CN114073995A - Microfluidic device and unit for analyzing single cells and method thereof - Google Patents

Abstract

The invention provides a sample introduction and mixing unit and a sample focusing unit for a micro-fluidic device. The invention also provides a micro-fluidic device for analyzing single cells or single nucleic acids by using the bar code particles, which is provided with the sample feeding and mixing unit and the sample focusing unit. The invention also provides a method for analyzing a sample from a single cell or a single nucleic acid using the barcode particles.

Description

Microfluidic device and unit for analyzing single cells and method thereof

Technical Field

The present invention relates to the field of biology and medical devices. In particular, the present invention relates to a microfluidic system, unit and method for analysing individual biological particles, such as cells or nucleic acids.

Background

The single cell sequencing tool has incomparable advantages in the research of embryo, tumor and various stem cells. With the continuous breakthrough of single cell sequencing technology and the wide use of sequencing instruments, single cell sequencing analysis is increasingly applied to various frontier biomedical researches. Without the genomes of both cells, the transcriptome levels are completely similar, so an average omics expression analysis of a large number of cells averages out expression signatures that would otherwise be quite significant in a single cell.

Most of the current single cell sequencing analysis platforms are carried out by adopting microfluidic equipment. In a microfluidic device, biological substances such as cells, nucleic acids and the like, and a part of particles (such as polymer microspheres, magnetic beads and the like) in a cavity can precipitate and aggregate even in a short time (such as a few minutes), so that the biological substances move in a micro-channel and even block the micro-channel. Conventional methods used in the prior art, such as magnetic rotors or motor-driven blades, suffer from device size and the problem of causing air bubbles in the liquid.

There is also a need in the art for better single cell sequencing analysis platforms and techniques.

Disclosure of Invention

The invention provides a sample introduction and mixing unit for a microfluidic device, which is used for stirring a sample solution in a sample cavity or a flow channel of the microfluidic device so as to keep the dispersed state of particles (such as biological particles and/or bar code particles) in a sample. The sample feeding and mixing unit comprises a bulk acoustic wave resonator arranged at the bottom of the sample cavity or the flow channel.

In one aspect of the present invention, the power of the bulk acoustic wave output by the bulk acoustic wave resonator in the sample-feeding and mixing unit of the microfluidic device is about 0.1-5mW, preferably about 0.5-2 mW.

In one aspect of the invention, the output power of the bulk acoustic wave resonator in the sample-feeding and mixing unit of the microfluidic device is pulse power.

In one aspect of the invention, the bulk acoustic wave resonator in the sample-feeding and mixing unit of the microfluidic device is an ultra-high frequency bulk acoustic wave resonator, and can generate a bulk acoustic wave with a frequency of about 0.1-50GHz, preferably 0.5-50GHz, in the solution.

In one aspect of the invention, the ultra-high frequency bulk acoustic wave resonator in the sample-feeding homogenizing unit of the microfluidic device is a film bulk acoustic wave resonator or a solid assembled resonator, for example, a thickness stretching vibration mode acoustic wave resonator.

The invention also provides a sample focusing unit for a microfluidic device, e.g. a microfluidic device for analyzing biological particles (e.g. cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles.

In one aspect of the present invention, the sample focusing unit includes:

a fluid channel having a solution inlet and an outlet;

a UHF bulk acoustic wave resonator disposed at a bottom of the flow channel, the UHF bulk acoustic wave resonator being capable of generating bulk acoustic waves in the flow channel at a frequency of about 0.5-50GHz propagating towards an opposite wall of the flow channel; wherein the UHF bulk acoustic wave resonator emits bulk acoustic waves that propagate to the opposite wall of the fluid channel, creating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generating region of the UHF bulk acoustic wave resonator; wherein the structure of the micro flow channel and the shape and position of the bulk acoustic wave action region of the UHF bulk acoustic wave resonator are configured such that particles (such as bio-particles and/or barcode particles) in the solution sample enter and move along the vortex channel when passing through the bulk acoustic wave region and leave the vortex channel at a specified position to enter the downstream channel.

In one aspect of the invention, the bulk acoustic wave action region of the ultra-high frequency bulk acoustic wave resonator corresponding to the release point in the sample focusing unit has a turning or curvature change.

In one aspect of the present invention, the boundary line stripe shape of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator in the sample focusing unit allows the bio-particles and barcode particles to remain moving in the vortex channel to the release point, for example, by reducing the occurrence of turns or curvature changes in the boundary line of the bulk acoustic wave generating region.

In one aspect of the present invention, the power of the bulk acoustic wave output by the UHF bulk acoustic wave resonator in the sample focusing unit is about 20-5000mW, preferably 50-2000mW, and more preferably 100-500 mW.

In one aspect of the present invention, the sample focusing unit has a flow rate adjusting device therein, which can adjust the flow rate of the solution through the bulk acoustic wave region to about 0.1-100mm/s, preferably about 0.5-50mm/s, and more preferably about 1-10 mm/s.

In one aspect of the present invention, the sample focusing unit has a flow rate adjusting device therein, which can adjust the flow rate of the solution through the bulk acoustic wave region to about 0.1-500. mu.L/min, preferably about 0.5-100. mu.L/min, and more preferably about 1-50. mu.L/min.

In one aspect of the invention, the height of the fluid channel in the sample focusing unit is about 10-300 μm, preferably about 25-100 μm, for example about 40-85 μm.

In one aspect of the present invention, the bulk acoustic wave generating region area of the UHF bulk acoustic wave resonator in the sample focusing unit is about 500-²Preferably about 5000-²Most preferably about 10000-25000 μm²。

In one aspect of the invention, the bulk acoustic wave generating region of the UHF bulk acoustic wave resonator in the sample focusing unit has a side length of about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm.

In one aspect of the present invention, the uhf bulk acoustic wave resonator of the sample focusing unit is a film bulk acoustic wave resonator or a solid-state mount type resonator, such as an acoustic wave resonator of a thickness extensional vibration mode.

In one aspect of the present invention, the bulk acoustic wave resonator of the sample focusing unit is an ultra high frequency bulk acoustic wave resonator, which can generate a bulk acoustic wave with a frequency of about 0.5-50GHz in the solution.

The invention also provides a microfluidic device for analyzing biological particles (e.g. cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles. The device is configured to pair individual bioparticles from the sample with barcode particles and form droplets containing the paired individual bioparticles-barcode particles therein.

In one of its aspects, the microfluidic device comprises the following units:

a bio-particle-barcode particle pairing and droplet encapsulation unit; for forming droplets containing one paired bioparticles-barcode particle therein;

and combinations of one or more of the following elements:

the sample feeding and mixing unit is used for the microfluidic device; and/or

A sample focusing unit for a microfluidic device as described previously.

In one of its aspects, the microfluidic device comprises the following units:

the sample feeding and mixing unit;

the sample focusing unit; and

the bio-particle-barcode particle pairing and droplet encapsulation unit.

In one of its aspects, the microfluidic device further comprises a processing and analysis unit which processes the individual paired bioparticles-barcodes contained within the droplets, for example such that the nucleic acids of the bioparticles are brought into contact with the barcodes and barcoded, and analyzes the information, in particular the nucleic acid information, of the individual bioparticles contained within the droplets.

In one aspect of the invention, the nanoparticle-barcode particle pairing and droplet encapsulation unit in the microfluidic device is configured to form individual biological particles and barcode particles from a sample into droplets containing the biological particles or the barcode particles therein, respectively, and then pair and combine the droplets containing the biological particles or the barcode particles, thereby forming droplets containing paired biological particle-barcode particles therein.

In one aspect of the invention, the nanoparticle-barcode particle pairing and droplet encapsulation unit in the microfluidic device is configured to pair a single nanoparticle from the sample with a barcode particle and then form a droplet containing the paired nanoparticle-barcode particle therein, e.g. by contacting a first liquid (typically an aqueous solution) containing the paired nanoparticle-barcode particle with a second liquid (typically an oil).

In one aspect of the present invention, the droplet generation element of the droplet encapsulation unit in the microfluidic device has the following structure:

a. a second liquid channel for transporting a second liquid, and a junction where the first liquid channel intersects the first liquid channel, the junction being configured such that the first liquid contacts the second liquid and is separated by the second liquid to produce substantially monodisperse droplets;

or is

b. A chamber containing a second liquid into which the first liquid enters to form substantially monodisperse droplets.

In one of its aspects, the processing and analysis unit in the microfluidic device is selected from the following devices or any combination thereof:

a sample application device, for example, for applying a reagent that disrupts cell membranes or increases permeability, or a stimulus that detaches the nucleic acid barcode from the particle, thereby allowing the nucleic acid fragments on the barcode particle to contact and react with the nucleic acid of the cell, including recognition and binding, etc.;

the light stimulation means is, for example, by cleavage of a photolabile bond of the releasable oligonucleotide. A thermally stimulated device, wherein an increase in the temperature of the bead environment may result in cleavage of the linkage or release of the oligonucleotide from the bead;

a nucleic acid amplification device (which includes a temperature controller and the like) for amplifying and pooling a nucleic acid of the cell using the nucleic acid fragment on the barcode particle;

a signal recognition device;

information analysis devices, including single analytes (e.g., RNA, DNA, or proteins) or multiple analytes (e.g., DNA and RNA, DNA and proteins, RNA and proteins, or RNA, DNA, and proteins) that process single cells, enable, for example, the analysis of proteomes, transcriptomes, and genomes of cells.

The present invention also provides a method for analyzing biological particles (e.g. cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles using the aforementioned microfluidic device, wherein individual biological particles from a sample are paired with barcode particles, and droplets containing the paired biological particle-barcode particles therein are formed;

the method comprises the following steps:

sample introduction and uniform mixing;

focusing a sample;

sample pairing and droplet encapsulation; the bio-particles and barcode particles entering the micro-channel are formed into droplets containing a pair of bio-particle-barcode particles therein.

