, stripping PDMS, and the like.
  • the above production process specifically includes:
  • the cleaned silicon substrate is placed in a clean place to dry;
  • SU-8 (50) negative photoresist was spin-coated with a glue applicator and spin-coated at 3000 rpm for 30 s.
  • the thickness of SU-8 (50) was about 50 ⁇ m.
  • the silicon wafer was placed horizontally at 90 ° C. 1h, the solvent in the photoresist is volatilized to enhance the adhesion between the photoresist and the silicon wafer;
  • the prepared film film is placed on the above-mentioned glued silicon wafer, and the photoresist is exposed through an exposure machine at an exposure dose of 600 CJ/cm 2 for 30 s.
  • the exposed silicon wafer is soaked with a developing solution, and is not exposed.
  • the area photoresist is dissolved, the exposed area photoresist remains, and after development, it is baked on a hot plate at 150 ° C for 10 min;
  • PD glue A glue and B glue according to the mass ratio of 10:1, mix well, pour into the Petri dish where the silicon wafer is located, vacuum the culture dish, remove the bubbles in the PDMS, and finally put the culture dish in 80
  • the PDMS is cured in an oven at °C for 30 minutes;
  • the PDMS containing the channels and microstructures was removed and completely stripped from the silicon wafer. Finally, the microchannels were perforated using a puncher to make an inlet and outlet.
  • the prepared interdigital transducer and the PDMS channel are plasma-treated, the plasma processing power is 150W, the duration is 70S, and then the PDMS channel is glued down to the two interdigital electrodes of the interdigital transducer. Bake between 80 ° C for 20 min.
  • FIG. 4 is a schematic diagram showing a possible specific structure of a microfluidic device provided by an embodiment of the present invention, including a PDMS cavity 410, a bulk wave transducer 420, and an interdigital transducer.
  • the interdigital transducer has two interdigital electrodes 320, wherein the PDMS cavity 410 and the bulk wave transducer 420 and the interdigital transducer can be integrated on the same chip.
  • the solution is injected into the PDMS channel 410.
  • arc-shaped bubbles are generated at the microstructure of the PDMS channel 410.
  • the bulk wave transducer 420 emits body waves outward during operation, and the emitted body The frequency of the wave coincides with the resonant frequency of the bubble in the PDMS cavity 410. Therefore, the body wave generated by the bulk wave transducer 420 causes vibration of the bubble in the PDMS cavity 410, and the vibration of the bubble drives the vibration of the solution in the PDMS cavity 410. , generating a flow field.
  • the PDMS cavity 410 is located in the middle of the two interdigital electrodes 320 on the interdigital transducer.
  • both of the interdigital electrodes 320 emit a surface acoustic wave signal, and the emitted sound is emitted.
  • the surface wave controls the position of the particles relative to the microstructure in the flow field.
  • Embodiments of the present invention provide a microfluidic device capable of accurately controlling the position of particles in a solution by a surface acoustic wave generating device, thereby achieving precise control of shear stress of particles in the solution.
  • the embodiment of the present invention provides a system for introducing a substance into a cell, as shown in FIG. 5, the system includes an injection device 510 and the microfluidic device 520 provided in the above embodiment 1;
  • the injection device 510 is configured to inject a solution containing cells into the channel of the microfluidic device 520, and the injected solution generates bubbles at the microstructure of the channel;
  • the microfluidic device 520 is configured to generate a body wave, resonate the bubble, cause vibration of the solution in the channel, generate a flow field in the channel, corresponding to different shear stress at each position in the flow field, and control the cell in
  • the position of the flow field relative to the microstructure causes the cells to flow through the preset position when flowing in the flow field, and the cells produce reversible perforation under the corresponding shear stress at the preset position, and the substance in the solution enters through the reversible perforation.
  • the cell is configured to generate a body wave, resonate the bubble, cause vibration of the solution in the channel, generate a flow field in the channel, corresponding to different shear stress at each position in the flow field, and control the cell in
  • the position of the flow field relative to the microstructure causes the cells to flow through the preset position when flowing in the flow field, and the cells produce reversible perforation under the corresponding shear stress at the preset position, and the substance in the solution enters through the reversible
  • the above substance may be a gene fragment, a drug molecule or the like.
  • the injection device 510 described above is connected to the lumen of the microfluidic device 520 via a conduit, which may be a microinjection pump.
  • the above system further includes a signal generator and a power amplifier
  • the signal generator is configured to generate a sine wave signal and send the sine wave signal to the power amplifier;
  • the power amplifier is configured to amplify the sine wave signal and send the amplified sine wave signal to the bulk wave transducer and the interdigital transducer.
  • the system in the embodiment of the present invention may include one signal generator, or may include two signal generators or multiple.
  • the embodiment of the present invention does not limit the number of signal generators, as long as sufficient number of signals can be obtained. can.
  • the above system further comprises a cell recovery container
  • the cell recovery container is connected to the lumen in the microfluidic device 520 through a conduit for containing cells into which the substance has been introduced.
  • FIG. 6 a specific structural diagram of a possible system for introducing a substance into a cell is shown, including: a micro syringe pump 610, a PDMS lumen 410, a bulk wave transducer 420, and a surface acoustic wave generating device.
  • the surface acoustic wave generating device has a pair of interdigital electrodes 320, a pipe 650, a cell recovery container 660, two power amplifiers 670, and a signal generator 680.
  • a signal generator 680 is included in the above FIG. 6, only the case where a signal generator 680 is included is shown.
  • the signal generators may also be two or more.
  • the specific number of the signal generators 680 may be set according to specific application scenarios.
  • the specific number of signal generators 680 is not limited.
  • microinjection pump 610 is coupled to PDMS lumen 410 via conduit 650, and a solution containing cells is injected into PDMS lumen 410 via conduit 650, after injection of solution into PDMS lumen 410, in PDMS lumen 410.
  • An arc-shaped bubble is generated at the microstructure, the bulk wave transducer 420 is connected to the power amplifier 670, the power amplifier 670 is connected to the signal generator 680, and the signal generator 680 outputs a sine wave signal and sends the output sine wave signal to the power.
  • the amplifier 670 amplifies the signal by the power amplifier 670, and sends the amplified signal to the bulk wave transducer 420 to drive the bulk wave transducer 420 to operate, and the bulk wave transducer 420 emits a body wave outward.
  • the frequency of the generated body wave coincides with the resonance frequency of the bubble in the PDMS cavity 410. Therefore, the body wave generated by the bulk wave transducer 420 causes vibration of the bubble in the PDMS cavity 410, and the vibration of the bubble drives the PDMS cavity 410.
  • the vibration of the fluid creates a flow field.
  • the PDMS cavity 410 is located in the middle of the two interdigital electrodes 320, both of the interdigital electrodes 320 are connected to the power amplifier 670, the power amplifier 670 is connected to the signal generator 680, and the signal generator 680 outputs a sine wave signal, and The output sine wave signal is sent to power amplifier 670, which amplifies the signal by power amplifier 670 and sends the amplified signal to interdigital electrode 320 to drive the operation of interdigital electrode 320.
