WO2020249131A1 - 利用超高频声波控制溶液中的微粒移动的方法及设备 - Google Patents

利用超高频声波控制溶液中的微粒移动的方法及设备 Download PDF

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WO2020249131A1
WO2020249131A1 PCT/CN2020/096178 CN2020096178W WO2020249131A1 WO 2020249131 A1 WO2020249131 A1 WO 2020249131A1 CN 2020096178 W CN2020096178 W CN 2020096178W WO 2020249131 A1 WO2020249131 A1 WO 2020249131A1
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acoustic wave
bulk acoustic
channel
vortex
cells
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PCT/CN2020/096178
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English (en)
French (fr)
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段学欣
杨洋
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安行生物技术有限公司
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Priority to US17/618,268 priority Critical patent/US20220333052A1/en
Priority to EP20823577.0A priority patent/EP3985096A4/en
Publication of WO2020249131A1 publication Critical patent/WO2020249131A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • B01L3/502707—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 manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/10—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing sonic or ultrasonic vibrations
    • 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
    • B01L3/502761—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • B01L2200/0652—Sorting or classification of particles or molecules
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1027—Determining speed or velocity of a particle

Definitions

  • the invention relates to the field of cell research methodology and medical equipment. Specifically, the present invention relates to a microfluidic system for separating and analyzing cells or microvesicles and a method for separating and analyzing cells or microvesicles using the system.
  • microfluidic systems to separate particles in samples is an emerging technology. Most of the methods that have been reported are separation based on the physical properties of particles such as cells, such as size, or differentiation and separation based on biological specificity. These methods have the problems of huge cost and low processing volume.
  • the present invention finds for the first time that the use of ultra-high frequency bulk acoustic waves can effectively control the moving position and direction of flexible particles such as cells or vesicles in solution, or biological macromolecules such as nucleic acids or proteins or polysaccharides in a microfluidic system, thereby providing A method and system for separating and obtaining target cells or vesicles or biological macromolecule particles, or obtaining a liquid after cells, vesicles, or biological macromolecule particles are removed.
  • the present invention provides a method for controlling the movement of flexible particles in a solution, including:
  • Fluid channel which has an inlet and an outlet channel
  • One or more ultra-high frequency bulk acoustic wave resonators which are arranged on a wall of the fluid channel, and the ultra-high frequency bulk acoustic wave resonator can generate in the fluid channel to the opposite side of the fluid channel
  • the wall frequency is about 0.5-50GHz bulk acoustic wave
  • the ultra-high frequency resonator emits a bulk acoustic wave transmitted to the wall on the opposite side of the fluid channel, and generates a vortex defined by the boundary of the bulk acoustic wave generation area of the ultra-high frequency resonator in the solution. aisle;
  • Flexible particles refer to nano or micro particles with deformable properties.
  • the flexible particles can be artificial or natural.
  • the particles may be micelles with a membrane structure, especially micelles with lipid bilayers or lipid bilayers, such as cells or vesicles, including exosomes, etc., or artificially prepared
  • the particles may also have irregular shapes, such as biological macromolecules such as nucleic acids or proteins.
  • the present invention provides a method for controlling the movement of cells or vesicles in a solution, including:
  • Flow a solution containing target cells or vesicles through a microfluidic device which includes;
  • Fluid channel which has an inlet and an outlet channel
  • One or more ultra-high frequency bulk acoustic wave resonators which are arranged on a wall of the fluid channel, and the ultra-high frequency bulk acoustic wave resonator can generate in the fluid channel to the opposite side of the fluid channel
  • the wall frequency is about 0.5-50GHz bulk acoustic wave
  • the UHF resonator emits a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and generates a vortex channel defined by the boundary of the bulk acoustic wave generation area of the UHF resonator in the solution;
  • the cells or vesicles can be artificial or natural.
  • the particles are micelles with membrane structure, especially micelles with lipid bilayers or lipid bilayers.
  • the flexible particles involved in the present invention usually have a diameter of about 0.01-30 um, preferably a diameter of 0.2-25 um, and more preferably 0.5-20 um.
  • the flexible particles are naturally occurring particles, such as cells or vesicles released by cells into the extracellular environment.
  • Cells include natural or cultured cells of higher plants or animals (for example, mammals including humans), and single-celled organisms such as bacteria and fungi or simple multicellular organisms.
  • Vesicles are microvesicles released by various animal cells into the extracellular environment.
  • microvesicles are vesicle-like bodies with a double-layer membrane structure that are shed from the cell membrane or secreted by the cell. They may have a diameter of about 30-1000 nm, about 30-800 nm, about 30-150 nm, or about 30-100 nm.
  • Microvesicles released by cells include exosomes, microvesicles, vesicles, membrane vesicles, vesicles, air bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosome-like vesicles or exocytotic vesicles Wait.
  • the present invention provides a method for controlling the movement of biological macromolecules (especially nucleic acids) such as target nucleic acids or proteins or polysaccharides in solution, including:
  • Flow a solution containing the target biological macromolecule through a microfluidic device the device including;
  • Fluid channel which has an inlet and an outlet channel
  • One or more ultra-high frequency bulk acoustic wave resonators which are arranged on a wall of the fluid channel, and the ultra-high frequency bulk acoustic wave resonator can generate in the fluid channel to the opposite side of the fluid channel
  • the wall frequency is about 0.5-50GHz bulk acoustic wave
  • the UHF resonator emits a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and generates a vortex channel defined by the boundary of the bulk acoustic wave generation area of the UHF resonator in the solution;
  • the biological macromolecule in the method refers to nucleic acid.
  • nucleic acid refers to a polymer of ribonucleosides or deoxyribonucleosides containing phosphodiester linkages between nucleotide subunits.
  • Nucleic acids include, but are not limited to, genetic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, microRNA, fragment nucleic acid, nucleic acid obtained from subcellular organelles such as mitochondria, and obtained from microorganisms or viruses that may appear on or in the sample Of nucleic acids.
  • Nucleic acids include natural or synthetic products, such as amplification reaction products using artificial or natural DNA or RNA as templates. Nucleic acids can be double-stranded or single-stranded, circular or linear. Samples that can be used to detect target nucleic acids include the following samples: from cell cultures, eukaryotic microorganisms or diagnostic samples such as body fluids, body fluid sediments, gastric lavage samples, fine needle aspirates, biopsy samples, tissue samples, cancer cells , Cells from patients, cells from tissues or cells cultured in vitro from individuals to be tested and/or treated for disease or infection, or forensic samples.
  • Non-limiting examples of body fluid samples include whole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, serum, plasma, urine, chyle, feces, ejaculation, sputum, nipple aspiration, saliva, swab samples, irrigation or irrigation Lotions and/or wipe samples.
  • the method of the present invention is particularly suitable for isolating nucleic acids with a length of ⁇ 300bp, preferably ⁇ 1kbp, more preferably ⁇ 10kbp, for example ⁇ 50kbp (such as any form of DNA and RNA, including natural or synthetic nucleic acids, such as DNA or RNA as a template) Increase reaction product).
  • the ultra-high frequency bulk acoustic wave resonator in the present invention refers to a resonator capable of generating a bulk acoustic wave with a frequency exceeding 0.5 GHz (preferably exceeding 1 GHz), for example, a frequency of 0.5-50 GHz.
  • the ultra-high frequency bulk acoustic wave resonator may be, for example, a thin film bulk acoustic wave resonator or a solid-state assembly type resonator.
  • the UHF resonator emits UHF bulk acoustic waves that are transmitted to the opposite wall of the fluid channel (for example, the top of the flow channel), and the sound waves are attenuated to the volumetric force generated in the fluid to make the flow through Acoustic jets appear in the solution, causing the liquid in the microchannels to produce local three-dimensional vortices.
  • the central axis of the vortex is above the boundary of the bulk acoustic wave; the continuous vortex caused by the UHF bulk acoustic wave forms the acoustic fluid vortex channel, and the shape of the vortex channel is basically It has the same shape as the bulk acoustic wave action area, which is located above the boundary of the bulk acoustic wave action area, that is, the shape and position of the vortex channel are defined by the boundary of the bulk acoustic wave generation area of the UHF resonator.
  • the fluid channel of the microfluidic device has an outflow channel for the controlled movement of flexible particles such as cells or vesicles, or biological macromolecules such as nucleic acids or proteins, etc., which can be called particle outflow. aisle.
  • the fluid channel also has other outflow channels, for example, an outflow channel for a solution that removes or contains fewer cells or vesicles that are controlled to move, which may be called a solution outflow channel.
  • the width ratio of the openings of the particle outflow channel and the solution outflow channel can be set to about 1:1-1:20, preferably about 1:2-1:15, for example, about 1:4-1:10.
  • the position where the flexible particles leave the vortex channel is close to the opening of the particle outflow channel.
  • the shape and position of the bulk acoustic wave action area of the UHF bulk acoustic wave resonator can be adjusted to make the flexible particles controlled to move in the solution such as cells or vesicles, or nucleic acids or proteins, etc.
  • Biological macromolecules enter and move along the vortex channel, and leave the vortex channel at a set position.
  • the flexible particles leave the bulk acoustic wave action area at a specified position and direction, and enter a desired outflow channel, for example, into the particle outflow channel.
  • the set position leaving the vortex channel is called the release point, that is, the position where the flexible particles leave the bulk acoustic wave action area.
  • the solution that removes the cells or vesicles that are controlled to move maintains the inflow direction and enters the solution outflow channel.
  • the release point is usually located in the downstream area of the vortex channel.
  • the release point is usually located at the place where the vortex channel turns or the curvature changes, that is, above the location where the turning or curvature changes occur at the boundary of the bulk acoustic wave action area, that is, the volume corresponding to the release point.
  • the boundary of the sound wave action area has a turning or curvature change.
  • the applicant believes that the reason for this phenomenon is that at the turning or corner of the vortex channel, the vortex direction and the direction of the acoustic radiation force suddenly change, and the flexible particles such as cells or In vesicles, or biological macromolecules such as nucleic acids or proteins, particles that meet the appropriate conditions (such as the appropriate size) have been focused to the center of the vortex, and under the action of the acoustic radiation force, they can change the direction of movement along with the vortex channel and Quickly refocus to the center of the vortex channel after turning; particles that do not meet the conditions (for example, with a smaller size) will be more affected by the jump in the laminar drag direction, and thus leave the vortex channel.
  • the flexible particles such as cells or In vesicles, or biological macromolecules such as nucleic acids or proteins
  • the above-mentioned method provided by the present invention is suitable for processing liquid samples containing a large number of flexible particles such as cells or vesicles, or nucleic acids or proteins; the large number of flexible particles can enter the vortex channel in a continuous movement He moves along the vortex channel and leaves the vortex channel at a set position, thereby achieving the purpose of rapid and large-throughput processing.
  • the above method provided by the present invention is suitable for processing samples containing a large number of cells or vesicles, such as whole blood or blood fractions.
  • the above-mentioned method provided by the present invention can be used to obtain or purify the required flexible particles in the sample.
  • the method can be used to enrich required flexible particles.
  • the above-mentioned method provided by the present invention can also be used to remove unwanted flexible particles in a sample to obtain a purified solution.
  • it can remove certain cells or vesicles from a blood sample, such as removing blood cells to obtain plasma.
  • the above method further includes adjusting the power of the bulk acoustic wave and/or adjusting the speed of the solution flowing through the bulk acoustic wave region to adjust the flexible particles entering the vortex channel.
  • the flexible particles that do not enter the vortex channel or the flexible particles that enter the vortex channel but leave the vortex channel before reaching the designated release point pass through the bulk acoustic wave region and flow out in the direction the sample enters the fluid channel.
  • the boundary line (that is, the shape of the corresponding vortex channel) of the bulk acoustic wave generation area of the UHF resonator in the above method is set to be suitable for the target flexible particles to follow in the vortex channel
  • the vortex channel moves to the release point. This prevents the target flexible particles from leaving the vortex channel without leaving the vortex channel from the release point as set.
  • the target flexible particles are kept in the vortex channel and moved to the release point by adjusting the boundary shape of the bulk acoustic wave generating region of the ultra-high frequency resonator.
  • the flexible particles can be kept moving in the vortex channel by reducing the turning or curvature change in the boundary line of the bulk acoustic wave generation area, that is, reducing the flexible particles leaving the vortex channel and improving the separation efficiency.
  • the flexible particles are kept in the vortex channel and moved to the release point.
  • the inventor unexpectedly found that the smaller the angle between the boundary line of the bulk acoustic wave generation area and the fluid channel, the easier it is to keep the flexible particles moving in the vortex channel, that is, to reduce the flexible particles leaving the vortex channel and improve the separation efficiency.
  • the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator in the microfluidic system has a focus zone and a sieve zone.
  • the focus zone is located upstream of the bulk acoustic wave action area (that is, the part near the inflow direction of the sample and far away from the release point), and the sieve area is located downstream of the bulk acoustic wave action area (that is, close to the sample outflow direction, closer to the release point or the part including the release point) ).
  • the setting of the bulk acoustic wave action area in the focus zone is more suitable for keeping the flexible particles moving in the vortex channel: the cells or vesicles in the vortex channel in the focus zone follow the same or similar direction as the laminar flow direction. Moving, the drag force of the vortex is relatively small, and it is easier for cells or vesicles to enter and remain in the vortex channel; in the downstream sieve section, the cells focused to the center of the vortex are more stable than unfocused cells Is moved in the vortex channel.
  • the angle between the boundary line of the bulk acoustic wave action area of the focal zone and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area of the screen section and the fluid channel.
  • the boundary of the bulk acoustic wave action area in the focal zone is basically the same or basically the same as the direction of the fluid channel (for example, the angle is less than 10°), and the vortex drag force on the cells in the vortex channel in this area basically does not change the cells along the path.
  • the state of motion in the laminar flow direction will only allow the cells to migrate laterally to the center of the vortex to achieve focusing on the cells; the boundary of the volumetric acoustic wave action area of the sieve section has a large angle with the fluid channel, which guides the cell movement direction to deviate from the fluid channel direction to the designated In the sieve zone, the cells focused to the center of the vortex can move more stably in the vortex channel than unfocused cells.
  • the microfluidic device usually includes a power adjusting device that adjusts the power of the bulk acoustic wave generated by the ultra-high frequency resonator.
  • the microfluidic device usually includes a flow rate adjusting device, which adjusts the speed of the solution flowing through the area affected by the bulk acoustic wave.
  • the cells include cell clusters.
  • the cell cluster usually consists of several, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells.
  • the vesicles comprise a group of vesicles.
  • the vesicle population usually consists of several, for example, 2-50 vesicles.
  • the output power of the power adjustment device is about 20-5000 mW, preferably 50-2000 mW, more preferably 100-1500 mW.
  • the flow rate adjusting device can adjust the velocity of the solution flowing through the bulk acoustic wave region to about 0.1-10mm/s, preferably about 0.3-5mm/s, more preferably about 0.5-3mm /s.
  • the flow rate adjusting device can adjust the speed of the solution flowing through the bulk acoustic wave zone to about 0.01-100 ⁇ L/min, preferably about 0.1-50 ⁇ L/min, more preferably about 0.5-30 ⁇ L /min.
  • the height of the fluid channel of the microfluidic device is about 5-200 m, preferably about 25-100 m, more preferably about 30-80 m, for example, about 40-60 m.
  • the UHF bulk acoustic resonator bulk acoustic wave generating area of about 500-200000 ⁇ m 2, preferably about 5000-50000 ⁇ m 2, and most preferably from about 10000-25000 ⁇ m 2.
  • the side length of the bulk acoustic wave generating area of the ultra-high frequency bulk acoustic wave resonator is about 30-500 ⁇ m, preferably about 40-300 ⁇ m, and most preferably about 50-200 ⁇ m. That is, the side length of the corresponding vortex channel (the distance from the upstream end closest to the sample to the downstream release point) is about 30-500 ⁇ m, preferably about 40-300 ⁇ m, and most preferably about 50-200 ⁇ m.