In one aspect of the invention, in the sample feeding and mixing step, the biological sample solution or the barcode particle solution in the sample cavity is stirred to maintain the dispersion state of the biological particles and/or the barcode particles. In one aspect of the invention, the method includes generating a bulk acoustic wave having a frequency of about 0.1-50GHz within the cavity with a bulk acoustic wave resonator disposed at the bottom of the sample cavity. In one aspect of the invention, the method outputs bulk acoustic waves in a pulse mode by the bulk acoustic wave resonator.

In one aspect of the invention, the sample focusing step in the method adjusts the movement of the biological particles or barcode particles in the flow channel to eliminate or reduce the random distribution in the width direction of the flow channel; preferably, sample pairing is performed after sample focusing.

In one aspect of the invention, the sample focusing step in the method is performed by passing the bio-particles or barcode particles through a microchannel of a bottom uhf baw resonator that generates baw in the flow channel at a frequency of about 0.5-50GHz to the opposite wall of the flow channel. Wherein the UHF bulk acoustic wave resonator emits bulk acoustic waves that propagate to the opposite wall of the fluid channel, creating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generating region of the UHF bulk acoustic wave resonator. Wherein the structure of the micro flow channel and the shape and position of the bulk acoustic wave action region of the UHF bulk acoustic wave resonator are configured such that bio-particles or barcode particles for pairing enter and move along the vortex channel when passing through the bulk acoustic wave region and leave the vortex channel at a specified position to enter the downstream channel. In one aspect of the invention, the action region of the bulk acoustic wave of the ultra-high frequency bulk acoustic wave resonator at the release point has a turning or curvature change. In one aspect of the present invention, wherein the boundary line stripe shape of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator allows the bio-particle and the barcode particle to keep moving in the vortex channel to the release point, for example, by reducing the occurrence of a kink or curvature change in the boundary line of the bulk acoustic wave generating region.

In one aspect of the present invention, the power of the bulk acoustic wave output by the UHF bulk acoustic wave resonator in the focusing step is about 20-5000mW, preferably 50-2000mW, and more preferably 100-500 mW.

In one aspect of the present invention, the speed of the solution flowing through the bulk acoustic wave region in the micro flow channel in the focusing step is adjusted to about 0.1 to 100mm/s, preferably about 0.5 to 50mm/s, and more preferably about 1 to 10 mm/s.

In one aspect of the present invention, the speed of the solution flowing through the bulk acoustic wave region in the focusing step is adjusted to about 0.1-500. mu.L/min, preferably about 0.5-100. mu.L/min, and more preferably about 1-50. mu.L/min.

In one of its aspects, the method further comprises a processing and analysis unit step, processing the single paired bioparticles-barcodes contained within the droplets, for example, bringing the nucleic acids of the bioparticles into contact with the barcodes and barcoding them, and analyzing the information, in particular the nucleic acid information, of the single bioparticles contained within the droplets.

In one aspect of the invention, the method wherein individual biological particles and barcode particles from the sample are formed into droplets containing the biological particles or the barcode particles therein, respectively, and then the droplets containing the biological particles or the barcode particles are paired and combined, thereby forming droplets containing paired biological particle-barcode particles therein.

In one aspect of the invention, in which a single bioparticles from a sample are paired with barcode particles and then droplets are formed that contain the paired bioparticles-barcode particles therein, for example, a first liquid (typically an aqueous solution) containing the paired bioparticles-barcode particles is contacted with a second liquid (typically an oil).

In one aspect of the invention, the method wherein droplet encapsulation is performed by:

a. contacting a first liquid channel carrying a first liquid with a second liquid channel carrying a second liquid at a junction such that the first liquid is separated by the second liquid to produce substantially monodisperse droplets;

or is

b. The first liquid is passed into a chamber containing a second liquid to form substantially monodisperse droplets within the second liquid.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 is a schematic diagram of steps involved in a method for analyzing biological particles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids, etc.) using barcode particles in a microfluidic device according to the present invention.

Fig. 2 is a schematic diagram of the method and the arrangement structure and the operation mode of the ultra-high frequency bulk acoustic wave resonator in the microfluidic device.

Fig. 3 is a schematic diagram of the principle and phenomenon that the ultrahigh frequency bulk acoustic wave resonator in the microfluidic device induces vortex tunnel in a micro channel through bulk acoustic wave and the particles in the solution move in the vortex tunnel in the method and microfluidic device provided by the invention.

Fig. 4 is a schematic diagram of the structure and arrangement of an exemplary sample homogenizing unit of the microfluidic device provided by the present invention, and a diagram of a working result of homogenizing a sample by the sample homogenizing unit.

Fig. 5 is a schematic diagram of the structure and arrangement of an exemplary sample focusing unit of the microfluidic device provided in the present invention, and a diagram of the working result of the sample focusing unit focusing and "queuing" biological particles or particles in a micro flow channel.

Fig. 6 shows the structure and arrangement of a further exemplary sample pairing unit of the microfluidic device of the present invention.

Fig. 7 is a schematic diagram of the structure and arrangement of an exemplary droplet encapsulating unit of the microfluidic device provided by the present invention, and a schematic diagram of the operation and experimental results of the droplet encapsulating unit to form a droplet containing a pair of bio-particle-barcode particles.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1 Experimental methods and materials

Preparing a micro-fluid channel:

microfluidic channels made of Polydimethylsiloxane (PDMS) were prepared by soft lithography.

Preparing an ultrahigh frequency bulk acoustic wave resonator:

the bulk acoustic wave resonator device is manufactured by performing methods such as chemical vapor deposition, metal sputtering, photolithography, and the like on a silicon-based wafer. The specific method comprises the following steps:

1. the surface of the silicon wafer is thoroughly cleaned by using the piranha solution with the volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:1, and organic matters and inorganic matters on the silicon wafer can be effectively removed by the method.

2. On the cleaned silicon chip, a layer of aluminum nitride film is formed by a surface sputtering method, and then a layer of silicon dioxide film is deposited by an ion enhanced chemical vapor deposition method. And then, using the same method, alternately depositing an aluminum nitride film and a silicon dioxide film to form a Bragg acoustic reflection layer structure in which the aluminum nitride and the silicon dioxide are alternately overlapped.

3. And sputtering a 600nm molybdenum film as a bottom electrode on the Bragg reflection layer structure. And then, photoetching the molybdenum electrode film by adopting a standard photoetching technology including glue spreading, exposure, development and the like, and then etching to form a bottom electrode with a target pattern.

4. And sputtering an aluminum nitride film on the molybdenum electrode as a piezoelectric layer. The aluminum nitride film is patterned using dry etching.

5. The negative photoresist is used for transferring the pattern on the mask plate, and then a layer of titanium-tungsten alloy with the thickness of 50nm is sputtered out and used as an adhesion layer to increase the adhesion of the gold electrode. Then, a layer of 300nm thick gold film is grown on the upper electrode by using an evaporation method. And finally, removing the gold film around the target pattern by using acetone to form a gold electrode with the target pattern.

And finally, the bulk acoustic wave resonator device is bonded and integrated with the PDMS micro-channel chip. The bulk acoustic wave resonator device is disposed at a middle position of the channel.

The bulk acoustic wave resonator device is connected with a network analyzer by a standard SMA connector, and the frequency of the bulk acoustic wave emitted by the bulk acoustic wave resonator device in a micro-channel can be measured by finding a resonance peak through testing a frequency spectrum. The bulk acoustic wave resonator frequency prepared and used in the examples of the present application is about 1.5GHz-2.0 GHz.

Instruments and materials

High-frequency signal generator: (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz

A power amplifier: Mini-Circuits with 35dBm enhancement of the original RF source power

An injection pump: new Era Pump Systems, Inc., NE-1000

Cell:

HeLa cell line: ATCC # CCL2, Inc. of Biotech, Inc. of Guinie, Guangzhou

Cell culture:

293T cells were cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100U/ml penicillin (Thermo) and 100ug/ml streptomycin (Thermo). Cultured cell density was 1X10⁵/mL～2x10⁶and/mL. When the micro flow channel experiment is carried out, the micro flow channel experiment can be carried out by diluting the micro flow channel experiment to1x 105/mL. PBS buffer (Gibco).

Dyeing agent:

Calcein-AM (Beijing Solaibao science and technology Co., Ltd., China)

4', 6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA)

Example 2

In a specific implementation of this embodiment, a method and a microfluidic device for analyzing biological particles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids, etc.) using barcode particles in the microfluidic device, in particular, for analyzing nucleic acids of the biological particles, are provided. The present invention provides methods and microfluidic devices that can pair individual bioparticles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids, etc.) from a biological sample with barcode particles, and then contact a first liquid (typically an aqueous solution) comprising the paired individual bioparticle-barcode particles with a second liquid (which is a liquid immiscible with the first liquid, typically an oily liquid) to form droplets (e.g., water-in-oil droplets) comprising one paired bioparticle-barcode particle therein. The method further comprises further processing and analysis of the nucleic acid of the individual biological particles encapsulated in the droplets, for example, amplification and pooling of nucleic acid.

The method and microfluidic device provided by the present invention are suitable for processing biological particles having a diameter of typically about 0.01-30um, preferably 0.2-25um, more preferably 0.5-20 um.

In one aspect of the invention, the biological particle is a cell or a vesicle released by the cell into the extracellular environment. The cells include natural or cultured cells of higher plants or animals (e.g., mammals including humans), and unicellular organisms or simple multicellular organisms such as bacteria and fungi. Vesicles are microvesicles that are released into the extracellular environment by various animal cells. These cell-associated microvesicles are vesicular bodies having a double-layer membrane structure, which are shed from cell membranes or secreted from cells. Microvesicles released from cells include exosomes, microvesicles, vesicles, membrane vesicles, gas bubbles, prostasomes, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytosis vesicles, and the like. The microvesicles released from the cells have a diameter of about 30-1000nm, about 30-800nm, about 30-150nm, or about 30-100 nm.

In one aspect of the invention, the biological particle is a virus, including a viroid, viroid and virion. The virus may be a DNA/RNA virus, as well as a protein virus. Although viruses do not have an intact cellular structure, in this context, cells also include viruses.