  • the surface acoustic wave signal is emitted outward, and the emitted surface acoustic wave controls the position of the cell relative to the microstructure in the flow field, so that each cell can pass the preset when the cell flows in the horizontal direction.
  • the position when the cells flow through the preset position, produces a reversible perforation under the shear stress at the preset position, and the substance in the solution enters the cell through the reversible perforation.
  • the embodiment of the present invention provides a system for introducing a substance into a cell, wherein the microfluidic device can accurately control the position of the cell in the flow field, so that each cell passes through a preset position when flowing in the flow field, thereby accurately Controls the shear stress experienced by cells.
  • Embodiments of the present invention provide a method for introducing a substance into a cell, which method uses the system for introducing a substance into a cell in Embodiment 2, which can precisely control cell flow by a body wave generating device in a microfluidic device
  • the position of the field relative to the microstructure allows each cell to pass through a predetermined position as it flows through the flow field, thereby precisely controlling the shear stress experienced by the cell.
  • the substance introduced into the cell in the embodiment of the present invention may be a gene fragment, a drug molecule or the like, and the user may put the substance to be introduced into the cell into a solution according to actual needs, and then introduce the substance into the cell by the method provided by the embodiment of the present invention.
  • the substance is introduced into the cells by the method provided by the embodiment of the present invention, as shown in FIG. 7, and includes steps S710-S730, as follows.
  • the aforementioned channel comprises at least one microstructure, and the solution produces an arcuate bubble at each microstructure.
  • the solution containing the cells is injected into the lumen of the microfluidic device by an injection device as described above, and the injection device may be a microinjection pump.
  • a solution comprising cells can be continuously injected into the lumen of the microfluidic device.
  • shear stress at each location can be determined as follows.
  • the corresponding shear stress at each position in the first flow field generated by the solution in the channel is determined before the solution containing the cell is injected into the channel.
  • the shear stress corresponding to each position in the flow field can be determined by adding tracer particles in the flow field and by tracing the flow of the particles in the flow field.
  • the solution containing the tracer particles is injected into the cavity, and the injected solution containing the tracer particles generates arc-shaped bubbles at the microstructure in the cavity; the bubble is resonated by the body wave generating device to cause the cavity
  • the vibration of the fluid generates a second flow field in the channel; according to the flow state of the tracer particles in the second flow field, the shear stress corresponding to each position in the second flow field is determined by a computer;
  • the corresponding shear stress at each location is determined as the corresponding shear stress at each location in the first flow field.
  • the second flow field generated by the solution containing the tracer particles in the channel and the first flow field generated by the solution containing the cells in the channel are the same flow field, and the body is generated by the same body wave generating device. Wave-induced, therefore, the corresponding shear stress at each location in the second flow field can be determined as the corresponding shear stress at each location in the first flow field.
  • the corresponding shear stress at each position in the second flow field is determined by a computer, including:
  • the state of the tracer particles in the second flow field is continuously captured by the high speed camera to obtain a state image of the tracer particles; and according to the state image, the flow velocity of the tracer particles at each position in the second flow field is determined by a computer; The shear stress corresponding to the position is determined by a computer based on the flow velocity of the tracer particles at each location.
  • the flow state image of the plurality of tracer particles in the second flow field is continuously captured by the high speed camera, and any two frame state images are taken out from the multi-frame state image, and the two frame state images are the same.
  • the method performs segmentation, divides into the same plurality of small windows, and performs cross-correlation calculation on the windows corresponding to the two frames before and after, and specifically calculates the cross-correlation value of the corresponding window of the two frames by the following formula;
  • R(m, n) is a value of cross-correlation at a position (m, n) on a window corresponding to two images before and after
  • M*N is a size of the above window
  • M and N may Represented by pixels
  • m and n can also be represented by pixels
  • f(i,j) is the pixel value at the (i,j) position on the image of the previous frame
  • g(i+m,j+m) is the latter.
  • ⁇ f is the mean value of the f(i, j) pixels corresponding to all the pixels in the window on the image of the previous frame
  • ⁇ g is the image of the next frame.
  • the mean of the g(i+m, j+m) pixels corresponding to all the pixels on the window wherein the values of m and n above are fixed for a R(m, n) to be calculated.
  • the calculated cross-correlation value of the corresponding window of the two frames is determined as the displacement of the window movement, the size of the segmented small window is gradually reduced, and the spatial resolution of the calculation is improved, and each of the second flow fields is calculated by the above method.
  • the displacement of the positions, the above bits are removed from the time interval between the previous two frames, and the flow velocity of the tracer particles at each position is calculated.
  • the corresponding shear stress at each position is determined by the following formula according to the flow velocity of the tracer particles at each position;
  • ⁇ is the density of the fluid in the second flow field
  • ⁇ is the viscosity of the fluid in the second flow field
  • V x is the velocity of the fluid in the horizontal direction in the second flow field
  • V y is the fluid in the flow field.
  • WSS is the corresponding shear stress at the above position.
  • the velocity of the tracer particles at a location in the second flow field is the velocity of the fluid at that location, so the velocity of the flow field at each location can be known by the speed of the tracer particles at each location.
  • the shear stress corresponding to the position is determined by the flow velocity of the fluid at a certain position in the flow field and its periphery, for example, the velocity of the fluid passing through the A position in the flow field and the periphery thereof Determine the shear stress corresponding to the A position.
  • the surface acoustic wave generating device in the microfluidic device controls the position of the cells in the solution relative to the microstructure in the first flow field, so that the cells flow through the preset position when the first flow field flows, and the cells are in The reversible perforation is generated by the corresponding shear stress at the preset position, and the substance in the solution enters the cell through the reversible perforation.
  • the surface acoustic wave generating device generates a surface acoustic wave signal during operation, and the surface acoustic wave signal controls cells in the channel to be arranged in a straight line, and can control the position of the cell in the first flow field relative to the microstructure, so that the channel is inside
  • the distance between the bubble at the microstructure and the cell is fixed, so that the cell is in the water in the first flow field
  • each cell flows through a preset position, and is subjected to shear stress at a predetermined position, resulting in a reversible opening, so that the substance in the solution enters the cell through the reversible perforation.
  • the width of the channel is larger than the width of the cell and smaller than the surface acoustic wave generated by the surface acoustic wave generating device. Half of the wavelength, so that only one row of cells can pass through the lumen.
  • the above preset position can be adjusted and determined as follows:
  • the sine wave signal is generated by the signal generator, and the sine wave signal is sent to the power amplifier; the sine wave signal is amplified by the power amplifier, and the amplified sine wave signal is sent to the body wave transducing And an interdigital transducer; adjusting the position of the cell relative to the microstructure in the first flow field by adjusting the phase of the sine wave signal generated by the signal generator.
  • three sinusoidal signals can be generated by one signal generator, and one sine wave signal is amplified by the first power amplifier and sent to the body wave generating device, and the other two sinusoidal signals are amplified by the second power amplifier and sent to A pair of interdigital electrodes in a surface acoustic wave generating device.