  • the inlet includes a sample inlet and auxiliary solution inlets arranged on one or both sides of the sample inlet.
  • the auxiliary solution can be used to control the flow direction and range of the sample liquid in the micro channel, so that the sample liquid fully flows through the bulk acoustic wave generation area of the UHF bulk acoustic wave resonator.
  • the flow direction and range of the sample liquid in the microchannel can be controlled by controlling the flow rate and inflow area of the auxiliary solution.
  • a plurality of ultra-high frequency resonators with the same bulk acoustic wave generating region are arranged in the microfluidic system in the aforementioned method.
  • the release points of the bulk acoustic wave generating areas of the plurality of ultra-high frequency resonators are the same, for example, the bulk acoustic wave generating areas thereof have the same shape.
  • the fluid channel is divided into different regions, and ultra-high frequency resonators for separating different cells or vesicles are arranged in different regions.
  • the UHF resonator for separating different cells or vesicles may have different shapes of sound wave generating regions, or apply different powers of bulk acoustic waves, or have different flow rates. Therefore, it can be used to separate different cells or vesicles in the solution and make them flow into different flow channels or outlets.
  • the aforementioned method can be used to separate (or separate) flexible particles of different types or properties (for example, different sizes or densities, etc.) such as cells or vesicles, or biological macromolecules such as nucleic acids or proteins.
  • the aforementioned method can be used to separate (or separate) white blood cells and red blood cells from blood, for example, separate (or separate) nucleated red blood cells.
  • the aforementioned method can be used to separate (or separate) trophoblast cells from blood.
  • the aforementioned method can be used to separate CTC from a sample.
  • the aforementioned method can be used to separate bacteria, especially pathogens, from a solution, or to separate (or separate) different bacteria, especially pathogens.
  • the separation (or separation) of different flexible particles such as cells or vesicles, or biological macromolecules such as nucleic acids or proteins
  • separation methods can be selected by one or any combination of the following methods:
  • the method further includes increasing the power of the bulk acoustic wave generated by the UHF resonator, destroying the cell membrane or vesicle membrane, so as to release substances in the cell or vesicle, such as protein or nucleic acid.
  • the solution in the method is a liquid containing cells or vesicles to be separated, such as body fluids, whole blood, any blood fraction containing cells, fragmented tumors, tumor cell suspensions, cells Culture or culture supernatant.
  • the solution is blood, including whole blood or diluted blood.
  • the cell is a eukaryotic animal cell, preferably a mammalian cell, and more preferably a human cell.
  • the invention also provides a microfluidic device for controlling the movement of flexible particles such as cells or vesicles, or biological macromolecules such as nucleic acids or proteins or polysaccharides in the solution.
  • the microfluidic device can be used to obtain or purify required cells or biological macromolecules.
  • the microfluidic device can also be used to purify a solution (such as blood), for example, by separating and removing cells or vesicles in the solution to obtain a purified liquid from which the cells or vesicles have been removed (for example, plasma from which blood cells have been removed).
  • the microfluidic device provided by the present invention is used for processing biologically active cells or molecules, and therefore has settings or materials for processing costly biologically active substances.
  • the inner surface of the flow channel can be made of biocompatible materials.
  • it has a design to prevent cross-contamination, especially causing contamination amplification.
  • a microfluidic device for separating cells or vesicles including:
  • Fluid channel which has an inlet and an outlet
  • One or more ultra-high frequency bulk acoustic wave resonators which are arranged on a wall of the fluid channel, and the ultra-high frequency bulk acoustic wave resonator can generate in the fluid channel to the opposite side of the fluid channel
  • the wall frequency is about 0.5-50GHz bulk acoustic wave
  • a power adjusting device that adjusts the power of the bulk acoustic wave generated by the ultra-high frequency resonator
  • a flow rate adjusting device which adjusts the speed of the solution flowing through the bulk acoustic wave region
  • the UHF resonator can emit a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and a vortex channel defined by the boundary of the bulk acoustic wave generation area of the UHF resonator is generated in the solution.
  • Cells or vesicles enter and move along the vortex channel, and leave the vortex channel at a set position, which is called the release point.
  • the boundary line of the bulk acoustic wave generation area of the ultra-high frequency resonator is set to be suitable for cells or vesicles to be held in the vortex channel and move along the vortex channel to the release point.
  • the shape of the boundary line of the bulk acoustic wave generating region of the ultra-high frequency resonator allows the cells or vesicles to remain in the vortex channel and move to the release point. For example, by reducing the bending or curvature change in the boundary line of the volume generated area.
  • the angle between the boundary line of the bulk acoustic wave generating region of the UHF resonator and the fluid channel is such that the cells or vesicles are kept in the vortex channel and move to the release point.
  • the angle between the boundary line of the bulk acoustic wave generation area and the fluid channel is made smaller.
  • the bulk acoustic wave action area of the UHF bulk acoustic wave resonator has a focus zone and a sieve zone, the focus zone is located upstream of the bulk acoustic wave action zone, and the sieve zone is located downstream of the bulk acoustic wave action zone,
  • the setting of the bulk acoustic wave action area in the focal zone is more suitable for keeping the cells or vesicles moving in the vortex channel than the setting of the sieve zone.
  • the angle between the boundary line of the bulk acoustic wave action area of the focal zone and the fluid channel is smaller than the angle of the boundary line of the bulk acoustic wave action area of the sieve zone and the fluid channel.
  • the liquid flow velocity of the bulk acoustic wave action area flowing through the focusing area is controlled to be lower than the liquid flow velocity of the bulk acoustic wave action area flowing through the screen section.
  • the output power of the power adjustment device is about 20-5000 mW, preferably 50-2000 mW, more preferably 100-1500 mW.
  • the flow rate adjusting device can adjust the velocity of the solution flowing through the bulk acoustic wave region to about 0.1-10mm/s, preferably about 0.3-5mm/s, more preferably about 0.5-3mm /s.
  • the flow rate adjusting device can adjust the speed of the solution flowing through the bulk acoustic wave area to about 0.1-100 ⁇ L/min, preferably about 0.1-50 ⁇ L/min, more preferably about 0.5-30 ⁇ L /min.
  • the height of the fluid channel of the microfluidic device is about 20-200 ⁇ m, preferably about 25-100 ⁇ m, more preferably about 30-80 ⁇ m, for example about 40-60 ⁇ m.
  • the UHF bulk acoustic resonator bulk acoustic wave generating area of about 500-200000 ⁇ m 2, preferably about 5000-50000 ⁇ m 2, and most preferably from about 10000-25000 ⁇ m 2.
  • the side length of the bulk acoustic wave generating area of the ultra-high frequency bulk acoustic wave resonator is about 30-500 ⁇ m, preferably about 40-300 ⁇ m, and most preferably about 50-200 ⁇ m.
  • the inlet of the fluid channel includes a sample inlet and auxiliary solution inlets arranged on one or both sides of the sample inlet.
  • the fluid channel has at least two outflow channels, one of which is the outflow channel of the cells or vesicles that are controlled to move, called the particle outflow channel;
  • the outflow channel of the solution that controls the moving cells or vesicles can be called the solution outflow channel.
  • a plurality of ultra-high frequency resonators with the same bulk acoustic wave generating region are provided.
  • the fluid channel is divided into different regions, and UHF resonators for separating different cells or vesicles are arranged in different regions.
  • the UHF resonator for separating different cells or vesicles may have different shapes of sound wave generating regions. Or, for example, applying bulk acoustic waves of different powers, or having different flow rates.
  • the ultra-high frequency bulk acoustic wave resonator is a thin-film bulk acoustic wave resonator or a solid assembled resonator, for example, a thickness stretching vibration mode acoustic wave resonator.
  • the thickness of the piezoelectric layer of the UHF bulk acoustic wave resonator of the device is in the range of 1nm to 2um.
  • Figure 1 is a schematic structural diagram of a microfluidic device system provided by an embodiment of the present application.
  • Fig. 2 is a schematic structural diagram of a UHF bulk acoustic wave resonator in a microfluidic device system provided by an embodiment of the present application; wherein, (a) shows a top view of the microfluidic channel of the microfluidic system shown in Fig.
  • FIG. 1 Left side and cross-sectional view of AA (right side);
  • (b) shows the top view (left side) of the UHF bulk acoustic wave resonator (the black pentagonal part is the acoustic wave action area of the UHF bulk acoustic wave resonator ) And the cross-sectional view of BB (right side);
  • (c) shows the top view (left side) and cross-sectional view (right side) of the micro-channel + UHF bulk acoustic wave resonator;
  • FIG. 3 shows that HeLa cells enter the vortex channel in a microfluidic device system provided in an embodiment of the application.
  • Figure 3(a) shows the trajectory image of a single Hela cell in the vortex channel formed by the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator of the microfluidic device system (the trajectory of a single cell at different times is superimposed);
  • Figure 3( b) Shows the schematic diagram of the motion trajectory of Hela cells (the motion trajectories of multiple cells are superimposed) and the analysis diagram;
  • Figure 3(c) is the time and velocity analysis diagram of Hela cells.
  • Figure 4 shows the movement of cells in a vortex channel in a microfluidic device system provided in an embodiment of the application.
  • Figure 4(a) shows the image of cell movement in the vortex;
  • Figure 4(b) shows the cell sedimentation after stopping the bulk acoustic wave;
  • Figure 4(c) shows the arrangement of the cells in the vortex tunnel.
  • Fig. 5 shows that a microfluidic device system provided by an embodiment of the present application can separate and capture Hela cells from a whole blood mixture containing Hela cells.
  • Figures 5(c) and (d) show photos of Hela cells being captured and blood cells released under two micro-channel settings.
  • Figures 5(a) and (b) are the photos of Figure 5(c) and (d), respectively Analysis and schematic diagram.
  • Figure 5(e) shows the distribution of blood cells upstream and downstream (the color lines in Figure 5(c) and (d)) of the bulk acoustic wave action area.
  • Figure 6 shows the effect of the setting of the BAW action area of different UHF BAW resonators on the separation of blood cells in whole blood.
  • Figure 7 shows the effect of the setting of the BAW action area of different UHF BAW resonators on the separation of blood cells in whole blood.
  • microfluidic channel made of polydimethylsiloxane (PDMS) was prepared by soft lithography.
  • the bulk acoustic wave resonator device is prepared by chemical vapor deposition, metal sputtering, and photolithography on a silicon-based wafer.
  • the specific method is as follows:
  • a layer of aluminum nitride film is formed by surface sputtering, and then a layer of silicon dioxide film is deposited by ion-enhanced chemical vapor deposition. Then use the same method to alternately deposit aluminum nitride films and silicon dioxide films to form a Bragg acoustic reflection structure in which aluminum nitride and silicon dioxide alternately overlap.
  • the bulk acoustic wave resonator device is bonded and integrated with the PDMS microchannel chip.
  • the bulk acoustic wave resonator device is placed in the middle of the channel.
  • the bulk acoustic wave resonator device is connected to a network analyzer with a standard SMA interface, and the resonance peak is found by testing the frequency spectrum, and the frequency of the bulk acoustic wave emitted by the bulk acoustic wave resonator device in the micro channel can be measured.
  • High frequency signal generator (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz
  • Hela cell line Guangzhou Jinio Biotechnology Co., Ltd., ATCC#CCL2
  • 293T cells were cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100U/ml penicillin (Thermo) and 100ug/ml streptomycin (Thermo).
  • the density of cultured cells is between 1x10 5 /mL and 2x10 6 /mL. When performing microfluidic experiments, it can be diluted to 1x10 5 /mL for experiments.
  • PBS buffer Gibco).
  • DAPI 4',6-diamidino-2-phenylindole
  • a microfluidic device which can be used to separate and capture flexible particles in a solution, especially flexible particles with a diameter of about 0.2-30 um.
  • the flexible particles may be artificial or natural.
  • the particles are micelles with a membrane structure, especially micelles with lipid bilayers or lipid bilayers.
  • the flexible particles involved in the present invention generally have a diameter of about 0.2-30 um.
  • the flexible particles suitable for processing of the present invention are usually about 0.8-25um in diameter, preferably about 1-20um in diameter.
  • the method and device of the present invention can be used to separate flexible particles from a solution, for example, to separate blood cells from blood to obtain purified plasma.
  • the microfluidic device 100 includes a fluid channel 101, an ultra-high frequency bulk acoustic wave resonator 202, a bulk acoustic wave drive and power adjustment device, and a liquid injection and flow rate adjustment device 400.
  • the microfluidic device provided by the present invention can exist alone or can be a part of a microfluidic system, for example, in the form of a removable chip.
  • the microfluidic system or device can be used to contain and transport fluid materials such as liquids, and the size of the flow channel is in the micron or even nanometer level.
  • Typical microfluidic systems and devices usually include structures and functional units with dimensions of millimeters or smaller.
  • the fluid channel of the microfluidic device is generally closed except for the opening for the fluid to enter and exit.
  • the cross-section of the fluid channel usually has a size of 0.1-500 ⁇ m, which can be in various shapes, including ellipse, rectangle, square, triangle, circle, etc.
  • Various known microfabrication techniques can be used to prepare the fluid channel, and its materials include but are not limited to silica, silicon, quartz, glass or polymer materials (for example, PDMS, plastic, etc.).
  • the channel can be coated with a coating.
  • the coating can change the characteristics of the channel and can be patterned.
  • the coating can be hydrophilic, hydrophobic, magnetic, conductive, or biologically functional.
  • the height of the fluid channel of the microfluidic device is about 20-200 ⁇ m, preferably about 25-100 ⁇ m, more preferably about 30-80 ⁇ m, for example about 40-60 ⁇ m.
  • the width of the fluid channel of the microfluidic device is about 50-1000 ⁇ m, preferably about 100-500 ⁇ m, more preferably about 150-300 ⁇ m.
  • the microfluidic channel 100 in this embodiment has an inlet and an outlet for fluid to enter and exit.
  • the inlet is connected with a liquid injection device for receiving liquid injection.
  • the inlet in this embodiment includes a sample inlet 101 and a buffer inlet 102.
  • the buffer inlets are two inlets arranged on both sides of the sample inlet, and are connected to the sample inlet.
  • the microfluidic inlet is set by the above-mentioned three-phase flow mode (the sample flow in the middle, the buffer flow on both sides), which is beneficial to passively focusing the sample passed through the middle sample inlet.
  • the microfluidic device of this embodiment includes a liquid injection and flow rate adjustment device 400 for controlling liquid injection and controlling the flow rate of the liquid.
  • the liquid may be a liquid containing a sample.
  • the sample is a liquid containing the cells to be captured.
  • the sample may include body fluids, whole blood, any blood fraction containing cells, fragmented tumors, tumor cell suspensions, cell cultures or culture supernatants, and the like.
  • the fluid may be various body fluids, including blood, tissue fluid, extracellular fluid, lymphatic fluid, cerebrospinal fluid, aqueous humor, urine, sweat and the like.
  • the flow rate of the injected liquid can be controlled by an external pressure source, an internal pressure source, electronic dynamics or magnetic field dynamics.
  • the external pressure source and the internal pressure source may be pumps, such as a peristaltic pump, a syringe pump, or a pneumatic pump.
  • a syringe pump fine-tuned by a computer is used to control the flow rate of liquid injection.
  • the flow rate of the liquid is in the range of about 0.1-10 mm/s, preferably about 0.3-5 mm/s, more preferably about 0.5-3 mm/s. In another aspect of the present invention, the flow rate of the liquid is in the range of about 0.1-100 ⁇ L/min, preferably about 0.1-50 ⁇ L/min, more preferably about 0.5-30 ⁇ L/min.
  • the channel may be a single channel, or a plurality of channels arranged in parallel or in other forms and having a common output and input, wherein the outflow and inflow of the fluid and the flow rate of each channel can be controlled jointly or independently as required.
  • the microfluidic device of the present invention has one or more ultra-high frequency bulk acoustic wave resonators 200, which are arranged on a wall of the fluid channel (usually arranged at the bottom of the flow channel).