In one of its aspects, the biological particle in the method is a nucleic acid, protein or polysaccharide molecule, in particular a nucleic acid molecule. As used herein, "nucleic acid" (and the equivalent term "polynucleotide") refers to a polymer of ribonucleosides or deoxyribonucleosides that contain phosphodiester linkages between nucleotide subunits. Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, microRNA, fragment nucleic acids, nucleic acids obtained from subcellular organelles such as mitochondria, and nucleic acids obtained from microorganisms or viruses that may be present on or in a sample. Nucleic acids include natural or synthetic, e.g., artificial or natural, DNA or RNA-templated amplification reaction products. The nucleic acid may be double-stranded or single-stranded, circular or linear. The method of the invention is particularly useful for isolating nucleic acids (e.g., DNA and RNA in any form, including natural or synthetic nucleic acids, e.g., amplification reaction products using DNA or RNA as a template) of 200bp or more, preferably 1kbp, more preferably 10kbp, e.g., 50 kbp.

Samples that can be used to detect biological particles include: from cell cultures, eukaryotic microorganisms or diagnostic samples such as body fluids, body fluid deposits, gastric lavage samples, fine needle aspirates, biopsy samples, tissue samples, cancer cells, cells from a patient, cells from tissue or in vitro cultured cells from an individual to be tested and/or treated for a disease or infection, or forensic samples. Non-limiting examples of body fluid samples include whole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph fluid, serum, plasma, urine, chyle, stool, ejaculation, sputum, nipple aspirate, saliva, cotton swab samples, irrigation or lavage fluids, and/or swabbing samples.

Barcode particles used in the methods of the invention are particles having nucleic acid barcodes attached to them. The nucleic acid fragments comprise barcodes. "barcode" or "barcode sequence" refers to any unique sequence tag that can be coupled to at least one nucleotide sequence for, e.g., later identification of the at least one nucleotide sequence. The nucleic acid fragments may also include a molecular identifier (UMI) sequence. The UMI sequence contains randomized nucleotides and is incorporated into the nucleic acid fragment independently of the barcode sequence. When nucleic acid fragments containing the same barcode sequence but different UMI sequences are added to RNA associated with one sample, each RNA sequence can be ligated to a different UMI sequence during barcoding. The nucleic acid fragments may also include binding sequences that recognize and bind to the nucleic acids of the biological particles to be analyzed, e.g., RNA binding and amplification that may be associated with the sample cells. The particles may be beads, chromatography resins, multi-well plates, microcentrifuge tubes, and the like, for example, beads, microbeads, microparticles, microspheres, nanoparticles, nanobeads, or hydrogels. In one of its aspects, the particles are beads, for example spherical beads made of metallic and/or polymeric material. In one of the aspects of the present invention, the particle is a hydrogel particle. The barcode particles employed in the methods and microfluidics of the present invention range in diameter from about 0.1 μm to 100 μm. The barcode containing nucleic acid can be bound to the particle using any desired mechanism such as a chemical bond or biotin-avidin, biotin-streptavidin, gold-thiol, etc. interactions.

Fig. 1 is a schematic diagram of the steps involved in the method of analyzing biological particles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles in a microfluidic device according to the present invention. As shown in fig. 1, the method of the present invention may comprise the steps of:

1. and (6) injecting and mixing the sample. Adding a sample containing biological particles (such as cells) to be detected into a sample cavity, adding bar code particles into another sample cavity, and stirring a biological sample solution or a particle solution positioned in the sample cavity to keep the dispersion state of the biological particles (such as cells) and/or particles and avoid precipitation and/or aggregation;

2. and focusing the sample. The movement of biological particles (such as cells) or bar code particles in the flow channel is adjusted, so that the biological particles and the bar code particles are converged, and the random distribution in the width direction of the flow channel is eliminated or reduced;

3. sample-barcode particle pairing and droplet encapsulation. Allowing the bioparticles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids) and barcode particles entering the micro flow channel to generate a pairing of a single bioparticle and a single barcode particle, and forming a droplet containing the single paired bioparticle-barcode therein;

4. and (4) processing and analyzing. The method comprises the steps of processing individual paired bioparticles-barcodes contained within the droplets such that the nucleic acids of the bioparticles are contacted and barcoded with the barcodes, and analyzing information, in particular nucleic acid information, of the individual bioparticles contained within the droplets.

The above steps can be used independently or in various combinations in the methods of the present invention. The combination includes various combinations and execution manners as illustrated in fig. 1 (arrows indicate execution order of each program).

In one aspect of the invention, the method of the invention may comprise all the aforementioned 4 steps, such as sample homogenization and sample focusing of the biological sample of interest, further sample homogenization and sample focusing of the barcode particles, followed by sample pairing and droplet packing, processing and analysis of the focused biological sample and the barcode-bearing particles.

In another aspect of the present invention, the method of the present invention may comprise some of all of the aforementioned 7 steps. For example, in the methods of the present invention, the biological sample of interest and the barcode particles can be directly subjected to sample pairing, droplet encapsulation, processing, and analysis; for another example, in the aforementioned method, the sample focusing may be performed on the biological sample and the barcode-bearing particles, respectively, prior to the step of sample pairing the biological sample and the barcode-bearing particles.

The present invention also provides an apparatus for processing and analyzing bio-particles using the barcode particles. In the present invention, the device is a microfluidic device. Microfluidic systems and devices are used to contain and transport fluid materials, such as liquids, with channel dimensions on the micrometer or even nanometer scale. Typical microfluidic systems and devices typically include structural and functional units that are on the order of millimeters or less.

Fig. 2 is a schematic diagram of the arrangement structure and the operation mode of the ultra-high frequency bulk acoustic wave resonator in the microfluidic device provided by the invention. Fig. 2(a) is a schematic (top view) of an ultra high frequency bulk acoustic wave resonator arranged in a microfluidic device. As shown in fig. 2(a), the microfluidic device 10 for processing and analyzing biological particles provided by the present invention may include the following units:

-asample homogenizing unit 100;

asample focusing unit 200;

-a pairing anddroplet encapsulation unit 300;

a processing and analysis unit 700 (not shown).

The fluid channel of the microfluidic device, or referred to as a microchannel, provided by the invention is generally closed except for the opening for the fluid to enter and exit. The cross-section of the fluid channel typically has a dimension of 0.1-500 μm, which may be of various shapes including oval, rectangular, square, triangular, circular, and the like. The fluidic channels can be fabricated using a variety of known microfabrication techniques, including but not limited to silica, silicon, quartz, glass, or polymeric materials (e.g., polydimethylsiloxane, i.e., PDMS, plastic, etc.). The channels may be coated with a coating. The coating may alter the characteristics of the channels and may be patterned. For example, the coating may be hydrophilic, hydrophobic, magnetic, conductive, or biologically functionalized.

In the microfluidic device provided by the invention, the ultrahigh frequency bulk acoustic wave resonator is adopted. As shown in fig. 2, the microfluidic device of the present embodiment has a plurality of ultra high frequency bulk acoustic wave resonators (shown in dark figures) disposed at the bottom of the cavity or flow channel. The movement of the biological particles or bar code particles is controlled by the bulk acoustic wave generated by the UHF bulk acoustic wave resonator in the solution. In the present invention, the ultra high frequency bulk acoustic wave resonator refers to a resonator that can generate a bulk acoustic wave having a frequency of about 0.5-50GHz (e.g., about 1-50 GHz). The ultra-high frequency bulk acoustic wave resonator used in the present invention may be a film bulk acoustic wave resonator or a solid-state mount type resonator, such as an acoustic wave resonator of a thickness extensional vibration mode.

The ultra-high frequency bulk acoustic wave resonator adopted by the invention can be well suitable for the material of the wall of the microfluidic device, and can be conveniently arranged in the wall of the microfluidic device, such as a cavity for containing liquid or the bottom of a microfluidic channel. The UHF bulk acoustic wave resonator can generate bulk acoustic waves in the solution which are transmitted to the opposite side.

Fig. 2(B) is a sectional view showing a schematic structure of a fluid channel (a-a section) of the microfluidic device and an ultra-high frequency bulk acoustic wave resonator (B-B section) disposed at the bottom of a flow channel of the microfluidic device or an ultra-high frequency bulk acoustic wave resonator (C-C section) disposed at the bottom of a cavity of the microfluidic device. Fig. 2(c) and (d) are schematic diagrams of how the uhf bulk acoustic wave resonator operates and the effects produced in solution. As shown in fig. 2(b), the uhf bulk acoustic wave resonator may be located in the wall (typically the bottom 13) of the microchannel 11 or thechamber 12 of the microfluidic device. The top of the ultra-high frequency bulk acoustic wave resonator is configured on the surface of the wall of the microfluidic device (such as the bottom surface of the cavity or the micro-channel), and bulk acoustic waves with the propagation direction perpendicular to the wall are generated towards the opposite side (such as towards the top 14 of the channel or above the cavity 12). The region formed by the top surface of the uhf bulk acoustic wave resonator is the bulk acoustic wave generating region, also referred to herein as the bulk acoustic wave region. As shown in fig. 2(b), the uhf bulk acoustic wave resonator includes an acousticwave reflection layer 15, abottom electrode layer 16, apiezoelectric layer 18, and atop electrode layer 17, which are sequentially disposed from bottom to top. The overlapped area of the bottom electrode layer, the piezoelectric layer, the top electrode layer and the acoustic wave reflecting layer forms a bulk acoustic wave generating area.

The inventors of the present application have found that in the device of the present invention, two modes of operation of the uhf bulk acoustic wave resonator are possible, as shown in fig. 2(c) and (d). As shown in fig. 2(d), in the cavity (height greater than 1000 μm, for example greater than 5000 μm), the uhf bulk acoustic resonator emits bulk acoustic waves that are directed to the top, creating an acoustic fluid in the solution, creating a perturbation in the solution, such that the particles in the solution remain dispersed. In this mode of operation, pulsed power is typically applied to the bulk acoustic wave resonator to achieve better dispersion of the particles in solution.