  • three signal generators are used, one for generating a sine wave signal required by the bulk wave generating device, and two for generating a sine wave signal required for the surface acoustic wave generating device.
  • the specific number of the signal generators is not set, and in a specific application scenario, it can be selected according to the actual application.
  • the position of the cells relative to the microstructure in the first flow field can be adjusted by adjusting the phase of the sinusoidal signal generated by the signal generator.
  • the surface acoustic wave generating device described above is an interdigital transducer.
  • the channel is placed between the two interdigital electrodes of the interdigital transducer such that the channel can simultaneously receive the surface acoustic wave signals emitted by the two interdigital electrodes.
  • the specific process by which the user adjusts the position of the cell in the first flow field by adjusting the signal generator includes: when the interdigital transducer is working, a one-dimensional standing wave field is formed in the cavity, and the interdigital transducer is transposed Under the action of the ultrasonic radiation force, the cells in the channel are arranged at the position of the standing wave node, and the surface acoustic wave generated by the device generates a surface acoustic wave generated by the surface acoustic wave generating device. Will move up or down, resulting in a standing wave node superimposed with the surface acoustic wave generated by another surface acoustic wave generating device Move up or down. Since the cells are locked in the position of the nodes, the cells in the channels move up and down as the nodes move up and down, so that the position of the cells in the first flow field in the channel relative to the microstructure can be adjusted.
  • ⁇ x is the displacement of the cell movement
  • ⁇ is the wavelength of the surface acoustic wave generated by the interdigital transducer.
  • the viability and perforation degree of the cells can be observed by scanning electron microscopy.
  • an FDA fluorescent probe and a PI fluorescent probe may be added to the above-mentioned solution containing the cells.
  • the phase of the sine wave signal outputted by the signal generator can be adjusted by adding the FDA fluorescent probe and the PI fluorescent probe to the solution at the beginning.
  • the adjustment is completed, it is not necessary to add the FDA fluorescent probe and the PI fluorescent probe to the solution.
  • the user can add the FDA fluorescent probe to the solution at intervals of time during the introduction of the substance into the cell. PI fluorescent probe to observe the current cell viability and degree of perforation.
  • the above FDA is a hydrophobic complex that penetrates intact cell membranes into cells and catalyzes the hydrolysis of diacetate groups by intracellular esterases to produce high-intensity fluorescent products. If the cell membrane is intact, the FDA fluorescent molecule will Intracellular accumulation, emitting green fluorescence, therefore, FDA can be used as a label for cell viability.
  • PI penetrates into the nucleus through the perforation on the surface of the cell membrane, and combines with DNA to produce red fluorescence.
  • the intensity of red fluorescence can directly reflect the degree of cell perforation. Therefore, it can be labeled by cells.