  • the ultra-high frequency bulk acoustic wave resonator can generate a bulk acoustic wave with a frequency of about 0.5-50 GHz that is transmitted to the opposite wall of the fluid channel (usually referred to as the top of the flow channel) in the fluid channel.
  • the ultra-high frequency bulk acoustic wave resonator that can be used in the present invention may be a thin film bulk acoustic wave resonator or a solid-state assembly type resonator, for example, a thickness stretching vibration mode acoustic wave resonator.
  • the microfluidic device of this embodiment has a plurality of ultra-high frequency bulk acoustic wave resonators 202 arranged at the bottom of the flow channel.
  • the ultra-high frequency bulk acoustic wave resonator is a bulk acoustic wave generating component, and can generate a bulk acoustic wave in the fluid channel that is transmitted to the wall on the opposite side of the fluid channel.
  • the UHF resonator can emit a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and a vortex channel defined by the boundary of the bulk acoustic wave generation area of the UHF resonator is generated in the solution. Cells or vesicles enter and move along the vortex channel, and leave the vortex channel at a set position, which is called the release point.
  • the UHF bulk acoustic wave resonator includes an acoustic wave reflection layer 206, a bottom electrode layer 205, a piezoelectric layer 204, and a top electrode layer 203 that are sequentially arranged from bottom to top. .
  • the overlapping area of the bottom electrode layer, the piezoelectric layer, the top electrode layer and the acoustic wave reflection layer constitutes a bulk acoustic wave generation area.
  • the top surface of the UHF bulk acoustic wave resonator is arranged on the wall of the fluid channel, and a bulk acoustic wave whose propagation direction is perpendicular to the wall is generated to the opposite wall;
  • the area formed by the top surface of the UHF bulk acoustic wave resonator is the bulk acoustic wave generating area, which is also called the bulk acoustic wave area or the bulk acoustic wave action area in this article.
  • the area of insonation is about 500-200000 ⁇ m 2, preferably about 5000-50000 ⁇ m 2, and most preferably from about 10000-25000 ⁇ m 2. As shown in FIG.
  • the bulk acoustic wave action area of this embodiment is a pentagonal shape with a side length of about 120 ⁇ m.
  • the continuous vortex generated by the UHF bulk acoustic wave in the solution forms an acoustic fluid vortex channel. Since the vortex is generated by the volume force caused by the attenuation of sound waves, the central axis of the vortex is above the boundary of the bulk acoustic wave, so the shape of the vortex channel is basically the same as the shape of the bulk acoustic wave, which is located at the boundary of the bulk acoustic wave. Above.
  • the shape of the bulk acoustic wave action area includes but is not limited to at least one of the following: circle, ellipse, semicircle, parabola, polygon with sharp or obtuse vertices, polygon with vertices replaced by arcs, vertices: Any combination of acute, semicircle, or parabolic polygons, or repeating square or circular arrays of the same shape.
  • This application provides the acoustic wave action area of the above-mentioned shape, but other acoustic wave action areas of any shape are also within the protection scope of this application.
  • the shape of the bulk acoustic wave generating region of a preferred UHF resonator is a spindle shape.
  • the boundary line of the bulk acoustic wave generation area of the UHF resonator (that is, the shape of the corresponding vortex channel) is set to be suitable for cells or vesicles in the vortex channel. Move along the vortex channel to the release point. This prevents cells or vesicles from leaving the vortex channel without leaving the vortex channel from the release point as set.
  • the cells or vesicles are kept in the vortex channel and moved to the release point.
  • twists or curvature changes in the boundary lines of the bulk acoustic wave generation area which may increase the probability of cells or vesicles leaving the vortex channel. Therefore, the cells or vesicles can be kept moving in the vortex channel by reducing the turning or curvature changes in the boundary lines of the bulk acoustic wave generation area, that is, reducing the cells or vesicles leaving the vortex channel and improving the separation efficiency.
  • the cells or vesicles are kept in the vortex channel and moved to the release point.
  • the inventor unexpectedly discovered that the smaller the angle between the boundary line of the bulk acoustic wave generation area and the fluid channel, the easier it is to keep the cells or vesicles moving in the vortex channel, that is, to reduce the cells or vesicles leaving the vortex channel and improve the separation. effectiveness.
  • the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator in the microfluidic system has a focus zone and a sieve zone.
  • the focus zone is located upstream of the bulk acoustic wave action area (that is, the part near the inflow direction of the sample and far away from the release point), and the sieve area is located downstream of the bulk acoustic wave action area (that is, close to the sample outflow direction, closer to the release point or the part including the release point) ).
  • the setting of the bulk acoustic wave action area in the focal zone is more suitable for keeping the cells or vesicles moving in the vortex channel: the cells or vesicles in the vortex channel in the focal zone are in the same or laminar flow direction Moving in a similar direction, the drag force of the vortex is relatively small, and it is easier for cells or vesicles to enter and remain in the vortex channel; in the downstream sieve area, the cells focused to the center of the vortex can be compared to unfocused cells. It moves more stably in the vortex channel.
  • the angle between the boundary line of the bulk acoustic wave action area of the focal zone and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area of the screen section and the fluid channel.
  • the boundary of the bulk acoustic wave action area in the focal zone is basically the same or basically the same as the direction of the fluid channel (for example, the angle is less than 10°), and the vortex drag force on the cells in the vortex channel in this area basically does not change the cells along the path.
  • the state of motion in the direction of laminar flow will only allow the cells to migrate laterally to the center of the vortex to achieve focus on the cells; the boundary of the bulk acoustic wave action area of the sieve section has a large angle with the fluid channel, which guides the cell movement direction to deviate from the direction of the fluid channel to the designated At the release point of the sieve zone, the cells focused to the center of the vortex can move more stably in the vortex channel than the unfocused cells.
  • the liquid flow velocity of the bulk acoustic wave action area flowing through the focus area is controlled to be lower than the liquid flow velocity of the bulk acoustic wave action area flowing through the screen section.
  • the fluid channel of this embodiment may have multiple UHF bulk acoustic wave resonators. In one aspect of the present invention, they are arranged linearly in a direction consistent with the direction of fluid movement.
  • the ultra-high frequency bulk acoustic wave resonator used in the present invention is a thickness stretching vibration mode, in which a piezoelectric material thin film layer is grown in a vertical direction, and is excited by coupling a vertical electric field with a d33 piezoelectric coefficient.
  • the ultra-high frequency bulk acoustic wave resonator used in the present invention can generate a localized sound flow at the interface between the device and the liquid, without the aid of a coupling medium or structure.
  • the ultra-high-frequency bulk acoustic wave generated by the ultra-high-frequency bulk acoustic wave resonator used in the present invention basically does not generate standing waves in the solution.
  • the UHF resonator emits a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel (such as the top of the flow channel).
  • the emergence of the acoustic jet 500 causes the liquid in the micro channel to generate a local three-dimensional vortex 501, and the continuous vortex caused by the UHF bulk acoustic wave forms an acoustic fluid vortex channel.
  • the central axis of the vortex is above the boundary of the bulk acoustic wave, so the shape of the vortex channel is basically the same as the shape of the bulk acoustic wave, which is located at the boundary of the bulk acoustic wave.
  • the vortex of the acoustic fluid is caused by the nonlinearity of the acoustic wave propagating in the liquid medium. The amplitude of the sound wave directly determines the strength of the vortex of the acoustic fluid.
  • the amplitude of the ultra-sonic device By adjusting the applied power, the amplitude of the ultra-sonic device, that is, the amplitude of the sound wave, can be adjusted, thereby controlling the flow velocity of the acoustic fluid vortex.
  • the forces on particles in the vortex include fluid drag force generated by vortex (Stokes drag force), and inertial drag generated by laminar flow Acoustic radiation force (inertial lift force) and acoustic radiation force caused by sound wave attenuation. Since the size of the fluid drag force has a positive relationship with the particle diameter, and the acoustic radiation force has a positive relationship with the square of the particle size.
  • the force they receive will change from being dominated by fluid drag to acoustic radiation, which pushes the particles to the center of the vortex.
  • Larger particles receive greater acoustic radiation force and move to the center of the vortex; while smaller particles rotate around the periphery under the action of the vortex drag force, and further, the lateral drag force generated by the laminar flow Under the action, it moves downstream of the area of effect of the bulk acoustic wave.
  • the fluid drag force of the particles in the vortex channel after the vortex and the acoustic radiation force caused by sound waves can be calculated according to certain formulas, but the inertial lift force generated by the acoustic fluid vortex can hardly be used simply Physics principles and formula calculations, especially in the case of fluids containing complex components.
  • the method and device of the present invention involve deformable cells or vesicles, which have more complicated force and movement trajectories in the acoustic fluid vortex and the channel formed by it.
  • the cells or vesicles in the solution pass through the vortex channel area of the acoustic fluid caused by the UHF bulk acoustic wave, they are at a suitable flow rate and bulk acoustic wave power. Under the conditions of, it will enter the vortex channel and move along the vortex channel; under the action of the lateral drag force generated by the laminar flow, at a certain position (s) of the vortex channel, the cells or vesicles will leave the body acoustic wave The area of action moves downstream and is released; the position where cells or vesicles leave the vortex channel is also called the release point. Since one of the important factors for cells or vesicles to leave the vortex channel is the influence of laminar flow along the direction of the fluid channel, the release point is usually located in the most downstream area of the vortex channel.
  • the position where cells or vesicles leave the vortex channel is usually at the transition point of the vortex and acoustic radiation force.
  • the position of the cell or vesicle leaving the vortex channel is usually at the place where the vortex channel has a turning or corner, that is, the corresponding bulk acoustic wave action area Above the position where the border turns or corners appear.
  • the applicant believes that the reason for this phenomenon is that at the turning or corner of the vortex channel, the vortex direction and the direction of the acoustic radiation force suddenly change, and larger cells or particles have been focused on The center of the vortex, and the greater acoustic radiation force, can change the direction of motion along with the vortex channel and quickly refocus to the center of the vortex channel after turning; while the smaller particles or cells will be dragged by the laminar flow The jump in direction is more affected and leaves the vortex channel.
  • the cells or vesicles in the solution enter and move along the vortex channel, and leave the vortex channel at a set position.
  • the cells or vesicles leave the body acoustic wave action area at a specified position and direction, and enter a desired outflow channel, for example, into the particle outflow channel.
  • the set position to leave the vortex channel is called the release point, that is, the position where the cell or vesicle leaves the body acoustic wave action area.
  • the solution that removes the cells or vesicles that are controlled to move maintains the inflow direction and enters the solution outflow channel.
  • the frequency of the film bulk acoustic resonator is mainly determined by the thickness and material of the piezoelectric layer.
  • the thickness of the piezoelectric layer of the film bulk acoustic resonator used in the present invention is in the range of 1 nm to 2 um.
  • the frequency of the ultra-high frequency bulk acoustic wave resonator of the present invention is about 0.5-50 GHz, preferably about 1-10 GHz.
  • the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator is driven by a signal from a high frequency signal generator.
  • the pulse voltage signal driving the resonator can be driven by pulse width modulation, which can generate any desired waveform, such as sine wave, square wave, sawtooth wave or triangle wave.
  • Pulse voltage signals can also have amplitude modulation or frequency modulation start/stop capabilities to start or eliminate bulk acoustic waves.
  • the microfluidic device of the present invention also includes a power adjusting device that adjusts the power of the bulk acoustic wave generated by the ultra-high frequency resonator.
  • the power adjustment device is a power amplifier with a power adjustment function.
  • the output power of the power adjustment device is about 20-5000 mW, preferably 50-2000 mW, more preferably 100-1500 mW. Since the film bulk acoustic wave resonator has high energy conversion efficiency and basically no loss, the output power of the power adjustment device can be basically regarded as the output power of the film bulk acoustic wave resonator to generate bulk acoustic waves in the fluid.
  • the power adjustment device can be connected to a high-frequency signal generator.
  • the output circuit of the power amplifier is respectively connected with the bottom electrode, the piezoelectric layer and the top electrode of the ultra-high frequency bulk acoustic wave resonator.
  • Example 3 Cell movement in the vortex channel caused by UHF bulk acoustic waves
  • Hela cells were dissolved in DMEM medium to prepare a sample (cell content is about 1*10 5 /mL).
  • Hela cells were labeled with Calcein-AM to observe their movement pathways.
  • the sample is injected into the microchannel from the sample inlet.
  • the height of the micro flow channel is 50 ⁇ m.
  • the frequency of the UHF bulk acoustic wave resonator is 1.83GHz and the output power is 30mW.
  • the control sample input flow rate is 1 ⁇ L/min, which is about 0.67mm/s.
  • Figure 3(a) shows the trajectory of a single Hela cell in the vortex channel formed by the bulk acoustic wave generated by the UHF bulk acoustic wave resonator of the microfluidic device system. It is the superposition of multiple images of the same single cell at different times to show its movement trajectory.
  • Figure 3(b) is a schematic diagram of the movement trajectories of different individual cells: the upper image is a top view, and the lower image is a side view.
  • Figure 3(c) is the time and velocity analysis diagram of Hela cells.
  • the bulk acoustic wave generated by the UHF bulk acoustic wave resonator causes the flowing fluid to generate vortices, and each vortex is connected to the adjacent vortex along the UHF bulk acoustic wave.
  • the boundary of the acoustic wave action area of the resonator forms an acoustic fluid vortex channel or vortex tunnel.
  • the image sequence in Figure 3(a) shows and proves the movement trajectory of a single HeLa cell.
  • the movement process can be divided into three stages that occur in the area a-b-c, as shown in Figure 3(a) and Figure 3(c).
  • area a the cells move at a uniform speed in the lateral flow.
  • area b the direction of cell movement is changed by the vortex array, and the cell enters the vortex tunnel generated by the sound wave, while focusing on the 3-D axis and moving along a fixed path.
  • the speed of cells with different initial positions when entering the vortex array varies greatly. Due to the difference in the relative direction of the vortex, the cells at the initial position in the lower layer of the microchannel accelerate, while the cells at the initial position in the upper layer slow down. In area c, the speed of cell movement gradually slows down. Since the trajectory of the cells is adjusted in the vortex tunnel, the process of deceleration and movement of the cells has good stability, and the cells that enter the initial position have good deceleration and
  • Figure 3(b) further illustrates the movement of cells in the flow channel and under the action of bulk acoustic waves.
  • Figure 3(b) is a superimposed schematic diagram of the movement trajectories of different single cells.
  • the upper figure of Figure 3(b) is a top view, and the lower figure is a side view. It can be seen from the top view that the trajectories of the cells are only distributed on the boundary of the acoustic wave action area of the UHF bulk acoustic wave resonator. From the side view, it can be seen that the cells are floating above the bottom of the channel without touching the bottom.
  • Hela cells labeled with Calcein-AM are used, and the movement of the cells on the z axis is observed and measured using a confocal microscope (Leica, Germany) .
  • the x-z-t mode is used. In this mode, the shooting speed can reach 37 frames per second.
  • Example 3 According to the same experimental setup of Example 3, but the input flow rate of the sample containing Hela cells was reduced to 0.1 ⁇ L/min. After the cell-containing sample and buffer are passed into the micro-channel, the signal generator is turned on, and the cells are captured in the sound wave action area. Then adjust the inlet and outlet pressures to be the same, and the fluid stagnates. Observe the movement of the cells in the vortex. Then the signal generator is turned off and the movement of the cells is observed.
  • Figure 4(a) is an image of cell movement in the vortex, which is a composite stacked image (6 images, 27ms apart), in which the red dot shows the center of the particle in each frame, the green dotted circle represents the range of particle movement and the red arrow represents The direction of particle movement.
  • the power of the signal generator is turned on, a bulk acoustic wave is generated, forming a vortex and a vortex tunnel in the flow channel, and the particles are relatively "stationary" suspended from the chip surface.
  • Figure 4(b) when the power is turned off, the bulk acoustic wave disappears, and the particles settle on the chip surface under the combined action of gravity and buoyancy. The results show that the particles are indeed trapped in the vortex tunnel.