In addition, as shown in fig. 2(c), the uhf bulk acoustic wave resonator can emit bulk acoustic waves in a microchannel (height not more than 500 μm, for example, not more than 300 μm) traveling toward the opposite wall of the flow channel, generating vortices distributed along the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator in the solution, the continuous vortices constituting a vortex channel defined by the boundary of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator. The forces experienced by the particles in the vortex include the fluid drag force (Stokes drag force) generated by the vortex, the inertial drag force (inertial lift force) generated by the laminar flow, and the acoustic radiation force (acoustic radiation force) caused by the attenuation of the sound waves. Since the magnitude of the fluid drag force is positively related to the particle diameter, for example, while the magnitude of the acoustic radiation force is positively related to the square of the particle size. As the particles grow, the force experienced will transition from predominantly fluid drag to predominantly acoustic radiation force, which pushes the particles towards the centre of the vortex. Larger particles are subjected to greater acoustic radiation force to move to the center of the vortex; while smaller particles rotate around the periphery under the drag of the vortex, and smaller particles even leave the vortex. In addition, the particles move downstream of the bulk acoustic wave action region under the lateral drag force created by the laminar flow. When the forces applied to the particles retained in the vortex reach a certain equilibrium, the particles stop at the relevant position of the vortex passage and do not move relative to the flow passage.

The inventors of the present application have unexpectedly found that in the bulk acoustic wave mode of the uhf bulk acoustic wave resonator in the microchannel shown in fig. 2(c), the particles move in the three moving modes described below under certain parameters when passing through the bulk acoustic wave region of the uhf bulk acoustic wave resonator disposed in the microchannel (the related findings are also described and disclosed in the filed patent applications PCT/CN2020/096131 and PCT/CN2020/096178, which are incorporated herein in their entirety). Fig. 3 is a schematic diagram showing the movement of particles in the bulk acoustic wave action region through the uhf bulk acoustic wave resonator disposed in the microchannel in this operation mode. As shown in fig. 3, the uhf bulkacoustic wave resonator 21 emits bulk acoustic waves in themicrochannel 11 that travel to the opposite wall of the flow channel (i.e., the top of the flow channel, not shown) to createvortices 22 in the solution that are distributed along theboundary 23 of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator, with the successive vortices forming avortex channel 24 defined by theboundary 23 of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator. When particles in thesample solution 25 pass through the bulk acoustic wave action region, the particles can (a) pass through the bulk acoustic wave action region (without entering a vortex channel) without being affected basically under different parameter conditions (height of a flow channel, flow speed, bulk acoustic wave power generated by an ultrahigh frequency bulk acoustic wave resonator, particle size, shape and property, self property of the solution, relative property of the solution to the particles and the like); (b) enter the vortex channel and move along the vortex channel and then leave the vortex channel from a certain position in the vortex channel, and the position can be called a release point; or (c) enter and follow the vortex channel and stay in the vortex channel at a position, which may be referred to as a capture point. Under certain device and fluid conditions (i.e., fixed flow channel height, bulk acoustic power, flow rate, solution properties, etc.),larger particles 251 are more likely to be "trapped," i.e., enter and follow the vortex channel and settle at a trapping point in the vortex channel,smaller particles 252 are more likely to enter and follow the vortex channel and then exit the vortex channel from a release point in the vortex channel, andsmaller particles 253 are less likely to enter the vortex channel. The release point and the capture point are typically the same location. The three moving modes of the same particles can be respectively generated by adjusting different parameter conditions (such as changing the height of a flow channel, the flow speed, the bulk acoustic wave power generated by an ultrahigh frequency bulk acoustic wave resonator and the like) within a certain range, namely, the particles do not enter a vortex channel, or enter the vortex channel and stay at a certain position of the vortex channel after moving along the vortex channel, or enter the vortex channel and leave the vortex channel at a certain position after moving along the vortex channel. It is also possible to adjust the relevant parameters such that the same particles are transformed in the three above-mentioned ways of movement, for example from not entering the vortex channel to entering the vortex channel, or from staying somewhere in the vortex channel to leaving the vortex channel. In addition, the above three moving modes can be generated respectively by setting certain parameter conditions for different particles entering the vortex channel. Therefore, the particles or a plurality of types of particles in the solution sample can be distinguished, captured, separated and enriched, and the movement of the particles in the micro-flow channel can be controlled by arranging the ultrahigh frequency bulk acoustic wave resonator in the micro-flow channel and adjusting various relevant parameters (the height of the flow channel, the flow speed, the bulk acoustic wave power generated by the ultrahigh frequency bulk acoustic wave resonator, the size, the shape and the property of the particles, the self property of the solution, the relative property of the solution and the particles and the like).

In one aspect of the present invention, in the method for analyzing biological particles using barcode particles and the microfluidic device according to the present invention, the biological particles (such as cells or nucleic acids) in the solution or the barcode particles can be controlled to enter and move along the vortex channel caused by the uhf bulk acoustic wave in the solution and stay at or leave the vortex channel at a set position by the uhf bulk acoustic wave resonator disposed in the microfluidic channel. In yet another aspect of the present invention, the shape and position of the bulk acoustic wave action region of the uhf bulk acoustic wave resonator can be adjusted so that the controlled-movement biological particles (such as cells or nucleic acids) or barcode particles in the solution enter and follow the vortex channel and leave the vortex channel at a set position. Whereby the biological particles (e.g. cells or nucleic acids) or barcode particles leave the bulk acoustic wave action region in a defined position and orientation into the desired outflow channel. This defined position away from the vortex channel is called the release point, i.e. the position where the biological particles (e.g. cells or nucleic acids) or barcode particles leave the bulk acoustic wave action region. The solution from which the controlled movement of the biological particles is removed is kept moving in the inflow direction.

Since one of the important factors for the detachment of biological particles (such as cells or nucleic acids) or barcode particles from the vortex channel is the influence of laminar flow in the direction of the fluid channel, the release point is usually located in the downstream region of the vortex channel. In another aspect of the invention, the release point is generally located where a turn or change in curvature occurs in the vortex channel, i.e., above the location where a turn or change in curvature occurs in the boundary of the bulk acoustic wave action region corresponding to the release point, i.e., where a turn or change in curvature occurs in the boundary of the bulk acoustic wave action region corresponding to the release point. Without being bound by theory, the inventors believe that the reason for this phenomenon is that, at the turns or corners of the vortex channel, the vortex direction and the direction of the acoustic radiation force change abruptly, and among the biological particles (such as cells or nucleic acids) or barcode particles entering the vortex channel, particles meeting appropriate conditions (such as appropriate size) can change the direction of motion with the vortex channel and refocus to the center of the turned vortex channel rapidly under the action of the acoustic radiation force as they have focused to the vortex center; while biological particles (e.g. cells or nucleic acids) or barcode particles that do not meet the criteria (e.g. have a smaller size) are more affected by the jump in the laminar flow drag direction and thus leave the vortex channel.

In one aspect of the invention, the above-described method is suitable for processing a liquid sample containing a plurality of biological particles (e.g., cells or nucleic acids) or barcode particles; the large amount of biological particles or bar code particles can enter and move along the vortex channel in a continuous moving mode and leave the vortex channel at a set position, and therefore the purpose of rapid and large-flux treatment is achieved.

In one aspect of the invention, the method further comprises adjusting the height of the micro flow channel, the power for generating the bulk acoustic wave and/or the speed of the solution flowing through the bulk acoustic wave region to adjust the biological particles or barcode particles entering the vortex channel. Biological particles or barcode particles that do not enter the vortex channel pass through the bulk acoustic wave region and exit in the direction of the sample entering the fluid channel.

In one aspect of the present invention, the boundary line of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator (i.e. the shape of the corresponding vortex channel) is set to be suitable for the bio-particles or barcode particles to move along the vortex channel to the release point in the vortex channel. This prevents the biological particles or barcode particles from leaving the vortex channel without leaving the vortex channel from the release point.

In yet another aspect of the present invention, the boundary shape of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator is adjusted such that the target bio-particle or barcode particle remains moving in the vortex channel to the release point. As mentioned above, the boundary line of the bulk acoustic wave generation region has a turn or curvature change, which may increase the probability of particles escaping from the vortex channel. Therefore, it is possible to keep the particles moving in the vortex channel by reducing the occurrence of turns or curvature changes in the boundary line of the bulk acoustic wave generating region, i.e., to reduce bio-particles or barcode particles escaping from the vortex channel.

In one aspect of the present invention, in the method or the microfluidic device of the present invention, the biological particles (such as cells or nucleic acids) or barcode particles in the bulk acoustic wave generation region of the uhf bulk acoustic wave resonator may be controllably held or released by the uhf bulk acoustic wave resonator disposed in the microchannel. In yet another aspect of the present invention, the method or the microfluidic device may control the types of the bio-particles or barcode particles staying or releasing in the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator, for example, after allowing bio-particles or barcode particles of different sizes to stay in the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator, bio-particles or barcode particles of different sizes are released, especially, bio-particles or barcode particles of different sizes are released in the order of size from small to large. In yet another aspect of the present invention, the sequential release of "trapped" particles or granules from small to large size can be controlled by adjusting the power of the bulk acoustic wave.

The ultra high frequency bulk acoustic wave resonator in the present invention means a resonator capable of generating an acoustic wave having a frequency exceeding 0.5GHz, for example, a frequency of 0.5 to 50GHz (preferably not less than 1 GHz). The UHF bulk acoustic wave resonator can be a film bulk acoustic wave resonator or a solid assembled resonator.

In the present invention, the shape of the bulk acoustic wave action region of the uhf bulk acoustic wave resonator (typically the top of the uhf bulk acoustic wave resonator) includes at least one of the following, but is not limited to: the array comprises a circle, an ellipse, a semicircle, a parabola, a polygon with acute or obtuse vertex, a polygon with circular arc replaced vertex, a polygon with acute, semicircular or parabolic vertex, or a repeated array of the same shape or a circular array. The present application provides the acoustic wave action region in the above-described shape, but any other shape of the acoustic wave action region is also within the scope of the present application.