  • the above two fluorescent probes measure the activity of the cells and the degree of perforation.
  • the intensity of the intracellular fluorescence is observed by scanning electron microscopy, and the viability of the cells in the first flow field relative to the various positions of the microstructure is determined according to the intensity of the fluorescence.
  • the degree of opening when the cell viability and the degree of opening are found to be optimal, the position of the cell in the first flow field relative to the microstructure, at this time, the phase of the signal output signal of the signal generator is no longer adjusted, the signal generator Sending the sinusoidal signal of the current phase to the interdigital transducer so that the cell does not change relative to the position in the first flow field, at which time each cell flows as the cell flows in the first flow field
  • the preset position, the shear stress received at the preset position is the optimal shear stress, resulting in reversible perforation, and the substance in the solution enters the cell through reversible perforation.
  • the process of introducing the substance into the cell is completed, and the cells that have been introduced into the substance are piped to the cell recovery container.
  • the method for introducing a substance into a cell provided by the embodiment of the invention can precisely control the position of the cell relative to the microstructure in the flow field, so that each cell passes through a preset position when flowing in the flow field, thereby accurately controlling the cell. Shear stress received.

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Abstract

一种微流控装置、将物质导入细胞的系统及方法，其中，微流控装置包括腔道（110）、体波产生器件（120）和声表面波产生器件（130）；所述腔道（110）的内壁上设置有微结构（112），微结构（112）构造成当溶液注入所述腔道（110）时，溶液在所述微结构（112）处形成气泡；所述体波产生器件（120），用于产生体波使所述气泡共振以产生流场；所述声表面波产生器件（130），用于产生声表面波并控制所述溶液中颗粒的位置。通过声表面波产生器件（130）能够精确的控制溶液中颗粒的位置，进而实现精确的控制溶液中的颗粒受到的剪切应力。

Description

微流控装置、将物质导入细胞的系统及方法
本申请要求申请日为2016年12月23日，申请号为201611208089.6，发明名称为“微流控装置、将物质导入细胞的系统及方法”的中国专利申请的优先权，在此通过引用将该申请的全部内容包括在内。
技术领域
本发明涉及生物技术领域，具体而言，涉及一种微流控装置、将物质导入细胞的系统及方法。
背景技术
随着基因治疗理论的提出和不断深入研究，使人类在彻底攻克恶性肿瘤、遗传性疾病、感染性疾病等研究上进入了一个崭新的发展阶段，基因治疗是指通过生物学、物理学、化学方法，将正常基因或者有治疗作用的基因导入人体靶细胞，纠正基因缺陷或功能上的错乱，从而达到治疗疾病的目的。如何高效、安全的将外源物质从细胞外输送至细胞内是基因治疗的关键。
目前，将物质导入细胞的方法有病毒性基因导入、注射法、基因枪法、电穿孔法和声致穿孔方法等，其中，声致穿孔是将大量细胞与气泡同时加入溶液中，并将气泡与细胞摇匀，使气泡均匀散落在细胞之间，利用超声对混合溶液进行辐照，气泡在超声的作用下将产生空化效应，产生膨胀、内爆、微声流、微射流和冲击波等物理现象，这些物理现象将会在细胞表面形成微孔，导致细胞膜通透性发生改变。
有研究表明，当在细胞溶液中加入超声造影气泡，由于气泡在超声作用下会产生径向振动，发生稳态或瞬态空化效应，可显著提高细胞膜的开孔效率，当气泡在细胞附近发生瞬态空化时，气泡非对称破碎形成的微激流、声微流以及冲击波是导致声致穿孔的重要物理机制，微激流对应的剪切应力大小直接决定细胞膜完整性及细胞活性，当气泡与细胞之间距离过大时，微射流对应的剪切力不足以破坏细胞膜结构的完整性，细胞开孔率低下，当气泡与细胞距离过小时，细胞开孔效率虽可得到显著提高，但过大的剪切应力可使贴壁细胞脱离基底，在细胞膜表面形成致死性损伤。
因此，为了使细胞的开孔率和穿孔效果较好，需要将气泡和细胞之间的距离控制在一个较佳的距离范围内，但是现有技术中用于声致穿孔的设备不能精确控制细胞与气泡之间的距离，因此使得细胞受到的剪切力不稳定。
发明内容
有鉴于此，本发明实施方式的目的在于提供一种微流控装置、将物质导入细胞的系统及方法，以解决现有技术中的用于声致穿孔的设备不能精确控制细胞与气泡之间的距离，因此使得细胞受到的剪切力不稳定的问题。
第一方面，本发明实施方式提供了一种微流控装置，所述装置包括：
腔道、体波产生器件和声表面波产生器件；
所述腔道的内壁上设置有微结构，当溶液注入所述腔道时，所述溶液在所述微结构处形成气泡；
所述体波产生器件，用于产生体波使所述气泡共振以产生流场；
所述声表面波产生器件，用于产生声表面波并控制所述溶液中颗粒的位置。
在一种实施方式中，所述微结构为一个或多个；
优选地，所述微结构为所述腔道的内壁上内凹的阱或孔。
在一种实施方式中，所述声表面波产生器件控制所述颗粒排列成一条直线并控制所述颗粒在所述流场中的位置。
在一种实施方式中，所述腔道由透明材料制成，所述腔道优选为硅氧烷腔道，更优选为聚二甲基硅氧烷腔道；
优选地，所述体波产生器件为体波换能器；
优选地，所述声表面波产生器件为叉指换能器。
第二方面，本发明实施方式提供了一种将物质导入细胞的系统，其中，所述系统包括第一方面提供的微流控装置；以及，
