WO2020249130A1 - 细胞或微囊泡的分离方法及设备 - Google Patents

细胞或微囊泡的分离方法及设备 Download PDF

Info

Publication number
WO2020249130A1
WO2020249130A1 PCT/CN2020/096176 CN2020096176W WO2020249130A1 WO 2020249130 A1 WO2020249130 A1 WO 2020249130A1 CN 2020096176 W CN2020096176 W CN 2020096176W WO 2020249130 A1 WO2020249130 A1 WO 2020249130A1
Authority
WO
WIPO (PCT)
Prior art keywords
acoustic wave
cells
bulk acoustic
cell
vesicles
Prior art date
Application number
PCT/CN2020/096176
Other languages
English (en)
French (fr)
Inventor
段学欣
杨洋
Original Assignee
安行生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 安行生物技术有限公司 filed Critical 安行生物技术有限公司
Priority to US17/618,677 priority Critical patent/US20230219086A1/en
Priority to EP20823576.2A priority patent/EP3985098A4/en
Publication of WO2020249130A1 publication Critical patent/WO2020249130A1/zh

Links

Images

Classifications

    • 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
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • G01N15/1433—
    • 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—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Electro-optical investigation, e.g. flow cytometers 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—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484—Electro-optical investigation, e.g. flow cytometers 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
    • G01N2015/1027—

