Microfluidic devices and units for analyzing single cells and methods thereof

Abstract

The application provides a sample injection and mixing unit for a microfluidic device and a sample focusing unit. The application also provides a microfluidic device for analyzing from a single cell or a single nucleic acid using barcode particles, which has the sample injection and mixing unit and the sample focusing unit. The application also provides a method for analyzing from a single cell or a single nucleic acid using barcode particles.

Description

Technical Field

[0001] This invention relates to the fields of biology and medical devices. Specifically, this invention relates to a microfluidic system, unit, and method for analyzing individual biological particles such as cells or nucleic acids. Background Technology

[0002] Single-cell sequencing offers unparalleled advantages in embryonic, tumor, and various stem cell studies. With continuous breakthroughs in single-cell sequencing technology and the widespread use of sequencing instruments, single-cell sequencing analysis is increasingly being applied to various cutting-edge biomedical research. No two cells have completely similar genomes and transcriptomes; therefore, averaging omics expression analysis across a large number of cells can average out expression characteristics that would be significant in a single cell.

[0003] Most current single-cell sequencing analysis platforms utilize microfluidic devices. Within these devices, biological materials such as cells and nucleic acids, as well as certain particles (e.g., polymer microspheres, magnetic beads), can precipitate and aggregate even within a short period (e.g., a few minutes), leading to movement issues and even blockages in the microchannels. Conventional methods used in existing technologies, such as magnetic rotors or motor-driven blades, suffer from limitations related to device size and the induction of air bubbles in the liquid.

[0004] There is still a need for better single-cell sequencing analysis platforms and technologies in this field. Summary of the Invention

[0005] This invention provides a sample introduction and mixing unit for a microfluidic device, which is used to stir the sample solution in the sample chamber or channel of the microfluidic device to maintain the dispersion of particles (such as biological particles and / or barcode particles) in the sample. The sample introduction and mixing unit includes a bulk acoustic resonator disposed at the bottom of the sample chamber or channel.

[0006] In one aspect of the invention, the power of the bulk acoustic wave output by the bulk acoustic resonator in the sample mixing unit of the microfluidic device is about 0.1-5mW, preferably about 0.5-2mW.

[0007] In one aspect of the invention, the output power of the bulk acoustic resonator in the sample feeding and mixing unit of the microfluidic device is pulsed power.

[0008] In one aspect of the invention, the bulk acoustic resonator in the sample mixing unit of the microfluidic device is an ultra-high frequency bulk acoustic resonator, which can generate bulk acoustic waves with a frequency of about 0.1-50 GHz, preferably 0.5-50 GHz, in the solution.

[0009] In one aspect of the invention, the ultra-high frequency bulk acoustic resonator in the sample feeding and mixing unit of the microfluidic device is a thin-film bulk acoustic resonator or a solid-state assembled resonator, such as a thickness-stretching vibration mode acoustic resonator.

[0010] The present invention also provides a sample focusing unit for a microfluidic device, such as a microfluidic device for analyzing biological particles (e.g., cells, microvesicles, biological macromolecules such as nucleic acids) using barcode particles.

[0011] In one aspect of the invention, the sample focusing unit includes:

[0012] A fluid channel having a solution inlet and an outlet;

[0013] An ultra-high frequency (UHF) bulk acoustic wave resonator is disposed at the bottom of the fluid channel. The UHF bulk acoustic wave resonator can generate bulk acoustic waves with a frequency of approximately 0.5-50 GHz that propagate towards the opposite wall of the fluid channel. The UHF bulk acoustic wave resonator emits bulk acoustic waves that propagate towards the opposite wall of the fluid channel, generating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generation region of the UHF bulk acoustic wave resonator. The structure of the microchannel 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 biological particles and / or barcode particles) in the solution sample enter the vortex channel and move along the vortex channel when passing through the bulk acoustic wave region, and exit the vortex channel at a designated position to enter the downstream channel.

[0014] In one aspect of the invention, the volume acoustic wave action region of the ultra-high frequency volume acoustic resonator corresponding to the release point in the sample focusing unit has a turning point or curvature change.

[0015] In one aspect of the invention, the shape of the boundary line of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator in the sample focusing unit enables biological particles and barcode particles to remain in the vortex channel and move to the release point, for example by reducing the occurrence of turns or curvature changes in the boundary line of the bulk acoustic wave generation region.

[0016] In one aspect of the present invention, the power of the ultra-high frequency bulk acoustic resonator outputting the bulk acoustic wave in the sample focusing unit is about 20-5000mW, preferably 50-2000mW, and more preferably 100-500mW.

[0017] In one aspect of the invention, the sample focusing unit has a flow rate regulating device that can adjust the speed at which the solution flows through the bulk acoustic region to about 0.1-100 mm / s, preferably about 0.5-50 mm / s, and more preferably about 1-10 mm / s.

[0018] In one aspect of the invention, the sample focusing unit has a flow rate regulating device that can adjust the flow rate of the solution through the bulk acoustic region to about 0.1-500 μL / min, preferably about 0.5-100 μL / min, and more preferably about 1-50 μL / min.

[0019] 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.

[0020] In one aspect of the invention, the volume acoustic wave generation region area of ​​the ultra-high frequency volume acoustic wave resonator in the sample focusing unit is approximately 500-200,000 μm. 2 Preferably, it is about 5000-50000μm 2 The optimal size is approximately 10,000-25,000 μm. 2 .

[0021] In one aspect of the invention, the side length of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator in the sample focusing unit is about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm.

[0022] In one aspect of the invention, the ultra-high frequency bulk acoustic resonator of the sample focusing unit is a thin-film bulk acoustic resonator or a solid-state assembled resonator, such as a thickness-stretching vibration mode acoustic resonator.

[0023] In one aspect of the invention, the bulk acoustic resonator of the sample focusing unit is an ultra-high frequency bulk acoustic resonator, which can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the solution.

[0024] The present invention also provides a microfluidic device for analyzing biological particles (such as cells, microvesicles, and biomacromolecules such as nucleic acids) using barcode particles. The device is configured to pair individual biological particles from a sample with barcode particles and form droplets containing the paired individual biological particle-barcode particles.

[0025] In one aspect of the invention, the microfluidic device includes the following units:

[0026] A bioparticle-barcode particle pairing and droplet encapsulation unit; used to form a droplet containing a paired bioparticle-barcode particle.

[0027] and combinations of one or more of the following units:

[0028] The sample feeding and mixing unit for microfluidic devices as described above; and / or

[0029] The sample focusing unit for microfluidic devices as described above.

[0030] In one aspect of the invention, the microfluidic device includes the following units:

[0031] The sample injection and mixing unit;

[0032] The sample focusing unit; and

[0033] The biological particle-barcode particle pairing and droplet encapsulation unit.

[0034] In one aspect of the invention, the microfluidic device further includes a processing and analysis unit that processes a single paired bioparticle-barcode contained within a droplet, for example, by contacting and barcoding the nucleic acid of the bioparticle with the barcode, and by analyzing information about the single bioparticle contained within the droplet, particularly nucleic acid information.

[0035] In one aspect of the invention, the bioparticle-barcode particle pairing and droplet encapsulation unit in the microfluidic device is configured to form droplets containing the bioparticle or the barcode particle from individual bioparticles and barcode particles from a sample, respectively, and then pair and merge the droplets containing the bioparticle or the barcode particle to form droplets containing paired bioparticle-barcode particles.

[0036] In one aspect of the invention, the bioparticle-barcode particle pairing and droplet encapsulation unit in the microfluidic device is configured to pair a single bioparticle from a sample with a barcode particle, and then form a droplet containing the paired bioparticle-barcode particle, for example, by contacting a first liquid (typically an aqueous solution) containing the paired bioparticle-barcode particle with a second liquid (typically oil).

[0037] In one aspect of the invention, the droplet generating element of the droplet encapsulation unit in the microfluidic device has the following structure:

[0038] a. A second liquid channel for conveying a second liquid, and a connector where a first liquid channel intersects with the second liquid channel, the connector being configured such that the first liquid contacts the second liquid and is separated by the second liquid to produce substantially monodisperse droplets;

[0039] Or for

[0040] b. A cavity containing a second liquid, into which the first liquid enters, forming substantially monodisperse droplets.

[0041] In one aspect of the invention, the processing and analysis unit in the microfluidic device is selected from the following devices or any combination thereof:

[0042] Sample loading devices, such as those used to add reagents that cause cell membrane rupture or increase permeability, or stimulants that cause nucleic acid barcodes to detach from particles, thereby enabling nucleic acid fragments on barcode particles to come into contact with and react with the nucleic acids of cells, including recognition and binding;

[0043] Photostimulation devices, for example, by breaking the photoinstantaneous bonds of oligonucleotides. Thermal stimulation devices, in which an increase in temperature in the bead environment may cause the cleavage of bonds or the release of oligonucleotides from the beads;

[0044] Nucleic acid amplification device (including temperature controller, etc.) is used to amplify and build libraries of cellular nucleic acids using nucleic acid fragments on barcode particles;

[0045] Signal recognition device;

[0046] Information analysis devices include those 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) from a single cell to enable analysis of, for example, the cell's proteome, transcriptome, and genome.

[0047] The present invention also provides a method for analyzing biological particles (such as cells, microvesicles, and biological macromolecules such as nucleic acids) using the aforementioned microfluidic device with barcode particles, wherein a single biological particle from a sample is paired with a barcode particle, and a droplet containing the paired biological particle-barcode particle is formed therein.