  • Figure 4(c) shows that Calcein-AM labeled Hela cells are trapped in the vortex tunnel, which has the same shape as the boundary of the acoustic wave action area of the UHF bulk acoustic wave resonator.
  • Blood is the most primitive and most suitable environment for cells. In the blood environment, cells have the best viability and complete functions, which are important for biological research, such as cell metabolism and proteomics. However, compared to a diluted blood sample, whole blood is a more challenging sample. In terms of physical parameters, whole blood is more viscous and turbid, which will have a serious negative impact on cell operation. Physical fields, such as dielectric electric fields, magnetic fields, and hydrodynamic fields, are disordered in blood samples. In addition, high-density cells, especially red blood cells (10 9 /mL), will cause strong interactions between cells, thereby changing the trajectory of the specimen and affecting the stability of cell movement.
  • the Hela cells were dissolved in DMEM culture medium, mixed with whole blood, and prepared as a test sample sample (the content of Hela cells was adjusted to about 1 ⁇ 10 5 cells/mL).
  • the sample is injected into the microchannel from the sample inlet. And inject PBS buffer into the micro flow channel from the buffer inlets on both sides. PBS is used as a clamping sheath to ensure the lateral extent of the sample fluid and ensure that all samples pass over the device. Passive focus is performed on the injected sample through a three-phase flow, so that the sample can better flow through the acoustic wave action area of the high-frequency bulk acoustic wave resonator.
  • Figures 5(c) and (d) show the photos of Hela cells entering the vortex channel and blood cells passing through the body acoustic wave area under two micro-channel settings.
  • Figure 5(a) and (b) are Figure 5(c) and ( d) Analysis and schematic diagram.
  • the bulk acoustic wave action area of the UHF bulk acoustic wave resonator is a pentagon.
  • the volumetric acoustic wave action area is set to a protruding corner (a corner of the pentagon) downstream of the liquid inflow direction: at this time, one of the points where the blood cell leaves the volumetric acoustic wave action area is at the corner of the pentagon nearby.
  • Figure 5(f-1) shows the input mode of three-phase flow to passively focus the injected sample, so that the sample can better flow through the acoustic wave action area of the high-frequency bulk acoustic wave resonator.
  • Figure 5(f-2) shows the flow of blood cells when the power is turned on and the body acoustic wave is generated in the microchannel.
  • Figure 5(f-3)-(f-5) shows that CTC is selectively entered into the vortex tunnel: the composite stacked image sequence shows the trajectory of CTC.
  • Figure 5(f-6) shows the result of selective CTC entering the vortex tunnel: the combined images show that the device of the present invention is stable while releasing blood cells so that CTC enters and remains in the vortex tunnel.
  • the release point is the jump point of the vortex and sound radiation force.
  • a regular pentagonal device after the cells are captured and focused by the vortex, since each side is a straight line, it means that the laminar flow generates a lateral drag force that moves downstream, and the vortex drag force is in the radial direction of the flow channel.
  • the components of are all stable, which makes it difficult for the focused cells to fall off when moving along the boundary.
  • both the direction of the vortex and the direction of the acoustic radiation force suddenly change. Since the larger cells have been focused to the center of the vortex and received greater acoustic radiation, they can change the direction of motion and quickly re-start with the vortex.
  • the microfluidic device such as the bulk acoustic wave action area of the UHF bulk acoustic wave resonator, non-target cells are taken away by laminar flow, and only target cells can enter and/or remain in the vortex channel.
  • Example 6 The effect of the setting of the BAW action area of different UHF BAW resonators on the separation of blood cells in whole blood
  • This experiment tested the effect of using the microfluidic system and method of the present invention to separate blood cells from plasma in whole blood.
  • the micro flow channel as shown in FIG. 6 was prepared and constructed.
  • Each of the micro-channels has three UHF resonators arranged in sequence with the same sound wave generating area. Using multiple UHF resonators in the direction of the flow channel can strengthen the control of the transfer of the same target particle.
  • the surface of the UHF resonator (that is, the area where the bulk acoustic wave is generated, shown as a triangular device in the figure) is located on the side of the micro-channel channel (left in the figure).
  • the micro-channel is equipped with two solution inlets, and the right inlet is open Whole blood sample.
  • the downstream angle of the triangular bulk acoustic wave generating area (that is, the vortex channel) is the set cell release point.
  • the released cells leave from the downstream angular position and enter the cell outflow channel in the direction of the microchannel channel (the left channel at the bottom of the figure).
  • the height of the micro channel is 50um.
  • the frequency of the UHF BAW resonator is 1.83GHz, and the three output powers tested are shown in Figure 6.
  • the control sample input flow rate is 0.5uL/min, and the PBS buffer input flow rate is 5uL/min.
  • the two leftmost pictures in Fig. 6, the two middle pictures and the two right pictures respectively show the effect of separating blood cells under UHF bulk acoustic wave power of 1660mW, 417mW and 208mW.
  • the angle formed by the boundary of the triangular bulk acoustic wave generation area (that is, the direction of the vortex channel) and the flow direction of the liquid in the microchannel (i.e. laminar flow) in the left picture in each two pictures is greater than the bulk acoustic wave generation area in the right picture
  • the angle formed by the boundary of and the flow direction of the liquid in the microchannel is relatively large.
  • the boundary line (that is, the shape of the corresponding vortex channel) of the bulk acoustic wave generation area of the UHF resonator in the microfluidic system is set to be suitable for cells or capsules.
  • the bubble moves in the vortex channel along the vortex channel to the release point. This prevents cells or vesicles from leaving the vortex channel without leaving the vortex channel from the release point as set.
  • the cells or vesicles are kept in the vortex channel and moved to the release point.
  • the inventor unexpectedly discovered that the smaller the angle between the boundary line of the bulk acoustic wave generation area and the fluid channel, the easier it is to keep the cells or vesicles moving in the vortex channel, that is, to reduce the cells or vesicles leaving the vortex channel and improve the separation. effectiveness.
  • Example 7 The effect of the setting of the BAW action area of different UHF BAW resonators on the separation of blood cells in whole blood
  • the surface of the UHF resonator (that is, the area where the bulk acoustic wave is generated, shown as a spindle-shaped device in the figure) is located on the side of the microchannel channel (the right side in the figure) and is inclined to the left.
  • the micro flow channel is provided with two solution inlets, and the right inlet is for blood samples.
  • the PBS solution is passed into the left side, and the flow rate of the PBS solution and the focusing effect of the sheath flow are adjusted to make the sample flow fully pass through the right side of the spindle-shaped sound wave generating area.
  • the downstream angle of the boundary of the spindle-shaped body acoustic wave generation area (that is, the location of the vortex channel) is the set cell release point.
  • the released cells leave from the downstream angular position and enter the cell outflow channel in the direction of the microchannel channel.
  • the height of the micro channel is 50um.
  • the frequency of the UHF BAW resonator is 1.83GHz, and the three output powers are shown in Figure 7.
  • the control sample input flow rate is 1uL/min, and the PBS buffer input flow rate is 10uL/min.
  • the acoustic wave generation area of the spindle-shaped body is divided into two areas, the focus area (the upper part of the spindle-shaped device, whose right boundary is the same as the laminar flow direction) and the sieve partition (the lower part of the spindle-shaped device, on the right).
  • the side boundary forms a larger angle with the laminar flow direction).
  • upstream of the device the direction of the vortex tunnel is the same as the laminar flow direction.
  • the vortex drag force does not change the movement state of the cells along the laminar flow direction, but only allows the cells to migrate laterally to the center of the vortex to achieve the Focusing of cells.
  • the cells focused to the center of the vortex can be moved more stably than unfocused cells. Fluorescence is added to the blood to characterize the movement of plasma: 2 ⁇ L of Calcein-AM is added to 1mL of blood. As shown in the figure, blood cells are efficiently migrated from the plasma along the right boundary of the spindle-shaped device to the cell release point on the left side of the flow channel, and leave the spindle-shaped device at the cell release point and enter the PBS buffer.
  • the boundary line (that is, the shape of the corresponding vortex channel) of the bulk acoustic wave generation area of the UHF resonator in the system is set to be suitable for cells or vesicles in the vortex channel Move along the vortex channel to the release point. This prevents cells or vesicles from leaving the vortex channel without leaving the vortex channel from the release point as set.
  • the cell or vesicle is kept in the vortex channel and moved to the release point by adjusting the shape of the boundary line of the bulk acoustic wave generating region of the UHF resonator.
  • the cells or vesicles can be kept moving in the vortex channel by reducing the turning or curvature changes in the boundary lines of the bulk acoustic wave generation area, that is, reducing the cells or vesicles leaving the vortex channel and improving the separation efficiency.
  • the shape of the bulk acoustic wave generating region of a preferred UHF resonator is a spindle shape.
  • the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator in the microfluidic system has a focus zone and a sieve zone.
  • the focus zone is located upstream of the bulk acoustic wave action area (that is, the part near the inflow direction of the sample and far away from the release point), and the sieve area is located downstream of the bulk acoustic wave action area (that is, close to the sample outflow direction, closer to the release point or the part including the release point) ).
  • the setting of the bulk acoustic wave action area in the focal zone is more suitable for keeping the cells or vesicles moving in the vortex channel: the cells or vesicles in the vortex channel in the focal zone are in the same or laminar flow direction Moving in a similar direction, the drag force of the vortex is relatively small, and it is easier for cells or vesicles to enter and remain in the vortex channel; in the downstream sieve area, the cells focused to the center of the vortex can be compared to unfocused cells. It moves more stably in the vortex channel.
  • the angle between the boundary line of the bulk acoustic wave action area of the focal zone and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area of the screen section and the fluid channel.
  • the boundary of the bulk acoustic wave action area in the focal zone is basically the same or basically the same as the direction of the fluid channel (for example, the angle is less than 10°), and the vortex drag force on the cells in the vortex channel in this area basically does not change the cells along the path.
  • the state of motion in the laminar flow direction will only allow the cells to migrate laterally to the center of the vortex to achieve focusing on the cells; the boundary of the volumetric acoustic wave action area of the sieve section has a large angle with the fluid channel, which guides the cell movement direction to deviate from the fluid channel direction to the designated In the sieve zone, the cells focused to the center of the vortex can move more stably in the vortex channel than unfocused cells.
  • Example 8 Controlling the movement of nucleic acid and separating and purifying nucleic acid
  • FIG. 8(a) is a top view, and the right side of the figure is the flow channel inlet for inputting PBS solutions containing nucleic acid fragments of different sizes.
  • the surface of the UHF resonator that is, the area where the bulk acoustic wave occurs, is shown as a spindle shape, and the left tip is the nucleic acid release point.
  • the left side of the microchannel includes three outflow channels, among which is the nucleic acid outflow channel in the middle, and the nucleic acid that flows out from the nucleic acid release point on the left tip of the spindle enters the nucleic acid outflow channel.
  • the upper and lower channels of the nucleic acid outflow channel are for the solution outflow channel after the target nucleic acid is removed.
  • Figure 8(a) shows the system setup and the observation after the fluorescent dye nucleic acid solution is introduced: the upper picture is a bright field, and the lower picture is a fluorescent signal observation picture. It shows that the fluorescent dye nucleic acid solution is evenly distributed in the microcomputer when the UHF resonator is not working. The situation in the runner.
  • Figure 8(b) shows a double-stranded nucleic acid sample with a size of about 20k in the device shown in Figure 8(a) at different flow rates (0, 0.1, 0.25, 0.5, 1 ⁇ L/min) and different bulk acoustic wave working power ( 50-1000mW) performance.
  • the fluid channel height is about 20 microns.
  • the nucleic acid is extracted from sheep whole blood genomic DNA, the size is about 20kbp.
  • Figure 8(c) shows the performance of a double-stranded nucleic acid sample with a size of about 20k in the device shown in Figure 8(a) under different flow channel heights (50 microns and 20 microns).
  • Figure 8(d) shows a double-stranded nucleic acid sample with a size of about 5k in the device shown in Figure 8(a) at different flow rates (0.1, 0.25, 0.5 ⁇ L/min) and different bulk acoustic wave operating powers (50-1000mW) Performance.
  • the fluid channel height is about 20 microns.
  • the 5k double-stranded nucleic acid sample is a DNA plasmid, which is a double-stranded circular DNA.
  • the device and method of the present invention have a good ability to control the movement of 5 kbp plasmid DNA, and the control ability is positively correlated with the applied power and negatively correlated with the lateral fluid velocity in the flow channel.
  • Figure 8(e) shows the performance of a circular nucleic acid and a chain nucleic acid sample with a size of about 5k in the device shown in Figure 8(a).
  • the fluid channel height is about 20 microns.
  • the 5k-stranded nucleic acid is the DNA plasmid of the 5k double-stranded nucleic acid sample in Figure 8(d) is digested and broken to form double-stranded DNA.