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.
  • the manipulation of biological particles, especially cells or microvesicles, is the most basic biotechnology, and it is also the cornerstone technology of cutting-edge research.
  • methods to control biological particles such as cells or microvesicles, including electrical methods, acoustic methods, optical methods, magnetic methods, chemical methods, and fluid manipulation methods.
  • Each method has a specific application range and is only effective for specific particle properties.
  • the control of surface acoustic wave and dielectrophoresis is related to the radius of the particle. Therefore, the control effect is better for micron-sized particles.
  • Class particles have poor discrimination; electrophoresis and other methods can only work when the particles are charged, so they have poor discrimination between different substances that are uncharged or have similar charges.
  • the optical manipulation method has high precision, but will generate more heat during the manipulation process, which will have a greater impact on the activity of biomolecules, and the optical system is more complex and difficult to integrate with microfluidic devices.
  • the use of each method alone has certain limitations, so in most cases, the combined use of different methods will make the distinction and manipulation of samples better.
  • Single-cell manipulation is an area that has received increasing attention in recent years.
  • the manipulation of single cells or microvesicles is very important for studies such as single cell analysis, drug development, organ microarrays, and cell-cell interactions.
  • studies based on cell-to-cell specificity (such as studies on cell morphology, surface adhesion, migration speed, protein expression and gene expression of single cells) rely heavily on single cell manipulation techniques.
  • the traditional method of extracting single cells is complicated, time-consuming, laborious and inefficient.
  • Acoustic fluid technology such as surface acoustic wave and acoustophoretic technology based on standing waves in fluids, have been used for the separation, extraction and manipulation of micron particles and cells.
  • the existing devices used in these technologies have the characteristics of being large in size and difficult to miniaturize.
  • the present invention finds for the first time that the use of ultra-high frequency bulk acoustic waves can effectively manipulate and separate flexible particles such as cells in a solution in a microfluidic system, thereby providing a method and system for separating and "capturing" target cells.
  • the method and system of the present invention can also be used to precisely control the type and number of captured cells, and is particularly suitable for obtaining a single or limited number of cells.
  • the present invention provides a method for separating flexible particles such as cells or vesicles from a solution, including the following steps:
  • Flow a solution containing flexible particles through a microfluidic device which includes;
  • 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
  • the UHF resonator emits a bulk acoustic wave transmitted to the wall on the opposite side of the fluid channel, and the solution in the area affected by the bulk acoustic wave generates a vortex;
  • it further includes the step of making the staying flexible particles leave the staying position, that is, "releasing" the flexible particles.
  • the captured flexible particles can be released into the required channel or container.
  • the ultra-high frequency bulk acoustic wave resonator in the present invention refers to a resonator capable of generating acoustic waves with a frequency exceeding 0.5 GHz (preferably exceeding 1 GHz), for example, 0.5 to 50 GHz.
  • the ultra-high frequency bulk acoustic wave resonator may be a thin film bulk acoustic wave resonator or a solid assembled resonator.
  • the microfluidic device usually includes a power adjustment 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 that adjusts the speed of the solution flowing through the area affected by the bulk acoustic wave.
  • Flexible particles refer to nano or micro particles with deformable properties.
  • 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 0.01-30um, preferably 0.5-25um, more preferably 0.8-20um.
  • the flexible particles are naturally occurring particles, such as cells or cell vesicles released by cells into the external environment, including exosomes, microvesicles, vesicles, membrane vesicles, vesicles, Air bubbles, prostate corpuscles, microparticles, intraluminal vesicles, endosome-like vesicles or exocytotic vesicles, etc.
  • These cell-related vesicles are vesicle-like bodies with a double-layer membrane structure that are shed from the cell membrane or secreted by the cell.
  • Vesicles generally have a diameter of about 30-1000 nm, for example, about 800-1000 nm.
  • 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 diameter of the cell or vesicle is about 0.8-30um, for example, 1-25um, and for example, 5-20um.
  • the output power of the power adjusting device is about 0.5-800 mW, preferably 0.5-500 mW, more preferably 0.5-350 mW.
  • the flow rate adjusting device can adjust the velocity of the solution flowing through the bulk acoustic wave region to about 0.1-10 mm/s, preferably about 0.3-5 mm/s, more preferably about 0.5-2.5 mm/s.
  • the flow rate adjusting device can adjust the speed of the solution flowing through the bulk acoustic wave zone to about 0.1-100 ⁇ L/min, preferably about 0.1-50 ⁇ L/min, more preferably about 0.5-20 ⁇ L /min.
  • the height of the fluid channel is about 1.5-10 times the diameter of the cell or vesicle, preferably about 1.5-6 times the diameter, and more preferably about 2-4 times the diameter.
  • the height of the fluid channel of the microfluidic device is about 25-200 ⁇ 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 inlet includes a sample inlet and auxiliary solution inlets arranged on one or both sides of the sample inlet.
  • the auxiliary solution may be a liquid such as a buffer solution.
  • the auxiliary solution can be used to resuspend the "captured" cells or vesicles, or to add reagents for processing the "captured" cells or vesicles, such as the fluorescence that specifically recognizes the cells or vesicles or their specific markers mark. Based on the sheath flow, the auxiliary solution can also be used to control the flow direction and range of the sample liquid in the microchannel.
  • the aforementioned method is used to isolate and obtain a single cell or several (for example, 2-100, preferably 2-10) cells, or to isolate and obtain a single or several (for example, 2 -100, preferably 2-10) cell clusters.
  • the aforementioned method further includes selectively capturing cells or vesicles of different types or properties (for example, different sizes or densities, etc.) in the bulk acoustic wave affected area. In yet another aspect of the present invention, it also includes controlling the number of cells or vesicles staying in the area affected by the bulk acoustic wave. In yet another aspect of the present invention, the cells or vesicles staying in the bulk acoustic wave affected area and the number thereof can be selected by one or any combination of the following methods:
  • the above method controls the cells or vesicles staying in the body acoustic wave affected area to be single or several (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Pieces, or 11-20 pieces).
  • the velocity of the adjustment solution flowing through the bulk acoustic wave region is about 0.1-10 mm/s, preferably about 0.5-3 mm/s.
  • the power of the adjusted bulk acoustic wave is about 0.1-500 mW, preferably about 0.5-200 mW.
  • the speed of the adjusting solution flowing through the bulk acoustic wave region is about 0.1-10 mm/s, preferably about 0.5-3 mm/s, and the power of adjusting the bulk acoustic wave is about 0.1-500 mW, preferably about 0.5-200 mW.
  • the aforementioned method further includes allowing the retained cells or vesicles to leave the retention position, that is, releasing the cells or vesicles.
  • the cells or vesicles can be released by one of the following methods or any combination thereof:
  • the method can controllably release the remaining cells or vesicles.
  • the method can control the types of released cells or vesicles, such as releasing cells or vesicles of different sizes. Cells or vesicles.
  • the "captured" cells are controlled to be released in order from small to large in size, for example, the power of the bulk acoustic wave is decreased by about 5-20%.
  • the method can control the number of released cells or vesicles, for example, by adjusting the power of the bulk acoustic wave and/or the flow rate of the solution to control the "captured" cells to be released in different numbers, In this way, the required number of cells or vesicles, especially single cells or vesicles, can be obtained in a controlled manner.
  • the method further includes the step of dispersing clustered cells. For example, by adjusting the power of the bulk acoustic wave generated by the UHF resonator, the clustered cells are dispersed.
  • 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 captured, 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 blood fractions.
  • the method is used to separate cells or vesicles to be separated from a solution containing other particles or cells.
  • the ratio of the content of the cells or vesicles to be separated in the solution to the other particles or cells is less than 1:10 4 , preferably less than 1:10 5 , and more preferably less than 1. : 10 6 .
  • the content of the cells or vesicles to be separated in the solution is less than 10 cells/ml or less.
  • the microfluidic device in the method is set to capture the desired cell or vesicle position (or called the capture point) with other cells or other cells when processing different particles or cells.
  • the location of vesicles leaving the body acoustic wave action area (or called release point) is different.
  • the position where other cells leave the body acoustic wave action area is arranged upstream of the position where the desired cells are captured.
  • the method of setting the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device is to capture the position of the required cell (or called the capture point) and other cells.
  • the position (or called the release point) leaving the body acoustic wave action area is different.
  • 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.
  • the cell is a eukaryotic animal cell, preferably a mammalian cell, and more preferably a human cell.
  • the cell is a blood cell, a stem cell, a somatic cell, or a tumor cell, for example, a circulating tumor cell (CTC).
  • the circulating tumor cells are selected from, but not limited to, the following epithelial cell cancers: prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, head and neck cancer, pancreatic cancer, and lung cancer.
  • the cells are placental trophoblast cells or nucleated red blood cells. More preferably, the cells are derived from the peripheral blood of pregnant women.
  • the method further includes using the isolated cells or vesicles for one or more of the following analyses: DNA and RNA amplification (such as rapid cDNA end amplification (RACE); degeneracy PCR with oligomer primers; PCR for mitochondrial DNA; genomic PCR, digital PCR, RT-PCR, adjacent ligation PCR; immunoPCR); sequencing; immunochemistry; metabolite analysis; enzymatic analysis; reporter gene expression analysis; and hybridization research.
  • DNA and RNA amplification such as rapid cDNA end amplification (RACE); degeneracy PCR with oligomer primers
  • PCR for mitochondrial DNA
  • genomic PCR genomic PCR
  • digital PCR digital PCR
  • RT-PCR adjacent ligation PCR
  • immunoPCR immunoPCR
  • the method is used for the combination of cells or vesicles and microcapsules, especially the combination of single cells or vesicles and single microcapsules.
  • it includes making single or several cells or vesicles stay in the acoustic wave affected area of the UHF resonator body, and also including making single or several microcapsules in the same UHF resonator body.
  • the sonic wave affected area stays, and then the single or several cells or vesicles form a combination with a single microcapsule, for example, a combination of the single or several cells or vesicles and a single microcapsule is formed at the capture point.
  • the method further includes changing the parameters of the microfluidic device so that the combined microcapsule-cell/vesicle is released.
  • Microcapsules are devices that include multiple partitions and are widely used in different molecular biology applications, including but not limited to: nucleic acid sequencing, protein sequencing, nucleic acid quantification, sequencing optimization, gene expression detection, gene expression quantification, and Single cell analysis of genomic markers or expressed markers.
  • the partitions of the microcapsules may contain one or more reagents (e.g., enzymes, unique identifiers such as barcodes, antibodies, etc.).
  • the materials used for the microcapsules especially the materials of the shells of the microcapsules, enable the microcapsules to be ruptured under applied stimulation.
  • the reagents encapsulated in the microcapsules can be various molecules, chemical substances, particles and elements suitable for the sample preparation reaction of the analyte.
  • a microcapsule used in a sample preparation reaction for DNA sequencing of a target may contain one or more of the following reagents: enzymes, restriction enzymes, ligases, polymerases, fluorophores, oligonucleotide barcodes, buffers Wait.
  • the microcapsules used in the sample preparation reaction for single-cell analysis may contain: lysis buffer, detergent, fluorophore, oligonucleotide barcode, ligase, protease, heat-activatable Protease, protease or nuclease inhibitors, buffers, enzymes, antibodies, nanoparticles, etc.
  • Microcapsules are usually encapsulated and prepared by emulsifiers or droplet-based microfluidics.
  • the emulsion or droplets include a water-in-oil formulation or a water-in-oil-in-water (W/O/W) double emulsion formulation, or the emulsion or droplets include agarose or polymers such as PEG.
  • Microcapsules generally have a size from 10 nm to 100 ⁇ m. Preferably, in the method of the present invention, the microcapsules have a size from 1 ⁇ m to 100 ⁇ m.
  • the above method includes the following steps:
  • step (b) Inject the gel microspheres from the sample inlet, and adjust the parameters of the UHF bulk acoustic wave resonator under the condition that the cells or vesicles staying in step (a) are not released, for example, by adjusting the bulk acoustic wave power and Flow velocity, so that single or several microcapsules stay in the bulk acoustic wave action area of the UHF bulk acoustic wave resonator;
  • the microcapsules and the staying cells or vesicles are combined in the bulk acoustic wave action area of the UHF bulk acoustic wave resonator.
  • the combination of the single or several cells or vesicles and a single microcapsule is usually formed at the capture point of the bulk acoustic wave action area of the UHF bulk acoustic wave resonator.
  • the above method further includes the following steps:
  • the present invention also provides a kit, which includes a microcapsule and an ultra-high frequency bulk acoustic wave resonator as defined above.
  • the kit can be used to combine microcapsules and cells or vesicles (especially single cells or vesicles), and for one or more of the following analysis of cells or vesicles (especially single cells or vesicles): DNA and RNA amplification (such as rapid amplification of cDNA ends (RACE); degenerate oligomer primer PCR; mitochondrial DNA PCR; genomic PCR, digital PCR, RT-PCR, adjacent ligation PCR; immune PCR); sequencing; immunization Chemistry; metabolite analysis; enzymatic analysis; reporter gene expression analysis; and hybridization studies.
  • the kit may also contain reagents for the analysis.
  • the invention also provides a microfluidic device for separating the required flexible particles from the solution.
  • 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 ultra-high frequency resonator can emit a bulk acoustic wave transmitted to the wall on the opposite side of the fluid channel, so that the solution flowing through the bulk acoustic wave region generates a vortex, and the power and/or the bulk acoustic wave is adjusted by the power regulator
  • the flow rate adjusting device adjusts the speed of the solution flowing through the bulk acoustic wave area, so that cells or vesicles stay in the bulk acoustic wave area.
  • 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.
  • the output power of the power adjusting device of the microfluidic device is about 0.5-800 mW, preferably 0.5-500 mW, more preferably 0.5-350 mW.
  • the flow rate adjusting device of the microfluidic device can adjust the velocity of the solution flowing through the bulk acoustic wave region to about 0.1-10 mm/s, preferably about 0.3-5 mm/s, more preferably It is about 0.5-2.5mm/s.
  • the flow rate adjusting device of the microfluidic device can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.1-100 ⁇ L/min, preferably about 0.1-50 ⁇ L/min, more preferably It is about 0.5-20 ⁇ L/min.
  • the height of the fluid channel of the microfluidic device is about 1.5-10 times the diameter of the desired cell or vesicle, preferably about 1.5-6 times the diameter, more preferably It is about 2-4 times.
  • the height of the fluid channel of the microfluidic device is about 25-200 ⁇ m, preferably about 25-100 ⁇ m, more preferably about 30-80 ⁇ m, such as about 40-60 ⁇ m.
  • the microfluidic bulk acoustic wave BAW resonator frequency control device generating area of about 500-200000 ⁇ m 2, preferably about 5000-50000 ⁇ m 2, and most preferably from about 10000-25000 ⁇ m 2 .
  • the inlet of the microfluidic device includes a sample inlet and auxiliary solution inlets arranged on one or both sides of the sample inlet.
  • the microfluidic device is set to capture the required cells or vesicles at a position different from the position where other cells or vesicles in the solution leave the body acoustic wave action area.
  • the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device is set in a way that the location where the required cells are captured is different from the location where other cells leave the bulk acoustic wave action area.
  • the position where other cells leave the body acoustic wave action area is arranged upstream of the position where the desired cell is captured.
  • the ultra-high frequency bulk acoustic wave resonator of the microfluidic device 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 thickness of the piezoelectric layer of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device is in the range of 1 nm to 2 um.
  • 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;
  • Figure 3 shows that HeLa cells are captured 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 presence of multiple cells in the vortex tunnel.
  • Figure 5 shows the controllable decomposition and release of cell clusters in a microfluidic device system provided in an embodiment of the application.
  • Figure 5(a) shows that cell clusters are captured and decomposed into individual cells in the acoustic vortex tunnel of the present invention under the condition of a bulk acoustic wave power of 66 mV.
  • Figure 5(b) shows that when the power of the bulk acoustic wave emitted by the UHF bulk acoustic wave resonator exceeds a certain level, the cells are destroyed under the action of the vortex.
  • Figure 5(c) shows a fluorescence image of cell clusters captured in the acoustic vortex tunnel of the present invention.
  • Figure 5(d) shows a fluorescence image of cell clusters decomposed into single cells and single cells released in the acoustic vortex tunnel of the present invention (power 15mV, flow rate 2 ⁇ L/min).
  • Fig. 6 shows that a microfluidic device system provided by an embodiment of the present application can separate and capture Hela cells from a blood mixture containing Hela cells and blood cells.
  • Figure 6(a) shows that Hela cells are trapped at the edge of the sound wave action area of the high-frequency bulk acoustic wave resonator, while white blood cells and red blood cells flow through the sound wave action area.
  • Figure 6(b) is a graph showing the relationship between cell capture efficiency and different bulk acoustic wave power and sample solution flow rate.
  • FIG. 7 shows that a microfluidic device system provided by an embodiment of the present application can separate and capture different cells from a blood mixture.
  • Figure 7(a) shows that Hela cells are trapped at the edge of the sound wave action area of the high-frequency bulk acoustic wave resonator and remain active at 560s.
  • Figure 7(b) shows that the device and method of the present invention can capture leukocytes and maintain their activity.
  • Figure 7(c) shows that the device and method of the present invention can capture Hela cells, white blood cells and red blood cells, respectively.
  • Figure 8 shows the temperature distribution around the UHF bulk acoustic wave resonator. Image processing uses Matlab software.
  • FIG. 9 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.
  • Figure 9(c) and (d) show the photos of Hela cells being captured and blood cells released under two micro-channel settings.
  • Figure 9(a) and (b) are the photos of Figure 9(c) and (d), respectively Analysis and schematic diagram.
  • Figure 9(e) shows the distribution of blood cells upstream and downstream (the color lines in Figures 9(c) and (d)) of the bulk acoustic wave action area.
  • FIG. 10 shows that a microfluidic device system provided by an embodiment of the present application can capture circulating tumor cells from a blood sample of a liver cancer patient.
  • Figure 10 (a-1) and (a-2) show the flow of liquid in the flow channel when electric power is applied and applied to generate a bulk acoustic wave and a vortex.
  • Figure 10(a-3) shows that a single captured cell is stained green by Calcein-A.
  • Figure 10 (a-4) and Figure 10 (a-5) are fluorescence and bright field images, respectively.
  • Figure 10(b) shows the staining of the captured cells. Calcein-A is stained green, and the red fluorescence labeled with anti-EpCAM represents the presence of the cancer marker EpCAM in the captured cells.
  • Figure 11 shows that under the condition of constant flow rate, the number of captured cells has a linear relationship with the power applied to the UHF bulk acoustic wave resonator.
  • FIG. 12 shows that a microfluidic device system provided by an embodiment of the present application combines single or several cells and microcapsules one to one.
  • 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
  • 3T3 cells Guangzhou Jinio Biotechnology Co., Ltd., ATCC#CRL-1658
  • 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 0.8-30 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 0.8-30um.
  • the flexible particles are naturally occurring particles, such as cells or vesicles released by cells into the extracellular environment.
  • the method and device of the present invention can be used to separate and capture flexible particles in solution, for example, to separate and capture target cells in blood.
  • Another example is the separation and capture of vesicles in 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 20-100 ⁇ m, more preferably about 30-80 ⁇ m, for example, about 40-60 ⁇ m.
  • the microfluidic device is used to capture cells, and the height of its fluid channel is usually about 1.5-10 times the diameter of the target cell, preferably about 1.5-6 times the diameter, more preferably About 2-4 times.
  • 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 0.5-2.5 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 0.5-20 ⁇ 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 at the bottom of the 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 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.
  • the bulk acoustic wave action area of this embodiment is a pentagonal shape with a side length of about 120 ⁇ m.
  • the shape of the sound wave action area includes but is not limited to at least one of the following: circle, ellipse, semicircle, parabola, polygon with acute or obtuse vertices, polygon with vertices replaced by arcs, and vertices with acute angles , Semicircle or parabola, or any combination of 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 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 it 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.
  • 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 and the acoustic radiation force caused by the particles in the vortex channel can be calculated according to certain formulas, but the inertial lift force generated by the acoustic fluid vortex can hardly be used with simple 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 position where the cell or vesicle reaches the stable state can be called the capture position
  • Other cells or vesicles in the solution consistent with the steady state such as cells or vesicles with a smaller volume
  • the position(s) leaves the body acoustic wave action area and moves downstream, which is released; the position where the cell or vesicle leaves the vortex channel is also called the release point.
  • the cells or vesicles in the center of the vortex usually move to the region where the vortex channel is located downstream of the fluid channel, that is, the capture position is usually in the region where the vortex channel is downstream of the fluid 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 direction of the vortex and the direction of the acoustic radiation force suddenly change and enter the cells or vesicles of the vortex channel.
  • the particles that meet the appropriate conditions have been focused to the center of the vortex, under the action of the sound radiation force, they can change the direction of motion along with the vortex channel and quickly refocus to the center of the vortex channel after turning ; Particles that do not meet the conditions (such as having a smaller size) will be more affected by the jump in the laminar drag direction, and thus leave the vortex channel.
  • the microfluidic device is configured to capture the target cells or the location of the vesicles (or called capture points) and non-target cells when processing different cells or vesicles. Or the position of the vesicle leaving the body acoustic wave action area (or called the release point) is different.