[0048] The method includes the following steps:

[0049] Inject and mix thoroughly;

[0050] Sample focusing;

[0051] Sample pairing and droplet encapsulation; enabling bioparticles and barcode particles entering the microfluidic channel to form droplets containing a paired bioparticle-barcode particle.

[0052] In one aspect of the invention, the sample mixing step in the method involves stirring the biological sample solution or barcode particle solution within the sample chamber to maintain the dispersion of the biological particles and / or barcode particles. In another aspect of the invention, the method includes generating bulk acoustic waves with a frequency of approximately 0.1-50 GHz within the chamber using a bulk acoustic resonator disposed at the bottom of the sample chamber. In another aspect of the invention, the bulk acoustic resonator in the method outputs pulsed bulk acoustic waves.

[0053] In one aspect of the invention, the sample focusing step in the method adjusts the movement of biological particles or barcode particles in the flow channel to eliminate or reduce random distribution in the width direction of the flow channel; preferably, sample pairing is performed after sample focusing.

[0054] In one aspect of the invention, the sample focusing step of the method involves passing bioparticles or barcode particles through a microchannel of a bottom ultra-high frequency (UHF) bulk acoustic wave resonator, which generates bulk acoustic waves with a frequency of approximately 0.5-50 GHz propagating towards the opposite wall of the fluid channel. The UHF bulk acoustic wave resonator emits bulk acoustic waves towards the opposite wall of the fluid channel, creating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generation region of the UHF bulk acoustic wave resonator. The structure of the microchannel and the shape and position of the bulk acoustic wave action region of the UHF bulk acoustic wave resonator are configured such that the bioparticles or barcode particles for pairing enter and move along the vortex channel as they pass through the bulk acoustic wave region, and exit the vortex channel at a designated location into a downstream channel. In one aspect of the invention, the bulk acoustic wave action region of the UHF bulk acoustic wave resonator at the release point has a bend or curvature change. In one aspect of the invention, the shape of the boundary line of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator causes biological particles and barcode particles to remain in the vortex channel and move to the release point, for example by reducing the occurrence of bends or curvature changes in the boundary line of the bulk acoustic wave generation region.

[0055] In one aspect of the invention, the power of the ultra-high frequency bulk acoustic resonator outputting the bulk acoustic wave in the focusing step is about 20-5000mW, preferably 50-2000mW, and more preferably 100-500mW.

[0056] In one aspect of the invention, the velocity of the solution flowing through the bulk acoustic region in the microchannel during the focusing step is adjusted to about 0.1-100 mm / s, preferably about 0.5-50 mm / s, and more preferably about 1-10 mm / s.

[0057] In one aspect of the invention, the velocity of the solution flowing through the bulk acoustic region in the focusing step is adjusted to about 0.1-500 μL / min, preferably about 0.5-100 μL / min, and more preferably about 1-50 μL / min.

[0058] In one aspect of the invention, the method further includes a processing and analysis unit step of processing a single paired biological particle-barcode contained within a droplet, for example, by contacting and barcoding the nucleic acid of the biological particle with the barcode, and analyzing information of the single biological particle contained within the droplet, particularly nucleic acid information.

[0059] In one aspect of the invention, the method involves forming droplets containing either a single biological particle or a barcode particle from a sample, and then pairing and merging the droplets containing the biological particle or the barcode particle to form droplets containing paired biological particle-barcode particles.

[0060] In one aspect of the invention, the method involves pairing individual biological particles from a sample with barcode particles, and then forming droplets containing the paired biological particle-barcode particles, for example, by contacting a first liquid (typically an aqueous solution) containing the paired biological particle-barcode particles with a second liquid (typically oil).

[0061] In one aspect of the invention, the droplet encapsulation in the method is performed in the following manner:

[0062] a. The first liquid channel for transporting the first liquid is brought into contact with the second liquid channel for transporting the second liquid at the joint, such that the first liquid is separated by the second liquid to produce substantially monodisperse droplets;

[0063] Or for

[0064] b. The first liquid is introduced into a cavity containing the second liquid, and substantially monodisperse droplets are formed within the second liquid. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 This is a schematic diagram illustrating the steps involved in the method for analyzing biological particles (such as cells, microvesicles, and biological macromolecules such as nucleic acids) using barcode particles in a microfluidic device, as provided by the present invention.

[0067] Figure 2 The present invention provides a schematic diagram of the setup structure and operation mode of the ultra-high frequency bulk acoustic resonator in the method and microfluidic device.

[0068] Figure 3 The diagram illustrates the principle and phenomenon of the ultra-high frequency bulk acoustic resonator in the microfluidic device and the movement of particles in the solution within the microchannel caused by bulk acoustic waves.

[0069] Figure 4This is a schematic diagram of the structure and configuration of an exemplary sample injection and mixing unit of the microfluidic device provided by the present invention, and a diagram showing the working result of the sample mixing unit mixing the sample.

[0070] Figure 5 This is a schematic diagram of the structure and configuration of an exemplary sample focusing unit of the microfluidic device provided by the present invention, and a diagram showing the working results of the sample focusing unit focusing and "queuing" biological particles or particles in the microchannel.

[0071] Figure 6 This is yet another exemplary structure and arrangement of the sample pairing unit for the microfluidic device of the present invention.

[0072] Figure 7 The diagram shows the structure and configuration of an exemplary droplet encapsulation unit for a microfluidic device provided by the present invention, as well as a working diagram and experimental results diagram of the droplet encapsulation unit forming droplets containing paired biological particles-barcode particles. Detailed Implementation

[0073] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0074] Example 1: Experimental Methods and Materials

[0075] Microfluidic channel fabrication:

[0076] Microfluidic channels made of polydimethylsiloxane (PDMS) were fabricated using soft photolithography.

[0077] Fabrication of ultra-high frequency bulk acoustic resonators:

[0078] Bulk acoustic wave resonator devices are fabricated on silicon-based wafers using methods such as chemical vapor deposition, metal sputtering, and photolithography. The specific methods are as follows:

[0079] 1. Thoroughly clean the surface of the silicon wafer using a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:1. This method can effectively remove organic and inorganic substances from the silicon wafer.

[0080] 2. On a cleaned silicon wafer, an aluminum nitride thin film is formed by surface sputtering, followed by the deposition of a silicon dioxide thin film using ion-enhanced chemical vapor deposition. Then, using the same method, aluminum nitride and silicon dioxide thin films are deposited alternately to form a Bragg acoustic reflection layer structure with alternating layers of aluminum nitride and silicon dioxide.

[0081] 3. A 600nm molybdenum thin film is sputtered onto the Bragg reflector structure as the bottom electrode. Then, standard photolithography techniques, including resist coating, exposure, and development, are used to photolithographically shape the molybdenum electrode film, followed by etching to form the bottom electrode with the target pattern.

[0082] 4. Sputter another aluminum nitride thin film onto the molybdenum electrode as a piezoelectric layer. Use dry etching to define the pattern of the aluminum nitride thin film.

[0083] 5. The pattern on the photomask is transferred using negative photoresist, and then a 50nm thick titanium-tungsten alloy layer is sputtered. This layer acts as an adhesion layer to increase the adhesion of the gold electrode. Next, a 300nm thick gold film is grown as the top electrode using vapor deposition. Finally, acetone is used to remove the gold film around the target pattern, forming the gold electrode with the target pattern.

[0084] Finally, the bulk acoustic wave resonator device is bonded and integrated with the PDMS microchannel chip. The bulk acoustic wave resonator device is positioned in the middle of the channel.

[0085] The bulk acoustic wave resonator device is connected to a network analyzer using a standard SMA connector. By testing the spectrum and finding the resonance peak, the frequency of the bulk acoustic waves emitted by the device in the microchannel can be measured. In the embodiments of this application, the frequency of the bulk acoustic wave resonator fabricated and used is approximately 1.5 GHz to 2.0 GHz.

[0086] Instruments and materials

[0087] High-frequency signal generator: (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz)

[0088] Power amplifier: Mini-Circuits, with 35dBm enhancement of the original RF source power.

[0089] Injection pump: New Era Pump Systems, Inc., NE-1000

[0090] cell:

[0091] HeLa cell line: Guangzhou Genio Biotechnology Co., Ltd., ATCC#CCL2

[0092] Cell culture:

[0093] 293T cells were cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100 U / ml penicillin (Thermo), and 100 μg / ml streptomycin (Thermo). The cell density was 1 x 10⁻⁶ cells / mL. 5 / mL~2x10 6 / mL. For microfluidic experiments, it can be diluted to 1x10⁵ / mL. PBS buffer (Gibco).

[0094] Dye:

[0095] Calcein-AM (Beijing Solarbio Technology Co., Ltd., China)

[0096] 4',6-Diamidinyl-2-phenylindole (DAPI) (Invitrogen, USA)

[0097] Example 2

[0098] In the specific implementation of this embodiment, a method and microfluidic device are provided for analyzing biological particles (such as cells, microvesicles, and biomolecules such as nucleic acids) using barcode particles in a microfluidic device, particularly analyzing the nucleic acids of said biological particles. The method and microfluidic device provided by this invention pair individual biological particles (such as cells, microvesicles, and biomolecules such as nucleic acids) from a biological sample with barcode particles. Then, a first liquid (usually an aqueous solution) containing the paired individual biological particle-barcode particle is contacted with a second liquid (which is immiscible with the first liquid, usually an oily liquid) to form a droplet (e.g., a water-in-oil droplet) containing one paired biological particle-barcode particle. The method further includes further processing and analysis of the nucleic acids of the individual biological particles encapsulated in the droplet, such as nucleic acid amplification and library construction.