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the height of the microfluidic channel (which may be simply referred to as a microchannel or a flow channel herein) of the bulk acoustic wave generation region of the uhf bulk acoustic wave resonator (distance between the top of the uhf bulk acoustic wave resonator and the wall opposite thereto, typically, the distance from the bottom to the top of the flow channel) is about 10 to 300 μm.

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the bulk acoustic wave generation region area of the UHF bulk acoustic wave resonator is about 500-²Preferably about 5000-²Most preferably about 10000-25000 μm²。

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the length (direction of fluid in the microchannel) or width (direction horizontally perpendicular to the direction of fluid in the microchannel) of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator is about 20 to 500 μm, preferably about 40 to 400 μm.

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the power of the bulk acoustic wave generated by the uhf bulk acoustic wave resonator is about 0.5 to 5000mW, preferably about 10 to 2000 mW.

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the speed of the solution flowing through the bulk acoustic wave region of the uhf bulk acoustic wave resonator is about 0.1 to 100mm/s, preferably about 0.5 to 5 mm/s.

In one aspect of the present invention, in the method for analyzing bio-particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the speed of the solution flowing through the bulk acoustic wave region of the uhf bulk acoustic wave resonator is about 0.1 to 500 μ L/min, preferably about 0.5 to 30 μ L/min.

In the microfluidic device of the present invention, the flow rate of the injected liquid can be controlled by an external pressure source, an internal pressure source, electro-dynamic or magnetic field-dynamic means. The external and internal pressure sources may be pumps, such as peristaltic, syringe, or pneumatic pumps. In this embodiment, a computer-tuned syringe pump is used to control the flow rate of the liquid.

The microfluidic device of the present invention further comprises a power adjusting means which adjusts the power of the bulk acoustic wave generated by the ultra high frequency bulk acoustic wave resonator. In this embodiment, the power adjusting device is a power amplifier having a power adjusting function. Because the film bulk acoustic resonator has high energy conversion efficiency and basically has no loss, the output power of the power regulating device can be basically regarded as the output power of the film bulk acoustic resonator for generating bulk acoustic waves in fluid. In the microfluidic device of the present invention, the power adjusting means may be connected to a high frequency signal generator. And the output circuit of the power amplifier is respectively connected with the bottom electrode, the piezoelectric layer and the top electrode of the ultrahigh frequency bulk acoustic wave resonator.

Example 3 sample introduction and mixing step and sample introduction and mixing unit

The method for analyzing biological particles using barcode particles in a microfluidic device provided by the present invention may include a step of homogenizing by a sample, and the microfluidic device provided by the present invention may include a homogenizing by a sample unit. The method provided by the invention comprises the steps of adding a sample containing biological particles (such as cells) to be detected into a sample cavity of the microfluidic device, and adding bar code particles into another sample cavity. In one aspect of the present invention, the method comprises stirring the biological sample solution or the particle solution in different sample chambers respectively to keep the dispersion state of the biological particles (such as cells) and/or the barcode particles (such as magnetic beads) and avoid precipitation and/or aggregation.

In one aspect of the invention, the biological sample solution or particle solution is stirred by bulk acoustic waves generated by an ultra-high frequency bulk acoustic wave resonator arranged at the bottom of a sample cavity of the microfluidic device.

As shown in fig. 2, the exemplary microfluidic device provided by the present invention includes a sample-homogenizing unit 100. Fig. 4 is a diagram of the structure and arrangement of an exemplary sample homogenizing unit of the microfluidic device provided by the present invention, and a working result of homogenizing a sample by the sample homogenizing unit.

As shown in fig. 4(a), thesample homogenizing unit 100 includes asample inlet 101 or a sample well 101 and asample chamber 102, and a solution of biological particles (e.g. cells) and/or barcode particles is added into thesample chamber 102 from thesample inlet 101 and then enters a downstream processing unit through asample outlet 103 and asample channel 104.

Thesample chamber 102 is a cylinder with a circular cross-section, a volume of about 0.2-2.0ml, and a height of about 0.5-2.0 cm. The sample addition port may or may not have a cap.

The sample introduction and homogenizing unit further comprises an ultra-high frequency bulkacoustic wave resonator 105 arranged at the bottom of the sample cavity, and the ultra-high frequency bulk acoustic wave resonator can generate bulk acoustic waves with the frequency of about 0.5-50GHz in the solution of the sample cavity. As shown in fig. 4(b) and (c), within the cavity (height typically greater than 0.2cm, for example greater than 0.5cm), the bulk acoustic wave acts in a manner as described in example 2 and as shown in fig. 2(d), i.e. the bulk acoustic wave generates a vortex in the solution, causing a vortex and a perturbation of the solution within the sample cavity, such that the cells or the barcode particles in the solution remain suspended and dispersed. Since the cavity is not provided with a top cover or the distance between the top cover and the bottom is large (more than 0.2cm), the vortex caused by the bulk acoustic wave generated by the bulk acoustic wave resonator arranged at the bottom of the cavity does not basically cause the accumulation of particles in the solution on the surface of the resonator. In the application of generating vortex and keeping the solution in a flowing state, pulse power is applied to the UHF bulk acoustic wave resonator, and the effect of dispersing particles in the solution can be better obtained. In one aspect of the present invention, in the sample homogenizing unit, the action power of the bulk acoustic wave resonator is about 0.1-5mW, preferably about 0.5-2 mW. When the solution contains cells, the applied action power can be lower than 0.5mW, so that excessive heat is prevented from generating and adverse effects on the cells are avoided.

Fig. 4(d) shows the effect of the homogenizing unit in the microfluidic device provided by the present invention. 100 μ l of PBS solution containing Polystyrene (PS) microspheres (particle size 10um, 1000-. The results show that when the microscope is focused on the liquid surface in the cavity, the suspension conditions of the particles shot on the same surface at 0min, 2min, 4min and 6min are recorded under the on state (power 630 mW; 250ms intermittent vibration (250ms non-vibration)) and the off state of the bulk acoustic wave resonator. The results show that in the static state, the photograph shows that the particles generate virtual coke, which indicates that sedimentation occurs, and the particles suspend well under the operation of the bulk acoustic wave resonator until the particles still present a uniform suspension state after 15 min. In the experiment, the phenomenon of sedimentation is found to be earlier when the continuous power output mode is adopted than when the pulse power mode is adopted.

In one of its aspects, the invention is as described aboveSample introduction and uniform stirring unitUltra high frequency bulk acoustic wave resonator producing bulk acoustic waves having a frequency of about 0.5-50GHz. In other aspects of the invention, lower frequency bulk acoustic wave resonators, such as at a frequency of about 0.1-0.5GHz, may also be used.

In a microfluidic device, biological substances such as cells, nucleic acids and the like, and a part of particles (such as polymer microspheres, magnetic beads and the like) in a cavity can precipitate and aggregate even in a short time (such as a few minutes), so that the biological substances move in a micro-channel and even block the micro-channel. Conventional methods used in the prior art, such as magnetic rotors or motor-driven blades, suffer from device size and the problem of causing air bubbles in the liquid. The method and the device provided by the invention can effectively solve the problems.

EXAMPLE 4 sample focusing procedure and sample focusing Unit

The method for analyzing biological particles using barcode particles in a microfluidic device provided in the present invention may include a step of sample focusing, and the microfluidic device provided in the present invention may include a sample focusing unit. The moving mode of the biological particles or the bar code particles in the micro-flow channel is adjusted to converge the biological particles or the bar code particles, and the random distribution in the width direction of the flow channel is eliminated or reduced, so that the biological particles or the bar code particles enter the downstream sample matching unit in a more consistent direction, position and speed. Therefore, when the biological particles or the barcode particles enter the acoustic fluid vortex of the matching unit, the biological particles or the barcode particles have the same initial relative position and state, and the matching efficiency of the biological particles or the barcode particles and the capture efficiency of the biological particles are improved.

In one aspect of the invention, bulk acoustic waves are generated in a solution by an ultra-high frequency bulk acoustic wave resonator arranged at the bottom of a flow channel of a microfluidic device, and a vortex channel defined by the boundary of a bulk acoustic wave generating area of the ultra-high frequency bulk acoustic wave resonator is formed, so that biological particles (such as cells or nucleic acids) or bar code particles in the micro-flow channel enter the vortex channel and move along the vortex channel when passing through a bulk acoustic wave action area, and when leaving a release point of the vortex channel, the biological particles or bar code particles in the solution move forwards at the same or similar speed and movement direction, and the random distribution condition in the width direction of the flow channel is eliminated or reduced. Therefore, the biological particles or the barcode particles enter the downstream sample matching unit in a relatively consistent direction, position and speed, so that when the biological particles or the barcode particles enter the acoustic fluid vortex of the matching unit, the biological particles or the barcode particles have the same initial relative position and state, and the matching efficiency of the biological particles or the barcode particles and the capture efficiency of the biological particles are improved.

As shown in fig. 2, the present invention provides an exemplary microfluidic device including a sample focusing unit 400. Fig. 5 is a diagram showing the structure and arrangement of an exemplary sample focusing unit of the microfluidic device provided by the present invention, and the working results of the sample focusing unit focusing and "queuing" biological particles or particles in a micro flow channel. Fig. 5(a) shows a schematic diagram of the arrangement and the action principle of the uhf bulk acoustic wave resonator in the exemplary sample focusing unit, and fig. 5(b) and (c) are a schematic diagram of the working and experimental results of the sample focusing unit on biological particles or barcode particles in a sample.