  • the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device is arranged in such a way as to capture cells or vesicles of different sizes.
  • the location of the cell or vesicle (or called the capture point) is different from the non-target cell or vesicle leaving the body acoustic wave action area (or called the release point).
  • the bulk acoustic wave action area of the UHF bulk acoustic wave resonator is pentagonal, set it so that one of its corners faces the direction of liquid inflow in the channel and is placed symmetrically along the center line of the channel: at this time, non-target cells Or the vesicle leaves the body acoustic wave action area, that is, the release point, located at both ends of the downstream edge along the fluid direction; and the location of the capture target cell or vesicle, that is, the capture point is in the middle of the downstream edge along the fluid direction; the two are not mutually exclusive coincide.
  • non-target cells have the least influence on the stability of the captured cells.
  • the location (or called the capture point) for capturing the target cell or vesicle is set at the point where the non-target cell or vesicle leaves the body acoustic wave action area (or called the release point) Upstream.
  • the setting mode of the microfluidic device capable of reaching the different capture point and release point can be obtained by theoretical calculation or actual experimental method.
  • the capture point is separated from the release point, for example, the release point is set before the capture point. Non-target red blood cells and white blood cells will be taken away by laminar flow without passing through the capture point, and only the target circulation Tumor cells can reach the capture location.
  • the inventors of the applicant have discovered and provided a more effective method for separating target cells or vesicles in a sample, especially for separating a very small number or a very low proportion of target cells from a sample containing other cells or vesicles. Or the vesicle method.
  • 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 GHz to 50 GHz, preferably more than 1 GHz to about 10 GHz.
  • the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator is driven by the signal of the 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 0.5-800 mW, preferably 0.5-500 mW, more preferably 0.5-350 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.
  • the microfluidic device of the present invention may also include a detection device for detecting the characteristic signal of the cell in the sample or the marker carried by it. These characteristics can include physical properties such as molecular size, molecular weight, molecular magnetic moment, refractive index, electrical conductivity, charge, absorbance, fluorescence, and polarity.
  • the detection equipment includes a detection electrical detection device, such as a Coulter counter, for cell counting.
  • the detection device may also be a photodetector, which includes an illumination source and an optical detection component for detecting physical parameters such as charge, absorbance, fluorescence, and polarity.
  • the device is based on the impedance meter 303, which is arranged in the micro flow channel from the sample inlet and the buffer. A designated distance from the confluence of the liquid inlets, and a designated distance from the outlet of the micro channel in the micro channel.
  • the Coulter counter is a sensor that uses the electrical characteristics of cells to be different from the culture medium (or buffer) to realize cell counting and detection. From the structural point of view, the Coulter counter is composed of multiple electrode strips, mostly two or three electrodes. Its working principle is that when cells pass through the electrodes, they will replace the same volume of electrolyte, resulting in dielectric impedance between the electrodes.
  • 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 makes the flowing fluid produce vortices, and the acoustic fluid vortex is formed along the boundary of the acoustic wave action area of the UHF bulk acoustic wave resonator
  • the channel is also called the vortex tunnel.
  • the cell enters the vortex tunnel when passing through the area of action of the bulk acoustic wave, and under the conditions of suitable flow velocity and bulk acoustic wave power, after reaching a stable state at a certain position (s) of the vortex channel, no movement relative to the flow channel occurs, namely Captured (the location where the cell or vesicle reaches a stable state can be called the capture location). Due to the interaction between the eddy current and the laminar flow, the cells in the capture position are subjected to very weak lateral flow forces, so they no longer move forward (horizontally), that is, they are captured. Moreover, the captured cells are floating above the bottom of the microfluidic channel and have no contact with the microfluidic channel.
  • the image sequence in Figure 3(a) shows and demonstrates the process of capturing a single HeLa cell.
  • the capture process can be divided into three stages that occur in areas 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.
  • 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. Each single cell finally stays at the capture point downstream of 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.
  • the cells in contact with the device may be killed by the negative effects triggered by the device, thereby degrading biocompatibility.
  • cell-cell adhesion and cell-device surface adhesion caused by contact are also a serious problem, especially for single-cell operations.
  • the method provided by the present invention can avoid the problems of cell-cell or cell-device surface contact and adhesion during cell capture and separation operations.
  • the inventor found in experiments that the excitation power of the ultra-high frequency bulk acoustic wave resonator in the micro-channel system is increased to more than 800mW, for example, at 1300mW, the high-speed vortex shear Under the combined action of the shear force and the volume force generated by the velocity gradient, the cell membrane will be destroyed, the cell will be lysed, and the substances in the cell such as protein and nucleic acid will be released (shown in red in the figure) and enter the system with the liquid flow Downstream, further research can be carried out.
  • the power that causes destruction of the cell or vesicle is related to the volume of the cell or vesicle.
  • the cell destroys the cell membrane, including the cell membrane and the nuclear membrane, so that the materials wrapped in the membrane can be released for downstream analysis.
  • DNA sequencing it is necessary to destroy the cell membrane through the lysis solution to obtain DNA and then participate in the polymerase chain reaction; the western blot method commonly used in protein detection also needs to obtain the protein from the cell.
  • the commonly used cell lysis methods are mainly chemical methods, and different lysis solutions are added to achieve cell lysis. Compared with physical lysis methods, chemical methods may have an impact on downstream analysis due to the introduction of foreign agents.
  • single cells can be lysed accurately and accurately, and information on the unique properties of individual cells can be obtained.
  • the force that affects the movement of cells in the acoustic vortex of the present invention includes fluid drag force (Stokes drag force), inertial lift force generated by the acoustic fluid vortex and acoustic radiation force caused by sound waves (acoustic force).
  • radiation force is closely related to the size of the cell. It is necessary to study whether the method of the present invention can be used in separating solutions, especially cells of different sizes in blood, and the accuracy of separation.
  • Hela cells are dissolved in DMEM culture medium and mixed with 200 times diluted whole blood to prepare a test sample (adjust the Hela cell content to about 1 ⁇ 10 5 /mL). Hela cells are labeled with Calcein-AM (green). The blood is stained with DAPI, so that the white blood cells appear blue under the microscope.
  • 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. 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.
  • Figure 6(a) shows that Hela cells (stained in green, indicated by the solid circle in the figure) stay in the sound wave action area of the high-frequency bulk acoustic wave resonator, while white blood cells (stained in blue, the figure is a dashed circle Indicates) and red blood cells continue to move along the direction of fluid movement.
  • the rightmost image in Figure 6(a) is an enlarged display of the left image. It can be seen from the figure that the capture of the 3 Hela cells (pointed by the arrow) is very stable, even if other cells in the blood (white blood cells and red blood cells) try to enter and occupy this position.
  • the cell capture efficiency is related to different bulk acoustic wave powers and sample solution flow rates.
  • the capture efficiency is defined as the number of cells captured in the sound wave action area of a single UHF bulk acoustic wave resonator when 10 target cells are input.
  • the cell capture efficiency increases with the applied power and decreases with the increase of the lateral flow rate. The reason is that an increase in lateral flow will trigger a stronger shear force, which causes the cells to escape.
  • the increased power produces a higher speed vortex, which can offset the negative impact of the lateral flow on the bulk acoustic wave vortex tunnel.
  • target cells can be extracted from a mixed sample (such as blood) into another solution (such as a buffer or a fluorescent dye).
  • a mixed sample such as blood
  • another solution such as a buffer or a fluorescent dye.
  • Hela cells stained in green
  • white blood cells stained in blue
  • red blood cells follow The fluid movement direction continues to move forward, leaving the microfluidic device; then, while maintaining the power and flow rate, stop the blood containing cells and input PBS solution, the Hela cells still stay in the sound wave action of the high-frequency bulk acoustic wave resonator Area, and the solution is converted from blood to PBS; then the power is turned down, the Hela cells are separated from the vortex, and flow to the next structure of the microchannel with the PBS solution, which completes the capture of Hela cells from the blood into the PBS buffer process.
  • HeLa cells can be transferred from blood
  • the device and method of the present invention can capture cells to meet the requirements of single cell separation. Because under the conditions of the method and device of the present invention, that is, when the height of the microchannel is about 1.5-6 times the diameter of the target cell, and more preferably about 2-4 times, the generated vortex channel is within the vertical height range Best for keeping a cell. In this way, the cells captured by the acoustic wave action area of each high-frequency bulk acoustic wave resonator are arranged side by side along the edge of the acoustic wave action area, basically no oscillation occurs, and the damage between cells due to contact and collision is reduced. And it is easy to proceed to the next step of single cell separation by changing the vortex forming conditions to make the cells leave the vortex tunnel.
  • the method and device of the present invention can distinguish and separate cells of different sizes, and the resolution can reach the separation of blood cells and other cells in the blood (such as circulating tumor cells such as Hela cells).
  • the size of the captured cells can be selected by adjusting the intensity of the acoustic flow and the lateral flow velocity.
  • White blood cells and red blood cells can also be selectively captured from blood samples, and the results are shown in Figure 7(b) and Figure 7(c).
  • the blood sample is stained with DAPI, so in the fluorescence view, the white blood cells WBC are stained blue.
  • Figure 7(b) shows that when the power is adjusted to 13.2mV, a single white blood cell is captured and good cell viability is maintained.
  • Figure 7(c) shows that under different powers, different cells were captured.
  • the inventor used software (COMSOL, USA) to build a model to analyze the separation process of the above three kinds of cells.
  • the resolution of cell separation in the simulation is far worse than the effect observed in the actual experiment. This may be because the interaction and deformation between cells are not reflected in the simulation model.
  • the method and device of the present invention can maintain the full viability of the captured cells.
  • trypan blue an important stain for selective labeling of dead tissues or cells
  • rhodamine B (a temperature-dependent fluorescent dye) was added to the solution to measure the temperature distribution around the UHF bulk acoustic wave resonator under different applied powers.
  • Fig. 8 shows that the thermal effect of the resonator is very weak, especially under the condition of selectively capturing cells, such as when the applied power is less than 100mW.
  • Blood is the most primitive and most suitable environment for cells.
  • 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. For these reasons, the separation of CTC and whole blood is an important requirement for research, and it is also a technical challenge.
  • whole blood is a more challenging sample.
  • 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.
  • high density of cells, especially red blood cells (10 9 /mL) will cause strong interactions between cells, which will change the trajectory of the specimen and affect the stability of cell capture.
  • 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 inlet of the microfluidic system is designed as a three-phase flow, which includes a central sample inlet and buffer inlets on both sides: inject the sample from the middle sample inlet into the microfluidic channel; and PBS buffer from both sides
  • the buffer inlet is injected into the micro flow channel. Therefore, the PBS buffer added on both sides can be used as a clamping sheath flow, which limits the lateral range of the sample fluid and ensures that all samples will pass over the UHF bulk acoustic wave resonator located in the center of the flow channel. 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.
  • the microfluidic device of the present invention is optimized. For example, the setting of the sound wave action area of the high-frequency bulk acoustic wave resonator is adjusted to separate the capture point of the target cell from the release point of the non-target cell.
  • the setting of the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator of this embodiment is such that when cells of different sizes are processed, the location of the target cell (or called the capture point) is captured and the non-target cell leaves the bulk acoustic wave.
  • the area of action (or called the release point) is different.
  • Hela cells in the whole blood containing Hela cells were selectively captured.
  • Figure 9(c) and (d) show the photos of Hela cells being captured and blood cells released under two micro-channel settings.
  • Figure 9(a) and (b) are the photos of Figure 9(c) and (d), respectively 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 as a raised 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; and the location of capturing Hela cells, that is, the capturing point is also near the same corner.
  • the BAW action area is adjusted to one of the corners facing the liquid inflow direction of the channel, and is placed symmetrically along the center line of the flow channel: at this time, the release point of blood cells is located at both ends of the downstream edge along the fluid direction.
  • FIG. 9(e) shows the distribution of blood cells upstream and downstream (the color lines in Figures 9(c) and (d)) of the bulk acoustic wave action area. The green curve and the blue curve indicate that blood cells are released at two symmetrical release points on average without passing through the capture point.
  • the microfluidic device such as the bulk acoustic wave action area of the UHF bulk acoustic wave resonator, is adjusted to distinguish the capture area of the target cell from the non- The release point of the target cell, which helps to capture the target cell.
  • the microfluidic device such as the bulk acoustic wave action area of the UHF bulk acoustic wave resonator, is adjusted to distinguish the capture area of the target cell from the non- The release point of the target cell, which helps to capture the target cell.
  • the microfluidic device such as the bulk acoustic wave action area of the UHF bulk acoustic wave resonator
  • the captured Hela cells may deviate from the original trajectory or capture point under the interaction of other cells. Separate the capture point from the release point by adjusting the bulk acoustic wave action area of the ultra-high frequency bulk acoustic wave resonator of the micro flow channel device, especially placing the release point before (upstream) the capture point, and non-target cells do not pass through the capture point Will be taken away by laminar flow, and only target cells can reach the capture position.
  • This application proves that the method and device of the present invention can separate and capture CTC from the blood of liver cancer patients.
  • 1ml blood samples from 3 stage IV liver cancer patients were injected into the micro flow channel from the sample inlet. And respectively inject PBS buffer and staining solution containing Calcein-AM and anti-EpCAM antibody labeled with red fluorescence into the micro flow channel from the inlets on both sides. 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.
  • the flow rate of the central sample is controlled to be about 1 ⁇ L/min, that is, about 0.67 mm/s, and the flow rate of PBS is about 5 ⁇ L/min.
  • Figure 10(a-1) shows the distribution of fluid in the microchannels when the power is not turned on: the PBS flow enters the channel from the left channel at the top, the blood sample enters from the right channel at the top, and the dye solution is input from the bottom right channel ; Under the clamping action of the PBS liquid stream and the staining solution, the blood sample flows through the bulk acoustic wave signal generating area of the pentagonal high-frequency bulk acoustic wave resonator arranged on the right side of the flow channel.
  • Figure 10(a-2) shows that when power is applied, the power is adjusted to 30mW, the high-frequency bulk acoustic wave resonator generates vortex in the micro flow channel, blood cells pass through the area where the bulk acoustic wave signal is generated, and the target cells are captured; then the blood sample and At the entrance of PBS buffer, the staining solution occupies the area where the volume acoustic signal is generated to stain the cells. After staining, close the dye inlet and inject PBS buffer to wash the remaining blood cells and dye.
  • Figure 10 (a3-5) shows the CTC captured in the area where the bulk acoustic wave signal is generated and blood cells that are not captured (passing through the area where the bulk acoustic wave signal is generated).
  • Figure 10(b) shows the staining of a single captured target cell, which is stained green by Calcein-AM, indicating that the captured cells retain activity, and the red fluorescence labeled with anti-EpCAM represents the presence of the cancer marker EpCAM in the captured cells.
  • 3T3 cell line (3T3 is a cell line with low expression of EpCAM) was also selected as a negative control.
  • the experiment process is the same as that in Figure 10(a), and the result is shown in Figure 10(c).
  • the results in Figure 10(c) show that both CTC and 3T3 were stained by Calcein-AM (green), CTC was stained by anti-EpCAM antibody, and 3T3 was not stained red because EpCAM on it was almost not expressed. The results prove that the captured single cells or cell clusters are cancer cells.
  • the method of the present invention can capture CTC in a patient's whole blood sample and perform in-situ staining. Compared with staining in a centrifuge tube by centrifugation and/or magnetic beads, the in-situ staining analysis method provided by the present invention has great advantages, including the steps of not requiring repeated centrifugation to wash and change dyes or solutions.
  • the inventor of the present application also unexpectedly discovered that in the method and device of the present invention, within a certain range, the number of cells captured in the vortex tunnel generated by the bulk acoustic wave of the UHF bulk acoustic wave resonator is different from the number of The power of the UHF bulk acoustic wave resonator is linear, thereby providing a controlled release of cells, especially a few or single cells.
  • the Hela cell sample and the label and the micro flow channel were prepared.
  • the sample is injected from the sample inlet into the micro flow channel and passes through the UHF bulk acoustic wave resonator to generate a vortex tunnel.
  • the inventor unexpectedly discovered that the number of captured cells (N), applied power (P) and lateral fluid velocity (V) have a linear relationship in a local range.
  • the present application also provides a controllable cell capture and release method, in which the number of captured cells and the number of released cells can be controlled, so that the cells are released one by one, that is, a single cell can be obtained.
  • the power is reduced by controlling the gradient and the vortex intensity is controlled so that the captured cells can be released in sequence.
  • the power for capturing multiple cells is about 35 mW.
  • the preferred power is less than 100mW, and higher power may cause adhesion between cells, which is not conducive to the operation of single cells.
  • Cell capture is performed at an input power of 30-35mW.
  • each reduced gradient is 5-20% of the total power, such as about 10%.
  • the smaller the gradient the more precise the manipulation, but the longer the cells are captured, the more likely it is that cell adhesion will occur.
  • the capture power when the capture power is about 33Mw, when two cells are captured, the single release power is about 13mW, and the complete release power is about 0.1mW; when three cells are captured, the first The cell release power is about 20mW, the second cell release power is about 16.5mW, the third cell release power is about 0.1mW; when four cells are captured, the first cell release power is about 21mW, and the second cell releases power about 21mW.
  • the released power was about 17mW, the third cell released about 6.6mW, and the fourth cell released about 0.1mW.
  • the number of cells captured in the vortex can be adjusted by adjusting the applied power.
  • the captured cells are arranged along the boundary of the bulk acoustic wave action area of the UHF bulk acoustic wave resonator. Under the action of the lateral fluid, the cells have a tendency to move downstream along the device boundary, so when the capturing conditions change, The first cell to shed is the cell at the most downstream boundary.
  • the ability of the vortex to capture cells is gradually reduced. When it is reduced to the point where it cannot capture all the current cells, the cells closest to the downstream will escape from the vortex, while other captured cells will move downstream by one cell in sequence. Fill in the vacant space by releasing cells. Therefore, the applied power can be reduced through a gradient to achieve a controlled release of the captured cells. It is worth emphasizing that this release can reach the single-cell level, which means that the captured cells can be released individually and sequentially.
  • the relationship between the increase in applied power and the number of captured cells or other flexible particles of similar nature is linear. Based on this, the captured power in the vortex can be adjusted by adjusting the applied power. The number of cells and/or other particles can be further reduced by applying a gradient to achieve a controlled release of the captured cells or other particles.
  • the cell sample to be processed from the sample inlet (the cell content is diluted to a BAW action area suitable for UHF BAW resonators to capture single to about ten cells), by adjusting the UHF BAW resonance
  • the bulk acoustic wave power and flow rate of the device enable the capture of single cells in the bulk acoustic wave action area of the UHF bulk acoustic wave resonator;
  • the inventors pass a single gel microsphere after the single cell is captured by the acoustic fluid system provided by the present invention.
  • the cells and microspheres can complete the preliminary Assembly. From this, it can be determined that the single cell that needs to be studied can be obtained, and this cell can be assembled with the gel microsphere.
  • the Hela cell sample and the label and the micro flow channel were prepared.
  • the gel microspheres used in the experiment are from Barcode Gelbead of 10X genomics, which are standard microspheres used for single-cell sequencing. The experiment and results are shown in Figure 12.
  • the applied power is 26.3mW and the flow rate is 1 ⁇ L/min.
  • the inlets of the microfluidic system can respectively pass buffer solution (PBS), HeLa cell solution or Barcode gel bead sample. Adjust the parameters of the system so that both a single cell and a single barcode gel bead can enter the bulk acoustic wave action area of the UHF bulk acoustic wave resonator and be captured.
  • the process of this method includes: first adding buffer; after the buffer has drained the air bubbles in the flow channel, only the cell flow channel is opened and the Hela cells are placed. After the bulk acoustic wave region of the UHF bulk acoustic wave resonator captures a Hela cell (as shown in Figure 12a), close the cell flow channel, open the Barcode gel beads flow channel, and put a Barcode gel bead (as shown in Figure 12b) ). A single cell and a single Barcode gel bead are captured in the BAW region of the same UHF BAW resonator (as shown in Figure 12c). Then the buffer flow channel is turned on and the applied power is turned off to achieve the release of the paired cells and gel microspheres (as shown in Figure 12d).
  • the particle capture system and method provided by the present application can achieve selective specific capture and controllable release of cells or vesicles of different sizes, and further perform single cell or vesicle analysis.