[0099] The method and microfluidic device provided by this invention are suitable for processing biological particles that typically have a diameter of about 0.01-30 μm, preferably 0.2-25 μm, and more preferably 0.5-20 μm.

[0100] In one aspect of the invention, the bioparticles are cells or vesicles released by cells into the extracellular environment. Cells include natural or cultured cells of higher plants or animals (e.g., mammals including humans), as well as single-celled or simple multicellular organisms such as bacteria and fungi. Vesicles are microvesicles released by various animal cells into the extracellular environment. These cell-associated microvesicles are vesicle-like bodies with a double-membrane structure that detach from the cell membrane or are secreted by the cell. Cell-released microvesicles include exosomes, microvesicles, vesicles, membrane vesicles, vesicles, gas bubbles, prostate bodies, microparticles, intraluminal vesicles, intranuclear body-like vesicles, or exocytic vesicles, etc. Cell-released microvesicles have a diameter of approximately 30-1000 nm, approximately 30-800 nm, approximately 30-150 nm, or approximately 30-100 nm.

[0101] In one aspect of the invention, the biological particles are viruses, including virusoids, viruses, and viral particles. The viruses can be DNA / RNA viruses, as well as protein viruses. Although viruses do not have a complete cellular structure, cells also include viruses herein.

[0102] In one aspect of the invention, the bioparticles in the method are nucleic acid, protein, or polysaccharide molecules, particularly nucleic acid molecules. As used herein, “nucleic acid” (and the equivalent term “polynucleotide”) refers to a polymer of ribonucleosides or deoxyribonucleosides containing phosphodiester bonds between nucleotide subunits. Nucleic acids include, but are not limited to, genetic 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, such as amplification reaction products using artificial or natural DNA or RNA as templates. Nucleic acids can be double-stranded or single-stranded, circular or linear. The method of the present invention is particularly suitable for separating nucleic acids ≥200 bp in length, preferably ≥1 kbp, more preferably ≥10 kbp, for example ≥50 kbp (e.g., any form of DNA and RNA, including natural or synthetic nucleic acids, such as amplification reaction products using DNA or RNA as templates).

[0103] Samples that can be used to detect biological particles include: cell cultures, eukaryotic microorganisms, or diagnostic samples such as body fluids, body fluid sediments, gastric lavage samples, fine needle aspirates, biopsy samples, tissue samples, cancer cells, cells from patients, cells from tissues, or in vitro cultured cells from individuals being tested and / or treated for diseases or infections, or forensic samples. Non-limiting examples of body fluid samples include whole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, serum, plasma, urine, chyle, feces, ejaculation, sputum, nipple aspiration, saliva, swab samples, irrigation or lavage fluid, and / or swab samples.

[0104] The barcode particles used in the method of the present invention are particles with nucleic acid barcodes, the particles having nucleic acid fragments bound thereto. The nucleic acid fragments include barcodes. A “barcode” or “barcode sequence” refers to any unique sequence marker that can be coupled to at least one nucleotide sequence for, for example, later identification of said at least one nucleotide sequence. The nucleic acid fragment 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 a sample, each RNA sequence may be linked to a different UMI sequence during barcoding. The nucleic acid fragments may also include binding sequences that recognize and bind nucleic acids to the biological particles to be analyzed, such as RNA that can bind and amplify to sample cells. The particles may be beads, chromatographic resins, multiwell plates, microcentrifuge tubes, etc., such as beads, microbeads, microparticles, microspheres, nanoparticles, nanobeads, or hydrogels. In one aspect of the invention, the particles are beads, such as spherical beads made of metallic and / or polymeric materials. In one aspect of the invention, the particles are hydrogel particles. The diameter of the barcode particles used in the methods and microfluidics of the present invention is in the range of about 0.1 μm to 100 μm. The barcode-containing nucleic acid can be bound to the particles using any desired mechanism such as chemical bonds or interactions such as biotin-avidin, biotin-streptavidin, gold-thiol, etc.

[0105] Figure 1 This is a schematic diagram illustrating the steps involved in the method of analyzing biological particles (such as cells, microvesicles, and biomolecules such as nucleic acids) using barcode particles in a microfluidic device according to the present invention. Figure 1 As shown, the method of the present invention may include the following steps:

[0106] 1. Sample injection and mixing. Add the sample containing the biological particles to be tested (such as cells) to the sample chamber, and add barcode particles to another sample chamber. Stir the biological sample solution or particle solution in the sample chamber to maintain the dispersion of biological particles (such as cells) and / or particles, and avoid precipitation and / or aggregation.

[0107] 2. Sample focusing. The movement of biological particles (such as cells) or barcode particles in the flow channel is adjusted to make the biological particles and barcode particles converge, eliminating or reducing random distribution in the width direction of the flow channel;

[0108] 3. Sample-barcode particle pairing and droplet encapsulation. This process enables biological particles (such as cells, microvesicles, and biomolecules such as nucleic acids) entering the microfluidic channel to pair with barcode particles, forming droplets containing individual paired biological particle-barcode particles.

[0109] 4. Processing and Analysis. The individual paired bioparticles contained within the droplet are processed to bring the nucleic acids of the bioparticles into contact with and barcode the codes. Information about the individual bioparticles contained within the droplet, particularly their nucleic acid information, is then analyzed.

[0110] The above steps can be used independently or in various combinations in the method of the present invention. Combinations include, for example: Figure 1 The various combinations and execution methods shown are illustrated (arrows indicate the execution order of each program).

[0111] In one aspect of the present invention, the method of the present invention may include all four steps described above, such as injecting and mixing the target biological sample, focusing the sample, injecting and mixing the barcode particles and focusing the sample, and then pairing the focused biological sample with the barcode particles and encapsulating the droplets, processing and analyzing them.

[0112] In another aspect of the invention, the method may include some of the steps described above. For example, in the method of the invention, the target biological sample and the barcode-coded particles may be directly sample-paired, droplet-encapsulated, processed, and analyzed; or, for another example, in the aforementioned method, the biological sample and the barcode-coded particles may be sample-focused separately before the step of sample-pairing the biological sample and the barcode-coded particles.

[0113] This invention also provides a device for processing and analyzing biological particles using barcode particles. In this 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 at the micrometer or even nanometer scale. Typical microfluidic systems and devices typically comprise structural and functional units at the millimeter scale or smaller.

[0114] Figure 2 The present invention provides a schematic diagram of the configuration structure and operation mode of the ultra-high frequency bulk acoustic resonator in the microfluidic device. Figure 2 (a) is a schematic diagram (top view) of an ultra-high frequency bulk acoustic resonator installed in a microfluidic device. Figure 2 As shown in (a), the microfluidic device 10 for processing and analyzing biological particles provided by the present invention may include the following units:

[0115] - Sample injection and mixing unit 100;

[0116] -Sample focusing unit 200;

[0117] - Pairing and droplet packaging unit 300;

[0118] - Processing and analysis unit 700 (not shown).

[0119] The fluid channels, or microchannels, of the microfluidic device provided by this invention are generally closed except for openings for fluid entry and exit. The cross-section of the fluid channel typically has dimensions of 0.1-500 μm and can be of various shapes, including elliptical, rectangular, square, triangular, and circular. The fluid channels can be fabricated using various known microfabrication techniques, and the materials include, but are not limited to, silica, silicon, quartz, glass, or polymeric materials (e.g., polydimethylsiloxane, i.e., PDMS, plastics, etc.). The channels can be coated. The coating can alter the properties of the channel and can be patterned. For example, the coating can be hydrophilic, hydrophobic, magnetic, conductive, or biofunctionalized.

[0120] The microfluidic device provided by this invention employs an ultra-high frequency bulk acoustic resonator. For example... Figure 2 As shown, the microfluidic device of this embodiment has multiple ultra-high frequency bulk acoustic wave resonators (shown in dark graphics) disposed at the bottom of the cavity or flow channel. The movement of biological particles or barcode particles is controlled by the bulk acoustic waves generated in the solution by the ultra-high frequency bulk acoustic wave resonators. In this invention, an ultra-high frequency bulk acoustic wave resonator refers to a resonator capable of generating bulk acoustic waves with a frequency of approximately 0.5-50 GHz (e.g., approximately 1-50 GHz). The ultra-high frequency bulk acoustic wave resonator used in this invention can be a thin-film bulk acoustic wave resonator or a solid-state assembled resonator, such as a thickness-stretching vibration mode acoustic wave resonator.

[0121] The ultra-high frequency bulk acoustic resonator used in this invention is well-suited to the material constituting the wall of a microfluidic device, allowing for convenient placement within the device's wall, such as in a liquid-containing cavity or at the bottom of a microfluidic channel. This ultra-high frequency bulk acoustic resonator can generate bulk acoustic waves propagating to the opposite side within the solution.

