Microvesicle separation method and equipment

Abstract

The present invention discloses a microfluidic system and method for separating flexible particles such as cell vesicles or biomacromolecules such as exosomes in a sample. The system of the present invention includes one or more ultra-high frequency acoustic wave resonators, which can generate a bulk acoustic wave with a frequency of about 0.5-50 GHz in a fluid channel and transmit to the wall on the opposite side of the fluid channel. By adjusting the power of the generated bulk acoustic wave and / or the speed at which the conditioned solution flows through the bulk acoustic wave region, the flexible particles in a specified range are pushed to the top of the flow channel and stay in the bulk acoustic wave region, while the flexible particles that do not belong to the specified range enter the downstream through the bulk acoustic wave region and are collected. The system and method of the present invention can capture or release flexible particles such as cell vesicles or biomacromolecules, especially exosomes, in a solution.

Description

[0001] This application claims priority to the following Chinese patent application: Application number 201910512713.9, filed on June 13, 2019, entitled “Microvesicle separation method and device”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of cell research methodology and medical devices. Specifically, the present invention relates to a microfluidic system for separating and analyzing cell microvesicles and a method for separating and analyzing cell microvesicles using the system. Background Art

[0003] Cells or subcellular particles, as well as biomacromolecules such as nucleic acids and proteins, present in human body fluids such as blood and tissue fluids are very important for physiological health and research, so there is a need to separate cells or subcellular particles or biomacromolecules in body fluids. In the human body, there are various cell vesicles released by cells into the extracellular environment, including exosomes, microvesicles, vesicles, membrane vesicles, blisters, bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytic vesicles. These subcellular particles, including exosomes, are important mediators of intercellular information transmission and play an important role in antigen presentation, cell apoptosis, inflammatory response, tumor development and metastasis. Subcellular particles are widely distributed in body fluids, including blood, saliva, urine, milk, pleural effusion, etc. Exosomes contain a variety of contents such as DNA, RNA and proteins, and can be used as non-invasive diagnostic markers for various diseases such as tumors. The amount of exosomes can also be used to determine the efficacy of treatment, the stage of a disease or condition, or the progression of a disease or condition.

[0004] The separation of subcellular particles such as exosomes or biomacromolecules such as nucleic acids and proteins is an important biotechnology. There are many methods to separate and manipulate subcellular particles, including electrical methods, acoustic methods, optical methods, magnetic methods, chemical methods, fluid manipulation methods, etc. However, these methods have problems such as adhesion, low efficiency, affected biological activity, unfavorable downstream experiments, low purity and recovery rate, and more impurities.

[0005] Microfluidics technology has been used to separate, extract and manipulate micron particles and cells, but there is no method for separating active nanoparticles, especially for controllable precision separation.

[0006] Therefore, there is an urgent need for a system and method to separate cell microvesicles or biological macromolecular particles such as nucleic acids and proteins, so as to facilitate further processing and analysis of the obtained cell microvesicles or biological macromolecular particles such as nucleic acids and proteins. Summary of the invention

[0007] The present invention discovers for the first time that ultrahigh frequency bulk acoustic waves can be used to effectively manipulate and separate flexible particles in solutions such as cell microvesicles or biological macromolecular particles such as nucleic acids and proteins in a microfluidic system, thereby providing a method and system for separating and purifying microvesicles or biological macromolecular particles such as nucleic acids and proteins.

[0008] Specifically, the present invention provides a method for separating flexible particles, comprising:

[0009] (1) allowing a solution sample containing flexible particles to flow through a microfluidic device, the device comprising:

[0010] Fluid channels;

[0011] One or more ultra-high frequency bulk acoustic wave resonators are disposed at the bottom of the fluid channel, and the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel;

[0012] (2) the ultra-high frequency resonator emits a bulk acoustic wave that travels toward the top of the fluid channel;

[0013] (3) By adjusting the power of the bulk acoustic wave (for example, through a power regulator) and / or adjusting the speed at which the solution flows through the area affected by the bulk acoustic wave (for example, through a flow rate regulating device), the specified flexible particles are pushed to the top of the fluid channel in the area affected by the bulk acoustic wave and stay there.