注射装置，连接至所述微流控装置的所述腔道的入口，用于将包含细胞的溶液注入所述微流控装置的腔道内，所述溶液在所述腔道的微结构处产生气泡；
所述微流控装置，用于产生体波，使所述气泡共振，引起所述腔道中溶液的振动，在所述腔道中产生流场，所述流场中每个位置处对应不同的剪切应力，并控制 所述细胞在所述流场中相对于所述微结构的位置，使所述细胞在所述流场中流动时流过预设位置，所述细胞在所述预设位置处对应的剪切应力的作用下产生可逆穿孔，所述溶液中的物质通过所述可逆穿孔进入所述细胞。
在一种实施方式中，所述系统还包括信号发生器和功率放大器；
所述信号发生器，用于产生正弦波信号，并将所述正弦波信号发送给所述功率放大器；
所述功率放大器，用于将所述正弦波信号进行放大，并将放大后的所述正弦波信号发送给所述体波换能器和所述叉指换能器。
在一种实施方式中，所述系统还包括细胞回收容器；
所述细胞回收容器连接至所述腔道的出口。
第三方面，本发明实施方式提供了一种将物质导入细胞的方法，其中，所述方法包括：
将包含细胞的溶液注入微流控装置的腔道内，所述包含细胞的溶液在所述腔道的微结构处产生气泡；
通过所述微流控装置中的体波产生器件产生体波，使所述气泡共振，引起所述腔道中溶液的振动，在所述腔道中产生第一流场，所述第一流场中每个位置处对应不同的剪切应力；
通过所述微流控装置中的声表面波产生器件控制所述细胞在所述第一流场中相对于所述微结构的位置，使所述细胞在所述第一流场中流动时流过预设位置，所述细胞在所述预设位置处对应的剪切应力的作用下产生可逆穿孔，所述溶液中的物质通过所述可逆穿孔进入所述细胞。
在一种实施方式中，所述第一流场中每个位置处对应的剪切应力通过如下方法确定：
将包含有示踪粒子的溶液注入PDMS腔道内，注入的包含有示踪粒子的溶液在所述PDMS腔道内的微结构处产生圆弧状气泡；
通过体波换能器使所述气泡共振，引起所述PDMS腔道中流体的振动，在所述PDMS腔道中产生第二流场；
根据所述第二流场中所述示踪粒子的流动状态，通过计算机确定所述第二流场中每个位置处对应的剪切应力；
将所述第二流场中每个位置处对应的剪切应力确定为所述第一流场中每个位置处对应的剪切应力。
在一种实施方式中，所述方法还包括：
通过信号发生器产生正弦波信号，并将所述正弦波信号发送给功率放大器；
通过所述功率放大器将所述正弦波信号进行放大，并将放大后的所述正弦波信号发送给所述体波换能器和所述叉指换能器；
通过调节所述信号发生器产生的正弦波信号的相位，调整所述细胞在所述第一流场中相对于所述微结构的位置。
在本发明实施方式提供的微流控装置、将物质导入细胞的系统及方法中，通过声表面波产生器件能够精确的控制溶液中颗粒的位置，进而实现精确的控制溶液中的颗粒受到的剪切应力。
为使本发明的上述目的、特征和优点能更明显易懂，下文特举较佳实施方式，并配合所附附图，作详细说明如下。
附图说明
为了更清楚地说明本发明实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施方式，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1示出了本发明实施方式1所提供的微流控装置的结构示意图；
图2示出了本发明实施方式1所提供的微流控装置中的腔道的结构示意图；
图3示出了本发明实施方式1提供的微流控装置中叉指换能器的结构示意图；
图4示出了本发明实施方式1所提供的微流控装置的一种具体结构示意图；
图5示出了本发明实施方式2所提供的将物质导入细胞的系统的结构示意图；
图6示出了本发明实施方式2所提供的将物质导入细胞的系统的第二种结构示意图；
图7示出了本发明实施方式3所提供的将物质导入细胞的方法的流程图。
图1附图标记说明：
110，腔道；120，体波产生器件；声表面波产生器件130；
图2附图标记说明：
111，进口；112，微结构；113，出口；
图3附图标记说明：
310，压电基底；320，叉指电极；
图4附图标记说明：
410，PDMS腔道；420，体波换能器；
图5附图标记说明：
510，注射装置；520，微流控装置；
图6附图标记说明：
610，微量注射泵；650，管道；660，细胞回收容器；670，功率放大器；680，信号发生器。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚，下面将结合本发明实施方式中附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明一部分实施方式，而不是全部的实施方式。通常在此处附图中描述和示出的本发明实施方式的组件可以以各种不同的配置来布置和设计。因此，以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施方式。基于本发明的实施方式，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施方式，都属于本发明保护的范围。
考虑到，当采用声致穿孔方法将物质导入细胞时，现有技术中用于声致穿孔的设备不能精确控制细胞与气泡之间的距离，因此使得细胞受到的剪切力不稳定。基于此，本发明实施方式提供了一种微流控装置、将物质导入细胞的系统及方法，下面通过实施方式进行描述。
实施方式1
本发明涉及一种微流控装置，包括：
腔道，其内壁上设置有微结构，该微结构构造成当溶液注入腔道时，溶液在微结构处形成气泡；以及，
体波产生器件，用于产生体波使上述气泡共振以产生流场。
在一种实施方式中，微流控装置进一步包括声表面波产生器件，用于产生声表面波并控制溶液中颗粒的位置。
本发明实施方式提供了一种微流控装置，如图1所示，该装置包括腔道110、体波产生器件120和声表面波产生器件130；
腔道110的内部上设置有微结构，当溶液注入腔道110时，溶液在微结构处形成气泡；
体波产生器件120，用于产生体波使上述气泡共振并产生流场；
声表面波产生器件130，用于产生声表面波并控制上述溶液中颗粒的位置。
根据一些实施方式，上述腔道110可以由透明材料制成，该腔道110可以是硅氧烷腔道，还可以是PDMS(polydimethylsiloxane，聚二甲基硅氧烷)腔道。体波产生器件120可以是体波换能器。声表面波产生器件130可以是叉指换能器。
根据一些实施方式，上述腔道110中的微结构为一个或多个。根据一些实施方式，所述微结构为腔道110的内壁上内凹的阱或孔。
如图2所示，画出了其中一种可能的腔道110的结构示意图。当溶液通过腔道110的进口111注入腔道后，溶液不会流到腔道110的微结构112内，而是在微结构112处形成一个气泡，且形成的气泡为圆弧状气泡。113为腔道110的出口，溶液中的颗粒或者溶液从出口113流出腔道110。
其中，上述图2只是画出了腔道110内包括5个微结构112的情况，但本发明的实施方式并不限于此。微结构112的具体个数可以根据具体应用场景进行设置。
上述体波产生器件120在工作时，向外发出体波，且发出的体波的频率为微结构处产生的气泡的共振频率，因此，产生的体波会引起气泡的共振，气泡共振带动腔道110中溶液的流动，从而在腔道110中产生流场。
上述共振频率能够根据圆弧状气泡的圆弧所在圆的半径确定，具体包括：
根据圆弧状气泡的圆弧所在圆的半径，通过如下公式确定上述共振频率；
Figure PCTCN2017000080-appb-000001
其中，在公式中，f为气泡的共振频率，σ为流场中流体的表面张力，p为流场中的流体压力，κ为气泡内部气体的多方指数，ρ为流场中流体的密度，a为气泡所在圆的半径。
上述声表面波产生器件130控制腔道内溶液中的颗粒排列成一条直线并控制颗粒在流场中的位置。
其中，上述声表面波产生器件130控制颗粒在流场中相对于微结构的位置，当腔道水平放置时，上述声表面波产生器件130可以控制颗粒在流场中的竖直方向上的位置。
其中，在本发明实施方式中，当腔道水平放置时，可以通过调节声表面波产生器件130的输入信号的相位，进而调节颗粒在流场中相对于微结构的位置，即在流场中竖直方向上的位置，具体包括：