[0122] Figure 2 (b) is a cross-sectional view showing the structure of the fluid channel (section AA) of the microfluidic device and the ultra-high frequency bulk acoustic resonator (section BB) located at the bottom of the flow channel of the microfluidic device or the ultra-high frequency bulk acoustic resonator (section CC) located at the bottom of the cavity of the microfluidic device. Figure 2 (c) and (d) are schematic diagrams illustrating the operation of an ultra-high frequency bulk acoustic resonator and its effects in solution. For example... Figure 2As shown in (b), an ultra-high frequency (UHF) bulk acoustic wave resonator can be disposed within the wall (typically the bottom 13) of the microchannel 11 or cavity 12 of the microfluidic device. The top of the UHF bulk acoustic wave resonator is disposed on the surface of the wall of the microfluidic device (such as the bottom surface of the cavity or microchannel), generating bulk acoustic waves propagating perpendicularly to the wall to the opposite side (such as above the top 14 of the channel or the cavity 12). The region formed by the top surface of the UHF bulk acoustic wave resonator is the bulk acoustic wave generation region, also referred to herein as the bulk acoustic wave region. Figure 2 As shown in (b), the ultra-high frequency bulk acoustic resonator includes, from bottom to top, an acoustic wave reflecting layer 15, a bottom electrode layer 16, a piezoelectric layer 18, and a top electrode layer 17. The overlapping area of ​​the bottom electrode layer, the piezoelectric layer, the top electrode layer, and the acoustic wave reflecting layer constitutes the bulk acoustic wave generating region.

[0123] The inventors of this application discovered that, as Figure 2 As shown in (c) and (d), in the device of the present invention, the ultra-high frequency bulk acoustic resonator can operate in two modes. Figure 2 As shown in (d), within a cavity (height greater than 1000 μm, for example, greater than 5000 μm), an ultra-high frequency bulk acoustic resonator emits bulk acoustic waves propagating towards the top, generating an acoustic fluid in the solution and creating disturbances that keep the particles in the solution dispersed. In this mode of operation, pulsed power is typically applied to the ultra-high frequency bulk acoustic resonator to achieve better dispersion of particles in the solution.

[0124] In addition, such as Figure 2As shown in (c), an ultra-high frequency (UHF) bulk acoustic wave resonator in a microchannel (height no greater than 500 μm, e.g., no greater than 300 μm) can emit bulk acoustic waves that propagate towards the opposite wall of the fluid channel, generating vortices in the solution along the bulk acoustic wave generation region of the UHF resonator. These continuous vortices form a vortex channel defined by the boundary of the bulk acoustic wave generation region of the UHF resonator. The forces acting on particles within the vortices include the stokes drag force generated by the vortex, the inertial lift force generated by laminar flow, and the acoustic radiation force caused by acoustic wave attenuation. The magnitude of the stokes drag force is positively correlated with particle size (e.g., particle diameter), while the magnitude of the acoustic radiation force is positively correlated with the square of the particle size. As the particle size increases, the forces shift from being dominated by stokes drag to being dominated by acoustic radiation, which pushes the particles towards the center of the vortex. Larger particles experience greater acoustic radiation forces and move to the center of the vortex; smaller particles rotate on the periphery under the drag force of the vortex, and even smaller particles may leave the vortex. Additionally, particles move downstream of the volumetric acoustic wave region under the lateral drag force generated by laminar flow. When the forces acting on particles retained in the vortex reach a certain equilibrium, the particles stop at their respective positions within the vortex channel and do not move relative to the flow channel.

[0125] The inventors of this application unexpectedly discovered that, in the aforementioned Figure 2 (c) shows the action mode of the ultra-high frequency bulk acoustic wave resonator in the microchannel. When the particles pass through the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator set in the microchannel, under certain parameter conditions, the following three movement modes will occur (the relevant findings are also described and disclosed in the filed patent applications PCT / CN2020 / 096131 and PCT / CN2020 / 096178. The full text of these patent applications is incorporated herein by reference). Figure 3 This diagram illustrates the movement of particles within the bulk acoustic wave (BAW) region of an ultra-high frequency BAW resonator located in the microchannel during this operation. Figure 3As shown, the ultra-high frequency bulk acoustic wave resonator 21 emits bulk acoustic waves in the microchannel 11 that propagate to the opposite wall (i.e., the top of the channel, not shown) of the fluid channel, generating vortices 22 in the solution that are distributed along the boundary 23 of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator. A series of vortices constitute a vortex channel 24 defined by the boundary 23 of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator. When particles in sample solution 25 pass through the bulk acoustic wave action area, under different parameter conditions (channel height, flow velocity, bulk acoustic wave power generated by the ultra-high frequency bulk acoustic wave resonator, particle size, shape and properties, as well as the properties of the solution itself and its relative properties with respect to the particles, etc.), they can (a) pass through the bulk acoustic wave action area with basically no impact (without entering the vortex channel); (b) enter the vortex channel and move along the vortex channel and then leave the vortex channel at a certain position in the vortex channel, which can be called the release point; or (c) enter the vortex channel and move along the vortex channel and stay at a certain position in the vortex channel, which can be called the capture point. Under certain device and fluid conditions (i.e., fixed channel height, bulk acoustic power, flow velocity, solution properties, etc.), larger particles 251 are more likely to be "captured," that is, enter the vortex channel, move along the vortex channel, and remain at the capture point in the vortex channel. Smaller particles 252 are more likely to enter the vortex channel, move along the vortex channel, and then leave the vortex channel from the release point. Even smaller particles 253 tend not to enter the vortex channel. The release point and the capture point are usually the same location. Within a certain range, by adjusting different parameter conditions (such as changing the channel height, flow velocity, and bulk acoustic power generated by the ultra-high frequency bulk acoustic resonator), the same particles can exhibit the above three movement patterns respectively: not entering the vortex channel, entering the vortex channel, moving along the vortex channel, and then remaining at a certain position in the vortex channel, or entering the vortex channel, moving along the vortex channel, and then leaving the vortex channel at a certain position. The same particles can also be made to switch between the three movement modes mentioned above by adjusting relevant parameters. For example, they can switch from not entering the vortex channel to entering the vortex channel, or from staying at a certain position in the vortex channel to leaving the vortex channel. In addition, by setting certain parameter conditions, different particles entering the vortex channel can produce the three movement modes mentioned above respectively. Thus, by setting an ultra-high frequency bulk acoustic resonator in the microchannel and adjusting various relevant parameters (channel height, flow velocity, bulk acoustic power generated by the ultra-high frequency bulk acoustic resonator, particle size, shape and properties, and the properties of the solution itself and its relative properties with respect to the particles, it is possible to distinguish, capture, separate, enrich one or more types of particles in a solution sample, and control the movement of particles in the microchannel.

[0126] In one aspect of the invention, in the method and microfluidic device for analyzing biological particles using barcode particles, biological particles (such as cells or nucleic acids) or barcode particles in the solution can be controlled by an ultra-high frequency (UHF) bulk acoustic wave resonator disposed in the microchannel to enter and move along the vortex channel induced by the UHF bulk acoustic wave in the solution, and to stop or leave the vortex channel at a predetermined position. In another aspect of the invention, by adjusting the shape and position of the bulk acoustic wave action area of ​​the UHF bulk acoustic wave resonator, controlled biological particles (such as cells or nucleic acids) or barcode particles in the solution can enter and move along the vortex channel, and leave the vortex channel at a predetermined position. Thus, the biological particles (such as cells or nucleic acids) or barcode particles leave the bulk acoustic wave action area at a specified position and direction, entering a desired outflow channel. This predetermined position of leaving the vortex channel is called the release point, which is the position where the biological particles (such as cells or nucleic acids) or barcode particles leave the bulk acoustic wave action area. The solution, after removing the controlled moving biological particles, continues to flow in the inflow direction.

[0127] One of the key factors contributing to the detachment of biological particles (such as cells or nucleic acids) or barcode particles from the vortex channel is the influence of laminar flow along the fluid channel direction; therefore, the release point is typically located downstream of the vortex channel. In another aspect of the invention, the release point is typically located where the vortex channel exhibits a turning point or a change in curvature, i.e., above the location where the boundary of the bulk acoustic wave action area exhibits a turning point or a change in curvature. In other words, the boundary of the bulk acoustic wave action area corresponding to the release point exhibits a turning point or a change in curvature. Without being constrained by relevant theories, the inventors believe that the reason for this phenomenon is that at the turning point or corner of the vortex channel, the direction of the vortex and the direction of the acoustic radiation force suddenly change. Among the biological particles (such as cells or nucleic acids) or barcode particles entering the vortex channel, those particles that meet the appropriate conditions (such as appropriate size) are already focused on the center of the vortex. Under the action of the acoustic radiation force, they can change their direction of motion with the vortex channel and quickly refocus on the center of the vortex channel after the turning point. On the other hand, biological particles (such as cells or nucleic acids) or barcode particles that do not meet the conditions (such as having a smaller size) will be more affected by the sudden change in the direction of laminar dragging and thus leave the vortex channel.

[0128] In one aspect of the present invention, the above-described method provided by the present invention is suitable for processing liquid samples containing a large number of biological particles (such as cells or nucleic acids) or barcode particles; the large number of biological particles or barcode particles can enter and move along the vortex channel in a continuous moving manner, and leave the vortex channel at a set position, thereby achieving the purpose of rapid and high-throughput processing.

[0129] In one aspect of the invention, the method further includes adjusting the amount of bioparticles or barcode particles entering the vortex channel by adjusting the height of the microchannel, the power of the generated bulk acoustic wave, and / or by adjusting the velocity of the solution flowing through the bulk acoustic wave region. Bioparticles or barcode particles that do not enter the vortex channel pass through the bulk acoustic wave region and flow out in the direction the sample enters the fluid channel.