[0014] In one aspect of this aspect, the method further comprises:

[0015] (4) obtaining a liquid sample that passes through the bulk acoustic wave region and enters the downstream. The liquid sample that enters the downstream contains other particles of the designated flexible particles that have not stayed in the bulk acoustic wave influence region in step (3), such as other flexible particles of different sizes from the designated flexible particles.

[0016] and / or

[0017] The parameters of step (3) are changed so that the designated flexible particles that are pushed to the top of the fluid channel and stay there are released. In one aspect of the present invention, the designated flexible particles are resuspended in a designated solution after being released, thereby being purified. The ultra-high frequency bulk acoustic wave resonator in the present invention refers to a resonator that can generate bulk acoustic waves with a frequency exceeding 0.5 GHz (preferably exceeding 1 GHz), for example, a frequency of 0.5-50 GHz. The ultra-high frequency bulk acoustic wave resonator can be a thin film bulk acoustic wave resonator or a solid-state assembled resonator.

[0018] In yet another aspect of the present invention, the distance from the ultrahigh frequency resonator to the top of the flow channel in the microfluidic device is about 5-60 um, preferably about 8-45 um, and more preferably about 10-30 um.

[0019] The microfluidic device generally includes a power regulating device, which regulates the power of the bulk acoustic wave generated by the ultrahigh frequency resonator.

[0020] The microfluidic device generally includes a flow rate regulating device, which regulates the speed at which the solution flows through the area affected by the bulk acoustic wave.

[0021] Flexible microparticles refer to nano or micro particles with deformation properties. Flexible particles can be artificial or natural. In one aspect of the present invention, the flexible particles are naturally occurring particles. In one aspect of the present invention, the flexible particles are subcellular particles, such as cell microvesicles released into the extracellular environment by various cells. These cell microvesicles are vesicle-like bodies with a double-layer membrane structure that fall off the cell membrane or are secreted by cells, usually with a diameter greater than about 10, 20 or 30 nm, but not limited to. They can have a diameter of about 30-1000 nm, about 30-800 nm, about 30-150 nm or about 30-100 nm. Microvesicles released by cells include exosomes, vesicles, membrane vesicles, blisters, bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytic vesicles. In the art, "exosomes" generally refer to membrane vesicles with a particle size between 30 and 250 nm that are secreted into the extracellular environment after the fusion of intracellular multivesicular bodies with cell membranes. Exosomes are important media for intercellular information transmission and play an important role in antigen presentation, cell apoptosis, inflammatory response, tumor development and metastasis. Exosomes are widely distributed in body fluids, including blood, saliva, urine, breast milk, pleural effusion, etc. Exosomes contain a variety of contents such as DNA, RNA and protein.

[0022] Therefore, in one aspect of the present invention, a method for isolating cell microvesicles is provided, comprising:

[0023] (1) allowing a solution sample containing cell microvesicles to flow through a microfluidic device, the device comprising:

[0024] Fluid channels;

[0025] One or more ultra-high frequency bulk acoustic wave resonators are disposed at the bottom of the fluid channel, and the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel;

[0026] (2) the ultra-high frequency resonator emits a bulk acoustic wave that travels toward the top of the fluid channel;

[0027] (3) or by adjusting the power of the bulk acoustic wave (e.g., by a power regulator) and / or adjusting the speed at which the solution flows through the bulk acoustic wave affected area (e.g., by a flow rate regulating device), so that the specified cell microvesicles are pushed to the top of the fluid channel in the bulk acoustic wave affected area and stay there.

[0028] In one aspect of this aspect, the method further comprises:

[0029] (4) obtaining a liquid sample that has passed through the BAW region and entered the downstream. The liquid sample that has entered the downstream contains other particles of the designated microvesicle that have not stayed in the BAW-affected region in step (3), such as other flexible particles of a size different from that of the designated cell microvesicle, such as exosomes that are relatively small in size relative to other cell microvesicles.

[0030] and / or

[0031] The parameters of step (3) are changed so that the designated cell microvesicles pushed to the top of the fluid channel and retained therein are released. In one aspect of the present invention, the designated cell microvesicles are released and then resuspended in a designated solution, thereby being purified.