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Abstract

一种控制流体中的目标颗粒移动的微流控***及方法，微流控***包括流体通道，其具有入口和多个出口，一个或多个超高频声波谐振器，所述超高频体声波谐振器可在所述流体通道产生频率为约0.5-50GHz的体声波；通过调节超高频体声波谐振器体声波产生区域的形状和设置方位，使得颗粒在进入体声波在溶液中引发的涡旋通道，按指定位置和方向移动，可以对溶液中的颗粒进行控制和分离，得到指定的颗粒，或得到分离颗粒后的纯化的溶液。

Description

利用超高频声波控制溶液中的微粒移动的方法及设备
本申请要求以下中国专利申请的优先权：2019年6月13日提交的、申请号为201910512148.6、发明名称为“利用超高频声波控制溶液中的微粒移动的方法及设备”，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及细胞研究方法学与医疗器械领域。具体的，本发明涉及一种对细胞或微囊泡进行分离和分析的微流控***和使用所述***来分离和分析细胞或微囊泡的方法。
背景技术
人体体液如血液和组织液中存在的细胞或亚细胞颗粒，以及核酸和蛋白质等生物大分子颗粒对生理健康和研究非常重要，因此存在将体液中的细胞或亚细胞颗粒或生物大分子颗粒进行分离的需求。现有技术中通过滤网的方法分离细胞等颗粒的方法很多，但都存在诸如操作复杂，容易出现堵塞，处理量低等缺点。
利用微流控***对样品中的颗粒进行分离是新兴的技术。已经报道的方法大多数是基于细胞等颗粒的物理性质如大小进行分离，或者是基于生物特异性进行区别和分离。这些方法存在花费巨大和处理量低的问题。
因此，目前亟需一种***及方法，以实现对溶液中的细胞或微囊泡或生物大分子颗粒的分离，获得需要的细胞或生物大分子颗粒，或是去除细胞等颗粒后得到的纯化的体液。
发明内容
本发明首次发现利用超高频体声波能够在微流控***中有效地操控溶液中的细胞或囊泡，或核酸或蛋白质或多糖等生物大分子等柔性颗粒的移动位置和方向，由此提供了分离和获得目标细胞或囊泡或生物大分子颗粒， 或者是获得去除细胞或囊泡或生物大分子等颗粒后的液体的方法和***。
具体的，本发明提供了一种控制溶液中柔性微粒的移动的方法，包括：
(1)使含有目标柔性微粒的溶液流经一个微流控设备，所述设备包括；
流体通道，其具有入口和流出通道；
一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
(2)所述超高频谐振器发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定(define)的涡旋通道；
(3)使得溶液中的目标柔性微粒进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道。
柔性微粒是指具有形变性质的纳米或微米颗粒。柔性颗粒可以是人工的或天然的。所述颗粒可以为带有膜结构的微团，特别是具有脂质双分子层或类脂质双分子层的微团，例如为细胞或囊泡，包括外泌体等，或者是人工制备的，例如为油包水制剂或者水包油包水(W/0/W)双乳液制剂形式的脂质体或微胶囊等。所述颗粒也可以是具有不规则形状的，例如为核酸或蛋白等生物大分子。
在其中一个方面，本发明提供了一种控制溶液中细胞或囊泡的移动的方法，包括：
(1)使含有目标细胞或囊泡的溶液流经一个微流控设备，所述设备包括；
流体通道，其具有入口和流出通道；
一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
(2)所述超高频谐振器发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定的涡旋通道；
(3)使得溶液中的目标细胞或囊泡进入涡旋通道和顺着涡旋通道移 动，并在设定的位置离开涡旋通道。
在本发明中，细胞或囊泡可以是人工的或天然的，通常所述颗粒为带有膜结构的微团，特别是具有脂质双分子层或类脂质双分子层的微团。本发明涉及的柔性颗粒通常具有约0.01-30um的直径，优选为0.2-25um的直径，更优选为0.5-20um。在本发明的其中一个方面，所述柔性颗粒为天然存在的颗粒，例如细胞或细胞释放到细胞外环境中的囊泡。细胞包括天然的或培养的高等植物或动物(例如包括人在内的哺乳动物)的细胞，以及细菌，真菌等单细胞生物或简单多细胞生物。囊泡为各种不同的动物细胞释放到细胞外环境中的微囊泡。这些细胞相关的微囊泡是从细胞膜上脱落或由细胞分泌的具有双层膜结构的囊泡状小体。它们可以具有大约30-1000nm、大约30-800nm、大约30-150nm或者大约30-100nm的直径。细胞释放的微囊泡包括外泌体、微囊泡、囊泡、膜小泡、水泡、气泡、***小体、微颗粒、管腔内囊泡、核内体样囊泡或胞吐囊泡等。
在其中一个方面，本发明提供了一种控制溶液中目标核酸或蛋白质或多糖等生物大分子(特别是核酸)的移动的方法，包括：
(1)使含有目标生物大分子的溶液流经一个微流控设备，所述设备包括；
流体通道，其具有入口和流出通道；
一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
(2)所述超高频谐振器发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定的涡旋通道；
(3)使得溶液中的目标生物大分子进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道。
在本发明的其中一个方面，所述方法中的生物大分子是指核酸。本文所用的“核酸”(以及等价术语“多核苷酸”)是指在核苷酸亚单位之间包含磷酸二酯键的核糖核苷或脱氧核糖核苷的聚合物。核酸包括，但不限于，基因DNA、cDNA、hnRNA、mRNA、rRNA、tRNA、微RNA、片段核酸、从 亚细胞器如线粒体获得的核酸，以及从可能出现在样品上或样品中的微生物或病毒获得的核酸。核酸包括天然或合成的，例如以人工或天然DNA或RNA为模板的扩增反应产物。核酸可以是双链或者单链、环状或线性的。可以用于检测目标核酸的样品包括下述样品：来自细胞培养物、真核微生物或诊断样品如体液、体液沉淀物、洗胃样本、细针抽取物、活组织检查样品、组织样品、癌细胞、来自病人的细胞、来自组织的细胞或来自待测试和/或治疗疾病或感染的个体的体外培养的细胞、或法医样品。体液样品的非限制性实例包括全血、骨髓、脑脊液、腹膜液、胸膜液、淋巴液、血清、血浆、尿、乳糜、粪便、***、痰、***吸液、唾液、棉签样本、冲洗或灌洗液和/或擦拭样本。本发明方法尤其适用于分离长度≥300bp，优选≥1kbp、更优选≥10kbp、例如为≥50kbp的核酸(例如任何形式的DNA和RNA，包括天然或合成核酸，例如以DNA或RNA为模板的扩增反应产物)。
本发明中的超高频体声波谐振器是指能够产生频率超过0.5GHz(优选为超过1GHz)，例如频率为0.5-50GHz的体声波的谐振器。所述超高频体声波谐振器例如可以为薄膜体声波谐振器或固态装配型谐振器等。
在本发明的方法中，超高频谐振器发射传向所述流体通道的对侧的壁(例如流道顶部)的超高频体声波，声波衰减到流体中产生的体积力使得流经的溶液中出现声射流，导致微流道中的液体产生局部的立体的旋涡。由于涡旋是由于声波衰减引发的体积力产生的，涡旋的中心轴在体声波作用边界的上方；超高频体声波引起的连续涡旋形成声流体涡旋通道，涡旋通道的形状基本与体声波作用区域的形状相同，位于体声波作用区域边界的上方，即涡旋通道的形状和位置是由超高频谐振器的体声波产生区域的边界限定(define)的。
在本发明的其中一个方面，所述微流控设备的流体通道具有所述被控制移动的柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子等的流出通道，可称为颗粒流出通道。在本发明的另一个方面，所述流体通道还具有其它流出通道，例如为除去或含有较少所述被控制移动的细胞或囊泡的溶液的流出通道，可称为溶液流出通道。所述颗粒流出通道和溶液流出通道的开口的宽度比例可设为约1:1-1:20，优选为约1:2-1:15，例如为约 1:4-1:10。在本发明的其中一个方面，所述柔性颗粒离开涡旋通道的位置接近所述颗粒流出通道的开口。
在上述方法的步骤(3)，可通过调节超高频体声波谐振器的体声波作用区域的形状和位置，使得溶液中的被控制移动的柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道。由此所述柔性颗粒以指定位置和方向离开体声波作用区域，进入希望的流出通道，例如进入所述颗粒流出通道。该设定的离开涡旋通道的位置称为释放点，也即所述柔性颗粒离开体声波作用区域的位置。除去所述被控制移动的细胞或囊泡的溶液则保持流入方向，进入到前述溶液流出通道。
由于柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子等脱离涡旋通道的其中一个重要因素是沿流体通道方向的层流的影响，释放点通常位于涡旋通道的下游区域。在本发明的另一个方面，释放点通常处于涡旋通道出现转折或曲率变化的地方，即对应体声波作用区域边界出现转折或曲率变化的位置的上方，也即，对应所述释放点的体声波作用区域的边界存在转折或曲率变化。在不受有关理论约束的情况下，申请人认为，这个现象的原因在于，在涡流通道的转折或转角处，涡旋方向和声辐射力方向突然变化，进入涡旋通道的柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子中，符合合适的条件(如合适的尺寸)的颗粒由于已经聚焦到涡旋中心，在声辐射力的作用下能够随着涡旋通道改变运动方向并且快速重新聚焦到转折后的涡旋通道的中心；而不符合条件(如具有较小尺寸)的颗粒则会因为层流拖拽方向的跳变而受到更大的影响，从而离开涡旋通道。
在本发明的其中一个方面，本发明提供的上述方法适于处理含有大量柔性颗粒如细胞或囊泡，或核酸或蛋白质等的液体样品；所述大量柔性颗粒可以连续移动的方式进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道，由此达到快速和大通量处理的目的。在本发明的其中一个方面，本发明提供的上述方法适于处理含有大量细胞或囊泡的样品，例如全血或血液级份。
本发明提供的上述方法可用于获得或纯化样品中的需要的柔性颗粒。 在本发明的其中一个方面，所述方法可用于富集需要的柔性颗粒。
本发明提供的上述方法还可用于去除样品中的不想要的柔性颗粒，获得纯化的溶液。在本发明的其中一个方面，其可以从血液样品中去除某种细胞或囊泡，例如去除血细胞，获得血浆。
在本发明的其中一个方面，上述方法中还包括通过调节体声波的功率和/或通过调节所述溶液流经体声波区域的速度，来调节进入涡旋通道的柔性颗粒。未进入涡旋通道的柔性颗粒或进入涡旋通道但未到达指定释放点就离开涡旋通道的柔性颗粒则穿过体声波区域，沿样品进入流体通道的方向流出。
在本发明的其中一个方面，上述方法中所述超高频谐振器的体声波产生区域的边界线条(即对应的涡旋通道的形状)设置为适于目标柔性颗粒在涡旋通道中顺着涡旋通道移动至释放点。由此可避免目标柔性颗粒脱离涡旋通道而不按设定从释放点离开涡旋通道。
在本发明的其中又一个方面，其中通过调节所述超高频谐振器的体声波产生区域的边界形状使得目标柔性颗粒保持在涡旋通道中移动至释放点。如前所述，体声波产生区域的边界线条中存在转折或曲率变化，可能会增加柔性颗粒脱离涡旋通道的几率。因此，可以通过减少体声波产生区域的边界线条中出现转折或曲率变化来使得柔性颗粒保持在涡旋通道中移动，即减少脱离涡旋通道的柔性颗粒，提高分离的效率。
在本发明的其中又一个方面，通过调节所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得柔性颗粒保持在涡旋通道中移动至释放点。发明人意外地发现，体声波产生区域的边界线条与流体通道的角度越小，越易于使得柔性颗粒保持在涡旋通道中移动，即减少脱离涡旋通道的柔性颗粒，提高分离的效率。
在本发明的其中又一个方面，所述微流控***中的超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区。聚焦区位于体声波作用区域上游(即靠近样品流入方向，离释放点较远的部分)，筛分区位于体声波作用区域下游(即靠近样品流出方向，离释放点较近或包括释放点的部分)。其中聚焦区的体声波作用区域的设置相对筛分区的设置更适于使得柔性颗 粒保持在涡旋通道中移动：在聚焦区的涡流通道中的细胞或囊泡沿着和层流方向相同或相似方向移动，受到的涡旋拖拽力相对较小，更易于细胞或囊泡进入和保留在涡旋通道；在下游的筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。在本发明的其中又一个方面，聚焦区的体声波作用区域边界线条与流体通道的角度相对筛分区的体声波作用区域边界线条与流体通道的角度较小。例如，聚焦区的体声波作用区域边界基本与流体通道方向一致或基本一致(例如角度小于10°)，在这个区域的涡流通道中的细胞受到的涡旋拖拽力基本上不改变细胞沿着层流方向的运动状态，只会让细胞横向迁移至涡旋中心，实现对细胞的聚焦；筛分区的体声波作用区域边界与流体通道角度较大，引导细胞移动方向偏离流体通道方向转移到指定的释放点，在筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。
在本发明中，所述微流控设备通常包括功率调节装置，其调节所述超高频谐振器产生的体声波的功率。
在本发明中，所述微流控设备通常包括流速调节装置，其调节所述溶液流经体声波影响区域的速度。
在本发明的其中一个方面，其中所述细胞包括细胞簇。所述细胞簇通常由数个，例如2、3、4、5、6、7、8、9或10个，细胞组成。在本发明的其中一个方面，其中所述囊泡包括囊泡群。所述囊泡群通常由数个，例如2-50个囊泡组成。
在本发明的其中一个方面，其中所述功率调节装置的输出功率为约20-5000mW，优选为50-2000mW，更优选为100-1500mW。
在本发明的其中一个方面，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.1-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-3mm/s。
在本发明的其中一个方面，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.01-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-30μL/min。
在本发明的其中一个方面，其中所述微流控设备的流体通道的高度为 约5-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
在本发明的其中一个方面，其中所述超高频体声波谐振器的体声波产生区域面积为约500-200000μm 2，优选为约5000-50000μm 2，最优选为约10000-25000μm 2。
在本发明的其中一个方面，其中所述超高频体声波谐振器的体声波产生区域的边长为约30-500μm，优选为约40-300μm，最优选为约50-200μm。即对应涡流通道的边长(从最接近样品的上游一端到下游的释放点的距离)为约30-500μm，优选为约40-300μm，最优选为约50-200μm。
在本发明的其中一个方面，其中所述入口包括样品入口和设置于所述样品入口的一侧或两侧的辅助溶液入口。基于鞘流作用，辅助溶液可以用于控制样品液体在微流道中的流动方向和范围，使得样品液体充分流经所述超高频体声波谐振器的体声波产生区域。例如可通过控制辅助溶液的流速和流入面积来控制样品液体在微流道中的流动方向和范围。
在本发明的其中一个方面，前述方法中的微流控***中设置多个具有相同的体声波产生区域的超高频谐振器。例如，所述多个超高频谐振器的体声波产生区域的释放点相同，例如其体声波产生区域的形状相同。由此可以将从上游超高频谐振器体声波产生区域发生不希望的脱离的细胞或囊泡，或核酸或蛋白质等生物大分子再次进行收集和按指定方向移动，增加分离的效率。
在本发明的其中一个方面，前述方法中的微流控***中将所述流体通道分为不同区域，在不同区域设置分离不同细胞或囊泡的超高频谐振器。例如所述分离不同细胞或囊泡的超高频谐振器可具有不同形状的声波产生区域，或者施加不同功率的体声波，或者具有不同的流速。由此，可用于分离溶液中不同的细胞或囊泡，使其流入不同的流道或出口。
在本发明的其中一个方面，前述方法可用于分离(或分开)不同种类或不同性质(例如不同大小或密度等)的柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子。在本发明的其中又一个方面，前述方法可用于从血液中分离(或分开)白细胞和红细胞，例如分离(或分开)有核红细胞。在本发明的其中又一个方面，前述方法可用于从血液中分离(或分开)滋 养层细胞。在本发明的其中又一个方面，前述方法可用于从样品中分离CTC。在本发明的其中一个方面，前述方法可用于从溶液中分离细菌尤其是病菌，或是分离(或分开)不同的细菌尤其是病菌。