[0130] In one aspect of the invention, the boundary lines (i.e., the shape of the corresponding vortex channel) of the ultra-high frequency bulk acoustic resonator in the above method are configured to allow biological particles or barcode particles to move along the vortex channel to the release point. This prevents biological particles or barcode particles from leaving the vortex channel instead of exiting from the release point as intended.

[0131] In another aspect of the invention, the target biological particle or barcode particle is kept within the vortex channel and moves to the release point by adjusting the boundary shape of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator. As previously mentioned, the presence of bends or curvature changes in the boundary lines of the bulk acoustic wave generation region may increase the probability of particles escaping the vortex channel. Therefore, particles can be kept within the vortex channel by reducing the occurrence of bends or curvature changes in the boundary lines of the bulk acoustic wave generation region, i.e., reducing the number of biological particles or barcode particles escaping the vortex channel.

[0132] In one aspect of the invention, in the method or microfluidic device of the invention, biological particles (such as cells or nucleic acids) or barcode particles can be controllably detained or released in the bulk acoustic wave generation region of the ultra-high frequency (UHF) bulk acoustic wave resonator disposed in the microchannel. In another aspect of the invention, the method or microfluidic device can control the types of biological particles or barcode particles detained or released in the bulk acoustic wave generation region of the UHF bulk acoustic wave resonator, for example, by allowing biological particles or barcode particles of different sizes to remain in the bulk acoustic wave generation region of the UHF bulk acoustic wave resonator, and then releasing biological particles or barcode particles of different sizes, particularly releasing biological particles or barcode particles of different sizes in ascending order of size. In yet another aspect of the invention, the release of "captured" particles or particles in ascending order of size can be controlled by adjusting the power of the bulk acoustic wave.

[0133] The ultra-high frequency bulk acoustic wave resonator in this invention refers to a resonator capable of generating sound waves with frequencies exceeding 0.5 GHz, for example, frequencies between 0.5 and 50 GHz (preferably not less than 1 GHz). The ultra-high frequency bulk acoustic wave resonator can be a thin-film bulk acoustic wave resonator or a solid-state assembled resonator.

[0134] In this invention, the shape of the bulk acoustic wave action region (typically the top of the UHF bulk acoustic wave resonator) of the UHF bulk acoustic wave resonator includes, but is not limited to, one of the following: circular, elliptical, semicircular, parabolic, polygonal with an acute or obtuse angle at the vertex, polygonal with an arc replacing the vertex, polygonal with an acute angle, semicircular, or parabolic vertex, or a square or ring array of the same shape arranged in repetition. This application provides acoustic wave action regions of the above shapes, but acoustic wave action regions of any other shape are also within the scope of protection of this application.

[0135] In one aspect of the invention, in the method for analyzing biological particles using barcode particles provided by the invention, or in the microfluidic device provided by the invention, the height of the microfluidic channel (hereinafter referred to as microchannel or channel) in the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator (the distance between the top of the ultra-high frequency bulk acoustic wave resonator and its opposite wall, typically the distance from the bottom to the top of the channel) is about 10-300 μm.

[0136] In one aspect of the invention, in the method for analyzing biological particles using barcode particles provided by the invention, or in the microfluidic device provided by the invention, the area of ​​the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is approximately 500-200,000 μm. 2 Preferably, it is about 5000-50000μm 2 The optimal size is approximately 10,000-25,000 μm. 2 .

[0137] In one aspect of the present invention, in the method for analyzing biological particles using barcode particles provided by the present invention or in the microfluidic device provided by the present invention, the length (fluid direction within the microchannel) or width (direction perpendicular to the fluid direction within the microchannel) of the ultra-high frequency bulk acoustic resonator is about 20-500 μm, preferably about 40-400 μm.

[0138] In one aspect of the invention, in the method for analyzing biological particles using barcode particles provided by the invention, or in the microfluidic device provided by the invention, the ultra-high frequency bulk acoustic resonator generates bulk acoustic waves with a power of about 0.5-5000mW, preferably about 10-2000mW.

[0139] In one aspect of the invention, in the method for analyzing biological particles using barcode particles provided by the invention, or in the microfluidic device provided by the invention, the velocity of the solution flowing through the bulk acoustic region of the ultra-high frequency bulk acoustic resonator is about 0.1-100 mm / s, preferably about 0.5-5 mm / s.

[0140] In one aspect of the invention, in the method for analyzing biological particles using barcode particles provided by the invention, or in the microfluidic device provided by the invention, the velocity of the solution flowing through the bulk acoustic region of the ultra-high frequency bulk acoustic resonator is about 0.1-500 μL / min, preferably about 0.5-30 μL / min.

[0141] In the microfluidic device of this invention, the flow rate of the injected liquid can be controlled by an external pressure source, an internal pressure source, electrodynamics, or magnetodynamics. The external and internal pressure sources can be pumps, such as peristaltic pumps, syringe pumps, or pneumatic pumps. In this embodiment, a computer-tuned syringe pump is used to control the liquid injection flow rate.

[0142] The microfluidic device of the present invention further includes a power adjustment device that adjusts the power of the bulk acoustic waves generated by the ultra-high frequency bulk acoustic resonator. In this embodiment, the power adjustment device is a power amplifier with power adjustment function. Since the thin-film bulk acoustic resonator has high energy conversion efficiency and virtually no loss, the output power of the power adjustment device can be essentially considered as the output power of the thin-film bulk acoustic resonator generating bulk acoustic waves in the fluid. In the microfluidic device of the present invention, the power adjustment device can be connected to a high-frequency signal generator. The output circuit of the power amplifier is connected to the bottom electrode, piezoelectric layer, and top electrode of the ultra-high frequency bulk acoustic resonator, respectively.

[0143] Example 3 Sample injection and mixing steps and sample injection and mixing unit

[0144] The method for analyzing biological particles using barcode particles in a microfluidic device provided by this invention may include a sample introduction and mixing step, and the microfluidic device provided by this invention may include a sample introduction and mixing unit. The method provided by this invention involves adding a sample containing the biological particles to be tested (such as cells) into a sample chamber of the microfluidic device, and adding barcode particles into another sample chamber. In one aspect of this invention, the method includes stirring the biological sample solution or particle solution located in different sample chambers respectively to maintain the dispersed state of the biological particles (such as cells) and / or barcode particles (such as magnetic beads) and avoid precipitation and / or aggregation.

[0145] In one aspect of the invention, the biological sample solution or particle solution is stirred by a bulk acoustic wave generated by an ultra-high frequency bulk acoustic resonator disposed at the bottom of the sample chamber of a microfluidic device.

[0146] like Figure 2 As shown, the exemplary microfluidic device provided by the present invention includes a sample injection and mixing unit 100. Figure 4 The diagram shows the structure and setup of an exemplary sample mixing unit for a microfluidic device provided by the present invention, as well as the working result of the sample mixing unit mixing the sample.

[0147] like Figure 4 As shown in (a), the sample feeding and mixing unit 100 includes a sample inlet or sample outlet 101 and a sample chamber 102. A solution of biological particles (such as cells) and / or barcode particles is added into the sample chamber 102 from the sample inlet 101 and then enters the downstream processing unit through the sample outlet 103 and the sample channel 104.

[0148] The sample chamber 102 is a cylindrical body with a circular cross-section, a volume of approximately 0.2-2.0 ml, and a height of approximately 0.5-2.0 cm. The sample dispensing port may or may not have a top cap.

[0149] The sample injection and mixing unit also includes an ultra-high frequency bulk acoustic resonator 105 disposed at the bottom of the sample chamber, which can generate bulk acoustic waves with a frequency of approximately 0.5-50 GHz within the solution in the sample chamber. Figure 4 As shown in (b) and (c), within the cavity (typically greater than 0.2 cm in height, for example, greater than 0.5 cm), the bulk acoustic waves act as described in Example 2. Figure 2 (d) illustrates the working mode, where bulk acoustic waves generate swirling currents in the solution, causing swirling and disturbance in the solution within the sample chamber, thus keeping the cells or barcode particles in the solution suspended and dispersed. Since the chamber does not have a top cover, or the distance between its top cover and the bottom is large (greater than 0.2 cm), the vortices caused by the bulk acoustic resonator located at the bottom of the chamber do not cause the accumulation of particles in the solution on the resonator surface. In applications used to generate vortices and maintain the flow of the solution, applying pulsed power to the ultra-high frequency bulk acoustic resonator can achieve better dispersion of particles in the solution. In one aspect of the invention, in the sample feeding and mixing unit, the operating power of the bulk acoustic resonator is approximately 0.1-5 mW, preferably approximately 0.5-2 mW. When the solution contains cells, the operating power can be lower than 0.5 mW to avoid generating excessive heat that could adversely affect the cells.

[0150] Figure 4(d) The effect of the mixing unit in the microfluidic device provided by this invention is shown. 100 μl of PBS solution containing polystyrene (PS) microspheres (10 μm particle size, 1000-3000 particles / μl) was added to the cavity (inner diameter 4 mm, height 10 mm). The results showed that when the microscope focused on the liquid surface inside the cavity, particle suspension was recorded at 0 min, 2 min, 4 min, and 6 min on the same surface under both the on and off states of the bulk acoustic wave resonator (power 630 mW; 250 ms intermittent vibration (250 ms non-vibration)). The results showed that in the static state, the photographs showed particles in a blurred focus, indicating sedimentation. Under the operation of the bulk acoustic wave resonator, the particles were well suspended, remaining uniformly suspended even after 15 min. The experiment found that sedimentation occurred earlier when using continuous power output compared to pulsed power output.