As shown in fig. 5(a), the microchannel includes a sample inlet and a plurality of outlets located downstream of the location where the uhf baw resonator is located, wherein one of the outlets is an outlet for the desired bio-particles or barcode particles, and the others are outlets for the solution from which the desired bio-particles or barcode particles have been removed. The lower diagram of fig. 5(a) is a block diagram of an exemplary sieving unit. The sieving unit is provided with a sample inlet, a buffer inlet, an ultrahigh frequency bulk acoustic resonator (shown in a five-pointed star-shaped dark figure) arranged at the bottom of the micro-channel, and two outlets at the downstream. As shown in fig. 5(a), the solution containing the biological particles or barcode particles flows through the action region of the uhf baw resonator, and when the vhf resonator does not generate baw and induce vortex tunnel, all the biological particles or barcode particles are distributed dispersedly along the width of the entire flow channel and move downstream in the flow direction (fig. 5(a) upper diagram). When the UHF bulk acoustic wave resonator generates bulk acoustic waves and induces vortex tunnels, by adjusting parameter conditions (for example, by adjusting the height of a flow channel, the flow velocity, the bulk acoustic wave power generated by the UHF bulk acoustic wave resonator or a combination of the flow channel height and the flow velocity), biological particles or bar code particles enter the vortex channel, move along the vortex channel, leave the vortex channel from a certain position (release point) in the vortex channel, and move forward to enter a downstream flow channel at the same or similar velocity and movement direction, so that the random distribution condition in the width direction of the flow channel is eliminated or reduced.

Fig. 5(b) and (c) show the placement of the uhf bulk acoustic wave resonators in the sample screening unit of the microfluidic device of the present invention, along with the corresponding experiments and results.

The upper diagram of fig. 5(b) shows that the uhf baw resonator is leaf-shaped, and the downstream tip of the uhf baw resonator is biased above the flow channel, so that the desired bio-particles or barcode particles can change their moving direction after passing through the baw action region of the hf baw resonator, and enter the opening at the upper end of the downstream flow channel along the edge of the hf baw resonator after leaving from the downstream tip of the leaf-shaped device along the edge of the hf baw resonator, and form a "queue" that eliminates or reduces the random distribution of bio-particles or barcode particles in the width direction of the flow channel. The upper panel of fig. 5(c) shows the corresponding experiment and results. After the PBS solution containing Hela cells (fluorescent staining) entered the flow channel (height of 50 μm, flow rate of about 1ul/min, power of bulk acoustic wave applied by UHF bulk acoustic resonator of 25mW), it exited from the downstream tip of the leaf-shaped device along the edge of the HF bulk acoustic resonator and entered the opening at the upper end of the downstream flow channel along with the horizontal flow, where Hela cells formed and maintained as a thin cell stream.

The lower diagram of fig. 5(b) shows that the uhf bulk acoustic wave resonator is leaf-shaped, and the downstream tip of the uhf bulk acoustic wave resonator faces the middle of the flow channel, so that the desired bio-particles or barcode particles can move away from the position of the downstream tip of the leaf-shaped device along the edge of the hf bulk acoustic wave resonator after passing through the bulk acoustic wave action region of the hf bulk acoustic wave resonator, and enter the opening arranged in the middle of the downstream flow channel along with the horizontal liquid flow, and form a "queue", thereby eliminating or reducing the random distribution of the bio-particles or barcode particles in the width direction of the flow channel. The lower panel of fig. 5(c) shows the corresponding experiment and results. After PBS solution containing poly (p-Phenylene Styrene) (PS) microspheres (with the particle size of 10um and 1000/ul) enters a flow channel (the height of the flow channel is 28 mu m, the flow rate is about 1ul/min, and the power of bulk acoustic waves applied by an ultrahigh frequency bulk acoustic wave resonator is 200mW), the PS microspheres exit from the position of the downstream tip of a leaf-shaped device along the edge trend of a high frequency bulk acoustic wave resonator and enter an opening arranged at the upper end of the downstream flow channel along with horizontal liquid flow, and in the downstream flow channel, the PS microspheres form and are kept into thin cell flow.

In one aspect of the present invention, in the sample focusing step in the method for analyzing bio-particles using barcode particles provided by the present invention and in the sample focusing unit of the microfluidic device provided by the present invention, the microfluidic channel height of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator is about 5 to 200 μm, preferably about 25 to 100 μm, for example, about 30 to 90 μm.

In one aspect of the present invention, in the sample focusing step in the method for analyzing bio-particles using barcode particles provided by the present invention or in the sample focusing unit of the microfluidic device provided by the present invention, the bulk acoustic wave generation area of the UHF bulk acoustic wave resonator is about 500-200000 μm²Preferably about 5000-²Most preferably about 10000-25000 μm²。

In one aspect of the present invention, in the sample focusing step in the method for analyzing bio-particles using barcode particles provided by the present invention or in the sample focusing unit of the microfluidic device provided by the present invention, the side length of the bulk acoustic wave generating region of the uhf bulk acoustic wave resonator is about 30 to 500 μm, preferably about 40 to 300 μm, and most preferably about 50 to 200 μm.

In one aspect of the present invention, in the sample disaggregation step in the method for analyzing biological particles using barcode particles provided by the present invention or in the sample focusing unit of the microfluidic device provided by the present invention, the power of the bulk acoustic wave generated by the uhf bulk acoustic wave resonator is about 20 to 5000mW, preferably 50 to 2000mW, and more preferably 100-1500 mW.

In one aspect of the present invention, in the sample disaggregation step in the method for analyzing biological particles using barcode particles provided by the present invention or in the sample focusing unit of the microfluidic device provided by the present invention, the speed of the solution flowing through the bulk acoustic wave region is about 0.1 to 100mm/s, preferably about 0.3 to 20mm/s, and more preferably about 0.5 to 5 mm/s.

In one aspect of the present invention, in the sample disaggregation step in the method for analyzing biological particles using barcode particles provided by the present invention or in the sample focusing unit of the microfluidic device provided by the present invention, the speed of the solution flowing through the bulk acoustic wave region is about 0.1 to 200 μ L/min, preferably about 0.1 to 50 μ L/min, and more preferably about 0.5 to 30 μ L/min.

Example 5 Bio-particle-barcode particle pairing and droplet encapsulation procedures and units

The method for analyzing biological particles by using barcode particles in the microfluidic device provided by the invention comprises the steps of biological particle-barcode particle pairing and droplet packaging, and the microfluidic device provided by the invention comprises a biological particle-barcode particle pairing and droplet packaging unit.

In one embodiment of the invention, the pairing of individual bioparticles and individual barcode particles is generated by bioparticles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids) and barcode particles entering the microfluidic channel, and the paired bioparticle-barcode particles are then suspended in a first liquid (typically an aqueous solution), and passed to a downstream droplet encapsulation unit and droplet encapsulation step to contact a second liquid (which is a liquid immiscible with the first liquid, typically an oily liquid) to form droplets containing the individual paired bioparticle-barcode particles therein.

In another embodiment of the present invention, the bioparticles-barcode particles pairing and droplet encapsulation unit is configured to form individual bioparticles and barcode particles from a sample into droplets containing the bioparticles or the barcode particles therein, respectively, and then pair and combine the droplets containing the bioparticles or the barcode particles, thereby forming droplets containing paired bioparticles-barcode particles therein.

As shown in fig. 2, the present invention provides an exemplary microfluidic device including a bio-particle-barcode particle pairing anddroplet encapsulation unit 300.

In one embodiment of the present invention, the method provided by the present invention may add a solution (e.g., a first liquid for use in a subsequent droplet encapsulation step) or a reagent for a subsequent working unit or step at the sample pairing step. In one embodiment of the present invention, the sample-matching unit of the microfluidic device provided by the present invention may comprise a flow channel and an inlet for adding a solution or a reagent for a subsequent working unit. In the present invention, the reagents that can be used in the subsequent working units or steps include reagents that facilitate cell lysis, nucleic acid amplification, or barcode particle dissociation, and the like, such as cell lysing agents, cell lysing enzymes, nucleic acid ligases, nucleic acid polymerases, transcriptases, and the like.

Fig. 6 shows the structure and arrangement of an exemplary sample pairing unit of the microfluidic device of the present invention. As shown in fig. 6(a), the sample pairing unit may have three sample introduction flow channels and inlets, wherein two sides are thebioparticles inlet 501 and thebarcode particles inlet 502, respectively, and the middle is the firstliquid inlet 507, and the first liquid for resuspending the paired bioparticles-barcode particles may be input. By inputting the first liquid at the first solution inlet, the paired bioparticles and barcode particles enter the downstream droplet encapsulating unit along with the first liquid flow direction, and form droplets containing one paired bioparticle-barcode particle with the second liquid.

Fig. 6(b) shows the structure and arrangement of another exemplary sample pairing unit of the microfluidic device of the present invention. As shown in fig. 6(b), one or more reagent flow channels andinlets 509 may be disposed downstream of the release point of the vortex channel of the sample-pairing unit, and reagents required by the downstream unit, such as reagents for cell lysis, nucleic acid amplification or barcode particle dissociation, etc., may be added to the fluid stream containing the paired bioparticles-barcode particles.

The method for analyzing biological particles using barcode particles in a microfluidic device provided by the present invention includes a step of droplet encapsulation, and the microfluidic device provided by the present invention includes a droplet encapsulation unit. Wherein a first liquid containing a single bioparticles or single barcode particles or paired bioparticle-barcode particles is contacted with a second liquid to form droplets containing one paired bioparticle-barcode particle therein.

The terms "droplet" and "droplet" are sometimes used interchangeably herein to refer to a small, generally spherical structure containing at least a first fluid phase, such as an aqueous phase (e.g., water), surrounded by a second fluid phase (e.g., oil) that is immiscible with the first fluid phase. In some embodiments, the second fluid phase will be an immiscible phase carrier liquid.

Various known methods can be used to controllably encapsulate the biological particles or individual barcode particles into droplets.

Where the droplets are contained in an emulsion, the carrier liquid may constitute the continuous phase and the droplets constitute the dispersed phase. The emulsion may further comprise a surfactant and optionally a co-surfactant. The surfactant and/or co-surfactant may be located at the interface of the dispersed and continuous phases. A variety of suitable surfactants are available and one skilled in the art will be able to select a suitable surfactant and/or co-surfactant according to the selected screening parameters. The surfactant is preferably biocompatible. For example, the surfactant may be selected to be non-toxic to the cells or enzymes used in the screening. The selected surfactant may also have good solubility for gases, which may facilitate growth and/or viability of the encapsulated cells.