Abstract

本发明公开了一种分离流体中的目标细胞或囊泡的微流控系统及方法。本发明的系统包括流体通道，其具有入口和出口；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；功率调节器，其调节所述超高频谐振器产生体声波的功率；流速调节装置，其调节所述溶液流经体声波区域的速度。通过所述功率调节器调节产生的体声波的功率和/或通过所述流速调节装置调节所述溶液流经体声波区域的速度，使得细胞或囊泡在体声波影响区域停留。本发明的系统和方法可以对溶液中的细胞或囊泡进行捕捉和释放，对得到的细胞或囊泡进行进一步的处理及分析。

Description

细胞或微囊泡的分离方法及设备
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201910512145.2、发明名称为“细胞或微囊泡的分离方法及设备”，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系统和使用所述系统来分离和分析细胞或微囊泡的方法。
背景技术
对生物颗粒物，特别是对细胞或微囊泡的操作是一项最基本的生物技术，同时也是前沿研究的基石技术。目前有很多种方法可以实现对生物颗粒物，例如细胞或微囊泡等物质的操控，其中包括电学方法、声学方法、光学方法、磁学方法、化学方法、流体操控方法等。每种方法的都有特定的应用范围，仅对特定的粒子属性有效，如声表面波和介电泳方式的操控是与粒子的半径大小相关，因此对于微米尺寸的粒子操控效果较好，对于纳米级的粒子区分度较差；电泳等方法只在粒子带有电性的情况下才能工作，因此对不带电性或着带电量相似的不同物质的区分度较差。光学操控方法精度高，但会在操控过程中产生较多的热，这样会对生物分子的活性有较大的影响，并且光学系统较复杂，难与微流体器件集成。每一种方法的单独使用都有一定的局限性，因此多数情况下，通过不同方法的结合使用，会使得样品的区分和操纵效果更好。
单细胞操控是近年来受到越来越多关注的领域。对单个细胞或微囊泡的操纵，对于例如单细胞分析，药物开发，器官芯片和细胞间相互作用等研究非常重要。其中基于细胞间特异性的研究(例如对单个细胞的细胞形态学、表面粘附性、迁移速度、蛋白表达和基因表达的研究)极度依赖单 细胞操控技术。但是，传统提取单细胞的方式繁复、耗时、费力且效率不佳。
声流体技术，例如表面声波和基于为流体中驻波的声泳技术都已经被用来微米粒子和细胞的分离、提取与操作。但是现有的这些技术采用的器件均有体积大、难以小型化的特点。
因此，目前亟需一种系统及方法，以实现对细胞或微囊泡，特别是单个细胞或囊泡的捕捉，以方便对捕捉后的单细胞或囊泡的进一步的处理及分析。
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系统中有效地操控和分离溶液中的细胞等柔性颗粒，由此提供了分离和“捕捉”目标细胞的方法和系统。本发明的方法和系统还可以用于精确控制捕捉到的细胞的种类和数量，特别适用于获得单个或有限数量的细胞。
具体的，本发明提供了一种将柔性微粒如细胞或囊泡从溶液中分离的方法，包括以下步骤：
(1)使含有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；
(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，在体声波影响区域的溶液产生涡旋；
(3)调节体声波的功率(例如通过功率调节器)和/或调节所述溶液流经体声波影响区域的速度(例如通过流速调节装置)，使得柔性微粒在体声波影响区域停留，即不发生相对流体通道向前的位移。
在本发明的其中一个方面，还包括使停留的所述柔性微粒离开停留位置的步骤，即“释放”所述柔性微粒。例如可以将所述捕捉到的柔性微粒释放到需要的通道或容器中。
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的声波的谐振器。所述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。
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。
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。
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，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，优选为0.5-25um，更优选为0.8-20um的直径。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外环境中的细胞囊泡，包括外泌体、微囊泡、囊泡、膜小泡、水泡、气泡、前列腺小体、微颗粒、管腔内囊泡、核内体样囊泡或胞吐囊泡等。这些细胞相关的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。囊泡通常具有大约30-1000nm的直径，例如具有大约800-1000nm的直径。
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，其中所述细胞或囊泡的直径约为0.8-30um，例如为1-25um，又例如为5-20um。
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输出功率为约0.5-800mW，优选为0.5-500mW，更优选为0.5-350mW。
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0.5-2.5mm/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0.5-20μL/min。
在本发明的其中一个方面，其中所述流体通道的高度为所述细胞或囊泡的直径的约1.5-10倍，优选为直径的约1.5-6倍，更优选为约2-4倍。
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25-200μm，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。辅助溶液可以为例如缓冲液等液体。辅助溶液可以用于重悬“捕获”的细胞或囊泡，或用于加入处理“捕获”的细胞或囊泡的试剂，如特异性识别所述细胞或囊泡或其特异性标记物的荧光标记。基于鞘流作用，辅助溶液也可以用于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，前述方法用于分离和得到单个细胞或数个(例如2-100个，优选为2-10个)细胞，或是用于分离和得到单个或数个(例如2-100个，优选为2-10个)细胞簇。
在本发明的其中一个方面，前述方法还包括在所述体声波影响区域选择性捕捉不同种类或不同性质(例如不同大小或密度等)的细胞或囊泡。在本发明的其中又一个方面，还包括控制在所述体声波影响区域停留的细胞或囊泡的数量。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在所述体声波影响区域停留的细胞或囊泡及其数量：
(a)调节体声波的功率；
(b)调节产生体声波的时间；
(c)调节所述溶液流经体声波区域的速度。
在本发明的其中一个方面，上述方法控制在所述体声波影响区域停留的细胞或囊泡为单个或数个(例如1、2、3、4、5、6、7、8、9、10个，或11-20个)。在其中又一个方面，调节溶液流经体声波区域的速度为约 0.1-10mm/s，优选为约0.5-3mm/s。在其中又一个方面，调节体声波的功率为约0.1-500mW，优选为约0.5-200mW。在其中又一个方面，调节溶液流经体声波区域的速度为约0.1-10mm/s，优选为约0.5-3mm/，以及调节体声波的功率为约0.1-500mW，优选为约0.5-200mW。
在本发明的其中一个方面，前述方法还包括使得停留的细胞或囊泡离开停留位置，即释放所述细胞或囊泡。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释放所述细胞或囊泡：
(a)停止体声波；
(b)降低所述体声波的功率；
(c)增大溶液流经体声波区域的速度。
在本发明的其中一个方面，所述方法可控地释放停留的细胞或囊泡，在本发明的其中又一个方面，所述方法可以控制释放的细胞或囊泡的种类，例如释放不同大小的细胞或囊泡。在本发明的其中又一个方面，通过调节体声波的功率，控制“捕获的”细胞按尺寸从小到大顺序释放，例如，所述体声波的功率按约5-20％的程度递减。在本发明的其中又一个方面，所述方法可以控制释放的细胞或囊泡的数量，例如通过调节体声波的功率和/或溶液的流速，控制“捕获的”细胞以不同的数量被释放，由此可控地得到需要的数量的细胞或囊泡，特别是单个细胞或囊泡。
在本发明的其中一个方面，所述方法还包括将成簇细胞分散的步骤。例如通过调节超高频谐振器产生的体声波的功率，将成簇细胞分散。
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捕捉细胞或囊泡的液体，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，包括全血或血液级份。
在本发明的其中一个方面，所述方法用于将需要分离的细胞或囊泡从含有其它颗粒或细胞的溶液中分离。在本发明的其中又一个方面，其中所 述需要分离的细胞或囊泡在溶液中的含量与所述其它颗粒或细胞的比例小于1∶10 4，优选小于1∶10 5，更优选小于1∶10 6。在本发明的其中又一个方面，其中所述需要分离的细胞或囊泡在溶液中的含量小于10个/ml或更少。
在本发明的其中一个方面，所述方法中的微流控设备的设置为在处理含有不同的颗粒或细胞时，捕获需要的细胞或囊泡的位置(或称为捕获点)与其它细胞或囊泡离开体声波作用区域的位置(或称为释放点)不同。优选的，其它细胞离开体声波作用区域的位置设置在捕获需要的细胞的位置的上游。在本发明的其中又一个方面，所述方法中微流控设备的超高频体声波谐振器的体声波作用区域的设置方式为捕获需要的细胞的位置(或称为捕获点)与其它细胞离开体声波作用区域的位置(或称为释放点)不同。
在本发明中，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。在本发明的其中一个方面，所述细胞为真核动物细胞，优选为哺乳动物细胞，更优选为人的细胞。在本发明的其中又一个方面，所述细胞为血细胞、干细胞、体细胞或肿瘤细胞，例如为循环肿瘤细胞(circulating tumor cell，CTC)。所述循环肿瘤细胞选自而不限于以下上皮细胞癌症：前列腺癌、乳腺癌、结肠癌、膀胱癌、卵巢癌、肾癌、头颈部癌、胰癌和肺癌。在本发明的其中又一个方面，所述细胞为胎盘滋养层细胞或有核红细胞，更优选的，所述细胞来自孕妇外周血。
在本发明的其中一个方面，所述方法中还包括把分离的细胞或囊泡用于以下一种或多种分析：DNA和RNA扩增(例如cDNA末端快速扩增(RACE)；简并性寡聚引物PCR；线粒体DNA PCR；基因组PCR、数字PCR、RT-PCR、邻位连接PCR；免疫PCR)；测序；免疫化学；代谢物分析；酶法分析；报告基因表达分析；和杂交研究。
在本发明的其中一个方面，所述方法用于细胞或囊泡与微胶囊的组合，特别是单个细胞或囊泡与单个微胶囊的组合。在本发明的其中一个方面，其中包括使得单个或数个细胞或囊泡在超高频谐振器体的声波影响区域停留，以及还包括使得单个或数个微胶囊在同一超高频谐振器体的声波影响区域停留，然后所述单个或数个细胞或囊泡与单个微胶囊形成组合， 例如在捕获点形成所述单个或数个细胞或囊泡与单个微胶囊的组合。所述方法还包括改变微流控设备的参数，使得组合后的微胶囊-细胞/囊泡被释放。
微胶囊是包括包含多个隔室(partitions)的装置，广泛用于不同的分子生物学应用，包括但不限于：核酸测序、蛋白质测序、核酸量化、测序优化、检测基因表达、量化基因表达以及基因组标记物或表达的标记物的单细胞分析。微胶囊的分区可以包含含有一种或多种试剂(例如，酶、独特标识符例如条形码、抗体等)。用于微胶囊的材料，尤其是微胶囊的壳的材料，可使微胶囊能够在施加的刺激下破裂。封装在微胶囊中的试剂可以是适合于分析物的样品制备反应的各种分子、化学物质、颗粒和元素。例如，在用于靶标的DNA测序的样品制备反应中使用的微胶囊可以包含一种或多种下列试剂：酶、限制酶、连接酶、聚合酶、荧光团、寡核苷酸条形码、缓冲液等。在另一实例中，在用于单细胞分析的样品制备反应中使用的微胶囊可以包含：裂解缓冲液、去污剂、荧光团、寡核苷酸条形码、连接酶、蛋白酶、可热活化的蛋白酶、蛋白酶或核酸酶抑制剂、缓冲液、酶、抗体、纳米颗粒等。微胶囊通常通过乳化剂或者基于液滴的微流控来封装和制备。所述乳液或液滴包含油包水制剂或者水包油包水(W/O/W)双乳液制剂，或者所述乳液或液滴包括琼脂糖或者PEG等聚合物。微胶囊一般具有从10nm到100μm的尺寸。优选的，在本发明的方法中，微胶囊具有从1μm到100μm的尺寸。
在本发明的其中一个方面，上述方法包括以下步骤：
(a)通过调节超高频体声波谐振器的参数，例如通过调节体声波功率和/或流速，使得单个或数个细胞或囊泡在超高频体声波谐振器的体声波作用区域停留；
(b)从样品入口注入凝胶微球，在保持步骤(a)中停留的细胞或囊泡不被释放的条件下，调节超高频体声波谐振器的参数，例如通过调节体声波功率和流速，使得单个或数个微胶囊在超高频体声波谐振器的体声波作用区域停留；
(c)所述微胶囊与所述停留的细胞或囊泡在超高频体声波谐振器的 体声波作用区域发生组合。通常在超高频体声波谐振器的体声波作用区域的捕获点形成所述单个或数个细胞或囊泡与单个微胶囊的组合。
在本发明的其中一个方面，上述方法还包括以下步骤：
(d)通过改变微流控设备的参数，例如通过减小体声波功率或停止体声波，使得组合后的微胶囊-细胞/囊泡被释放。
本发明还提供了一种试剂盒，其中包括微胶囊和如前定义的超高频体声波谐振器。所述试剂盒可用于组合微胶囊和细胞或囊泡(特别是单个细胞或囊泡)，以及用于对细胞或囊泡(特别是单个细胞或囊泡)的以下一种或多种分析：DNA和RNA扩增(例如cDNA末端快速扩增(RACE)；简并性寡聚引物PCR；线粒体DNA PCR；基因组PCR、数字PCR、RT-PCR、邻位连接PCR；免疫PCR)；测序；免疫化学；代谢物分析；酶法分析；报告基因表达分析；和杂交研究。所述试剂盒还可含有用于所述分析的试剂。
本发明还提供了将需要的柔性颗粒从溶液中分离的微流控设备。
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，使得流经体声波区域的溶液产生涡旋，通过所述功率调节器调节体声波的功率和/或通过所述流速调节装置调节所述溶液流经体声波区域的速度，使得细胞或囊泡在体声波区域停留。
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。
在本发明的其中又一个方面，所述微流控设备的功率调节装置的输出功率为约0.5-800mW，优选为0.5-500mW，更优选为0.5-350mW。
在本发明的其中又一个方面，所述微流控设备的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0.5-2.5mm/s。
在本发明的其中又一个方面，所述微流控设备的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0.5-20μL/min。
在本发明的其中又一个方面，所述微流控设备的流体通道的高度为所述需要的细胞或囊泡的直径的约1.5-10倍，优选为直径的约1.5-6倍，更优选为约2-4倍。
在本发明的其中又一个方面，所述微流控设备的流体通道的高度为约25-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
在本发明的其中又一个方面，所述微流控设备的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。
在本发明的其中又一个方面，所述微流控设备的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。
在本发明的其中又一个方面，所述微流控设备设置为捕获需要的细胞或囊泡的位置与溶液中其它细胞或囊泡离开体声波作用区域的位置不同。
在本发明的其中又一个方面，所述微流控设备的超高频体声波谐振器的体声波作用区域的设置方式为捕获需要的细胞的位置与其它细胞离开体声波作用区域的位置不同。在本发明的其中又一个方面，其它细胞离开体声波作用区域的位置设置在捕获需要的细胞的位置的上游。
在本发明的其中又一个方面，所述微流控设备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。
在本发明的其中又一个方面，所述微流控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系统的结构示意图；