[0151] In one aspect of the invention, as previously described, in the The sample injection and mixing unit uses An ultra-high frequency bulk acoustic resonator generates bulk acoustic waves with a frequency of approximately 0.5-50 GHz. In other aspects of the invention, a lower frequency bulk acoustic resonator, for example, with a frequency of approximately 0.1-0.5 GHz, may also be used.

[0152] In microfluidic devices, biological substances such as cells and nucleic acids, as well as some particles (such as polymer microspheres and magnetic beads), can precipitate and aggregate within the cavity, even for a short period (e.g., a few minutes), leading to movement or even blockage in the microchannels. Conventional methods used in the prior art, such as magnetic rotors or motor-driven blades, suffer from issues related to device size and the induction of air bubbles in the liquid. The method and apparatus provided by this invention effectively solve these problems.

[0153] Example 4: Sample Focusing Step and Sample Focusing Unit

[0154] The method for analyzing biological particles using barcode particles in a microfluidic device provided by this invention may include a sample focusing step, and the microfluidic device provided by this invention may include a sample focusing unit. Specifically, the movement of biological particles or barcode particles within the microchannel is adjusted to cause them to converge, eliminating or reducing random distribution along the channel width, so that the biological particles or barcode particles enter the downstream sample pairing unit with a more consistent direction, position, and velocity. Therefore, when the biological particles or barcode particles enter the acousto-fluid vortex of the pairing unit, they have the same initial relative position and state, which is beneficial for improving the pairing efficiency and capture efficiency of the biological particles.

[0155] In one aspect of the invention, a high-frequency bulk acoustic wave resonator located at the bottom of the flow channel of a microfluidic device generates bulk acoustic waves in the solution and forms a vortex channel defined by the boundary of the bulk acoustic wave generation region of the high-frequency bulk acoustic wave resonator. This allows biological particles (such as cells or nucleic acids) or barcode particles within the microfluidic channel to enter the vortex channel and move along it when passing through the bulk acoustic wave action region. Upon leaving the release point of the vortex channel, the biological particles or barcode particles in the solution move forward with essentially the same or similar speed and direction of motion, eliminating or reducing random distribution along the width of the flow channel. Consequently, the biological particles or barcode particles enter the downstream sample pairing unit with a more consistent direction, position, and speed. This ensures that when entering the acoustic-fluid vortex of the pairing unit, the biological particles or barcode particles have the same initial relative position and state, which is beneficial for improving the pairing efficiency and capture efficiency of the biological particles or barcode particles.

[0156] like Figure 2 As shown, the exemplary microfluidic device provided by the present invention includes a sample focusing unit 400. Figure 5 This invention shows the structure and configuration of an exemplary sample focusing unit of a microfluidic device, as well as the working results of the sample focusing unit focusing and "queuing" biological particles or particles in a microchannel. Figure 5 (a) A schematic diagram showing the setup and working principle of the ultra-high frequency bulk acoustic resonator in the exemplary sample focusing unit. Figure 5 (b) and (c) are schematic diagrams of the working process and experimental results of the sample focusing unit on biological particles or barcode particles in the sample.

[0157] like Figure 5 As shown in (a), the microchannel includes an inlet and multiple outlets located downstream of the ultra-high frequency bulk acoustic resonator, one of which is the outlet for the desired biological particles or barcode particles, and the others are outlets for the solution from which the desired biological particles or particles have been removed. Figure 5 (a) The following figure is a structural diagram of an exemplary sieving unit. The sieving unit has a sample inlet and a buffer inlet, an ultra-high frequency bulk acoustic resonator (shown as a dark pentagram in the figure) located at the bottom of the microchannel, and two downstream outlets. Figure 5 As shown in (a), a solution containing biological particles or barcode particles is allowed to flow through the active region of an ultra-high frequency bulk acoustic resonator. When the ultra-high frequency bulk acoustic resonator does not generate bulk acoustic waves or induce vortex tunneling, all biological particles or barcode particles are dispersed along the width of the entire flow channel and move downstream along the liquid flow direction. Figure 5(a) Figure above). When the UHF bulk acoustic resonator generates bulk acoustic waves and induces vortex tunneling, by adjusting the parameters (e.g., by adjusting the channel height, flow velocity, bulk acoustic wave power generated by the UHF bulk acoustic resonator, or a combination thereof), biological particles or barcode particles enter the vortex channel and move along the vortex channel before leaving the vortex channel at a certain position (release point) in the vortex channel. They then move forward into the downstream channel at the same or similar speed and direction of motion, eliminating or reducing the random distribution in the channel width direction.

[0158] Figure 5 (b) and (c) show the configuration of the ultra-high frequency bulk acoustic resonator in the sample screening unit of the exemplary microfluidic device of the present invention, as well as the corresponding experiments and results.

[0159] Figure 5 The figure above (b) shows that the ultra-high frequency bulk acoustic resonator is leaf-shaped with its downstream tip biased towards the upper part of the flow channel. This allows the desired biological particles or barcode particles to change their direction of movement after passing through the bulk acoustic wave action area of ​​the high frequency bulk acoustic resonator. They then leave from the downstream tip of the leaf-shaped device along the edge of the high frequency bulk acoustic resonator and enter the opening set at the upper end of the downstream flow channel with the horizontal liquid flow, forming a "queue". This eliminates or reduces the random distribution of biological particles or barcode particles in the width direction of the flow channel. Figure 5 The figure above (c) shows the corresponding experiment and results. After the PBS solution containing HeLa cells (fluorescently stained) entered the flow channel (flow channel height 50 μm, flow rate approximately 1 μL / min, and 25 mW of volume acoustic power applied by the ultra-high frequency volume acoustic resonator), it left from the downstream tip of the leaf-shaped device along the edge of the high frequency volume acoustic resonator and entered the opening set at the upper end of the downstream flow channel with the horizontal liquid flow. In the downstream flow channel, HeLa cells formed and were maintained as a thin cell stream.

[0160] Figure 5 (b) The figure below shows that the ultra-high frequency bulk acoustic resonator is leaf-shaped with its downstream tip facing the middle of the flow channel. This allows the desired biological particles or barcode particles to leave the leaf-shaped device from the downstream tip position after passing through the bulk acoustic wave action area of ​​the high frequency bulk acoustic resonator, and then enter the opening set in the middle of the downstream flow channel with the horizontal liquid flow, forming a "queue". This eliminates or reduces the random distribution of biological particles or barcode particles in the width direction of the flow channel. Figure 5(c) The figure below shows the corresponding experiment and results. After a PBS solution containing polystyrene (PS) microspheres (10 μm in diameter, 1000 microspheres / µl) was introduced into the flow channel (flow channel height 28 μm, flow rate approximately 1 µl / min, and a 200 mW power of the ultra-high frequency bulk acoustic wave resonator), the PS microspheres left the leaf-shaped device at the downstream tip along the edge of the high frequency bulk acoustic wave resonator and entered the opening at the upper end of the downstream flow channel with the horizontal liquid flow. In the downstream flow channel, the PS microspheres formed and remained as a thin stream of cells.

[0161] In one aspect of the present invention, in the sample focusing step of the method for analyzing biological 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 height of the microfluidic channel in the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is about 5-200 μm, preferably about 25-100 μm, for example about 30-90 μm.

[0162] In one aspect of the invention, in the sample focusing step of the method for analyzing biological particles using barcode particles provided by the invention, or in the sample focusing unit of the microfluidic device provided by the invention, the area of ​​the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is approximately 500-200,000 μm. 2 Preferably, it is about 5000-50000μm 2 The optimal size is approximately 10,000-25,000 μm. 2 .

[0163] In one aspect of the present invention, in the sample focusing step of 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 side length of the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm.

[0164] In one aspect of the present invention, in the sample deagglomeration step of 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 ultra-high frequency bulk acoustic resonator generating bulk acoustic waves is about 20-5000mW, preferably 50-2000mW, and more preferably 100-1500mW.

[0165] In one aspect of the invention, in the sample deagglomeration step of the method for analyzing biological particles using barcode particles provided by the invention, or in the sample focusing unit of the microfluidic device provided by the invention, the velocity of the solution flowing through the bulk acoustic region is about 0.1-100 mm / s, preferably about 0.3-20 mm / s, and more preferably about 0.5-5 mm / s.

[0166] In one aspect of the invention, in the sample deagglomeration step of the method for analyzing biological particles using barcode particles provided by the invention, or in the sample focusing unit of the microfluidic device provided by the invention, the velocity of the solution flowing through the bulk acoustic region is about 0.1-200 μL / min, preferably about 0.1-50 μL / min, and more preferably about 0.5-30 μL / min.

[0167] Example 5: Steps and Units for Bioparticle-Barcode Particle Pairing and Droplet Encapsulation

[0168] The method for analyzing biological particles using barcode particles in a microfluidic device provided by the present invention includes the steps of biological particle-barcode particle pairing and droplet encapsulation, and the microfluidic device provided by the present invention includes a biological particle-barcode particle pairing and droplet encapsulation unit.

[0169] In one embodiment of the invention, biological particles (such as cells, microvesicles, biomolecules such as nucleic acids) and barcode particles entering the microfluidic channel are paired with individual biological particles and individual barcode particles. The paired biological particle-barcode particles are then suspended in a first liquid (usually an aqueous solution) and enter a downstream droplet encapsulation unit and droplet encapsulation step to contact a second liquid (which is an immiscible liquid with the first liquid, usually an oily liquid) to form droplets containing individual paired biological particle-barcode particles.