A wide variety of different emulsification methods are known to those skilled in the art, any of which may be used to create the droplets of the present invention. Many emulsification techniques involve bulk mixing of two liquids, often using turbulence to enhance droplet break-up. Such methods include vortexing, sonication, homogenization, or combinations thereof. For example, in a microfluidic device, an emulsion may be formed by colliding an oil and water stream at a T-connection: the size of the droplets produced varies depending on the flow rate of each stream. A preferred method of preparing droplets for use according to the present invention comprises flow focusing: the continuous phase fluid (focusing or sheath fluid) beside or around the dispersed phase (focused or core fluid) creates droplet breakup near the orifice where both fluids are extruded. The flow focusing apparatus consists of a pressure chamber that is pressurized using a continuous supply of focusing liquid. Internally, one or more focusing fluids are injected through a capillary feed tube, the end of which opens in front of an orifice connecting the pressure chamber with the external environment. The focused fluid stream molds a fluid meniscus into a sharp tip, producing a steady micro or nano jet out of the chamber through the orifice; the jet size is much smaller than the exit orifice. Capillary instability breaks a stable jet into uniform droplets or bubbles. The feed tube may consist of two or more concentric needles and different immiscible liquids or gases injected resulting in composite droplets. Flow focusing ensures that millions of droplets are produced every second very quickly and controllably as the jet breaks.

The size of the droplets follows a probability distribution, such as a gaussian distribution. It will be further appreciated that the parameters used to prepare the microfluidic droplets may be selected to obtain a plurality of microfluidic droplets having a particular volume. Preferably, the droplets in the present invention are monodisperse, having substantially the same shape and/or size. The volume or size of a droplet refers to the average volume or size of a plurality of droplets. One of ordinary skill in the art will be able to determine the average diameter of the population of droplets, for example using laser light scattering or other known techniques. The droplets may be spherical or, in some cases, non-spherical. The diameter of a droplet (particularly an aspherical droplet) can be taken as the diameter of a perfect mathematical sphere having the same volume as the aspherical droplet.

Various methods known in the art may be used to form a one-to-one paired combination of a first droplet and a second droplet. For example, the first droplet and the second droplet may be combined at the channel interface in a one-to-one pairing in the form of an "ABAB" by feeding the first droplet and the second droplet separately through two intersecting microchannels, by controlling the microchannel dimensions and flow rate to cause an ordered periodic spacing of the droplets, and then the period of flow into the first droplet may be matched to the period of flow into the second droplet.

In one aspect of the present invention, the droplet encapsulation unit of the microfluidic device has:

a first liquid channel for inputting a first liquid comprising a single biological particle or a single barcode particle or a paired biological particle-barcode particle;

a droplet-generating element for contacting first and second immiscible liquids and forming droplets containing a pair of bio-particle-barcode particles therein;

a droplet outlet, the droplets formed may be carried by the continuous second liquid phase (e.g. an oil phase) to the outlet, and the droplets leaving the outlet channel may be distributed into the pores for further processing, e.g. heating.

Wherein, the liquid drop generating element can be in the following structure:

a. a second liquid channel for transporting the second liquid (typically an oily liquid), and a junction where the first liquid channel intersects the first liquid channel, the junction being arranged such that the first liquid contacts the second liquid and is separated by the second liquid to produce substantially monodisperse droplets;

or is

b. A chamber containing a second liquid into which the first liquid containing the paired cell-particles enters to form substantially monodisperse droplets.

In the method of the present invention, it is advantageous that the droplets formed contain a high proportion of paired bioparticles-barcode particles. In one aspect of the present invention, the generation of droplets is regulated by adjusting the cross-sectional flow area or the feed flow rate of the first liquid channel and/or the second liquid channel. In one aspect of the invention, the feed flow rate of the first fluid channel and/or the second fluid channel is about 0.1-200 mm/s.

Fig. 7 shows the structure and arrangement of an exemplary droplet encapsulation unit of the microfluidic device provided by the present invention, and a working schematic diagram and experimental result diagram of the droplet encapsulation unit forming a droplet containing a single biological particle or a single barcode particle or a paired biological particle-barcode particle.

Fig. 7(a) and (b) show the structure and arrangement of an exemplary droplet encapsulating unit of the microfluidic device provided by the present invention, and the experiment and results thereof for droplet encapsulation.

As shown in fig. 7(a), the droplet encapsulating unit of the microfluidic device has a firstliquid channel 701 into which a first liquid containing particles is input from an upstream sample pairing unit. In one aspect of the invention, the first liquid is an aqueous solution. The droplet encapsulation unit of the microfluidic device also has a secondliquid channel 702 and ajunction 703 intersecting and communicating with the first liquid channel. The second liquid is a liquid that is insoluble in the first liquid. In one aspect of the invention, the second liquid is an oil. The first liquid contacts the second liquid and is separated by the second liquid to produce substantially monodisperse droplets and enters thedownstream droplet outlet 704. Fig. 7(b) shows an experiment and a result of the droplet encapsulating unit of the microfluidic device shown in fig. 7 (a). A solution containing cells and microbeads is input from a left liquid channel (the sample injection flow rate is about 5mm/s), Oil (QX200 Droplet Generation Oil #1864005, Bio-Rad) is input from liquid channels at the upper end and the lower end (the sample injection flow rate is about 8mm/min), the Oil and the solution containing the cell microbeads form a liquid drop at the intersection, and the liquid drop contains paired single cell-microbeads. It was also observed that droplets containing a single microbead, as well as "empty-packed" droplets, were included in the droplets.

In the structure of fig. 7(a), the secondliquid passage 702 is a structure which intersects the firstliquid passage 701 perpendicularly from both the upper and lower ends and forms a cross with thedownstream passage 704. Fig. 7(c) shows the structure and arrangement of another exemplary droplet encapsulating unit of the microfluidic device provided by the present invention, wherein the secondliquid channel 702 perpendicularly intersects the firstliquid channel 701, and forms a T-shaped structure with thedownstream channel 704.

Fig. 7(d) shows the structure and arrangement of another exemplary droplet encapsulating unit of the microfluidic device according to the present invention, wherein a firstliquid channel 701 is connected to achamber 705 containing a second liquid, and the first liquid containing the paired cell-particles enters the second liquid through the first liquid channel to form substantially monodisperse droplets in the chamber.

Example 6 processing and analysis procedures and units

The method for analyzing biological particles using barcode particles in a microfluidic device provided in the present invention may further include the step of a processing and analyzing unit, and the microfluidic device provided in the present invention includes the processing and analyzing unit therein. Wherein individual paired bioparticles-barcodes contained within the droplets are processed such that the nucleic acids of the bioparticles are contacted and barcoded with the barcodes, and information, in particular nucleic acid information, of the individual bioparticles contained within the droplets is analyzed.

In the method of the present invention, the nucleic acid of a single biological particle contained in each droplet can be subjected to a coding reaction with a barcode particle contained in the same droplet, and when nucleic acids from a plurality of biological particles are mixed, the nucleic acids derived from these biological particles (and containing a barcode sequence) can be traced back to a cell. When the biological particles are cells, the individual cells contained in each droplet are lysed, their nucleic acids are released and a coding reaction takes place with the barcode particles. RNA from cells can be barcoded within the droplets, and when nucleic acids from multiple cells are mixed, nucleic acids derived from these RNAs (and containing barcode sequences) can be traced back to one cell.

The cells contained in the drops may be treated for cell disruption and nucleic acid release. The bar code particles can be decomposed and released. Single cells within the drop may also be made to take up reagents relevant to subsequent reactions. In some cases, the barcode particle may be provided with a light stimulus, causing cleavage of the photolabile bond releasing the oligonucleotide. In some cases, a thermal stimulus may be provided to the barcode particle, and an increase in temperature may result in cleavage of the linkage or release of the oligonucleotide from the particle. In some cases, a chemical stimulus may be provided to the barcode particle, thereby cleaving the linkage of the oligonucleotide to the particle. Or otherwise may cause the oligonucleotide to be released from the bead. In the case of light or thermal stimulation, a heat or light source is introduced through an opening in the microfluidic channel to the body cavity containing the drop.

Various recognition, binding and amplification reactions can be performed on the nucleic acid or barcode sequence of the cell.

The resulting nucleic acid (e.g., amplification product) can be signal labeled and the associated signal detected.

As shown in fig. 2, the present invention provides an exemplary microfluidic device including a processing andanalysis unit 200. The processing and analysis unit may be selected from the following devices or any combination thereof:

a sample application device, for example, for applying a reagent that disrupts cell membranes or increases permeability, or a stimulus that detaches the nucleic acid barcode from the particle, thereby allowing the nucleic acid fragments on the barcode particle to contact and react with the nucleic acid of the cell, including recognition and binding, etc.;

the light stimulation means is, for example, by cleavage of a photolabile bond of the releasable oligonucleotide. A thermally stimulated device, wherein an increase in the temperature of the bead environment may result in cleavage of the linkage or release of the oligonucleotide from the bead;

a nucleic acid amplification device (which includes a temperature controller and the like) for amplifying and pooling a nucleic acid of the cell using the nucleic acid fragment on the barcode particle;

a signal recognition device;

information analysis devices, and the like, include devices that process a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA, and protein) of a single cell, enabling, for example, analysis of the proteome, transcriptome, and genome of the cell.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