当两个声表面波产生器件130同时工作时，会在腔道110内形成一个一维驻波场，并且，在声表面波产生器件130产生的超声辐射力的作用下，腔道110内的颗粒会排列在驻波波节的位置，通过调整其中一个声表面波产生器件130输入信号的频率，使得该声表面波产生器件130产生的声表面波会上移或下移，进而导致与另一声表面波产生器件130产生的声表面波叠加出来的驻波节点上移或下移。由于颗粒被锁定在波节的位置，因此，腔道110内的颗粒随着波节的上下移动而整体上下移动，这样，可以调节颗粒在腔道内流场中竖直方向上的位置。
而颗粒在竖直方向上移动的距离和声表面波产生器件130输入信号的相位满足如下关系：
Figure PCTCN2017000080-appb-000002
其中，在上述公式中，Δx为颗粒移动的位移，λ为声表面波产生器件130产生的声表面波的波长，
Figure PCTCN2017000080-appb-000003
为声表面波产生器件130相对相位的改变量，n为声表面波产生器件130输入信号的相位由0°调整到360°的重复次数，其中，n＝1,2,3…。
当声表面波产生器件130输入信号的相位固定时，颗粒在流场竖直方向上的位置就不再变化，因此，可以精确控制颗粒在流场中竖直方向上的位置。
其中，上述声表面波产生器件130可以包括一对叉指换能器，即在压电基底上镀上一对叉指电极，结构示意图如图3所示，声表面波产生器件130的其中一种可能的结构示意图如图3所示。
如图3所示，声表面波产生器件130包括压电基底310和一对叉指电极320，叉指换能器是通过在压电基底310上镀入一对叉指电极320形成的，为了获得较大的机电耦合系数，本发明实施方式中的叉指换能器可以选用128°YX双面抛光的铌酸锂作为压电基底。
而叉指换能器则是包括压电基底和一个叉指电极，即通过在压电基底上镀入一个叉指电极形成。
可以通过涂胶、光刻、镀膜、剥离等工艺流程制作叉指换能器。
首先对压电基底进行涂胶，在完全清洗干净的压电基底材料的表面，将正面刻胶AZ4620以5000rpm旋涂30s，将芯片放置在120°加热板上烘烤3min，利用台阶仪对光刻胶的厚度进行测试，测试结果为光刻胶的厚度大约为5μm；
之后，对涂胶后的压电基底进行曝光和显影，将制作好的菲林片覆盖在涂胶后的压电基底上面进行曝光，有光透过的地方会固化，在采用AZ400进行显影的时候上述固化的部分不会被溶解，非固化部分会被溶解，显影之后放在150℃的加热板上烘烤10min；
对烘烤之后的压电基底进行磁控溅射，使其生长厚度约200nm的金属层；
将上述生长有金属层的铝膜的基底放在丙酮溶液中，利用超声清洗机的超声波震动剥离光刻胶，得到叉指换能器。
本发明实施方式中的腔道110可以是PDMS腔道，而PDMS腔道是通过预处理、涂胶和前烘、曝光和显影、浇筑PDMS、剥离PDMS等工艺流程制作的。
上述制作过程具体包括：
首先预处理，通过酸洗、醇洗和水洗等方法出去硅基片表面残留杂质，比如说，灰尘、有机吸附物等，将清洗后的硅基片置于洁净处晾干；
利用涂胶机旋涂SU-8(50)负光刻胶，以3000rpm旋涂30s，SU-8(50)的厚度大约为50μm，涂胶完后，将硅片水平放置在90℃加热板1h，让光刻胶中的溶剂挥发，以增强光刻胶与硅片之间的粘附力；
将制作好的菲林片放置在上述涂胶后的硅片上，通过曝光机对光刻胶进行曝光，曝光剂量为600CJ/cm2，持续时间30s，用显影液浸泡曝光过的硅片，未曝光区域光刻胶被溶解，曝光区域光刻胶继续保留，显影之后放在150℃的加热板上烘烤10min；
将PDMS的A胶和B胶按质量比10:1进行配比，混合均匀，倒入硅片所在的培养皿中，将培养皿抽真空，除去PDMS中的气泡，最后将培养皿放在80℃的烘箱内30min，使PDMS固化；
切除含有腔道和微结构的PDMS，并使其从硅片上完全剥离，最后利用打孔器对微腔道打孔，制作进出口。
将已经制作好的叉指换能器和PDMS腔道进行等离子处理，等离子处理的功率为150W，持续时间70S，然后将PDMS腔道朝下黏贴在叉指换能器的两个叉指电极之间，80℃中烘烤20min。
如图4所示，图4示出了本发明实施方式提供的微流控装置的一种可能的具体结构示意图，其中，包括PDMS腔道410、体波换能器420、叉指换能器，叉指换能器上有两个叉指电极320，其中，PDMS腔道410和体波换能器420、叉指换能器可以集成在同一个芯片上。
溶液注入PDMS腔道410内，当溶液注入PDMS腔道410后，在PDMS腔道410的微结构处产生圆弧状气泡，体波换能器420在工作时向外发射体波，发射的体波的频率和PDMS腔道410内的气泡的共振频率一致，因此，体波换能器420发生的体波引起PDMS腔道410内气泡的振动，气泡的振动带动PDMS腔道410中溶液的振动，产生流场。
另外，PDMS腔道410位于叉指换能器上的两个叉指电极320的中间，叉指换能器在工作时，两个叉指电极320均向外发射声表面波信号，发射的声表面波控制颗粒在流场中相对于微结构的位置。
本发明实施方式提供了一种微流控装置，通过声表面波产生器件能够精确的控制溶液中颗粒的位置，进而实现精确的控制溶液中的颗粒受到的剪切应力。
实施方式2
本发明实施方式提供了一种将物质导入细胞的系统，如图5所示，该系统包括注射装置510和上述实施方式1提供的微流控装置520；
上述注射装置510，用于将包含细胞的溶液注入微流控装置520的腔道内，注入的溶液在腔道的微结构处产生气泡；
上述微流控装置520，用于产生体波，使上述气泡共振，引起腔道中溶液的振动，在腔道中产生流场，流场中每个位置处对应不同的剪切应力，并控制细胞在流场中相对于微结构的位置，使细胞在流场中流动时流过预设位置，细胞在预设位置处对应的剪切应力的作用下产生可逆穿孔，溶液中的物质通过可逆穿孔进入所述细胞。
根据一些实施方式，上述物质可以是基因片段，也可以是药物分子等等。
上述注射装置510通过管道和微流控装置520的腔道连接，上述注射装置510可以是微量注射泵。
根据一些实施方式，上述系统还包括信号发生器和功率放大器；
上述信号发生器，用于产生正弦波信号，并将正弦波信号发送给功率放大器；
上述功率放大器，用于将正弦波信号进行放大，并将放大后的正弦波信号发送给体波换能器和叉指换能器。
本发明实施方式中的系统可以包括一个信号发生器，也可以包括两个信号发生器或者多个，本发明实施方式并不对信号发生器的个数进行限定，只要能够得到足够的信号个数即可。
根据一些实施方式，上述系统还包括细胞回收容器；
上述细胞回收容器通过管道和微流控装置520中的腔道连接，用于盛装已经导入物质的细胞。
如图6所示，画出了一种可能的将物质导入细胞的系统的具体结构图，包括：包括：微量注射泵610，PDMS腔道410，体波换能器420，声表面波产生器件，声表面波产生器件上有一对叉指电极320，管道650，细胞回收容器660，2个功率放大器670，信号发生器680。
上述图6中只是画出了包括一个信号发生器680的情形，上述信号发生器还可以是两个或者多个，信号发生器680的具体个数可以根据具体应用场景进行设置，上述图6并没有限定信号发生器680的具体个数。
在图6中，微量注射泵610通过管道650和PDMS腔道410连接，通过管道650将包含有细胞的溶液注入PDMS腔道410内，当溶液注入PDMS腔道410后，在PDMS腔道410的微结构处产生圆弧状气泡，体波换能器420和功率放大器670连接，功率放大器670和信号发生器680连接，信号发生器680输出正弦波信号，并将输出的正弦波信号发送给功率放大器670，由功率放大器670对该信号进行放大，并将放大后的信号发送给体波换能器420，以驱使体波换能器420工作，体波换能器420向外发射体波，发生的体波的频率和PDMS腔道410内的气泡的共振频率一致，因此，体波换能器420发生的体波引起PDMS腔道410内气泡的振动，气泡的振动带动PDMS腔道410中流体的振动，产生流场。