[0170] In another embodiment of the invention, the bioparticle-barcode particle pairing and droplet encapsulation unit is configured to form droplets containing the bioparticle or the barcode particle from individual bioparticles and barcode particles from the sample, respectively, and then pair and merge the droplets containing the bioparticle or the barcode particle to form droplets containing paired bioparticle-barcode particles.

[0171] like Figure 2 As shown, the exemplary microfluidic device provided by the present invention includes a biological particle-barcode particle pairing and droplet encapsulation unit 300.

[0172] In one embodiment of the present invention, the method provided by the present invention may add a solution (e.g., a first liquid for a subsequent droplet encapsulation step) or reagent for subsequent working units or steps in the sample pairing step. In one embodiment of the present invention, the sample pairing unit of the microfluidic device provided by the present invention may include a flow channel and an inlet for adding a solution or reagent for subsequent working units. In the present invention, the reagents that can be used in subsequent working units or steps include reagents that facilitate cell lysis, nucleic acid amplification, or barcode particle dissociation, such as cell lysis agents, cell lysis enzymes, nucleic acid ligases, nucleic acid polymerases, transcriptases, etc.

[0173] Figure 6 This invention illustrates the structure and arrangement of an exemplary sample pairing unit in the microfluidic device of the present invention. For example... Figure 6 As shown in (a), the sample pairing unit may have three inlet channels and inlets, with a bioparticle inlet 501 and a barcode particle inlet 502 on either side, and a first liquid inlet 507 in the middle, into which a first liquid for resuspending the paired bioparticle-barcode particles can be introduced. By introducing the first liquid into the first liquid inlet, the paired bioparticles and barcode particles enter the downstream droplet encapsulation unit along the flow direction of the first liquid, forming a droplet containing a paired bioparticle-barcode particle with the second liquid.

[0174] Figure 6 (b) shows the structure and arrangement of another exemplary sample pairing unit of the microfluidic device of the present invention. Figure 6 As shown in (b), one or more reagent channels and inlets 509 can be provided downstream of the release point of the vortex channel of the sample pairing unit to add reagents required by the downstream unit, such as reagents that help cell lysis, nucleic acid amplification or barcode particle dissociation, into the liquid flow containing paired biological particles-barcode particles.

[0175] The method for analyzing biological particles using barcode particles in a microfluidic device provided by the present invention includes a droplet encapsulation step, and the microfluidic device provided by the present invention includes a droplet encapsulation unit. Specifically, a first liquid containing a single biological particle, a single barcode particle, or a pair of biological particle-barcode particles is contacted with a second liquid to form a droplet containing a pair of biological particle-barcode particles.

[0176] The terms “droplet” and “microdroplet” are sometimes used interchangeably herein to refer to small, typically spherical structures 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 carrier liquid.

[0177] Various known methods can be used to controllably encapsulate biological particles or individual barcode particles into droplets.

[0178] When droplets are contained in an emulsion, the carrier liquid can form the continuous phase, and the droplets can form the dispersed phase. The emulsion may further contain a surfactant and optionally a co-surfactant. The surfactant and / or co-surfactant can be located at the interface between the dispersed and continuous phases. A variety of suitable surfactants are available, and those skilled in the art can select suitable surfactants and / or co-surfactants based on 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 in gases, which is beneficial to the growth and / or viability of the encapsulated cells.

[0179] A wide range of emulsification methods are well known to those skilled in the art, any of which can be used to generate the microdroplets of the present invention. Many emulsification techniques involve the large-volume mixing of two liquids, often using turbulence to enhance droplet breakup. Such methods include vortexing, sonication, homogenization, or combinations thereof. For example, in microfluidic devices, an emulsion can be formed by colliding oil and water flows at a T-junction: the resulting droplet size varies depending on the flow rate of each liquid flow. A preferred method for preparing the microdroplets used according to the invention involves flow focusing: a continuous phase fluid (focusing or sheath fluid) is placed next to or around a dispersed phase (focused or core fluid), generating droplet breakup near an orifice where both fluids are extruded. The flow focusing device consists of a pressure chamber pressurized using a continuous focusing liquid supply. Inside, 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 and the external environment. The focusing fluid flow molds the fluid meniscus into a pointed tip, generating a stable micron or nanojet flowing out of the chamber through the orifice; the jet size is much smaller than the outlet orifice. Capillary instability breaks a stable jet into uniform droplets or bubbles. The feed tube can consist of two or more concentric needles and different immiscible liquids or gases that result in composite droplets. Flow focusing ensures the extremely rapid and controlled generation of millions of droplets per second during jet breakup.

[0180] The size of the droplets follows a probability distribution, such as a Gaussian distribution. It will also be further understood that parameters used to prepare the microfluidic droplets can be selected to obtain multiple microfluidic droplets with a specific volume. Preferably, the droplets in this invention are monodisperse and have substantially the same shape and / or size. The volume or size of a droplet refers to the average volume or size of multiple droplets. Those skilled in the art will be able to determine the average diameter of a swarm of droplets, for example, using laser scattering or other known techniques. Droplets may be spherical, or in some cases, non-spherical. The diameter of a perfect mathematical sphere having the same volume as a non-spherical droplet can be used as the diameter of the droplet (especially the non-spherical droplet).

[0181] Various methods known in the art can be used to form a one-to-one pairing of the first and second droplets. For example, the first and second droplets can be introduced separately through two intersecting microchannels. By controlling the size and flow rate of the microchannels, an orderly periodic interval of the droplets can be induced. Then, the period of the liquid flow into the first droplet can be matched with the period of the liquid flow into the second droplet, so that the first and second droplets form a one-to-one pairing of the form "ABAB" at the channel interface.

[0182] In one aspect of the present invention, the droplet encapsulation unit of the microfluidic device has:

[0183] A first liquid channel is used to input a first liquid containing a single biological particle or a single barcode particle or a pair of biological particle-barcode particles;

[0184] A droplet generating element for contacting and forming a droplet containing a paired biological particle-barcode particle in a first and second immiscible liquid.

[0185] The droplet outlet allows the formed droplets to be transported to the outlet via a continuous second liquid phase (e.g., an oil phase). Droplets leaving the outlet channel can be distributed into orifices for further processing, such as heating.

[0186] The droplet generating element can have the following structure:

[0187] a. A second liquid channel for conveying the second liquid (typically an oily liquid), and a connector where the first liquid channel intersects with the second liquid channel, the connector being configured such that the first liquid contacts the second liquid and is separated by the second liquid to produce substantially monodisperse droplets;

[0188] Or for

[0189] b. A cavity containing a second liquid, into which a first liquid containing paired cell-particles enters, forming substantially monodisperse droplets.

[0190] In the method of the present invention, it is advantageous to form droplets containing a high proportion of paired biological particles-barcode particles. In one aspect of the invention, droplet formation is adjusted by regulating the cross-sectional area of ​​the first and / or second liquid channels or the injection flow rate. In one aspect of the invention, the injection flow rate of the first and / or second liquid channels is approximately 0.1-200 mm / s.

[0191] Figure 7The diagram illustrates the structure and arrangement of an exemplary droplet encapsulation unit for a microfluidic device provided by the present invention, as well as a schematic diagram and experimental results of the droplet encapsulation unit forming droplets containing a single biological particle, a single barcode particle, or a pair of biological particle-barcode particles.

[0192] Figure 7 (a) and (b) show the structure and arrangement of an exemplary droplet encapsulation unit for a microfluidic device provided by the present invention, as well as experiments and results thereof for droplet encapsulation.

[0193] like Figure 7 As shown in (a), the droplet encapsulation unit of the microfluidic device has a first liquid channel 701 into which a first liquid containing particles from an upstream sample pairing unit is introduced. In one aspect of the invention, the first liquid is an aqueous solution. The droplet encapsulation unit of the microfluidic device also has a second liquid channel 702 and a connector 703 that intersects with and communicates with the first liquid channel. The second liquid is a liquid insoluble in the first liquid. In one aspect of the invention, the second liquid is oil. The first liquid contacts the second liquid and is separated by the second liquid to form substantially monodisperse droplets that enter a downstream droplet outlet 704. Figure 7 (b) Display Figure 7 (a) Experiments and results of the droplet encapsulation unit of the microfluidic device shown. A solution containing cells and microbeads was introduced through the left liquid channel (injection flow rate of approximately 5 mm / s), while oil (QX200 DropletGeneration Oil #1864005, Bio-Rad) was introduced through the liquid channels at both ends (injection flow rate of approximately 8 mm / min). At the intersection of the oil and the cell-microbead-containing solution, droplets formed, each containing paired single cell-microbead pairs. Droplets containing single microbeads and "empty" droplets were also observed.

[0194] exist Figure 7 In structure (a), the second liquid channel 702 is a structure that intersects the first liquid channel 701 perpendicularly from both the upper and lower ends and forms a cross with the downstream channel 704. Figure 7 (c) Another exemplary structure and arrangement of a droplet encapsulation unit for a microfluidic device provided by the present invention, wherein the second liquid channel 702 intersects the first liquid channel 701 perpendicularly and forms a T-shaped structure with the downstream channel 704.