Translated fromChinese

1.一种用于微流控设备的进样混匀单元，其用于对微流控设备的样品腔或流道内的样品溶液进行搅拌以保持样品中颗粒(如生物颗粒和/或条形码粒子)的分散状态，所述进样混匀单元包括设置在样品腔或流道底部的体声波谐振器。1. A sample introduction and mixing unit for a microfluidic device, which is used to agitate a sample solution in a sample chamber or a flow channel of a microfluidic device to keep particles (such as biological particles and/or barcode particles) in the sample. ) in a dispersed state, the sample injection and mixing unit includes a bulk acoustic wave resonator arranged at the bottom of the sample cavity or flow channel.2.如权利要求1所述的微流控设备的进样混匀单元，其中所述体声波谐振器输出体声波的功率为约0.1-5mW，优选为约0.5-2mW。2. The sample introduction and mixing unit of the microfluidic device according to claim 1, wherein the power of the bulk acoustic wave output from the bulk acoustic wave resonator is about 0.1-5 mW, preferably about 0.5-2 mW.3.如权利要求1所述的微流控设备的进样混匀单元，其中所述进样混匀单元中所述体声波谐振器输出功率为脉冲式功率。3 . The sample introduction and mixing unit of the microfluidic device according to claim 1 , wherein the output power of the bulk acoustic wave resonator in the sample introduction and mixing unit is pulsed power. 4 .4.如权利要求1所述的微流控设备的进样混匀单元，其中所述体声波谐振器为超高频体声波谐振器，可在溶液内产生频率为约0.5-50GHz，优选为0.1-50GHz的体声波。4. The sample injection mixing unit of the microfluidic device as claimed in claim 1, wherein the bulk acoustic wave resonator is an ultra-high frequency bulk acoustic wave resonator, and the frequency that can be generated in the solution is about 0.5-50 GHz, preferably 0.1-50GHz bulk acoustic wave.5.一种用于微流控设备的样品聚焦单元，例如为用于利用条形码粒子分析生物颗粒(如细胞、微囊泡、生物大分子如核酸)的微流控设备，所述样品聚焦单元包括：5. A sample focusing unit for a microfluidic device, such as a microfluidic device for analyzing biological particles (such as cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles, the sample focusing unit include:流体通道，其具有溶液入口和出口；a fluid channel having a solution inlet and outlet;超高频体声波谐振器，其设置于所述流体通道的底部，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的频率为约0.5-50GHz的体声波；其中，所述超高频体声波谐振器发射传向所述流体通道的对侧的壁的体声波，在溶液中产生由超高频体声波谐振器的体声波产生区域的边界限定的涡旋通道；其中所述微流道的结构和所述超高频体声波谐振器的体声波作用区域的形状和位置，配置为使得溶液样品中的颗粒(如生物颗粒和/或条形码粒子)在经过体声波区域时进入涡旋通道和顺着涡旋通道移动，并在指定位置离开涡旋通道进入下游通道，An ultra-high frequency bulk acoustic wave resonator, which is arranged at the bottom of the fluid channel, the ultra-high frequency bulk acoustic wave resonator can generate a frequency of about 0.5 in the fluid channel to the wall on the opposite side of the fluid channel -50GHz bulk acoustic wave; wherein, the UHF bulk acoustic wave resonator emits the bulk acoustic wave transmitted to the wall on the opposite side of the fluid channel, generating a region in the solution by the bulk acoustic wave generating area of the UHF bulk acoustic wave resonator A vortex channel defined by a boundary of (or barcoded particles) enter and move along the vortex channel when passing through the bulk acoustic wave region, and leave the vortex channel at a designated location to enter the downstream channel,优选的，其中所述体声波谐振器为超高频体声波谐振器，可在溶液内产生频率为约0.5-50GHz的体声波。Preferably, the bulk acoustic wave resonator is an ultra-high frequency bulk acoustic wave resonator, which can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the solution.6.如权利要求5所述的样品聚焦单元，其中所述样品聚焦单元中所述超高频体声波谐振器输出体声波的功率为约20-5000mW，优选为50-2000mW，更优选为100-500mW。6. The sample focusing unit according to claim 5, wherein the power of the output bulk acoustic wave from the UHF BAW resonator in the sample focusing unit is about 20-5000mW, preferably 50-2000mW, more preferably 100 -500mW.7.一种用于利用条形码粒子分析生物颗粒(如细胞、微囊泡、生物大分子如核酸)的微流控设备，所述设备设置为将来自样品的单个生物颗粒与条形码粒子配对和形成其内包含配对的单个生物颗粒-条形码粒子的液滴；7. A microfluidic device for analyzing biological particles (such as cells, microvesicles, biological macromolecules such as nucleic acids) using barcoded particles, the device being configured to pair and form individual biological particles from a sample with barcoded particles A droplet containing paired single bioparticle-barcoded particles within it;其中，所述微流控设备包括以下单元：Wherein, the microfluidic device includes the following units:生物颗粒-条形码粒子配对和液滴封装单元；其用于形成其内包含一个配对的生物颗粒-条形码粒子的液滴；Bioparticle-barcode particle pairing and droplet encapsulation unit; for forming a droplet containing a paired bioparticle-barcode particle therein;以及以下单元中的一个或多个的组合：and a combination of one or more of the following units:如权利要求1-5中任一项定义的用于微流控设备的进样混匀单元；和/或A sample introduction and mixing unit for a microfluidic device as defined in any one of claims 1-5; and/or如权利要求6-16定义的用于微流控设备的样品聚焦单元，A sample focusing unit for a microfluidic device as defined in claims 6-16,优选的，preferably,其中所述生物颗粒-条形码粒子配对和液滴封装单元设置为将来自样品的单个生物颗粒和条形码粒子分别形成其内包含所述生物颗粒或所述条形码粒子的液滴，然后将包含所述生物颗粒或所述条形码粒子的液滴配对和合并，由此形成内包含配对的生物颗粒-条形码粒子的液滴。wherein the bioparticle-barcode particle pairing and droplet encapsulation unit is configured to form a single bioparticle and barcode particle from a sample into droplets containing the bioparticle or the barcode particle, respectively, and then to form droplets containing the bioparticle or the barcode particle therein, respectively. Particles or droplets of the barcode particles are paired and merged, thereby forming droplets containing paired bioparticle-barcode particles within.或者，or,其中所述生物颗粒-条形码粒子配对和液滴封装单元设置为将来自样品的单个生物颗粒与条形码粒子配对，然后形成其内包含配对的生物颗粒-条形码粒子的液滴，例如将包含配对的生物颗粒-条形码粒子的第一液体(通常为水性溶液)与第二液体(通常为油)接触。wherein the bioparticle-barcode particle pairing and droplet encapsulation unit is configured to pair a single bioparticle from a sample with a barcode particle and then form a droplet containing paired bioparticle-barcode particles therein, eg Particles - A first liquid (usually an aqueous solution) of the barcode particles is contacted with a second liquid (usually an oil).8.权利要求7的微流控设备，其中还包括处理和分析单元，所述处理和分析单元对包含在液滴内的单个配对的生物颗粒-条形码进行处理，例如使得生物颗粒的核酸与条形码接触并且条形码化，以及对包含在液滴内的单个生物颗粒的信息，特别是核酸信息进行分析。8. The microfluidic device of claim 7, further comprising a processing and analysis unit that processes a single paired biological particle-barcode contained within the droplet, for example, such that the nucleic acid of the biological particle is associated with the barcode Contacting and barcoding, and analysis of information, particularly nucleic acid information, of individual biological particles contained within the droplets.9.一种采用如权利要求7或8所述微流控设备来利用条形码粒子分析生物颗粒(如细胞、微囊泡、生物大分子如核酸)的方法，其中将来自样品的单个生物颗粒与条形码粒子配对，和形成其内包含配对的生物颗粒-条形码粒子的液滴；9. A method for the analysis of biological particles (such as cells, microvesicles, biological macromolecules such as nucleic acids) using barcoded particles using a microfluidic device as claimed in claim 7 or 8, wherein a single biological particle from a sample is combined with pairing of barcode particles, and forming droplets containing the paired bioparticle-barcode particles therein;所述方法包括以下步骤：The method includes the following steps:进样混匀；例如，其中进样混匀步骤对样品腔内的生物样品溶液或条形码粒子溶液进行搅拌以保持生物颗粒和/或条形码粒子的分散状态。Sample injection mixing; for example, wherein the sample injection mixing step agitates the biological sample solution or the barcode particle solution in the sample chamber to maintain the dispersed state of the biological particles and/or barcode particles.样品聚焦；例如，其中样品聚焦步骤对流道中的生物颗粒或条形码粒子的运动进行调整，消除或减少在流道宽度方向的随机分布；Sample focusing; for example, where the sample focusing step adjusts the motion of biological or barcoded particles in the flow channel to eliminate or reduce random distribution across the width of the flow channel;样品配对和液滴封装；使进入微流道的生物颗粒和条形码粒子形成其内包含一个配对的生物颗粒-条形码粒子的液滴，例如，其中将来自样品的单个生物颗粒和条形码粒子分别形成其内包含所述生物颗粒或所述条形码粒子的液滴，然后将包含所述生物颗粒或所述条形码粒子的液滴配对和合并，由此形成内包含配对的生物颗粒-条形码粒子的液滴，或将来自样品的单个生物颗粒与条形码粒子配对，然后形成其内包含配对的生物颗粒-条形码粒子的液滴，例如将包含配对的生物颗粒-条形码粒子的第一液体(通常为水性溶液)与第二液体(通常为油)接触。Sample pairing and droplet encapsulation; bioparticles and barcode particles entering a microfluidic channel are formed into droplets containing a paired bioparticle-barcode particle, eg, where individual bioparticles and barcode particles from a sample are formed into separate droplets containing said bioparticles or said barcode particles, and then pairing and merging droplets containing said bioparticles or said barcode particles, thereby forming droplets containing paired bioparticles-barcode particles, Alternatively, a single bioparticle from a sample is paired with a barcode particle and then a droplet is formed containing the paired bioparticle-barcode particle within it, for example by combining a first liquid (usually an aqueous solution) containing the paired bioparticle-barcode particle with A second liquid (usually oil) comes into contact.10.如权利要求9所述的方法，其中还包括处理和分析单元步骤，对包含在液滴内的单个配对的生物颗粒-条形码进行处理，例如使得生物颗粒的核酸与条形码接触并且条形码化，以及对包含在液滴内的单个生物颗粒的信息，特别是核酸信息进行分析。10. The method of claim 9, further comprising a processing and analysis unit step of processing the single paired bioparticle-barcodes contained within the droplets, such as contacting the nucleic acids of the bioparticles with the barcodes and barcoding, As well as the analysis of information, especially nucleic acid information, contained within the droplets of individual biological particles.

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