图2本申请实施例提供的一种微流控设备系统中的超高频体声波谐振器的结构示意图；其中，(a)表示图1所示的微流控系统的微流道的俯视图(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系统中被捕捉。图3(a)显示的单个Hela细胞在微流控设备系统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系统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)显示涡旋隧道存在多个细胞。
图5显示细胞簇在本申请实施例提供的一种微流控设备系统中可控分解和释放。图5(a)显示，细胞簇在体声波功率为66mV下的条件下在本发明的声涡旋隧道中被捕捉和分解成单个细胞。图5(b)显示，当超高频体声波谐振器发出的体声波的功率大过一定水平后，细胞在涡旋的作用下被破坏。图5(c)显示细胞簇在本发明的声涡旋隧道中被捕捉的荧光图。图5(d)显示细胞簇在本发明的声涡旋隧道中分解成单个细胞和单个细胞被释放的荧光图(功率15mV，流速2μL/min)。
图6显示本申请实施例提供的一种微流控设备系统能够从含有Hela细胞和血液细胞的血液混合液中分离和捕获Hela细胞。图6(a)显示，Hela细胞被捕获在高频体声波谐振器的声波作用区域边缘，白细胞和红细胞则流过声波作用区。图6(b)为细胞的捕获效率与不同体声波功率和样品溶液流速的关系曲线图。
图7显示本申请实施例提供的一种微流控设备系统能够从血液混合液中分离和捕获不同细胞。图7(a)显示，Hela细胞被捕获在高频体声波谐振器的声波作用区域边缘并在560s仍保持活性。图7(b)显示，本发明的设备和方法可捕获白细胞并保持活性。图7(c)显示，本发明的设备和方法可分别捕获Hela细胞、白细胞和红细胞。
图8显示超高频体声波谐振器周围的温度分布。图像处理采用Matlab软件。
图9显示本申请实施例提供的一种微流控设备系统能够从含有Hela细胞的全血混合液中分离和捕获Hela细胞。图9(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9(a)和(b)分别是图9(c)和(d)的分析和示意图。图9(e)显示体声波作用区域上游和下游(图9(c)和(d)中的颜色线)的血细胞分布。
图10显示本申请实施例提供的一种微流控设备系统能够从肝癌患者血液样品中捕捉循环肿瘤细胞。
图10(a-1)和(a-2)分别显示为施加和施加电力产生体声波和涡流时流道里液流的情况。图10(a-3)显示捕获的单个细胞被Calcein-A染成绿色。图10(a-4)和图10(a-5)分别是荧光和亮视场像。图10(b)显示被捕获的细胞的染色。Calcein-A染成绿色，而抗-EpCAM标记的红色荧光代表捕获细胞存在癌症标记物EpCAM。
图11显示在流速不变的条件下，捕捉到的细胞数目与施加给超高频体声波谐振器的功率是线性关系。
图12显示本申请实施例提供的一种微流控设备系统将单个或数个细胞与微胶囊一对一组合。
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 35 dBm enhancement of the original RF source power
注射泵：New Era Pump Systems，Inc.，NE-1000
细胞：
Hela细胞株：广州吉妮欧生物科技有限公司，ATCC#CCL2
3T3细胞：广州吉妮欧生物科技有限公司，ATCC#CRL-1658
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1x 10 5/mL～2x 10 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0.8-30um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0.8-30um的直径。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。
本发明的方法和设备可用于溶液中分离和捕捉柔性颗粒，例如在血液中对目标细胞进行分离和捕捉。又例如在血浆中对囊泡进行分离和捕捉。
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系统的一部分，例如以可装卸的芯片形式存在。微流控系统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。典型的微流控系统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20-100μm，更优选为约30-80μm，例如为约40-60μm。 在本发明的其中一个方面，所述微流控设备用于捕获细胞，其流体通道的高度通常为目标细胞的直径的约1.5-10倍，优选为直径的约1.5-6倍，更优选为约2-4倍。
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，与所述样品入口交汇相通。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0.5-2.5mm/s。在本发明的另一个方面，所述液体的流量流速范围在约0.1-100μL/分钟，优选为约0.1-50μL/分钟，更优选为约0.5-20μL/分钟。
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。
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。
在本发明中，所述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。
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)。当流体中的颗粒受到的力达到一定的平衡时，颗粒停止在涡旋通道相关位置，不产生相对流道的运动。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外围旋转，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。
颗粒在进入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，在涡旋通道的某个(些)位置达到稳定状态后不产生相对流道的运动，即被捕捉(该细胞或囊泡达到稳定状态的位置可称为捕获位置)；而不符合稳定状态的溶液中的其它细胞或囊泡(例如体积尺寸较小的细胞或囊泡)在层流产生的侧向拖拽力作用下，不进入涡旋通道或是在涡旋通道的某个(些)位置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。在涡旋通道中，由于层流的作用，涡旋中心的细胞或囊泡通常会移动到涡旋通道位于流体通道下游的区域，即捕获位置通常处于涡旋通道位于流体通道下游的区域。
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，进入涡旋通道的细胞或囊泡中，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。
在本发明的其中一种优选实施方式中，所述微流体装置的设置为令在处理含有不同细胞或囊泡时，捕获目的细胞或囊泡的位置(或称为捕获点)与非目的细胞或囊泡离开体声波作用区域的位置(或称为释放点)不同。
在本发明的其中一种优选实施方式中，所述微流体装置的超高频体声波谐振器的体声波作用区域的设置方式，为令在处理含有不同尺寸的细胞或囊泡时，捕获目的细胞或囊泡的位置(或称为捕获点)与非目的细胞或囊泡离开体声波作用区域(或称为释放点)不同。例如，在超高频体声波谐振器的体声波作用区域为五边形时，将其设置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，非目的细胞或囊泡离开体声波作用区域，即释放点，位于沿流体方向的下游边线的两端；而捕获目的细胞或囊泡的位置，即捕获点在沿流体方向的下游边线中间；二者互不重合。由此，非目的细胞对被捕获的细胞的稳定影响最小。在本发明的其中又一种优选实施方式中，将捕获目的细胞或囊泡的位置(或称为捕获点)设置在非目的细胞或囊泡离开体声波作用区域(或称为释放点)的上游。
可以通过理论计算推断或实际实验方式获得能够达到所述捕获点和释放点不同的微流体装置的设置方式。
例如，从血液中筛分循环肿瘤细胞，由于血液中细胞密度极大，若是释放位点与捕捉位点重合，则所有的细胞都会通过捕捉位点，这会导致强的细胞间相互作用，在捕捉位点的循环肿瘤细胞存在由于大量的红细胞和白细胞的碰撞下脱落的可能性。在本发明的一个实施例中，将捕捉点与释放点分离，例如将释放点设置在捕捉位点之前，非目标的红细胞和白细胞不经过捕捉位点就会被层流带走，只有目标循环肿瘤细胞能够到达捕捉位置。
由此，本申请人的发明人发现和提供了更有效地分离样品中目标细胞或囊泡的方法，特别是从含有其它细胞或囊泡的样品中分离数量极少或比例极低的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0.5GHz-50GHz，优选为超过1GHz-约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0.5-800mW，优选为0.5-500mW，更优选为0.5-350mW。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。
本发明的微流控设备还可以包括检测设备，用于检测样品中的细胞或其携带的标记物的特征的信号。这些特征可包括分子尺寸、分子重量、分子磁矩、折射率、电导率、电荷、吸光率、荧光性、极性等物理性质。例如，检测设备包括检测电学检测装置，例如库尔特计数器，用于细胞计数。检测设备还可以是光电检测器，其包括照明源和光学检测部件，用于检测电荷、吸光率、荧光性、极性等物理参数。
在如图1和图2所示的本发明的微流控设备中，具有库尔特计数器(其器件基础为阻抗仪303)，设置于所述微流道内距离所述样品入口与所述缓冲液入口交汇处的一指定距离处，以及设置于所述微流道内距离所述微流道的出口的一指定距离处。库尔特计数器是利用细胞的电学特性与培养液(或者缓冲液)不同实现细胞计数和检测的传感器。从结构上看，由库尔特计数器由多个电极条组成，大多是两个或者三个电极，其工作原理是当细胞通过电极时会取代掉相同体积的电解液，导致电极之间介质阻抗发生变化，会产生一个瞬时的电位脉冲，可以被外接的阻抗仪检测到。从而可以实现细胞的计数。同时，由于其加工工艺与体声波器件(超高频体声波谐振器)加工工艺相兼容，可集成在同一个基底上，实现对细胞和囊泡等的检测。
实施例3分离缓冲液中的细胞
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细胞在微流控设备系统的超高频体声波谐振器发生体声波形成的涡旋通道中的的运动轨迹。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，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称为涡旋隧道。细胞在经过体声波作用区域时进入涡旋隧道，并在适合的流速和体声波功率的条件下，在涡旋通道的某个(些)位置达到稳定状态后不产生相对流道的运动，即被捕捉(该细胞或囊泡达到稳定状态的位置可称为捕获位置)。在该捕获位置的细胞，由于涡流和层流之间的相互作用，其受到的横向流动的力附近非常弱，因此不再发生向前(水平)的移动，即被捕获。而且捕获的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细胞捕获的过程。可以将捕获过程分为在区域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。最后，细胞被困在固定的点，在该点，拖曳力和声辐射力 相互平衡。由于在涡旋隧道调整了细胞的轨迹，细胞的减速和俘获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俘获的一致性。
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。每一个单个细胞最后都停留在超高频体声波谐振器的声波作用区域的边界的下游的捕获点。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。
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，在本发明的微流体系统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系统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。
对于细胞操作，与装置接触的细胞可能被装置触发的负面效应杀死，从而使生物相容性变差。此外，由接触引起的细胞-细胞粘附和细胞-设备表面粘附也是一个严重的问题，特别是对于单细胞操作。本发明提供的方法能够避免细胞捕获和分离操作中出现的细胞-细胞或细胞-设备表面接触和粘附的问题。
实施例5细胞的破碎
如图5(b)所示，发明人在实验中发现，微流道系统内所述超高频体声波谐振器的激发功率被提高到800mW以上，例如在1300mW时，在高速涡旋的剪切力和速度梯度产生的体积力共同作用下，细胞的细胞膜会被破坏，细胞被裂解，细胞内的物质如蛋白质和核酸等被释放(图中红色图所示)，随液流进入到系统的下游，可以进行进一步的研究。引起细胞或囊泡破坏的功率的与细胞或囊泡的体积相关。由于细胞碎裂主要是受声辐射力挤压的影响，而声辐射力一般与细胞或囊泡的体积的立方成正比，因此令具有较小体积的细胞或囊泡受到破坏和裂解的超高频体声波谐振器的激发功率较高。
在细胞研究当中，为了获得细胞内的信息，例如DNA，RNA，蛋白质的情况，细胞破坏细胞膜，包括细胞膜和细胞核膜，使得膜内包裹的物质能够释放出来进行下游分析。例如，DNA测序中，就需要通过裂解液破坏细胞膜获得DNA再参与聚合酶链式反应；蛋白检测中常用的蛋白质印迹法也需要获得从细胞中获得蛋白。目前常用的细胞裂解方法以化学法为主，加入不同的裂解液实现对细胞裂解。相比于物理裂解方法，化学法由于会引入外源药剂，有可能对下游分析产生影响。而物理方法中，有超声裂解，热裂解，激光裂解等方法，能做到少量细胞精准裂解方法目前只 有激光裂解，而激光裂解由于激光器和光路限制，成本高，与微流控芯片的兼容性也较差。
利用本发明的方法和系统，可以做到单细胞精度的精准裂解，获得个别细胞的独特性质信息。
实施例6在稀释的血液样品中分离不同的细胞
影响细胞在本发明的声波涡旋中的运动的力，包括流体拖拽力(Stokes drag force)，声流体涡旋产生的惯性拖拽力(inertial lift force)和声波引起的声辐射力(acoustic radiation force)，都与细胞的尺寸有密切关系。需要研究本发明的方法是否可以用于分离溶液中，特别是在血液中不同大小的细胞，以及分离的精度。
将Hela细胞溶于DMEM培养液，与稀释200倍的全血混合，制备成测试样品(调整Hela细胞含量为约1x10 5/mL)。Hela细胞用Calcein-AM标记(绿色)。以DAPI对血液进行染色，由此白细胞在观察镜下显示为蓝色。
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。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。
图6显示含有Hela细胞和血液细胞的血液混合液中的Hela细胞，通过本发明的设备和方法，被选择性地捕获。
具体的，图6(a)显示，Hela细胞(染为绿色，图中为实线圈指示)停留在高频体声波谐振器的声波作用区域，而白细胞(染为蓝色，图中为虚线圈指示)和红细胞则沿流体运动方向继续前进。图6(a)最右侧图为其左侧图的放大显示。从图中可以看出，3个Hela细胞(箭头所指)捕获非常稳定，即使血液中其它细胞(白细胞和红细胞)试图进入和占领该位置。
另外，如图6(b)所示，细胞的捕获效率与不同体声波功率和样品溶液流速相关。在本实验中，捕捉效率定义为输入10个目标细胞，在单个超高频体声波谐振器的声波作用区域被捕捉到的细胞数目。在一定范围 内，细胞捕获效率随施加的功率而增加，并随着横向流速的增加而降低。原因是横向流量的增加会引发更强的剪切力，从而导致细胞逃逸。增加的功率产生较高速度的涡流，可抵消横向流量对体声波涡旋隧道的负面影响。为了得到具有高分辨率的稳定捕获区域，可以平衡所施加的功率和液体侧向流速。