[0032] In yet another aspect of the present invention, the cell microvesicles include a vesicle population. The vesicle population is usually composed of several, for example, 2-50 cell microvesicles.

[0033] In yet another aspect of the present invention, the diameter of the cell microvesicle is about 0.02-1 um, preferably about 0.03-0.8 um, for example about 0.05-0.5 um.

[0034] In yet another aspect of the present invention, the distance from the ultrahigh frequency resonator to the top of the flow channel in the microfluidic device is about 10-60 um, preferably about 8-45 um, and more preferably about 10-20 um.

[0035] In one aspect of the invention, the flexible particles in the method are nucleic acids. "Nucleic acid" (and the equivalent term "polynucleotide") as used herein refers to a polymer of ribonucleosides or deoxyribonucleosides containing phosphodiester bonds between nucleotide subunits. Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, microRNA, fragmented 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 with artificial or natural DNA or RNA as templates. Nucleic acids can be double-stranded or single-stranded, circular or linear. Samples that can be used to detect target nucleic acids include the following samples: from cell culture, eukaryotic microorganisms or diagnostic samples such as body fluids, body fluid sediments, gastric lavage samples, fine needle aspirates, biopsy samples, tissue samples, cancer cells, cells from patients, cells from tissues, or cells cultured in vitro from individuals to be tested and / or treated for 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 aspirate, saliva, cotton swab samples, flushing or lavage fluid and / or swab samples. The inventive method can be used to separate nucleic acids with a length of about 50bp-50kbp, preferably about 50bp-10kbp, and more preferably about 60bp-1kbp. The inventive method is particularly suitable for separating short-chain nucleic acids. For example, the inventive method is suitable for separating short-chain nucleic acids with a length of ≤1500bp, preferably ≤500bp, more preferably ≤200bp, such as ≤100bp. The key field of short-chain nucleic acids is prenatal diagnosis. In addition to endogenous free circulating DNA, the blood of pregnant women also contains free circulating DNA of the fetus.

[0036] Therefore, in one aspect of the present invention, a method for isolating nucleic acid is provided, comprising:

[0037] (1) allowing a solution sample containing nucleic acid to flow through a microfluidic device, the device comprising:

[0038] Fluid channels;

[0039] One or more ultra-high frequency bulk acoustic wave resonators are disposed at the bottom of the fluid channel, and the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel;

[0040] (2) the ultra-high frequency resonator emits a bulk acoustic wave that travels toward the top of the fluid channel;

[0041] (3) By adjusting the power of the bulk acoustic wave (for example, through a power regulator) and / or adjusting the speed at which the solution flows through the area affected by the bulk acoustic wave (for example, through a flow rate regulating device), the specified nucleic acid is pushed to the top of the fluid channel in the area affected by the bulk acoustic wave and stays there.

[0042] In one aspect of this aspect, the method further comprises:

[0043] (4) obtaining a liquid sample that has passed through the BAW region and entered the downstream. The liquid sample that has entered the downstream contains other nucleic acids that have not remained in the BAW-affected region in step (3), such as other nucleic acids of different sizes from the designated nucleic acid.

[0044] and / or

[0045] The parameters of step (3) are changed so that the designated nucleic acid pushed to the top of the fluid channel and retained therein is released. In one aspect of the present invention, the designated nucleic acid is released and resuspended in a designated solution, thereby being purified.

[0046] In yet another aspect of the present invention, the distance from the ultrahigh frequency resonator to the top of the flow channel in the microfluidic device is about 5-25 um, preferably about 6-25 um, and more preferably about 7-20 um.

[0047] In one aspect of the present invention, the output power of the power regulating device in the aforementioned method is about 0.5-2000 mW, preferably about 5-1500 mW, more preferably about 15-900 mW, for example 70-300 mW.

[0048] In one aspect of the present invention, the flow rate regulating device in the aforementioned method can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.1-10 mm / s, preferably about 0.3-5 mm / s, and more preferably about 0.5-2.5 mm / s.