[0195] Figure 7 (d) Another exemplary structure and arrangement of a droplet encapsulation unit for a microfluidic device provided by the present invention, wherein a first liquid channel 701 is connected to a cavity 705 containing a second liquid, and the first liquid containing paired cell-particles enters the second liquid through the first liquid channel to form substantially monodisperse droplets in the cavity.

[0196] Example 6: Processing and Analysis Steps and Units

[0197] The method for analyzing biological particles using barcode particles in a microfluidic device provided by the present invention may further include the steps of a processing and analysis unit, and the microfluidic device provided by the present invention includes a processing and analysis unit. Specifically, the method involves processing a single paired biological particle-barcode contained within a droplet, such that the nucleic acid of the biological particle contacts and barcodes the barcode, and analyzing the information of the single biological particle contained within the droplet, particularly the nucleic acid information.

[0198] In the method of this invention, the nucleic acid of a single biological particle contained in each droplet can encode with barcode particles contained in the same droplet. When nucleic acids from multiple biological particles are mixed, the nucleic acids derived from these biological particles (and containing barcode sequences) can be traced back to a single cell. When the biological particle is a cell, the single cell contained in each droplet is lysed, its nucleic acid is released and encodes with the barcode particles. RNA from the cell can be barcoded within the droplet, and when nucleic acids from multiple cells are mixed, the nucleic acids derived from these RNAs (and containing barcode sequences) can be traced back to a single cell.

[0199] Cells contained in the droplets can be subjected to processes such as cell lysis and nucleic acid release. Barcode particles can be subjected to processes such as decomposition and barcode release. Individual cells within the droplets can also take up reagents relevant to subsequent reactions. In some cases, photostimulation can be applied to the barcode particles, causing the cleavage of photoinstantaneous bonds that release oligonucleotides. In some cases, thermal stimulation can be applied to the barcode particles; increased temperature may lead to the cleavage of bonds or the release of oligonucleotides from the particles. In some cases, chemical stimulation can be applied to the barcode particles, thereby cleaving the bonds between oligonucleotides and the particles. Alternatively, oligonucleotides can be released from the beads in other ways. In the case of photo or thermal stimulation, a heat source or light source is introduced into the body cavity containing the droplets through openings in the microfluidic channel.

[0200] Various reactions can be performed to recognize, bind to, and amplify the nucleic acid or barcode sequences of cells.

[0201] The generated nucleic acids (such as amplification products) can be used for signal labeling and detection of related signals.

[0202] like Figure 2 As shown, the exemplary microfluidic device provided by the present invention includes a processing and analysis unit 200. The processing and analysis unit can be selected from the following devices or any combination thereof:

[0203] Sample loading devices, such as those used to add reagents that cause cell membrane rupture or increase permeability, or stimulants that cause nucleic acid barcodes to detach from particles, thereby enabling nucleic acid fragments on barcode particles to come into contact with and react with the nucleic acids of cells, including recognition and binding;

[0204] Photostimulation devices, for example, by breaking the photoinstantaneous bonds of oligonucleotides. Thermal stimulation devices, in which an increase in temperature in the bead environment may cause the cleavage of bonds or the release of oligonucleotides from the beads;

[0205] Nucleic acid amplification device (including temperature controller, etc.) is used to amplify and build libraries of cellular nucleic acids using nucleic acid fragments on barcode particles;

[0206] Signal recognition device;

[0207] Information analysis devices, etc., include those 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) from a single cell to achieve, for example, analysis of the cell's proteome, transcriptome, and genome.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic device for analyzing biological particles with barcoded particles, the device configured to pair a single biological particle from a sample with a barcoded particle and form a droplet containing the paired single biological particle-barcode particle therein; wherein the microfluidic device comprising the following units: (1) a sample-in mixing unit, the sample-in mixing unit comprising a bulk acoustic wave resonator disposed at the bottom of a sample chamber or flow channel of the microfluidic device; (2) a sample focusing unit, wherein the sample focusing unit comprises: a fluidic channel having a solution inlet and an outlet; a ultrahigh frequency bulk acoustic wave resonator disposed at the bottom of the fluidic channel, the ultrahigh frequency bulk acoustic wave resonator configured to generate a bulk acoustic wave in the fluidic channel that travels toward the opposite wall of the fluidic channel at a frequency of 0.5-50 GHz; wherein the ultrahigh frequency bulk acoustic wave resonator emits a bulk acoustic wave that travels toward the opposite wall of the fluidic channel, generating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generation region of the ultrahigh frequency bulk acoustic wave resonator; wherein the structure of the fluidic channel and the shape and position of the bulk acoustic wave action region of the ultrahigh frequency bulk acoustic wave resonator are configured such that a particle in a solution sample enters the vortex channel and moves along the vortex channel when passing through the bulk acoustic wave region, and exits the vortex channel at a designated location into a downstream channel, wherein the bulk acoustic wave resonator is a ultrahigh frequency bulk acoustic wave resonator configured to generate a bulk acoustic wave in the solution at a frequency of 0.5-50 GHz, (3) a biological particle-barcode particle pairing and droplet encapsulation unit; the biological particle-barcode particle pairing and droplet encapsulation unit configured to form a droplet containing a paired biological particle-barcode particle therein, wherein the biological particle-barcode particle pairing and droplet encapsulation unit is configured to form droplets containing the biological particle or the barcode particle therein, respectively, from a single biological particle and a barcode particle from a sample, and then pair and merge the droplets containing the biological particle or the barcode particle, thereby forming a droplet containing a paired biological particle-barcode particle therein, or wherein the biological particle-barcode particle pairing and droplet encapsulation unit is configured to pair a single biological particle from a sample with a barcode particle, and then form a droplet containing the paired biological particle-barcode particle therein.

2. The microfluidic device of claim 1, wherein the biological particle-barcode particle pairing and droplet encapsulation unit is configured to pair a single biological particle from a sample with a barcode particle, and then form a droplet containing the paired biological particle-barcode particle therein, wherein the droplet containing the paired biological particle-barcode particle therein is formed by contacting an aqueous solution containing the paired biological particle-barcode particle with oil.

3. The microfluidic device of claim 1, wherein the boundary lines of the bulk acoustic wave generation region of the ultrahigh frequency bulk acoustic wave resonator in the sample focusing unit are shaped such that the biological particle and the barcode particle remain in the vortex channel moving to a release point.

4. The microfluidic device of claim 3, wherein the boundary lines of the body acoustic wave generating region are designed to have no turning or curvature change to keep the biological particles and the barcode particles moving in the vortex channel to the release point.

5. The microfluidic device of claim 1, wherein the power of the body acoustic wave outputted by the ultrahigh frequency body acoustic wave resonator in the sample focusing unit is 20-5000 mW.

6. The microfluidic device of claim 1, wherein the flow rate adjusting device in the sample focusing unit is configured to adjust the flow rate of the solution through the body acoustic wave region to be 0.1-100 mm / s.

7. The microfluidic device of claim 1, wherein the flow rate adjusting device in the sample focusing unit is configured to adjust the flow rate of the solution through the body acoustic wave region to be 0.1-200 μL / min.

8. The microfluidic device of claim 1, further comprising a processing and analyzing unit configured to process the single paired biological particle-barcode contained in the droplet.

9. The microfluidic device of claim 8, wherein the nucleic acid of the biological particle is contacted with the barcode and barcoded, and the nucleic acid information of the single biological particle contained in the droplet is analyzed.

10. The microfluidic device of claim 1, wherein the power of the body acoustic wave outputted by the body acoustic wave resonator in the sample injection and mixing unit is 0.1-5 mW.

11. The microfluidic device of claim 1, wherein the power outputted by the body acoustic wave resonator in the sample injection and mixing unit is pulsed power.

12. The microfluidic device of claim 1, wherein the body acoustic wave resonator in the sample injection and mixing unit is an ultrahigh frequency body acoustic wave resonator configured to generate body acoustic wave in the solution with a frequency of 0.5-50 GHz.

13. The microfluidic device of claim 1, wherein the biological particles are cells, microvesicles, biological macromolecules.

14. The microfluidic device of claim 13, wherein the biological macromolecules are nucleic acids.

15. A method of analyzing biological particles with barcode particles using the microfluidic device of any one of claims 1-14, wherein a single biological particle from a sample is paired with a barcode particle, and a droplet containing the paired biological particle-barcode particle is formed; the method comprising the following steps: sample injection and mixing; wherein the sample injection and mixing step agitates the biological sample solution or the barcode particle solution in the sample chamber to keep the biological particles and / or the barcode particles in a dispersed state; sample focusing; wherein the sample focusing step adjusts the movement of the biological particles or the barcode particles in the flow channel, eliminating or reducing the random distribution in the width direction of the flow channel; Sample pairing and droplet encapsulation; forming a droplet within a fluidic channel containing a paired biological particle-barcode particle therein, wherein a single biological particle and a barcode particle from a sample are each formed into a droplet containing the biological particle or the barcode particle therein, and then the droplets containing the biological particle or the barcode particle are paired and merged, thereby forming a droplet containing a paired biological particle-barcode particle therein, or a single biological particle from a sample is paired with a barcode particle, and then a droplet containing the paired biological particle-barcode particle is formed.

16. The method of claim 15, further comprising a processing and analyzing unit step, processing the single paired biological particle-barcode contained within the droplet.

17. The method of claim 16, wherein the nucleic acid of the biological particle is contacted with the barcode and barcoded, and the nucleic acid information of the single biological particle contained within the droplet is analyzed.

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