通过本发明的微流控系统和方法，可以将目标细胞从混合样品(例如血液)中提取到另一种溶液(例如缓冲液或荧光染料)中。具体的，如图7(a)所示，通过调节功率和流速，Hela细胞(染为绿色)停留在高频体声波谐振器的声波作用区域，而白细胞(染为蓝色)和红细胞则沿流体运动方向继续前进，留出微流体设备；然后，在保持所述功率和流速的情况下，停止输入含有细胞的血液，输入PBS液，Hela细胞仍然停留在高频体声波谐振器的声波作用区域，而溶液由血液转化为PBS；然后将功率调低，Hela细胞从涡旋中脱离，随PBS液流到微流道的下一结构，即完成Hela细胞从血液捕捉到PBS缓冲液中的过程。类似的，可以将Hela细胞从血液或PBS缓冲液转移到其它溶液，如Trypan染料溶液中。
而且，本发明的设备和方法对细胞的捕捉可以达到单细胞分离的要求。由于在本发明的方法和设备的条件下，即微流道的高度为目标细胞的直径的约1.5-6倍，更优选为约2-4倍时，产生的涡旋通道在垂直高度范围内最适合保持一个细胞。这样，每个高频体声波谐振器的声波作用区域捕捉到的细胞沿着声波作用区域边线并列排布，基本不发生振荡，减少细胞之间因接触和碰撞导致的破坏。并且易于进行下一步的单个细胞通过改变涡流形成条件达到令细胞离开涡流隧道而分离的操作。
实验证明，本发明的方法和设备能够区分和分离不同大小的细胞，并且分辨率能够达到分离血细胞和血液中的其它细胞(例如Hela细胞等循环肿瘤细胞)。
另外，可以通过调整声流和侧向流速的强度来选择捕获细胞的大小。白细胞和红细胞也可以从血液样本中选择性地捕获，结果显示在图7(b)和图7(c)中。图7(b)中，血液样品用DAPI染色，因此在荧光视图中，白细胞WBC染成蓝色。图7(b)显示，在当功率调整为13.2mV的条件 下，捕捉到单个的白细胞并保持良好细胞活性。图7(c)显示，不同功率下，捕捉到不同的细胞。例如，在功率为6.6mV时，白细胞和红细胞都未被捕捉；在功率为13.2mV时，捕捉到白细胞，而红细胞都未被捕捉；在功率为20.9mV时，白细胞和红细胞都被捕捉。
发明人使用软件(COMSOL，USA)构建模型来分析上述三种细胞的分离过程。模拟中细胞分离的分辨率远差于实际实验中观察到的效果。这可能是因为在模拟模型中未能体现细胞之间的相互作用和形变等因素。
实施例7捕获细胞的活性的保持
另外，细胞活性的保留对细胞研具有重要的意义。但大部分现有技术的细胞分离方法都会对细胞活性有不良影响，甚至是杀死细胞。现有细胞操作技术中，对细胞有影响的因素包括机械剪切力，焦耳热或电子场。当超高频体声波谐振器产生的声波被吸收到液体中时，机械能转换成流体和热量的动能。热效应可能导致生物效应，可能导致构象变化，蛋白质错误折叠，纠缠和/或蛋白质的非特异性聚集等。
然而，本发明的方法和设备能够保持捕获的细胞的完全活性。
在本实验中，采用台盼蓝(一种用于选择性标记死亡组织或细胞的重要染色剂)注入捕获的细胞作为活性细胞的探针。
实验和结果如图7(a)所示。根据前述方法和条件对HeLa细胞进行捕获。然后用台盼蓝检测其活性。实验证明，在生存力测试期间，对细胞进行在缓冲液和台盼蓝溶液间的交换，重复3次。在560秒时，细胞仍具有活性：细胞膜仍保持对台盼蓝的选择性透过。
另外，在如上描述的实验设置中，在溶液中加入罗丹明B(一种温度依赖性荧光染料)，测量不同应用功率下超高频体声波谐振器周围的温度分布。结果如图8所示，表明谐振器的热效应非常弱，特别是在选择性捕获细胞的条件，例如施加的功率小于100mW下的时候。
实施例8全血中分离和捕捉不同的细胞
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。基于这些原因，CTC与全血分离是研究的重要要求，同时也是技术的挑战。
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捕获的稳定性。
发明人证明了本发明的设备和方法能够在全血中分离和捕获目标细胞。
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)。
将微流道系统的入口设计为三相流的方式，即包括中央的样品入口和两侧的缓冲液入口：将样品从中间的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。由此，两侧加入的PBS缓冲液可作为夹持鞘流，限制了样品流体的横向范围，确保所有样品都会经过位于流道中央的超高频体声波谐振器的上方。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。
为了更好地分离目标细胞，增加分离效率，优化了本发明的微流道设备。例如，对高频体声波谐振器的声波作用区域的设置进行调整，把目标细胞的捕获点和非目标细胞的释放点分开。
本实施例的超高频体声波谐振器的体声波作用区域的设置方式，为令在处理含有不同尺寸的细胞时，捕获目的细胞的位置(或称为捕获点)与非目的细胞离开体声波作用区域(或称为释放点)不同。例如，在超高频体声波谐振器的体声波作用区域为五边形时，将其设置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，非目的细胞离开体声波作用区域的位置，即释放点，位于沿流体方向的下游边线的两端；而捕获目的细胞的位置，即捕获点在沿流体方向的下游边线中间；二者互 不重合。由此，非目的细胞对被捕获的细胞的稳定影响最小。
结果如图9所示，含有Hela细胞的全血中的Hela细胞被选择性地捕获。
图9(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9(a)和(b)分别是图9(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9(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；而捕获Hela细胞的位置，即捕获点也在同一个角的附近。由此，血细胞对被捕获的Hela细胞的稳定有所影响。图9(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，而且处于捕获点的上游(即细胞先经过的位置)；而Hela细胞的捕获点，在沿流体方向的下游边线中间；二者互不重合。由此，血细胞对被捕获的Hela细胞的稳定影响最小。图9(e)显示体声波作用区域上游和下游(图9(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而不通过捕获点。图9(f-1)显示采用三相流的输入方式对注入样品进行被动聚焦，即将样品从中间的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，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9(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9(f-3)-(f-5)显示CTC被选择性捕获：复合堆叠图像序列展示了捕获的CTC的轨迹。图9(f-6)显示CTC选择性捕获的结果：合并图像显示，在本发明的装置在释放血细胞的同时稳定地捕获三个CTC。
在本发明的更佳方法中，为了减少细胞-细胞相互作用引起的负面影响，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，以区分目的细胞的捕获区和非目的细胞的释放点，其有助于捕获目的细胞。在从稀释血液样品中分离加入的Hela细胞的情况下，由于血细胞密度相对较低，因此由捕获细胞(Hela细胞)和释放细胞(血细胞)之间的相互作 用引发的负面影响是可接受的。但是，在与未稀释的全血分离的情况下，血液的高粘度限制了流体的速度梯度，并且极高的细胞密度触发了细胞之间的碰撞和摩擦。被捕获的Hela细胞可能在其他细胞的相互作用下偏离原始轨迹或捕获点。通过调整调整微流道装置的超高频体声波谐振器的体声波作用区域，将捕捉点与释放点分离，特别是将释放点放在捕捉点之前(上游)，非目标细胞不经过捕捉点就会被层流带走，只有目标细胞能够到达捕捉位置。
实施例9病人血液样品中CTC的捕捉
循环肿瘤细胞或CTC，是指在肿瘤患者的循环中发现的肿瘤细胞。该术语一般不包括血液肿瘤细胞。在患者的循环中，CTC含量极少，每毫升血液中的CTC可以少于1个。循环肿瘤细胞对癌症的诊断具有重要临床意义。由于CTC的数量稀少，以及血液中存在大量物理性质相近的细胞，检测血液中的CTC或是从血液中分离CTC具有重大挑战。
本申请证明了本发明的方法和设备能够从肝癌患者的血液中分离和捕获CTC。
将来自3名IV期肝癌患者的1ml血液样品从样品入口注入至微流道中。以及分别将PBS缓冲液和含有Calcein-AM和用红色荧光标记的抗-EpCAM抗体的染色溶液分别从两侧的入口注入至微流道中。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。控制中央样品流速范围在约1μL/min，即约0.67mm/s，而PBS流速为约5μL/min。
结果如图10所示。图10(a-1)显示未打开电力，流体在微流道的分布：PBS液流从顶部的左侧通道进入流道，血液样品从顶部的右侧通道进入，底部右侧通道输入染色溶液；在PBS液流和染色溶液的夹持作用下，血液样品流经设置在靠近流道右侧的五角形高频体声波谐振器的体声波信号发生区域。图10(a-2)显示当施加电力时，功率调节为30mW，高频体声波谐振器在微流道产生涡旋，血细胞通过体声波信号发生区域，靶细胞被捕获；然后关闭血液样品和PBS缓冲液入口，染色溶液占领体声波 信号发生区域，对细胞进行染色。染色后，关闭染料入口并注入PBS缓冲液以洗涤剩余的血细胞和染料。图10(a3-5)显示在体声波信号发生区域被捕获的CTC和未被捕获(通过体声波信号发生区域)的血细胞。图10(b)显示被捕获的单个目标细胞的染色，该单个细胞被Calcein-AM染成绿色，显示捕获细胞保持活性，而抗-EpCAM标记的红色荧光代表捕获细胞存在癌症标记物EpCAM。
实验中还选择3T3细胞株(3T3是一种低表达EpCAM的细胞株)作为阴性对照。实验过程与前述图10(a)中的相同，结果见图10(c)。图10(c)中的结果表明，CTC和3T3均被Calcein-AM(绿色)染色，CTC被抗-EpCAM抗体染色，而3T3没有被染成红色，因为其上的EpCAM几乎不表达。结果证明捕获的单细胞或细胞簇是癌细胞。
实验证明，本发明的方法可以在病人全血样品中捕获CTC并做原位染色。与通过离心和/或磁珠等方式在离心管中进行染色比较，本发明提供的原位染色分析方法具有很大的优势，包括无需重复离心以洗涤和换染料或溶液的步骤。
实施例10细胞的可控释放
本申请的发明人还出乎意料地发现，在本发明的方法和设备中，在在一定范围内，超高频体声波谐振器体声波产生的涡旋隧道中捕捉到的细胞数目与施加给超高频体声波谐振器的功率是线性关系，由此提供了对细胞，特别是少数或单个细胞的可控释放。
根据实施例3的方法制备Hela细胞样品和标记以及微流道。将样品从样品入口注入至微流道中并通过超高频体声波谐振器体声波产生涡旋隧道。
如图11(a)所示，在超高频体声波谐振器体声波产生的涡旋或涡旋隧道中捕捉到的细胞数目受到细胞的流速和施加给超高频体声波谐振器的功率的影响。
发明人出乎意料地发现，捕捉细胞数目(N)，施加功率(P)和侧向流体速度(V)之间，在局部范围内具有线性关系。发明人通过实验发现， 当V＝0.33mm/s时，N＝0.4*P；当V＝0.67mm/s时，N＝0.28*P；当V＝1.34mm/s时，N＝0.18*P；当V＝2mm/s时，N＝0.15*P。其中系数k≈0.47*exp(-V/0.83)+0.14。这个公式适用于细胞间相互作用较少和较弱时，不发生细胞之间黏连团聚形成细胞簇的时候。
发明人通过实验发现，优选的条件为：在V＜1mm/s时，P＜100mW；在1mm/s＜V＜3mm/s时，P＜300mW。对Hela细胞而言，在保证筛分的准确性的前提下，侧向流速最优选为约0.5-1mm/s，施加功率推荐在5～20mW，此时单个器件捕捉的细胞数目在1～6个之间。如果需要更多的细胞数目，可以通过阵列化排布器件实现。
下表为捕捉数目结果：
Figure PCTCN2020096176-appb-000001
由此，本申请还提供可控的细胞捕捉和释放的方法，其中可以控制捕捉到的细胞的数量，以及控制释放的细胞的数量，从而控制让细胞逐个释放，即获得单个细胞。以图8(b)为例，通过控制梯度减小功率，控制涡旋强度，使得被捕捉的细胞能够按照顺序释放。例如，针对直径在15～20μm的细胞，在0.67min/s的侧向流速下，捕捉多个细胞的功率为约35mW。其中，优选功率低于100mW，更高的功率可能导致细胞间黏连出现，不利于单细胞的操作。在30～35mW的输入功率下进行细胞捕捉，当达到目标细胞数目的时候，开始梯度减小功率，例如，每一个减小的梯度为总功 率的5-20％，例如约10％。梯度越小操控越精确，但是细胞被捕捉的时间越长，越有可能发生细胞间黏连。
如图8(b)所示，捕捉功率为约33Mw的情况下，两个细胞被捕捉时，单个释放功率为约13mW，完全释放功率为约0.1mW；三个细胞被捕捉时，第一个细胞释放功率为约20mW，第二个细胞释放功率为约16.5mW，第三个细胞释放功率为约0.1mW；四个细胞被捕捉时，第一个细胞释放功率为约21mW，第二个细胞释放功率为约17mW，第三个细胞释放功率为约6.6mW，第四个细胞释放功率为约0.1mW。
可见。本发明的系统和方法中，可以通过调控施加功率来调整涡旋中被捕捉到的细胞的数目。被捕捉的细胞沿着超高频体声波谐振器的体声波作用区域边界排列，在侧向流体的作用下，细胞有沿着器件边界向下游运动的趋势，所以在捕捉条件变化的情况下，最先脱落的细胞是处于最靠下游边界的细胞。通过逐渐降低功率，涡旋捕捉细胞的能力逐渐降低，当降低到无法捕捉当前全部细胞的时刻，最靠近下游的细胞就会脱离涡旋，而其他捕捉细胞会顺次向下游移动一个细胞的位置填补释放细胞而空缺的空间。所以，可以通过梯度减小施加功率，实现对捕捉细胞的可控释放。值得强调的是，这种释放可以达到单细胞水平，即实现对捕获细胞单个依次释放。
实施例11细胞与凝胶微球的组合
由于发现本发明的系统和方法中，施加功率的提高与捕捉细胞或其它类似性质的柔性微粒的数目之间关系是线性的，基于此，可以通过调控施加功率来调整涡旋中被捕捉到的细胞和/或其它颗粒的数目，进而还可以通过梯度减小施加功率，实现对捕捉到的细胞或其它颗粒的可控释放。
在本实验中，测试了捕捉单个细胞以及将其与单个凝胶微球组装，以便对单细胞进行测序等进一步分析的方法。
本实施例的方法包括以下步骤：
(1)从样品入口注入待处理的细胞样品(细胞含量稀释至适于超高频体声波谐振器的体声波作用区域可捕获单个至十个左右的细胞)，通过 调节超高频体声波谐振器的体声波功率和流速，使得在超高频体声波谐振器的体声波作用区域捕获单个细胞；
(2)从样品入口注入凝胶微球，调节超高频体声波谐振器的体声波功率和流速，在保持步骤(1)中捕获的单个细胞不被释放的条件下，使得单个凝胶微球进入超高频体声波谐振器的体声波作用区域并被捕获，以使所述凝胶微球与所述被捕捉细胞一对一配对；
(3)通过控制梯度减小功率，控制涡旋强度，使得配对后的凝胶微球-细胞释放。
在单细胞测序等单细胞研究中，需要将凝胶微球(其中包裹有引物，裂解液等)与单细胞组装，目前的组装方式都是基于泊松分布的随机方式，需要大量的凝胶微球和浓度极低的细胞，才能保证每个细胞能够与单个凝胶微球被包裹在同一个滴液当中，完成后续反应。这样的技术会导致大量的凝胶微球浪费，更重要的是，组装过程是随机的，不可控制的，无法对需要的细胞做前期筛选，也不能保证某个指定细胞一定被凝胶微球包裹。利用声流体涡旋的单细胞捕捉能力，发明人在本发明提供的声流体系统捕捉到单细胞之后，再通入单个凝胶微球，在声流体隧道中，细胞和微球能够完成前期的组装。由此可以确定得到需要研究的单细胞，也保证了这个细胞能够与凝胶微球组装。
根据实施例3的方法制备Hela细胞样品和标记以及微流道。实验采用的凝胶微球来自10X genomics公司的Barcode Gel bead，其为用于单细胞测序的标准微球。实验和结果如图12所示。其中施加的功率为26.3mW，流速为1μL/min。微流控系统的入口分别可分别通入缓冲液(PBS)，HeLa细胞溶液或Barcode gel bead样品。调节系统的参数，使得单个细胞和单个barcode gel bead都可进入超高频体声波谐振器的体声波作用区域以及被捕获。本方法的流程包括：首先加入缓冲液；当缓冲液排干净流道内气泡后，仅开启细胞流道和放入Hela细胞。当超高频体声波谐振器的体声波区域捕捉到一个Hela细胞之后(如图12a所示)，关闭细胞流道，开启Barcode gel beads流道，放入一个Barcode gel bead(如图12b所示)。单个细胞与单个Barcode gel beads被捕获在同一个超高频体声波谐振器的体 声波区域(如图12c所示)。然后开启缓冲液流道，关闭施加功率，实现配对好的细胞和凝胶微球的释放(如图12d所示)。
综上所述，本申请提供的微粒捕捉系统及方法，能够实现选择性的对不同尺寸的细胞或囊泡的特异性捕捉以及可控释放，并进一步做单细胞或囊泡分析。
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 (25)