[0049] In one aspect of the present invention, the flow rate regulating device in the aforementioned method can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.1-100 μL / min, preferably about 0.1-50 μL / min, and more preferably about 0.5-20 μL / min.

[0050] In one aspect of the present invention, the power of the bulk acoustic wave generated by the ultra-high frequency resonator is adjusted to about 0.5-2000 mW, preferably about 5-1500 mW, more preferably about 15-900 mW, for example, 70-300 mW. That is, the adjustable power range of the power regulator includes about 0.5-2000 mW, preferably about 5-1500 mW, more preferably about 15-900 mW, for example, 70-300 mW.

[0051] In one aspect of the present invention, the area of ​​the bulk acoustic wave generating region of the ultra-high frequency bulk acoustic wave resonator is about 500-200000 μm 2 , preferably about 5000-50000 μm 2 , most preferably about 10000-25000 μm 2 .

[0052] In one aspect of the present invention, the inlet includes a sample inlet and an auxiliary solution inlet disposed on one or both sides of the sample inlet. The auxiliary solution may be a liquid such as a buffer. The auxiliary solution may be used to resuspend "captured" flexible particles such as cell microvesicles or biomacromolecule particles such as nucleic acids and proteins, or to add reagents for processing "captured" flexible particles, such as fluorescent markers that specifically recognize the flexible particles or their specific markers. The auxiliary solution may also be used to control the flow direction and range of the sample liquid in the microfluidic channel based on the sheath flow effect.

[0053] In one aspect of the present invention, the solution sample contains different flexible particles, such as flexible particles of different sizes; for example, flexible particles of different densities. In another aspect of the present invention, the aforementioned method also includes controlling the flexible particles pushed to the top of the fluid channel and staying (for example, called the first flexible particles), and obtaining the flexible particles that are not pushed to the top of the fluid channel and staying downstream of the area affected by the bulk acoustic wave (for example, called the second flexible particles). For example, the downstream of the area affected by the bulk acoustic wave is controlled to obtain exosomes with a smaller size than other cell microvesicles, and the cell microvesicles with a larger size that stay at the top of the fluid channel. In another aspect of the present invention, the aforementioned method also includes releasing the flexible particles of different sizes, such as nucleic acids, that are pushed to the top of the fluid channel and stay in sequence, such as from small to large. In another aspect of the present invention, the nucleic acids of different sizes that are pushed to the top of the fluid channel and stay are released in sequence, such as from small to large, by adjusting the power of the bulk acoustic wave and / or adjusting the speed at which the added solution flows through the bulk acoustic wave area.

[0054] In yet another aspect of the present invention, the flexible particles (such as microvesicles or nucleic acids) that stay in the area affected by the bulk acoustic wave can be selected by one or any combination of the following methods:

[0055] (a) regulating the power of the bulk acoustic wave;

[0056] (b) regulating the time of generating body sound waves;

[0057] (c) adjusting the velocity of the solution flowing through the bulk acoustic wave region.

[0058] In one aspect of the present invention, the method can controllably obtain different flexible particles, such as microvesicles or nucleic acids of different sizes, staying or passing through the area affected by the bulk acoustic wave.

[0059] In one aspect of the present invention, the method divides the fluid channel into different regions, and sets ultra-high frequency resonators for separating different flexible particles such as microvesicles or biological macromolecular particles such as nucleic acids and proteins in different regions. For example, the ultra-high frequency resonators for separating different flexible particles can have sound wave generating areas of different shapes, or apply body sound waves of different powers, or have different flow velocities, or have different flow channel heights, or a combination thereof.

[0060] In one aspect of the present invention, the solution sample in the method is a liquid containing flexible particles to be captured, such as microvesicles or biomacromolecule particles such as nucleic acids and proteins, such as body fluids, blood, cell culture or culture supernatant. In another aspect of the present invention, the solution is a sample without cells, such as body fluids, culture supernatant, plasma without blood cells by gradient centrifugation, etc.