在本发明的其中又一个方面，可通过以下方式的一种或其任意组合来选择分离(或分开)不同的柔性颗粒如细胞或囊泡，或核酸或蛋白质等生物大分子：
(a)调节体声波的功率；
(b)调节产生体声波的时间；
(c)调节溶液流经体声波区域的速度。
在本发明的其中一个方面，所述方法还包括提高超高频谐振器产生的体声波的功率，破坏细胞膜或囊泡膜，以释放细胞或囊泡内物质，如蛋白质或核酸等。
在本发明的其中一个方面，所述方法中的溶液为含有待分离细胞或囊泡的液体，例如体液、全血、任何含有细胞的血液级份、片段化的肿瘤、肿瘤细胞悬浮液、细胞培养物或培养物上清。在本发明的其中又一个方面，所述溶液为血液，包括全血或稀释的血液。
在本发明中，所述细胞为真核动物细胞，优选为哺乳动物细胞，更优选为人细胞。
本发明还提供了控制溶液中细胞或囊泡，或核酸或蛋白质或多糖等生物大分子等柔性颗粒的移动的微流控设备。所述微流控设备可用于获得或纯化需要的细胞或生物大分子。所述微流控设备也可用于纯化溶液(如血液)，例如通过分离去除溶液中的细胞或囊泡，获得清除了所述细胞或囊泡的纯化的液体(例如除去血细胞的血浆)。本发明提供的微流控设备用于处理生物活性细胞或分子，因此具有处理费生物活性物质的设置或材料。例如，其流道内表面可采用生物相容性的材料制成。又例如，其具有防止交叉污染特别是导致污染扩增的设计。
在本发明的一个方面，提供了一种用于分离细胞或囊泡的微流控设备，包括：
流体通道，其具有入口和出口；
一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
功率调节装置，其调节所述超高频谐振器产生的体声波的功率；
流速调节装置，其调节所述溶液流经体声波区域的速度，
所述超高频谐振器可发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定的涡旋通道，溶液中的细胞或囊泡进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道，该位置称为释放点。
在本发明的其中一个方面，其中对应所述释放点的体声波作用区域存在转折或曲率变化。
在本发明的其中一个方面，其中所述超高频谐振器的体声波产生区域的边界线条设置为适于细胞或囊泡保持在涡旋通道中顺着涡旋通道移动至释放点。
在本发明的其中一个方面，其中所述超高频谐振器的体声波产生区域的边界线条形状使得细胞或囊泡保持在涡旋通道中移动至释放点。例如通过减少体声波产生区域的边界线条中出现转折或曲率变化。
在本发明的一个方面，其中所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得细胞或囊泡保持在涡旋通道中移动至释放点。例如，使得体声波产生区域的边界线条与流体通道的角度较小。
在本发明的其中一个方面，其中超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区，所述聚焦区位于体声波作用区域上游，所述筛分区位于体声波作用区域下游，其中聚焦区的体声波作用区域的设置相对筛分区的设置更适于使得细胞或囊泡保持在涡旋通道中移动。在本发明的其中一个方面，其中聚焦区的体声波作用区域边界线条与流体通道的角度相对筛分区的体声波作用区域边界线条与流体通道的角度较小。在本发明的其中一个方面，其中控制流经聚焦区的体声波作用区域的液流速度小于流经筛分区的体声波作用区域的液流速度。
在本发明的其中一个方面，其中所述功率调节装置的输出功率为约 20-5000mW，优选为50-2000mW，更优选为100-1500mW。
在本发明的其中一个方面，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.1-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-3mm/s。
在本发明的其中一个方面，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.1-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-30μL/min。
在本发明的其中一个方面，其中所述微流控设备的流体通道的高度为约20-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
在本发明的其中一个方面，其中所述超高频体声波谐振器的体声波产生区域面积为约500-200000μm 2，优选为约5000-50000μm 2，最优选为约10000-25000μm 2。
在本发明的其中一个方面，其中所述超高频体声波谐振器的体声波产生区域的边长为约30-500μm，优选为约40-300μm，最优选为约50-200μm。
在本发明的其中一个方面，其中流体通道的所述入口包括样品入口和设置于所述样品入口的一侧或两侧的辅助溶液入口。
在本发明的其中一个方面，其中所述流体通道具有至少两个流出通道，其中一个为所述被控制移动的细胞或囊泡的流出通道，称为颗粒流出通道；另一个为除去所述被控制移动的细胞或囊泡的溶液的流出通道，可称为溶液流出通道。
在本发明的其中一个方面，其中设置多个具有相同的体声波产生区域的超高频谐振器。
在本发明的其中一个方面，其中将所述流体通道分为不同区域，在不同区域设置分离不同细胞或囊泡的超高频谐振器。例如所述分离不同细胞或囊泡的超高频谐振器可具有不同形状的声波产生区域。或者例如施加不同功率的体声波，或者具有不同的流速。
在本发明的其中一个方面，其中所述超高频体声波谐振器为薄膜体声波谐振器或固态装配型谐振器，例如为厚度伸缩振动模式的声波谐振器。
在本发明的其中一个方面，其中所述设备的超高频体声波谐振器的压 电层的厚度范围为1nm～2um。
附图标记
100微流道装置 101流体通道
200芯片外壳 201库尔特细胞计数器 202超高频体声波谐振器 203顶电极层 204压电层 205底电极层 206声波反射层声阻抗层 207底衬层 208顶部
300 PCL控制器 301高频信号发生器 302功率放大器 303阻抗仪
400液体注入和流速调节装置
500声射流 501涡旋
600较大尺寸颗粒 601中等尺寸颗粒 602较小尺寸颗粒
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1本申请实施例提供的一种微流控设备***的结构示意图；
图2本申请实施例提供的一种微流控设备***中的超高频体声波谐振器的结构示意图；其中,(a)表示图1所示的微流控***的微流道的俯视图(左侧)及A-A的剖面图(右侧)；(b)表示超高频体声波谐振器的俯视图(左侧)(其中的黑色五边形部分为超高频体声波谐振器的声波作用区域)以及B-B的剖视图(右侧)；(c)表示微流道+超高频体声波谐振器的俯视图(左侧)以及剖视图(右侧)；
图3显示Hela细胞在本申请实施例提供的一种微流控设备***中进入涡流通道。图3(a)显示的单个Hela细胞在微流控设备***超高频体声波谐振器发生体声波形成的涡旋通道中的运动轨迹图像(单个细胞在不同时间的轨迹叠加)；图3(b)显示Hela细胞的运动轨迹示意图(多个细胞的运动轨迹叠加)和分析图；图3(c)为Hela细胞的时间和速度分析图。
图4显示细胞在本申请实施例提供的一种微流控设备***中的涡旋通道中的运动。图4(a)显示涡旋中细胞移动的图像；图4(b)显示停止体声波后细胞沉降；图4(c)显示涡旋隧道中细胞的排布。
图5显示本申请实施例提供的一种微流控设备***能够从含有Hela细胞的全血混合液中分离和捕获Hela细胞。图5(c)和(d)显示两种微流道设置方式下Hela细胞被捕捉和血细胞被释放的照片，图5(a)和(b)分别是图5(c)和(d)的分析和示意图。图5(e)显示体声波作用区域上游和下游(图5(c)和(d)中的颜色线)的血细胞分布。
图6显示不同超高频体声波谐振器的体声波作用区域设置方式对分离全血中血细胞的影响。
图7显示不同超高频体声波谐振器的体声波作用区域设置方式对分离全血中血细胞的影响。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的区间。
实施例1实验方法和材料
微流体通道和超高频体声波谐振器制备：
通过软光刻制备由聚二甲基硅氧烷(PDMS)制成的微流体通道。
体声波谐振器装置通过在硅基的晶圆上进行化学气相沉积、金属溅射、光刻等方法制备。具体的方法如下：
1.使用浓硫酸与双氧水体积比为3:1的食人鱼溶液对硅片的表面进行彻底的清洗，该方法可以有效地去除硅片上的有机物和无机物。
2.在清洗过的硅片上，通过表面溅射的方法形成一层氮化铝薄膜，再使用离子增强型化学气相沉积的方法，沉积一层二氧化硅薄膜。接着使用 同样的方法，交替沉积氮化铝薄膜和二氧化硅薄膜，形成氮化铝和二氧化硅交替重叠的布拉格声反射结构。
3.在布拉格反射层结构上，溅射出一层600nm的钼薄膜作为底电极。接着采用标准光刻技术，包括涂胶、曝光、显影等，对钼电极薄膜进行光刻，之后进行刻蚀，形成有目标图案的底电极。
4.在钼电极上再溅射一层氮化铝薄膜作为压电层。使用干法刻蚀对氮化铝薄膜定义图案。
5.使用负光刻胶对掩模版上的图案进行转移，再溅射出一层50nm厚的钛钨合金，它作为粘附层可以增加金电极的粘附性。之后使用蒸镀的方法长出一层300nm厚的金薄膜的上电极。最后使用丙酮去除掉目标图案周围的金薄膜，形成有目标图案的金电极。
最后，体声波谐振器装置与PDMS微通道芯片粘合集成。体声波谐振器装置设置在通道的中间位置。
将体声波谐振器装置用标准SMA接口与网络分析仪连接，通过测试频谱找到谐振峰，可测得体声波谐振器装置在微流道中发出的体声波的频率。
仪器和材料
高频信号发生器：(MXG Analog Signal Generator,Agilent,N5181A 100kHz-3GHz
功率放大器：Mini-Circuits,with 35dBm enhancement of the original RF source power
注射泵：New Era Pump Systems,Inc.,NE-1000
细胞：
Hela细胞株：广州广州吉妮欧生物科技有限公司，ATCC#CCL2
细胞培养：
在补充有10％FBS(Thermo)，100U/ml青霉素(Thermo)和100ug/ml链霉素(Thermo)的DMEM培养基(Thermo)中培养293T细胞。培养的细胞密度在1x10 5/mL～2x10 6/mL。在进行微流道实验时，可稀释为 1x10 5/mL进行实验。PBS缓冲液(Gibco)。
细胞或肿瘤标记物或染色剂：
Calcein-AM(北京索莱宝科技有限公司，中国)
Anti-EpCAM(Biolegend，USA)
4',6-二脒基-2-苯基吲哚(DAPI)(Invitrogen,USA)
实施例2
在本实施例的具体实施过程中，提供了一种微流控设备，其可用于在溶液中分离和捕捉柔性颗粒，特别是直径约为0.2-30um的柔性颗粒。柔性颗粒可以是人工的或天然的，通常所述颗粒为带有膜结构的微团，特别是具有脂质双分子层或类脂质双分子层的微团。本发明涉及的柔性颗粒通常具有约0.2-30um的直径。本发明适合处理的柔性颗粒通常约0.8-25um的直径，优选为约1-20um的直径。
本发明的方法和设备可用于溶液中分离柔性颗粒，例如在血液中分离血细胞，得到纯化的血浆。
如图1所示，所述微流控设备100包括流体通道101，超高频体声波谐振器202，体声波驱动和功率调节装置，液体注入和流速调节装置400。
本发明提供的微流控设备可以单独存在，也可以是一个微流控***的一部分，例如以可装卸的芯片形式存在。微流控***或装置可用于容纳和运输液体等流体材料，其流道尺寸在微米甚至纳米级别。典型的微流控***和设备通常包括毫米级或更小尺寸的结构和功能单位。
所述微流控设备的流体通道，或称为微流道，除了供流体进入和流出的开口以外，一般是封闭的。流体通道的截面通常具有0.1-500μm的尺寸，其可以为各种形状，包括椭圆、矩形、方形、三角形、圆形等。可以用各种已知的微制备技术来制备流体通道，其材料包括但不限于硅石、硅、石英、玻璃或聚合材料(例如PDMS、塑料等)。可以用涂层涂覆所述通道。涂层可改变通道的特性，并且可以图案化。例如，涂层可以是亲水的，疏水的，磁性的，传导的，或生物性功能化的。
在本发明的其中一个方面，所述微流控设备的流体通道的高度为约20-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
在本发明的其中一个方面，所述微流控设备的流体通道的宽度为约50-1000μm，优选为约100-500μm，更优选为约150-300μm。
本实施例中的微流道100具有供流体出入的入口和出口。所述入口与液体注入装置连接，用于接收液体的注入。本实施方式的所述入口包括样品入口101及缓冲液入口102。其中，所述缓冲液入口为设置于所述样品入口的两侧的两个入口，与所述样品入***汇相通。所述微流道入口通过上述三相流方式(中间的样品流，两边的缓冲液流)的设置，有利于通过对中间的样品入口通入的样品进行被动聚焦。
如图1所示，本实施例的微流控设备包括液体注入和流速调节装置400，用于控制液体注入及控制液体的流速。所述液体可以为含有样品的液体。例如，所述样品为含有待捕捉细胞的液体。所述样品可以包含体液、全血、任何含有细胞的血液级份、片段化的肿瘤、肿瘤细胞悬浮液、细胞培养物或培养物上清等。所述液体可以为各种体液，包括血液、组织液、细胞外液、淋巴液、脑脊液、房水、尿液、汗液等。
可以通过外部压力源、内部压力源、电子动力学或磁场动力学方式来控制注入液体的流速。外部压力源和内部压力源可以是泵，例如蠕动泵、注射泵或气动泵等。本实施例中采用由电脑微调的注射泵来控制液体注入的流速。
在本发明中，液体的流速范围在约0.1-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-3mm/s。在本发明的另一个方面，所述液体的流量流速范围在约约0.1-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-30μL/min。
所述通道可以为单条通道，或是多个平行或以其它形式共同排布、具有共同输出和输入的通道，其中可以根据需要共同或独立控制各通道的流体的流出流入和其流速。
本发明的微流控设备具有一个或多个超高频体声波谐振器200，其设置于流体通道的一个壁上(通常是设置在流道的底部)。所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁(通常是指流 道的顶部)的频率为约0.5-50GHz的体声波。
可使用于本发明的超高频体声波谐振器可以为薄膜体声波谐振器或固态装配型谐振器，例如为厚度伸缩振动模式的声波谐振器。
如图1所示，本实施方式的微流控设备具有多个设置在流道的底部的超高频体声波谐振器202。
所述超高频体声波谐振器是体声波产生部件，可在所述流体通道产生传向所述流体通道的对侧的壁的体声波。所述超高频谐振器可发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定的涡旋通道，溶液中的细胞或囊泡进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道，该位置称为释放点。
如图2(b)右侧的剖面图所示，所述超高频体声波谐振器包括由下往上依次设置的声波反射层206、底电极层205、压电层204及顶电极层203。所述底电极层、压电层、顶电极层及声波反射层相重叠区域构成体声波产生区域。如图2(b)左侧的俯视图所示，所述超高频体声波谐振器的顶部表面配置在流体通道的壁上，向对侧的壁产生传播方向与所述壁垂直的体声波；一般来说，超高频体声波谐振器的顶部表面构成的区域即为体声波产生区域，在本文中也称为体声波区域或体声波作用区域。在本发明的其中一个方面，所述声波作用区域面积为约500-200000μm 2，优选为约5000-50000μm 2，最优选为约10000-25000μm 2。如图2所示的本实施例的体声波作用区域为五角形，其边长为约120μm微米。超高频体声波在溶液中产生的连续涡旋形成声流体涡旋通道。由于涡旋是由于声波衰减引发的体积力产生的，涡旋的中心轴在体声波作用边界的上方，因此涡旋通道的形状基本与体声波作用区域的形状相同，位于体声波作用区域边界的上方。
在本发明中，所述体声波作用区域的形状至少包括但不限于以下其一：圆形，椭圆形、半圆、抛物线、顶点为锐角或者钝角的多边形、顶点用圆弧替代的多边形、顶点为锐角、半圆或抛物线任一组合的多边形，或者同样形状的重复排列的方阵式或圆环式阵列。本申请提供上述形状的声波作用区域，但其他任意形状的声波作用区域也在本申请的保护范围之内。在本发明中，一种优选的超高频谐振器的体声波产生区域的形状为纺锤形。