  1. 一种分离目标细胞或囊泡的方法，包括：
    (1)使含有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；
    (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，在体声波影响区域的溶液产生涡旋；
    (3)通过调节体声波的功率和/或调节所述溶液流经体声波影响区域的速度，使得细胞或囊泡在体声波影响区域停留。
  2. 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0.5-800mW，优选为0.5-500mW，更优选为0.5-350mW。
  3. 权利要求1的方法，其中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0.5-2.5mm/s。
  4. 权利要求1的方法，其中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0.5-20μL/min。
  5. 权利要求1的方法，其中所述细胞或囊泡的直径约为0.8-30um，例如为约1-25um，又例如为约5-20um。
  6. 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25-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
  7. 权利要求1的方法，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。
  8. 权利要求1的方法，其用于分离单个或数个细胞或细胞簇。
  9. 权利要求1的方法，其中包括控制在所述体声波影响区域停留的细胞或囊泡和/或其数量，例如可通过以下方式的一种或其任意组合：
    (a)调节体声波的功率；
    (b)调节产生体声波的时间；
    (c)调节所述溶液流经体声波区域的速度。
  10. 权利要求1的方法，其中还包括使得停留的细胞或囊泡离开停留位置，例如可通过以下方式的一种或其任意组合：
    (a)停止体声波；
    (b)降低所述体声波的功率；
    (c)增大溶液流经体声波区域的速度。
  11. 权利要求1的方法，其为用于将需要分离的细胞或囊泡从含有其它细胞或囊泡的溶液中分离，优选的，其中所述需要分离的细胞或囊泡在溶液中的含量与所述其它细胞或囊泡的比小于1∶10 4，优选小于1∶10 5，更优选小于1∶10 6。
  12. 权利要求11的方法，其中所述设备的超高频体声波谐振器的体声波作用区域的设置方式为捕获需要的细胞或囊泡的位置与其它细胞离开体声波作用区域的位置不同，优选的，其它细胞或囊泡离开体声波作用区域的位置设置在捕获需要的细胞或囊泡的位置的上游。
  13. 权利要求1的方法，其中所述细胞为真核细胞，优选为哺乳动物细胞，更优选为人细胞。
  14. 权利要求1的方法，其中所述细胞为血细胞、干细胞、体细胞或肿瘤细胞，例如为循环肿瘤细胞(circulating tumor cell，CTC)。
  15. 权利要求1-14中任一项的方法，其还包括使得单个细胞或囊泡在体声波影响区域停留，然后将所述单个细胞或囊泡与单个微胶囊组合。
  16. 权利要求15的方法，其中包括以下步骤：
    (a)通过调节超高频体声波谐振器的参数，例如通过调节体声波功率和流速，使得单个或数个细胞或囊泡在超高频体声波谐振器的体声波作用区域停留；
    (b)从样品入口注入凝胶微球，在保持步骤(a)中停留的细胞或囊泡不被释放的条件下，调节超高频体声波谐振器的参数，例如通过调节体声波功率和流速，使得单个或数个微胶囊也在超高频体声波谐振器的体声波作用区域停留；
    (c)所述微胶囊与所述停留的细胞或囊泡发生组装，
    优选的，其中还包括(d)通过减小体声波功率或停止体声波，使得组合后的微胶囊-细胞/囊泡被释放。
  17. 一种用于分离目标的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，使得流经体声波区域的溶液产生涡旋，通过所述功率调节器调节体声波的功率和/或通过所述流速调节装置调节所述溶液流经体声波区域的速度，使得细胞或囊泡在体声波区域停留。
  18. 权利要求17的微流控设备，其中所述功率调节装置的输出功率为约0.5-800mW，优选为0.5-500mW，更优选为0.5-350mW。
  19. 权利要求17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0.1-10mm/s，优选为约0.3-5mm/s，更优选为约0.5-2.5mm/s。
  20. 权利要求17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0.1-100μL/min，优选为约0.1-50μL/min，更优选为约0.5-20μL/min。
  21. 权利要求17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25-200μm，优选为约25-100μm，更优选为约30-80μm，例如为约40-60μm。
  22. 权利要求17的微流控设备，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。
  23. 权利要求17的微流控设备，其中所述微流控设备的设置为捕获需要的细胞或囊泡的位置与溶液中其它细胞或囊泡离开体声波作用区域的 位置不同。
  24. 权利要求23的微流控设备，其中其它细胞离开体声波作用区域的位置设置在捕获需要的细胞的位置的上游。
  25. 权利要求17所述的微流控设备，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。
PCT/CN2020/096176 2019-06-13 2020-06-15 细胞或微囊泡的分离方法及设备 WO2020249130A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/618,677 US20230219086A1 (en) 2019-06-13 2020-06-15 Method and device for cell or microvesicle isolation
EP20823576.2A EP3985098A4 (en) 2019-06-13 2020-06-15 Method and device for cell or microvesicle isolation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910512145 2019-06-13
CN201910512145.2 2019-06-13