另外，PDMS腔道410位于两个叉指电极320的中间，两个叉指电极320均与功率放大器670连接，功率放大器670和信号发生器680连接，信号发生器680输出正弦波信号，并将输出的正弦波信号发送给功率放大器670，由功率放大器670将该信号进行放大，并将放大后的信号发送给叉指电极320，以驱动叉指电极320的工作。叉指电极320在工作时，向外发射声表面波信号，发射的声表面波控制细胞在流场中相对于微结构的位置，使得细胞在水平方向流动时，每个细胞都能够经过预设位置，当细胞流过预设位置时在预设位置处的剪切应力的作用下产生可逆穿孔，溶液中的物质通过可逆穿孔进入细胞。
本发明实施方式提供的将物质导入细胞的系统，其中的微流控装置能够精确的控制细胞在流场中的位置，使得每个细胞在流场中流动时均经过预设位置，进而精确的控制细胞受到的剪切应力。
实施方式3
本发明实施方式提供了一种将物质导入细胞的方法，该方法应用上述实施方式2中的将物质导入细胞的系统，该方法通过微流控装置中的体波产生器件可以精确控制细胞在流场中相对于微结构的位置，使得每个细胞在流场中流动时均经过预设位置，进而精确的控制细胞受到的剪切应力。
其中，本发明实施方式中导入细胞的物质可以是基因片段、药物分子等，用户可以根据实际需要将需要导入细胞的物质放入溶液中，再采用本发明实施方式提供的方法将物质导入细胞。
采用本发明实施方式提供的方法将物质导入细胞，如图7所示，包括步骤S710-S730，具体如下。
S710，将包含细胞的溶液注入微流控装置的腔道内，包含细胞的溶液在腔道的微结构处产生气泡。
根据一些实施方式，上述腔道包括至少一个微结构，溶液在每个微结构处产生一个圆弧状气泡。
上述通过注射装置将包含细胞的溶液注入微流控装置的腔道内，该注射装置可以是微量注射泵。
在一些具体实施方式中，可以将包含细胞的溶液连续注入到微流控装置的腔道内。
S720，通过微流控装置中的体波产生器件产生体波，使上述气泡共振，引起腔道中流体的振动，在腔道内产生第一流场，第一流场中每个位置处对应不同的剪切应力。
在第一流场中每个位置处对应不同的剪切应力，其中，每个位置处的剪切应力可以通过如下方式确定。
在采用本发明实施方式提供的方法将物质导入细胞之前，即将包含有细胞的溶液注入腔道之前，先确定出溶液在腔道中产生的第一流场中每个位置处对应的剪切应力。
在本发明实施方式中，可以通过在流场中加入示踪粒子，通过示踪粒子在流场中的流动，确定出流场中每个位置对应的剪切应力。
具体包括：将包含有示踪粒子的溶液注入腔道内，注入的包含有示踪粒子的溶液在腔道内的微结构处产生圆弧状气泡；通过体波产生器件使上述气泡共振，引起腔道中流体的振动，在腔道中产生第二流场；根据第二流场中示踪粒子的流动状态，通过计算机确定第二流场中每个位置对应的剪切应力；将第二流场中每个位置处对应的剪切应力确定为第一流场中每个位置处对应的剪切应力。
上述包含有示踪粒子的溶液在腔道内产生的第二流场和包含有细胞的溶液在腔道内产生的第一流场为相同的流场，都是通过相同的体波产生器件产生的体波引起的，因此，可以将第二流场中每个位置处对应的剪切应力确定为第一流场中每个位置处对应的剪切应力。
根据第二流场中示踪粒子的流动状态，通过计算机确定第二流场中每个位置处对应的剪切应力，包括：
通过高速相机连续拍摄第二流场中示踪粒子的流动状态，得到示踪粒子的状态图像；根据上述状态图像，通过计算机确定第二流场中每个位置处的示踪粒子的流动速度；根据每个位置处的示踪粒子的流动速度，通过计算机确定该位置对应的剪切应力。
在本发明实施方式中，通过高速相机连续拍摄多张第二流场中示踪粒子的流动状态图像，从上述多帧状态图像中，取出任意两帧状态图像，将两帧状态图像按照相同的方式进行分割，分割成相同的多个小窗口，对前后两帧图像相对应的窗口进行互相关计算，具体可以通过如下公式计算两帧图像对应窗口的互相关的值；
Figure PCTCN2017000080-appb-000004
其中，在上述公式中，R(m，n)为前后两帧图像对应的窗口上(m，n)位置处的互相关的值，M*N为上述窗口的大小，其中，M和N可以用像素点表示，m和n也可以用像素表示，f(i,j)为前一帧图像上(i,j)位置处的像素值，g(i+m,j+m)为后一帧 图像在(i+m,j+n)位置处的像素值，μf为前一帧图像上窗口内所有像素点对应的f(i，j)像素的均值，μg为后一帧图像对应窗口上所有像素点对应的g(i+m,j+m)像素的均值，其中，对于一个要计算的R(m，n)，上述m和n的值是固定的。
将计算出的两帧图像对应窗口的互相关的值确定为该窗口移动的位移，逐渐降低分割的小窗口的大小，提高计算的空间分辨率，通过上述方法计算出了第二流场中每个位置的位移，将上述位移除以前后两帧图像之间的时间间隔，计算出每个位置处示踪粒子的流动速度。
当确定了第二流场中每个位置处的示踪粒子的流动速度后，根据每个位置处示踪粒子的流动速度，通过如下公式确定每个位置处对应的剪切应力；
Figure PCTCN2017000080-appb-000005
在上述公式中，ρ为第二流场中流体的密度，μ为第二流场中流体的粘度，Vx为第二流场中流体在水平方向的速度，Vy为流场中流体的竖直方向的速度，WSS为上述位置处对应的剪切应力。
在第二流场中在某个位置处的示踪粒子的速度就是该位置处流体的速度，因此，通过各个位置处示踪粒子的速度就可以知道各个位置处流场的速度。
在本发明实施方式中，通过流场中某个位置和其周边处流体的流动速度，确定出该位置对应的剪切应力，比如说，通过流场中A位置和其周边处流体的流动速度，确定出A位置对应的剪切应力。
S730，通过微流控装置中的声表面波产生器件控制溶液中的细胞在第一流场中相对于微结构的位置，使细胞在第一流场中流动时流过预设位置，细胞在预设位置处对应的剪切应力的作用下产生可逆穿孔，溶液中的物质通过可逆穿孔进入细胞。
上述声表面波产生器件在工作时会产生声表面波信号，声表面波信号控制腔道内的细胞排列成一条直线，并且可以控制细胞在第一流场中相对于微结构的位置，使腔道内在微结构处的气泡与细胞之间的距离固定，这样，细胞在第一流场中的水 平方向上移动时，每个细胞都会流过预设位置，在预设位置处受到剪切应力，产生可逆开孔，使得溶液中的物质通过可逆穿孔进入细胞。
为了让腔道中的细胞只排列成一排，可以通过设置腔道的宽度来实现，因此，在本发明实施方式中，腔道的宽度大于细胞的宽度且小于声表面波产生器件产生的声表面波波长的一半，这样，在腔道内只能允许一排细胞通过。
上述预设位置可以通过如下方式进行调节确定：
在本发明实施方式中，通过信号发生器产生正弦波信号，并将正弦波信号发送给功率放大器；通过功率放大器将正弦波信号进行放大，并将放大后的正弦波信号发送给体波换能器和叉指换能器；通过调节所述信号发生器产生的正弦波信号的相位，调整所述细胞在所述第一流场中相对于微结构的位置。
其中，可以通过一个信号发生器产生三个正弦波信号，一个正弦波信号经过第一功率放大器放大后，发送给体波产生器件，另外两个正弦波信号经过第二功率放大器放大后，发送给声表面波产生器件中的一对叉指电极。
或者，采用三个信号发生器，一个信号发生器用于产生体波产生器件所需的正弦波信号，另外两个信号发生器用于产生声表面波产生器件所需的正弦波信号。
本发明实施方式中，并不对信号发生器的具体个数进行设置，在具体应用场景中，用于可以根据实际应用进行选择。
在本发明实施方式中，用于可以通过调节信号发生器产生的正弦波信号的相位，调整细胞在第一流场中相对于微结构的位置。
上述声表面波产生器件为叉指换能器。
在本发明实施方式中，将腔道置于叉指换能器的两个叉指电极之间，这样，腔道可以同时接收到两个叉指电极发射的声表面波信号。
用户通过调节信号发生器调整细胞在第一流场中的位置的具体过程包括：当叉指换能器在工作时，会在腔道内形成一个一维驻波场，并且，在叉指换能器产生的超声辐射力的作用下，腔道内的细胞会排列在驻波波节的位置，通过调整其中一个声表面波产生器件输入信号的频率，使得该声表面波产生器件产生的声表面波会上移或下移，进而导致与另一声表面波产生器件产生的声表面波叠加出来的驻波节点 上移或下移。由于细胞被锁定在波节的位置，因此，腔道内的细胞随着波节的上下移动而整体上下移动，这样，可以调节细胞在腔道内第一流场中相对于微结构上的位置。