在本发明的其中一个方面，上述方法中所述超高频谐振器的体声波产生区域的边界线条(即对应的涡旋通道的形状)设置为适于细胞或囊泡在涡旋通道中顺着涡旋通道移动至释放点。由此可避免细胞或囊泡脱离涡旋通道而不按设定从释放点离开涡旋通道。
在本发明的其中又一个方面，其中通过调节所述超高频谐振器的体声波产生区域的边界线条形状使得细胞或囊泡保持在涡旋通道中移动至释放点。如前所述，体声波产生区域的边界线条中存在转折或曲率变化，可能会增加细胞或囊泡脱离涡旋通道的几率。因此，可以通过减少体声波产生区域的边界线条中出现转折或曲率变化来使得细胞或囊泡保持在涡旋通道中移动，即减少脱离涡旋通道的细胞或囊泡，提高分离的效率。
在本发明的其中又一个方面，通过调节所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得细胞或囊泡保持在涡旋通道中移动至释放点。发明人意外地发现，体声波产生区域的边界线条与流体通道的角度越小，越易于使得细胞或囊泡保持在涡旋通道中移动，即减少脱离涡旋通道的细胞或囊泡，提高分离的效率。
在本发明的其中又一个方面，所述微流控***中的超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区。聚焦区位于体声波作用区域上游(即靠近样品流入方向，离释放点较远的部分)，筛分区位于体声波作用区域下游(即靠近样品流出方向，离释放点较近或包括释放点的部分)。其中聚焦区的体声波作用区域的设置相对筛分区的设置更适于使得细胞或囊泡保持在涡旋通道中移动：在聚焦区的涡流通道中的细胞或囊泡沿着和层流方向相同或相似方向移动，受到的涡旋拖拽力相对较小，更易于细胞或囊泡进入和保留在涡旋通道；在下游的筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。在本发明的其中又一个方面，聚焦区的体声波作用区域边界线条与流体通道的角度相对筛分区的体声波作用区域边界线条与流体通道的角度较小。例如，聚焦区的体声波作用区域边界基本与流体通道方向一致或基本一致(例如角度小于10°)，在这个区域的涡流通道中的细胞受到的涡旋拖拽力基本上不改变细胞沿着层流方向的运动状态，只会让细胞横向迁移至涡旋中心，实现对细 胞的聚焦；筛分区的体声波作用区域边界与流体通道角度较大，引导细胞移动方向偏离流体通道方向转移到指定的释放点，在筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。在本发明的其中又一个方面，控制流经聚焦区的体声波作用区域的液流速度小于流经筛分区的体声波作用区域的液流速度。
如图2右侧的剖面图所示，本实施例的流体通道可具有多个超高频体声波谐振器。在本发明的其中一个方面，它们以与流体运动方向一致的方向直线排列。
本发明采用的超高频体声波谐振器是厚度伸缩振动模式，其中的压电材料薄膜层在垂直方向上生长而制成，通过d33压电系数耦合垂直电场而被激发。本发明采用的超高频体声波谐振器可以在装置和液体的界面产生局部化的声流，不需要耦合介质或结构的帮助。
本发明采用的超高频体声波谐振器产生的超高频体声波，在溶液中基本上不产生驻波。如图1右图所示，超高频谐振器发射传向所述流体通道的对侧的壁(例如流道顶部)的体声波，声波衰减到流体中产生的体积力使得流经的溶液中出现声射流500，导致微流道中的液体产生局部的立体的旋涡501，超高频体声波引起的连续涡旋形成声流体涡旋通道。由于涡旋是由于声波衰减引发的体积力产生的，涡旋的中心轴在体声波作用边界的上方，因此涡旋通道的形状基本与体声波作用区域的形状相同，位于体声波作用区域边界的上方。声流体涡旋是由于声波在液体介质中传播的非线性引起的。而声波的振幅的强弱直接决定了声流体涡旋的强度。通过调节施加功率可以调控特超声器件的振幅，即声波的振幅，进而控制了声流体涡旋的流速。在涡旋中的颗粒(包括较大尺寸颗粒600、中等尺寸颗粒601，较小尺寸颗粒602)受到的力包括涡旋产生的流体拖拽力(Stokes drag force)，层流产生的惯性拖拽力(inertial lift force)和声波衰减引起的声辐射力(acoustic radiation force)。由于流体拖拽力的大小与颗粒如颗粒直径成正向关系，而声辐射力的大小与颗粒尺寸的平方成正向关系。随着颗粒的增大，受到的力会从以流体拖拽为主导转变到以声辐射力为主导，声辐射力将颗粒推向涡旋中心。较大的颗粒受到更大的声辐射力从而移动到涡 旋中心；而较小的颗粒则在涡旋拖拽力的作用下在***旋转，进一步的，在层流产生的侧向拖拽力作用下向体声波作用区域的下游移动。
颗粒在涡旋后涡旋通道中受到的流体拖拽力和声波引起的声辐射力可以根据某些公式推算，但声流体涡旋产生的惯性拖拽力(inertial lift force)几乎无法用简单的物理学原理和公式计算，特别是在含有复杂成份的流体的情况下。与现有技术中的二维的颗粒捕获相比，本发明的方法和设备涉及可变形的细胞或囊泡，其在声流体旋涡及其形成的通道中的受力情况和运动轨迹更复杂，因为各个涡旋之间的相互作用和颗粒在涡旋之间的迁移都对流体中的的捕获有影响，特别是在流体中存在大量的所述细胞或囊泡的情况下，因为细胞或囊泡相互之间由于碰撞等现象存在相互作用和影响，其在涡旋中的受力和运动与理论计算和模拟的受力和运动存在不同，在涡旋通道中的运动方式和轨迹更是无法根据理论计算和模拟预测。
申请人通过实验出乎意料地发现，在本发明的方法和装置中，溶液中的细胞或囊泡经过超高频体声波造成的声流体涡旋通道区域时，在适合的流速和体声波功率的条件下，会进入涡旋通道和沿着涡旋通道运动；在层流产生的侧向拖拽力作用下，在涡旋通道的某个(些)位置，细胞或囊泡会离开体声波作用区域，向下游移动，即被释放；细胞或囊泡离开涡旋通道的位置也称为释放点。由于细胞或囊泡脱离涡旋通道的其中一个重要因素是沿流体通道方向的层流的影响，释放点通常位于涡旋通道的最下游区域。
发明人还出乎意料地发现，细胞或囊泡离开涡旋通道的位置通常是在涡旋和声辐射力的跳变点。在产生涡流的体声波功率以及溶液流速、流体通道形状和尺寸不变的条件下，细胞或囊泡离开涡旋通道的位置通常处于涡旋通道出现转折或转角的地方，即对应体声波作用区域边界出现转折或转角的位置的上方。在不受有关理论约束的情况下，申请人认为，这个现象的原因在于，在涡流通道的转折或转角处，涡旋方向和声辐射力方向突然变化，较大的细胞或粒子由于已经聚焦到涡旋中心，并且受到更大的声辐射力，能够随着涡旋通道改变运动方向并且快速重新聚焦到转折后的涡旋通道的中心；而较小的粒子或细胞则会因为层流 拖拽方向的跳变而受到更大的影响，从而离开涡旋通道。
通过调节超高频体声波谐振器的体声波作用区域的形状和位置，使得溶液中的细胞或囊泡进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道。由此所述细胞或囊泡以指定位置和方向离开体声波作用区域，进入希望的流出通道，例如进入所述颗粒流出通道。该设定的离开涡旋通道的位置称为释放点，也即所述细胞或囊泡离开体声波作用区域的位置。除去所述被控制移动的细胞或囊泡的溶液则保持流入方向，进入到前述溶液流出通道。
由此，本申请人的发明人发现和提供了更有效地分离目标细胞或囊泡的方法。
在本发明中，薄膜体声波谐振器的频率主要由压电层的厚度和材料决定。本发明采用的薄膜体声波谐振器的压电层的厚度范围为1nm～2um。本发明的超高频体声波谐振器的频率在约0.5-50GHz，优选为约1-10GHz。
所述超高频体声波谐振器产生的体声波由高频信号发生器的信号驱动。驱动谐振器的脉冲电压信号可以用脉冲宽度调制驱动，脉冲宽度调制可以产生任何期望的波形，例如正弦波、方波、锯齿波或三角波。脉冲电压信号也可以具有调幅或调频开始/停止能力，以开始或消除体声波。
本发明的微流控设备还包括功率调节装置，其调节所述超高频谐振器产生的体声波的功率。在本实施例中，所述功率调节装置为具有功率调节功能的功率放大器。在本发明的其中一个方面，所述功率调节装置的输出功率为约20-5000mW，优选为50-2000mW，更优选为100-1500mW。由于薄膜体声波谐振器能量转换效率高，基本没有损耗，所述功率调节装置的输出功率可基本上视为薄膜体声波谐振器在流体中产生体声波的输出功率。本发明的微流控设备中，所述功率调节装置可与高频信号发生器连接。所述功率放大器的输出电路分别与所述超高频体声波谐振器的底电极、压电层、顶电极连接。
实施例3细胞在超高频体声波造成的涡流通道中的移动
将Hela细胞溶于DMEM培养液，制备成样品(细胞含量为约 1*10 5/mL)。另外，用Calcein-AM标记Hela细胞，以便观察其运动途径。将样品从样品入口注入至微流道中。
所述微流道的高度为50μm。超高频体声波谐振器频率1.83GHz，输出功率为30mW。控制样本输入流速为1μL/min，即约0.67mm/s。
结果如图3所示。图3(a)显示单个Hela细胞在微流控设备***的超高频体声波谐振器发生体声波形成的涡旋通道中的运动轨迹。其为同一个单个细胞在不同时间的多幅图像的叠加，以显示其运动轨迹。图3(b)为不同单个细胞的运动轨迹的示意图：上图为顶视图，下图为侧视图。图3(c)为Hela细胞的时间和速度分析图。
在本发明的微流控设备中，超高频体声波谐振器产生的体声波使得流经的流体产生涡旋，每个涡旋与相邻的涡旋相连通，沿着超高频体声波谐振器的声波作用区域的边界形成声流体涡旋通道或涡旋隧道。多个涡旋的组合效应以及涡流和层流之间的相互作用，使得进入涡旋通道的细胞沿涡旋通道移动，而且细胞是漂浮在微流道底部的上方，与微流道没有接触。
图3(a)中的图像序列显示和证明了单个HeLa细胞的移动轨迹。可以将移动过程分为在区域a-b-c中发生的三个阶段，如图3(a)和图3(c)所示。在区域a中，细胞在侧流中以均匀的速度移动。在区域b中，细胞运动的方向被涡流阵列改变，并且细胞进入声波产生的涡流隧道，同时在3-D轴上聚焦并沿固定路径移动。具有不同初始位置的细胞在进入涡流阵列时的速度差异很大。由于与涡流的相对方向的差异，初始位置在微通道下层的细胞加速，而初始位置在上层的细胞减慢。在区域c中细胞运动的速度逐渐减慢。由于在涡旋隧道调整了细胞的轨迹，细胞的减速和移动的过程具有良好的稳定性，不同的进入初始位置的细胞具有良好的减速和移动的一致性。
图3(b)示意图进一步说明了细胞在流道中和体声波作用下的运动情况。图3(b)是不同的单个细胞的运动轨迹的叠加示意图。图3(b)的上图为顶视图，下图为侧视图。从顶视视角可见，细胞的轨迹仅分布在超高频体声波谐振器的声波作用区域的边界。从侧视角度，可以看出细胞都漂浮在通道底部的上方，与底部没有接触。
在实验和模拟计算上发现，流体，特别是涡流中的细胞受力和运动情况与模拟公式推导的理论值相差甚大，无法预测。细胞具有与聚苯乙烯颗粒等具有明显不同的机械特性，例如惰性，刚性，具有多种物质的复杂结构等。在声波动力学和涡旋流体力学范畴，在理论计算涡流中的物体的运动时，该物体相对于溶液介质的密度和声速差是非常重要的因素。而且，由于细胞在声波力和拖曳力的变形，使得涡流对其所施加的总的力会有明显的不可预测的影响。
实施例4细胞在微流道中的悬浮和沉降
在现有技术的声流涡旋方法中，颗粒或细胞在微流道的z轴(即流道的垂直方向)的运动被忽略，因为平面内声流是主要的涡旋。然而，z轴的轨迹对于单细胞操作和高精度的感测非常重要。由于本发明采用的超高频体声波的独特的TE振动模式，在本发明的微流体***中主导的涡旋平面外(out-of-plane)的。为了更好地研究本发明的微流体***中超高频体声波产生的涡旋隧道，采用Calcein-AM标记的Hela细胞，使用共聚焦显微镜(Leica，Germany)观察和测量细胞在z轴上的运动。为了捕获涡流隧道内的粒子轨迹，使用了x-z-t模式。在此模式下，拍摄速度可达到每秒37帧。
根据实施例3的相同实验设置，但将样本含有Hela细胞的输入流速降低为0.1μL/min。在含有细胞的样品及缓冲液通入微流道之后，开启信号发生器，细胞被捕捉在声波作用区域。然后调整入口和出口的压力至相同，流体停滞。观察细胞在涡旋中的运动。然后信号发生器关闭，观察细胞的运动。
结果如图4所示。图4(a)是涡旋中细胞移动的图像，其为复合堆叠图像(6幅图像，相距27ms)，其中红点显示每帧中粒子的中心，绿色虚线圆圈表示范围粒子运动和红色箭头表示粒子运动的方向。当信号发生器电源打开，体声波产生，在流道内形成涡旋和涡旋隧道，颗粒相对“静止”地悬浮在距离芯片表面。如图4(b)所示，当电源关闭时，体声波消失，颗粒在重力和浮力的共同作用下沉淀到芯片表面。结果表明，粒子确实被困在涡旋隧道中。
图4(c)显示，Calcein-AM标记的Hela细胞被捕获在涡旋隧道中，涡旋隧道具有和超高频体声波谐振器的声波作用区域的边界一样的形状。
实施例5全血中分离不同的细胞
血液是最原始和最适合细胞的环境。在血液环境中，细胞具有最佳的活力和完整的功能，这对于生物学研究很重要，例如细胞代谢，蛋白质组学。然而，与稀释的血液样本相比，全血是更具挑战性的样本。从物理参数来看，全血更粘稠和浑浊，这是都会对细胞操作有严重的负面影响。物理场，如介电场，磁场，流体动力场，在血液样本中是紊乱的。此外，高密度的细胞，特别是红细胞(10 9/mL)，会引起细胞之间的强烈相互作用，从而改变标本的轨迹并影响细胞运动的稳定性。
发明人证明了本发明的设备和方法能够在全血中选择性控制和分离目标细胞。
将Hela细胞溶于DMEM培养液，与全血混合，制备成测试样品样品(Hela细胞含量调节为约1x10 5个/mL)。
将样品从样品入口注入至微流道中。以及将PBS缓冲液从两侧的缓冲液入口注入至微流道中。PBS作为夹持鞘流，作用是保证样品流体的横向范围，确保所有样品都会经过器件上方。通过三相流的方式对注入样品进行被动聚焦，使得样品更好地流经高频体声波谐振器的声波作用区域。
结果如图5所示，含有Hela细胞的全血中的Hela细胞被选择性地进入涡流通道和在涡流通道内移动。
图5(c)和(d)显示两种微流道设置方式下Hela细胞进入涡流通道和血细胞通过体声波区域的照片，图5(a)和(b)分别是图5(c)和(d)的分析和示意图。本实施例中，超高频体声波谐振器的体声波作用区域为五边形。其中图5(c)中，体声波作用区域设置为液体流入方向下游存在突起拐角(五边形的一个角)：此时，血细胞离开体声波作用区域的其中一个点在该五边形的角附近。图5(d)中体声波作用区域调整为其中一个角对着通道液体流入方向，沿流道中线对称的放置方式：此时，血细胞的释放点，位于沿流体方向的下游边线的两端。图5(e)显示体声波作用区 域上游和下游(图5(c)和(d)中的颜色线)的血细胞分布。绿色曲线和蓝色曲线表示血细胞平均在两个对称释放点处释放。图5(f-1)显示三相流的输入方式对注入样品进行被动聚焦，使得样品更好地流经高频体声波谐振器的声波作用区域。图5(f-2)显示电源打开，在微流道内产生体声波时血细胞的流动。图5(f-3)-(f-5)显示CTC被选择性进入涡流隧道：复合堆叠图像序列展示了CTC的运动轨迹。图5(f-6)显示选择性CTC进入涡流隧道的结果：合并图像显示，在本发明的装置在释放血细胞的同时稳定使得CTC进入和保留在涡流隧道。
释放点为涡旋和声辐射力的跳变点。在器件是正五边形的情况中，细胞被涡旋捕捉聚焦后，由于每个边都是直线，意味着层流产生向下游移动的横向拖拽力，涡旋拖拽力在流道径向的分量都是稳定的，这使得被聚焦的细胞在沿着边界运动是不容易脱落的。而到了顶点处，涡旋方向和声辐射力方向都突然变化，较大的细胞由于已经聚焦到涡旋中心，并且受到更大的声辐射力，是能够随着涡旋改变运动方向并且快速重新聚焦到新边的涡旋中心的；而较小的细胞则会因为层流拖拽力为主导而从器件上脱落。通过调整调整微流道装置，例如超高频体声波谐振器的体声波作用区域，使得非目标细胞被层流带走，只有目标细胞能够进入和/或保留在涡流通道。
实施例6不同超高频体声波谐振器的体声波作用区域设置方式对分离全血中血细胞的影响
本实验检测采用本发明的微流控***和方法将全血中血细胞与血浆分开的效果。
根据实施例1和2的描述的方法，制备和构建如图6所示的微流道。其中每个微流道具有3个体声波发生区域相同的按顺序排列的超高频谐振器。在流道方向上采用多个超高频谐振器可加强对同一目标颗粒转移的控制。其中超高频谐振器的表面(即体声波发生区域，图中显示为三角形器件)位于微流道通道一侧(图中左侧)，微流道设置两个溶液入口，右侧入口通入全血样品。左侧通入PBS溶液，通过调节PBS溶液流速和鞘流聚焦作用，使样品流充分通过体声波发生区域。三角形体声波发生区域(即涡流 通道)的下游角为设定的细胞释放点，释放的细胞从下游角位置离开，顺微流道通道方向进入细胞流出通道(图下方左边的通道)。
所述微流道的高度为50um。超高频体声波谐振器频率1.83GHz，检测的三种输出功率如图6所示。控制样本输入流速为0.5uL/min，PBS缓冲液输入流速为5uL/min。