Publications (1)

Publication Number Publication Date
WO2020249130A1 true WO2020249130A1 (zh) 2020-12-17

Family

ID=73735012

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/096176 WO2020249130A1 (zh) 2019-06-13 2020-06-15 细胞或微囊泡的分离方法及设备

Country Status (4)

Country Link
US (1) US20230219086A1 (zh)
EP (1) EP3985098A4 (zh)
CN (1) CN112080385A (zh)
WO (1) WO2020249130A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113189059A (zh) * 2021-01-07 2021-07-30 西安交通大学 基于适配体和声表面波的病毒分选富集检测系统及方法
CN114112826A (zh) * 2021-11-25 2022-03-01 天津大学 用于荧光微粒的声光互联微流控检测系统和检测方法
CN114522649A (zh) * 2022-02-15 2022-05-24 浙江大学 一种基于磁流体重构的声学微粒捕获及轨迹操控方法
SE2150546A1 (en) * 2021-04-29 2022-10-30 Bico Group Ab A method of culturing at least one cell in a microchamber configured to allow for optical inspection of the at least one cell, a device for use in the method and a system for performing one or more of

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210345917A1 (en) * 2020-04-15 2021-11-11 Iowa State University Ressearch Foundation, Inc. Resonance frequency shift sensors
CN113926221A (zh) * 2021-10-11 2022-01-14 天津大学 一种量子点溶液纯化方法及纯化系统

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107614700A (zh) * 2015-03-11 2018-01-19 布罗德研究所有限公司 基因型和表型偶联
CN107979352A (zh) * 2016-10-24 2018-05-01 天津大学 一种薄膜体声波微流控混合装置
US20180298371A1 (en) * 2015-04-29 2018-10-18 Flodesign Sonics, Inc. Separation Using Angled Acoustic Waves

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104968417B (zh) * 2012-08-01 2017-08-18 宾夕法尼亚州立大学研究基金会 颗粒和细胞的高效率分离和操作
CN103667051A (zh) * 2013-12-20 2014-03-26 河南省医药科学研究院 用于肿瘤细胞分离的表面声波微流控芯片
WO2019081521A1 (en) * 2017-10-25 2019-05-02 Universite De Lille ACOUSTIC KNIVES
CN108823065A (zh) * 2018-07-05 2018-11-16 中国科学院苏州生物医学工程技术研究所 基于间歇式倾斜声表面波的微颗粒分选装置

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107614700A (zh) * 2015-03-11 2018-01-19 布罗德研究所有限公司 基因型和表型偶联
US20180298371A1 (en) * 2015-04-29 2018-10-18 Flodesign Sonics, Inc. Separation Using Angled Acoustic Waves
CN107979352A (zh) * 2016-10-24 2018-05-01 天津大学 一种薄膜体声波微流控混合装置

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CUI,WEIWEI ET AL.: "Bulk Acoustic Wave Resonator Integrated Microfluidics for Rapid and High Efficience Fluids Mixing and Bioparticle Trapping", 2016 IEEE INTERNATIONAL ULTRASONICS SYMPOSIUM(IUS), 31 December 2016 (2016-12-31), XP032988257, ISSN: 1948-5727 *
CUI,WEIWEI ET AL.: "Hypersonic-Induced 3D Hydrodynamic Tweezers for Versatile Manipulations of Micro/Nanoscale Objects", PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION, vol. 35, no. 8, 6 June 2018 (2018-06-06), pages 1800068, XP055764592, ISSN: 0934-0866 *
HE,MEIHANG ET AL.: "IN-LINE TRAPPING AND ROTATION OF BIO-PARTICLES VIA 3-D MICRO-VORTICES GENERATED BY LOCALIZED ULTRAHIGH FREQUENCY ACOUSTIC RESONATORS", 2017 19TH INTERNATIONAL CONFERENCE ON SOLID-STATE SENSORS, ACTUATORS AND MICROSYSTEMS (TRANSDUCERS), 22 June 2017 (2017-06-22), pages 1789 - 1792, XP033131076 *
HE,MEIHANG ET AL.: "IN-LINE TRAPPING AND ROTATION OF BIO-PARTICLES VIA 3-D MICRO-VORTICES GENERATED BY LOCALIZED ULTRAHIGH FREQUENCY ACOUSTIC RESONATORS", 2017 19TH INTERNATIONAL CONFERENCE ON SOLID-STATE SENSORS, ACTUATORS AND MICROSYSTEMS (TRANSDUCERS), 22 June 2017 (2017-06-22), XP033131076 *
See also references of EP3985098A4
WEI, XUEYONG ET AL.: "Research progress of microfluidic technology based on surface acoustic waves", SCIENCE & TECHNOLOGY REVIEW, vol. 36, no. 16, 28 August 2018 (2018-08-28), pages 8 - 19, XP055764598, ISSN: 1000-7857 *
ZHENG,GRACE X.Y. ET AL.: "Massively parallel digital transcriptional profiling of single cells", NATURE COMMUNICATIONS, vol. 8, 16 January 2017 (2017-01-16), pages 14049, XP055503732, ISSN: 2041-1723 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113189059A (zh) * 2021-01-07 2021-07-30 西安交通大学 基于适配体和声表面波的病毒分选富集检测系统及方法
CN113189059B (zh) * 2021-01-07 2023-03-28 西安交通大学 基于适配体和声表面波的病毒分选富集检测系统及方法
SE2150546A1 (en) * 2021-04-29 2022-10-30 Bico Group Ab A method of culturing at least one cell in a microchamber configured to allow for optical inspection of the at least one cell, a device for use in the method and a system for performing one or more of
CN114112826A (zh) * 2021-11-25 2022-03-01 天津大学 用于荧光微粒的声光互联微流控检测系统和检测方法
CN114112826B (zh) * 2021-11-25 2024-04-19 天津大学 用于荧光微粒的声光互联微流控检测系统和检测方法
CN114522649A (zh) * 2022-02-15 2022-05-24 浙江大学 一种基于磁流体重构的声学微粒捕获及轨迹操控方法

Also Published As

Publication number Publication date
EP3985098A1 (en) 2022-04-20
US20230219086A1 (en) 2023-07-13
CN112080385A (zh) 2020-12-15
EP3985098A4 (en) 2023-06-28

Similar Documents

Publication Publication Date Title
WO2020249130A1 (zh) 细胞或微囊泡的分离方法及设备
Mao et al. Enriching nanoparticles via acoustofluidics
Yun et al. Cell manipulation in microfluidics
Huang et al. Recent advancements in optofluidics-based single-cell analysis: optical on-chip cellular manipulation, treatment, and property detection
Cetin et al. Microfluidic bio-particle manipulation for biotechnology
Li et al. An on-chip, multichannel droplet sorter using standing surface acoustic waves
WO2020249131A1 (zh) 利用超高频声波控制溶液中的微粒移动的方法及设备
Nilsson et al. Review of cell and particle trapping in microfluidic systems
Hu et al. On-chip hydrogel arrays individually encapsulating acoustic formed multicellular aggregates for high throughput drug testing
Ahmed et al. Sheathless focusing and separation of microparticles using tilted-angle traveling surface acoustic waves
Akther et al. Submicron particle and cell concentration in a closed chamber surface acoustic wave microcentrifuge
US20190101537A1 (en) Microfluidic determination of immune and other cells
Ling et al. Enhanced single-cell encapsulation in microfluidic devices: From droplet generation to single-cell analysis
Laubli et al. Embedded microbubbles for acoustic manipulation of single cells and microfluidic applications
Weiz et al. Microsystems for Single‐Cell analysis
Zhou et al. based acoustofluidics for separating particles and cells
Lee et al. Continuous medium exchange and optically induced electroporation of cells in an integrated microfluidic system
Bayareh Active cell capturing for organ-on-a-chip systems: a review
Yin et al. Effective cell trapping using PDMS microspheres in an acoustofluidic chip
Tay et al. Research highlights: Manipulating cells inside and out
WO2020249127A1 (zh) 微囊泡的分离方法及设备
JP4679847B2 (ja) 細胞の解析方法
Fakhfouri et al. Fully Microfabricated Surface Acoustic Wave Tweezer for Collection of Submicron Particles and Human Blood Cells
Dong et al. Advanced microfluidic devices for cell electroporation and manipulation
Li et al. Cell manipulation and cellular analysis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20823576

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020823576

Country of ref document: EP

Effective date: 20220113