而细胞移动的距离和信号发生器输出的正弦波信号的相位满足如下关系：
Figure PCTCN2017000080-appb-000006
在上述公式中，Δx为细胞移动的位移，λ为叉指换能器产生的声表面波的波长，
Figure PCTCN2017000080-appb-000007
为叉指换能器相对相位的该变量，n为信号发生器输出的正弦波信号的相位由0°调整到360°的重复次数，其中，n＝1,2,3…。
当信号发生器输出信号的相位固定时，细胞在第一流场相对于微结构的位置就不再变化。
在调节的过程中，为了使细胞在第一流场中流动时流过预设位置，在调节信号发生器输出的正弦波信号的相位的过程中，可以通过扫描电镜观察细胞的活力和穿孔程度。
为了能够观察细胞的活力和穿孔程度，可以在上述包含细胞的溶液中加入FDA荧光探针和PI荧光探针。其中，在采用本发明实施方式提供的方法将物质导入细胞时，可以在刚开始进行的时候在溶液中加入FDA荧光探针和PI荧光探针对信号发生器输出的正弦波信号的相位进行调节，当调节完成后，则没必要再在溶液中加入FDA荧光探针和PI荧光探针，当然，用户在将物质导入细胞的过程中，可以每间隔一段时间在溶液中加入FDA荧光探针和PI荧光探针，以观察当前细胞的活力和穿孔程度。
上述FDA是一种疏水性复合物，它可以穿透完整的细胞膜进入细胞，通过细胞内酯酶催化水解二乙酸酯基团产生具有高强度的荧光产物，若细胞膜完整，FDA荧光分子将在细胞内积累，发出绿色荧光，因此，FDA可以作为细胞活力的标签。
当细胞在剪切应力的作用下产生可逆穿孔后，PI会透过细胞膜表面的穿孔进入细胞核，与DNA结合产生红色荧光，红色荧光的强度可以直接反应细胞穿孔程度，因此，可以通过对细胞标记上述两种荧光探针，检测细胞的活性和穿孔程度。
因此，在调节信号发生器输出的正弦波信号的相位的过程中，通过扫描电镜观察细胞内荧光的强度，根据荧光的强度确定细胞在第一流场中相对于微结构各个位置处细胞的活力和开孔程度，找到细胞的活力和开孔程度都最佳时，细胞在第一流场中的相对于微结构的位置，这时，不再调节信号发生器输出信号的相位，信号发生器将当前相位的正弦波信号发送给叉指换能器，使得细胞在第一流场中相对于的位置不再发生变化，这时，细胞在第一流场中流动时每个细胞都流过预设位置，在预设位置受到的剪切应力为最佳剪切应力，产生可逆穿孔，溶液中的物质通过可逆穿孔进入细胞。
当溶液中的物质通过可逆穿孔进入细胞后，完成了将物质导入细胞这一过程，将已经导入进物质的细胞通过管道输送至细胞回收容器。
本发明实施方式提供的将物质导入细胞的方法，能够精确的控制细胞在流场中相对于微结构的位置，使得每个细胞在流场中流动时均经过预设位置，进而精确的控制细胞受到的剪切应力。
应注意到：相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步定义和解释，此外，术语“第一”、“第二”、“第三”等仅用于区分描述，而不能理解为指示或暗示相对重要性。
最后应说明的是：以上所述实施方式，仅为本发明的具体实施方式，用以说明本发明的技术方案，而非对其限制，本发明的保护范围并不局限于此，尽管参照前述实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，其依然可以对前述实施方式所记载的技术方案进行修改或可轻易想到变化，或者对其中部分技术特征进行等同替换；而这些修改、变化或者替换，并不使相应技术方案的本质脱离本发明实施方式技术方案的精神和范围。都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。

Claims (15)

  1. 一种微流控装置，其特征在于，所述微流控装置包括：腔道、体波产生器件和声表面波产生器件；
    所述腔道的内壁上设置有微结构，所述微结构构造成当溶液注入所述腔道时，所述溶液在所述微结构处形成气泡；
    所述体波产生器件，用于产生体波使所述气泡共振以产生流场；
    所述声表面波产生器件，用于产生声表面波并控制所述溶液中颗粒的位置。
  2. 根据权利要求1所述的装置，其特征在于，所述微结构为一个或多个。
  3. 根据权利要求1所述的装置，其特征在于，所述微结构为所述腔道的内壁上内凹的阱或孔。
  4. 根据权利要求1所述的装置，其特征在于，所述声表面波产生器件控制所述颗粒排列成一条直线并控制所述颗粒在所述流场中的位置。
  5. 根据权利要求1所述的装置，其特征在于，所述腔道由透明材料制成。
  6. 根据权利要求5所述的装置，其特征在于，所述腔道为硅氧烷腔道。
  7. 根据权利要求6所述的装置，其特征在于，所述腔道为聚二甲基硅氧烷腔道。
  8. 根据权利要求1所述的装置，其特征在于，所述体波产生器件为体波换能器。
  9. 根据权利要求1所述的装置，其特征在于，所述声表面波产生器件为叉指换能器。
  10. 一种将物质导入细胞的系统，其特征在于，所述系统包括权利要求1-9中的任意一项所述的微流控装置；以及，
    注射装置，连接至所述微流控装置的所述腔道的入口，用于将包含细胞的溶液注入所述微流控装置的腔道内，所述溶液在所述腔道的微结构处产生气泡；
    所述微流控装置，用于产生体波，使所述气泡共振，引起所述腔道中溶液的振动，在所述腔道中产生流场，所述流场中每个位置处对应不同的剪切应力，并控制所述细胞在所述流场中相对于所述微结构的位置，使所述细胞在所述流场中流动时流过预设位置，所述细胞在所述预设位置处对应的剪切应力的作用下产生可逆穿孔，所述溶液中的物质通过所述可逆穿孔进入所述细胞。
  11. 根据权利要求10所述的系统，其特征在于，所述系统还包括信号发生器和功率放大器；
    所述信号发生器，用于产生正弦波信号，并将所述正弦波信号发送给所述功率放大器；
    所述功率放大器，用于将所述正弦波信号进行放大，并将放大后的所述正弦波信号发送给所述微流控装置。
  12. 根据权利要求10所述的系统，其特征在于，所述系统还包括细胞回收容器；
    所述细胞回收容器连接至所述腔道的出口。
  13. 一种将物质导入细胞的方法，应用根据权利要求10-12中的任一项所述的系统，其特征在于，所述方法包括：
    将包含细胞的溶液注入微流控装置的腔道内，所述包含细胞的溶液在所述腔道的微结构处产生气泡；
    通过所述微流控装置中的体波产生器件产生体波，使所述气泡共振，引起所述腔道中溶液的振动，在所述腔道中产生第一流场，所述第一流场中每个位置处对应不同的剪切应力；
    通过所述微流控装置中的声表面波产生器件控制所述细胞在所述第一流场中相对于所述微结构的位置，使所述细胞在所述第一流场中流动时流过预设位置，所述细胞在所述预设位置处对应的剪切应力的作用下产生可逆穿孔，所述溶液中的物质通过所述可逆穿孔进入所述细胞。
  14. 根据权利要求13所述的方法，其特征在于，所述第一流场中每个位置处对应的剪切应力通过如下方法确定：
    将包含有示踪粒子的溶液注入腔道内，注入的包含有示踪粒子的溶液在所述腔道内的微结构处产生圆弧状气泡；
    通过体波换能器使所述气泡共振，引起所述腔道中流体的振动，在所述腔道中产生第二流场；
    根据所述第二流场中所述示踪粒子的流动状态，通过计算机确定所述第二流场中每个位置处对应的剪切应力；
    将所述第二流场中每个位置处对应的剪切应力确定为所述第一流场中每个位置处对应的剪切应力。
  15. 根据权利要求13所述的方法，其特征在于，所述方法还包括：
    通过信号发生器产生正弦波信号，并将所述正弦波信号发送给功率放大器；
    通过所述功率放大器将所述正弦波信号进行放大，并将放大后的所述正弦波信号发送给所述微流控装置；
    通过调节所述信号发生器产生的正弦波信号的相位，调整所述细胞在所述第一流场中相对于所述微结构的位置。
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