图6最左侧两幅图、中间两幅图和右边两幅图分别显示在超高频体声波功率分别为1660mW、417mW和208mW下分离血细胞的效果。其中每两幅图中的左图中的三角形体声波发生区域的边界(即涡流通道的走向)与微流道中液体(即层流)的流向形成的夹角比右图中的体声波发生区域的边界与微流道中液体的流向形成的夹角较大。
结果如图6所示，涡流通道与层流的流向形成的夹角越小，涡旋通道中脱落的细胞数目就越少，即到达三角形涡流通道的下游角(即细胞释放点)和脱离的细胞越多。可见，涡流通道与层流的流向形成的夹角越小，越易于使得细胞保持在涡旋通道中移动，即减少脱离涡旋通道的细胞，提高分离的效率。
由此，在本发明的其中一个方面，将微流控***中的所述超高频谐振器的体声波产生区域的边界线条(即对应的涡旋通道的形状)设置为适于细胞或囊泡在涡旋通道中顺着涡旋通道移动至释放点。由此可避免细胞或囊泡脱离涡旋通道而不按设定从释放点离开涡旋通道。
在本发明的其中又一个方面，通过调节所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得细胞或囊泡保持在涡旋通道中移动至释放点。发明人意外地发现，体声波产生区域的边界线条与流体通道的角度越小，越易于使得细胞或囊泡保持在涡旋通道中移动，即减少脱离涡旋通道的细胞或囊泡，提高分离的效率。
另外，如图6所示，在其它***参数条件相同的情况下，功率越高，到达三角形涡流通道的下游角(即细胞释放点)和脱离的细胞越多，分离效率越高。
实施例7不同超高频体声波谐振器的体声波作用区域设置方式对分离 全血中血细胞的影响
构建如图7所示的微流道。其中超高频谐振器的表面(即体声波发生区域，图中显示为纺锤形器件)位于微流道通道一侧(图中右侧)并向左倾斜。微流道设置两个溶液入口，右侧入口通入血液样品。左侧通入PBS溶液，通过调节PBS溶液流速和鞘流聚焦作用，使样品流充分通过纺锤形体声波发生区域的右侧。纺锤形体声波发生区域的边界(即为涡流通道所在位置)的下游角为设定的细胞释放点，释放的细胞从下游角位置离开，顺微流道通道方向进入细胞流出通道。
所述微流道的高度为50um。超高频体声波谐振器频率1.83GHz，三种输出功率如图7所示。控制样本输入流速为1uL/min，PBS缓冲液输入流速为10uL/min。
在如图所示的纺锤形体声波发生区域分为两个区域，聚焦区(纺锤形器件上半部分，其右侧边界与层流方向相同)与筛分区(纺锤形器件下半部分，其右侧边界与层流方向形成较大角度)。如图所示，在器件上游，涡旋隧道方向与层流方向相同，涡旋拖拽力并不改变细胞沿着层流方向的运动状态，只会让细胞横向迁移至涡旋中心，实现对细胞的聚焦。而在筛分区部分，聚焦到涡旋中心的细胞相比于未聚焦的细胞，能够更稳定的被移动。将荧光为加入到血液中来表征血浆的运动情况：1mL血液中加入2μL Calcein-AM。如图所示，血细胞被高效率的从血浆中沿着纺锤形器件右侧边界迁移到位于流道左侧的细胞释放点，并在细胞释放点离开纺锤形器件，进入PBS缓冲液中。
可以看到除了少部分血浆由于粘附在细胞中随着血细胞迁移到细胞流出通道，大部分的血浆都沿着原液流方向进入血浆回收通道而被回收。
由此，在本发明的其中一个方面，将***中的超高频谐振器的体声波产生区域的边界线条(即对应的涡旋通道的形状)设置为适于细胞或囊泡在涡旋通道中顺着涡旋通道移动至释放点。由此可避免细胞或囊泡脱离涡旋通道而不按设定从释放点离开涡旋通道。
在本发明的其中又一个方面，其中通过调节所述超高频谐振器的体声波产生区域的边界线条形状使得细胞或囊泡保持在涡旋通道中移动至释放 点。如前所述，体声波产生区域的边界线条中存在转折或曲率变化，可能会增加细胞或囊泡脱离涡旋通道的几率。因此，可以通过减少体声波产生区域的边界线条中出现转折或曲率变化来使得细胞或囊泡保持在涡旋通道中移动，即减少脱离涡旋通道的细胞或囊泡，提高分离的效率。在本发明中，一种优选的超高频谐振器的体声波产生区域的形状为纺锤形。
在本发明的其中又一个方面，所述微流控***中的超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区。聚焦区位于体声波作用区域上游(即靠近样品流入方向，离释放点较远的部分)，筛分区位于体声波作用区域下游(即靠近样品流出方向，离释放点较近或包括释放点的部分)。其中聚焦区的体声波作用区域的设置相对筛分区的设置更适于使得细胞或囊泡保持在涡旋通道中移动：在聚焦区的涡流通道中的细胞或囊泡沿着和层流方向相同或相似方向移动，受到的涡旋拖拽力相对较小，更易于细胞或囊泡进入和保留在涡旋通道；在下游的筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。在本发明的其中又一个方面，聚焦区的体声波作用区域边界线条与流体通道的角度相对筛分区的体声波作用区域边界线条与流体通道的角度较小。例如，聚焦区的体声波作用区域边界基本与流体通道方向一致或基本一致(例如角度小于10°)，在这个区域的涡流通道中的细胞受到的涡旋拖拽力基本上不改变细胞沿着层流方向的运动状态，只会让细胞横向迁移至涡旋中心，实现对细胞的聚焦；筛分区的体声波作用区域边界与流体通道角度较大，引导细胞移动方向偏离流体通道方向转移到指定的释放点，在筛分区，聚焦到涡旋中心的细胞相比于未聚焦的细胞能够更稳定的在涡流通道中被移动。
另外，如图7所示，在其它***参数条件相同的情况下，功率越高，从血浆中沿着纺锤形器件右侧边界迁移到位于流道左侧的细胞释放点，并在细胞释放点离开纺锤形器件的血细胞越多，分离效率越高。
实施例8控制核酸的移动和分离纯化核酸
根据实施例1和2的描述的方法，制备和设置如图8(a)所示的微流道和超高频谐振器***。图8(a)为俯视图，图右侧为流道入口，用于输 入含有不同大小的核酸片段的PBS溶液。超高频谐振器的表面，即体声波发生区域，如图中显示为纺锤形，其左边尖端为核酸释放点。微流道左侧包括三个流出通道，其中中间为核酸流出通道，从纺锤形左边尖端的核酸释放点流出的核酸进入核酸流出通道。核酸流出通道的上下两个通道为供去除目标核酸后的溶液流出通道。
核酸通过Qubit sDNA HS试剂盒进行染色和定量，用PBS溶液溶解。图8(a)显示***设置和通入荧光染色核酸溶液后观察现象：上图为亮场，下图为荧光信号观察图，显示超高频谐振器没有工作时荧光染色核酸溶液均匀分布在微流道中的情况。
图8(b)显示大小为约20k的双链核酸样品在如图8(a)所示装置中，在不同流速(0,0.1,0.25,0.5,1μL/min)和不同体声波工作功率(50-1000mW)下的表现。流体通道高度为约20微米。
核酸为提取的绵羊全血基因组DNA，大小为约20kbp。
如图8(b)所示，在从流道入口不断输入核酸溶液，超高频谐振器持续工作的情况下，在合适的流速和体声波功率条件下，核酸顺着纺锤形超高频谐振器器件边缘移动到释放点。结果证明，本发明的装置和方法对20kbp双链DNA具有良好的控制移动的能力，且控制能力与施加功率正相关，与流道中的侧向流体速度负相关。本实验是连续工作实验结果，可以看出，持续工作情况下核酸没有在超高频谐振器器件和流道中产生粘附，也不会在涡旋中出现聚团。
从荧光信号来看，在0.1μL/min流速下，功率1000mW，可对体声波工作范围内的所有核酸进行连续式富集，富集效率(即进入核酸流出通道的核酸的量占)超过90％。在体声波区域边缘的涡旋通道中，20kbp DNA被浓缩在约35微米直径的涡旋隧道中。在释放位点处，核酸脱离涡旋释放后，在次生涡旋和侧向流体的共同作用下扩散为宽度约为125μm的核酸条带流向下游。
图8(c)显示大小为约20k的双链核酸样品在如图8(a)所示装置中，在不同流道高度(50微米和20微米)下的表现。
结果显示，在相同的流速和体声波作用功率下，20微米高度的流道的 ***的作用明显比50微米高度的流道的***更加有效。这证明微流道高度降低，能够提高声流体捕捉小尺寸颗粒的效果。流道高度降低，会提高声流体涡旋的速度梯度，目标颗粒在涡旋中心的聚集与涡旋的梯度力正相关，从而提高了声流体的捕捉效率。可以从上图看出，50μm高度的流道中，体声波边缘形成的涡旋的范围明显大于20μm流道中的涡旋。
图8(d)显示大小为约5k双链核酸样品在如图8(a)所示装置中，在不同流速(0.1,0.25,0.5μL/min)和不同体声波工作功率(50-1000mW)下的表现。流体通道高度为约20微米。
5k双链核酸样品为DNA质粒，为双链环状DNA。
结果证明，本发明的装置和方法对5kbp的质粒DNA具有良好的控制移动的能力，且控制能力与施加功率正相关，与流道中的侧向流体速度负相关。
图8(e)显示大小为约5k的环状核酸与链状核酸样品在如图8(a)所示装置中的表现。流体通道高度为约20微米。
5k链状核酸为将图8(d)中5k双链核酸样品的DNA质粒进行酶切，断裂形成双链链状DNA。
结果显示，在相同的流速和体声波作用功率下，***对5k环状核酸的控制明显比同样大小的链状核酸作用明显。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (24)

  1. 一种控制溶液中目标柔性颗粒如细胞微囊泡或核酸和蛋白质等生物大分子颗粒的移动的方法，包括：
    (1)使含有柔性颗粒如细胞微囊泡或核酸和蛋白质等生物大分子颗粒的溶液流经一个微流控设备，所述设备包括；
    流体通道，其具有入口和流出通道；
    一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
    (2)所述超高频谐振器发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定(define)的涡旋通道；
    (3)通过调节超高频体声波谐振器的体声波作用区域的形状和位置，使得溶液中的柔性颗粒进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道，该位置称为释放点。
  2. 权利要求1的方法，其中还包括通过调节体声波的功率和/或通过调节所述溶液流经体声波区域的速度，来调节进入涡旋通道的柔性颗粒。
  3. 权利要求1的方法，其中对应所述释放点的体声波作用区域存在转折或曲率变化。
  4. 权利要求1-3中任一项的方法，其中所述超高频谐振器的体声波产生区域的边界线条设置为适于目标柔性颗粒保持在涡旋通道中顺着涡旋通道移动至释放点，例如通过减少体声波产生区域的边界线条中出现转折或曲率变化。
  5. 权利要求4的方法，其中通过调节所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得柔性颗粒保持在涡旋通道中移动至释放点，例如，使得体声波产生区域的边界线条与流体通道的角度较小。
  6. 权利要求4的方法，其中超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区，所述聚焦区位于体声波作用区域上游，所述筛分区位于体声波作用区域下游，其中聚焦区的体声波作用区域的设置相对筛分区 的设置更适于使得柔性颗粒保持在涡旋通道中移动，
    例如，聚焦区的体声波作用区域边界线条与流体通道的角度相对筛分区的体声波作用区域边界线条与流体通道的角度较小；
    又例如，控制流经聚焦区的体声波作用区域的液流速度小于流经筛分区的体声波作用区域的液流速度。
  7. 权利要求1的方法，其中所述超高频谐振器产生的体声波的功率为约20-5000mW，优选为50-2000mW，更优选为100-1500mW。
  8. 权利要求1的方法，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.01-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-3mm/s，或者
    其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.01-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-30μL/min。
  9. 权利要求1的方法，其中所述柔性颗粒为细胞或细胞囊泡，优选的，所述细胞或囊泡的直径约为0.01-30um，优选为0.2-25um，更优选为0.5-20um。
  10. 权利要求1的方法，其中所述柔性颗粒为核酸，优选的，所述核酸分子长度≥300bp，优选≥1kbp、更优选≥10kbp、例如为≥50kbp。
  11. 权利要求1的方法，其中所述微流控设备的流体通道的高度为约20-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
  12. 权利要求1的方法，其用于分离溶液中不同的柔性颗粒。
  13. 权利要求1的方法，其中将所述流体通道分为不同区域，在不同区域设置分离不同柔性颗粒的超高频谐振器，例如所述分离不同柔性颗粒的超高频谐振器可具有不同形状的声波产生区域，或者施加不同功率的体声波，或者具有不同的流速，或其组合。
  14. 权利要求1的方法，其中所述微流控设备的流体通道具有所述被控制移动的柔性颗粒的流出通道，即颗粒流出通道；优选的，所述流体通道还具有其它流出通道，例如为除去或含有较少所述被控制移动的细胞或囊泡的溶液的流出通道，即溶液流出通道，
    优选的，其中所述颗粒流出通道和溶液流出通道的开口的宽度比例为 约1:1-1:20，优选为约1:2-1:15，例如为约1:4-1:10。
  15. 一种控制溶液中目标柔性颗粒如细胞微囊泡或核酸和蛋白质等生物大分子颗粒的移动的微流控设备，包括：
    流体通道，其具有入口和出口；
    一个或多个超高频体声波谐振器，其设置于所述流体通道的一个壁上，所述超高频体声波谐振器可在所述流体通道产生传向所述流体通道的对侧的壁的频率为约0.5-50GHz的体声波；
    功率调节装置，其调节所述超高频谐振器产生的体声波的功率；
    流速调节装置，其调节所述溶液流经体声波区域的速度，
    所述超高频谐振器可发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频谐振器的体声波产生区域的边界限定的涡旋通道，溶液中的细胞或囊泡进入涡旋通道和顺着涡旋通道移动，并在设定的位置离开涡旋通道，该位置称为释放点。
  16. 权利要求15的微流控设备，其中对应所述释放点的体声波作用区域存在转折或曲率变化。
  17. 权利要求15的微流控设备，其中所述超高频谐振器的体声波产生区域的边界线条设置为适于柔性颗粒保持在涡旋通道中顺着涡旋通道移动至释放点，例如通过减少体声波产生区域的边界线条中出现转折或曲率变化。
  18. 权利要求18的微流控设备，其中所述超高频谐振器的体声波产生区域的边界线条与流体通道的角度使得柔性颗粒保持在涡旋通道中移动至释放点，例如，使得体声波产生区域的边界线条与流体通道的角度较小。
  19. 权利要求16的微流控设备，其中超高频体声波谐振器的体声波作用区域具有聚焦区与筛分区，所述聚焦区位于体声波作用区域上游，所述筛分区位于体声波作用区域下游，其中聚焦区的体声波作用区域的设置相对筛分区的设置更适于使得细胞或囊泡保持在涡旋通道中移动。
  20. 权利要求15的微流控设备，其中所述功率调节装置的输出功率为约20-5000mW，优选为50-2000mW，更优选为100-1500mW。
  21. 权利要求15的微流控设备，其中所述流速调节装置可调节所述溶 液流经体声波区域的速度为约0.01-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-3mm/s，
    或，其中所述流速调节装置可调节所述溶液流经体声波区域的速度为约0.01-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-30μL/min。
  22. 权利要求15的微流控设备，其中所述微流控设备的流体通道的高度为约20-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
  23. 权利要求15的微流控设备，其中所述流体通道具有至少两个流出通道，其中一个为所述被控制移动的柔性颗粒的流出通道，称为颗粒流出通道；另一个为除去所述被控制移动的柔性颗粒的溶液的流出通道，可称为溶液流出通道。
  24. 权利要求15的微流控设备，其中将所述流体通道分为不同区域，在不同区域设置分离不同柔性颗粒的超高频谐振器，
    例如所述分离不同柔性颗粒的超高频谐振器可具有不同形状的声波产生区域，
    或者例如施加不同功率的体声波，或者具有不同的流速。
PCT/CN2020/096178 2019-06-13 2020-06-15 利用超高频声波控制溶液中的微粒移动的方法及设备 WO2020249131A1 (zh)

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