[0061] In one aspect of the present invention, the method further comprises using the obtained flexible particles such as microvesicles or biomacromolecule particles such as nucleic acids and proteins, such as exosomes, for one or more of the following analyses: DNA and RNA amplification (e.g., rapid amplification of cDNA ends (RACE); degenerate oligo-primer PCR; mitochondrial DNA PCR; genomic PCR, digital PCR, RT-PCR, proximity ligation PCR; immuno-PCR); sequencing; immunochemistry; metabolite analysis; enzymatic analysis; reporter gene expression analysis; and hybridization studies.

[0062] The present invention also provides a microfluidic device for separating required flexible particles such as cell microvesicles or biological macromolecular particles such as nucleic acids and proteins from a solution.

[0063] In one aspect of the present invention, the present invention also provides a microfluidic device for separating desired flexible particles (such as cell microvesicles, such as exosomes) from a solution.

[0064] In one aspect of the present invention, a microfluidic device for separating flexible particles such as cell microvesicles or biomacromolecule particles such as nucleic acids and proteins is provided, comprising:

[0065] a fluid channel having an inlet and an outlet;

[0066] One or more ultra-high frequency bulk acoustic wave resonators are disposed on a wall of the fluid channel, the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of about 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel;

[0067] A power regulating device for regulating the power of the bulk acoustic wave generated by the ultrahigh frequency resonator;

[0068] a flow rate regulating device, which regulates the speed at which the solution flows through the bulk acoustic wave region,

[0069] The ultra-high frequency resonator can emit a bulk acoustic wave that is transmitted to the top of the fluid channel, so that the solution flowing through the bulk acoustic wave area generates an acoustic jet, and the microfluidic device is configured to adjust the power of the bulk acoustic wave through the power regulator and / or adjust the speed of the solution flowing through the bulk acoustic wave influence area through the flow rate regulating device, so that the specified flexible particles are pushed to the top of the fluid channel and stay in the bulk acoustic wave influence area. In another aspect of the present invention, the microfluidic device also has a specific outlet or channel, so that after removing the specified flexible particles pushed to the top of the fluid channel and staying, the liquid sample entering the downstream through the bulk acoustic wave area can be collected through the specific outlet or channel. In another aspect of the present invention, the microfluidic device also has a specific outlet or channel, and the flexible particles of different sizes (such as nucleic acids) pushed to the top of the fluid channel and staying are released in sequence, for example, from small to large, and then enter the specific outlet or channel.

[0070] In yet another aspect of the present invention, the distance from the ultrahigh frequency resonator to the top of the flow channel in the microfluidic device is about 5-60 um, preferably about 8-45 um, and more preferably about 10-30 um.

[0071] In another aspect of the present invention, the microfluidic device is used to separate cell microvesicles. In another aspect of the present invention, the flexible particles are exosomes, vesicles, membrane vesicles, blisters, bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytic vesicles. In another aspect of the present invention, the distance from the ultra-high frequency resonator to the top of the flow channel is about 10-60um, preferably about 8-45um, and more preferably about 10-20um.

[0072] In yet another aspect of the present invention, the microfluidic device is used for separating nucleic acids. In yet another aspect of the present invention, the distance from the ultrahigh frequency resonator to the top of the flow channel is about 5-25 um, preferably about 6-25 um, more preferably about 7-20 um.

[0073] In yet another aspect thereof, the output power of the power regulating device of the microfluidic device of the present invention is 0.5-2000 mW, preferably about 5-1500 mW, more preferably about 15-900 mW, for example 70-300 mW.

[0074] In yet another aspect thereof, the flow rate regulating device of the microfluidic device of the present invention can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.1-10 mm / s, preferably about 0.3-5 mm / s, and more preferably about 0.5-2.5 mm / s.

[0075] In yet another aspect thereof, the flow rate regulating device of the microfluidic device of the present invention can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.1-100 μL / min, preferably about 0.1-50 μL / min, and more preferably about 0.5-20 μL / min.

[0076] In another aspect, the area of ​​the BAW generation region of the UHF BAW resonator of the microfluidic device of the present invention is about 500-200000 μm 2 , preferably about 5000-50000 μm 2 , most preferably about 10000-25000 μm 2 .

[0077] In yet another aspect thereof, the ultrahigh frequency bulk acoustic wave resonator of the microfluidic device of the present invention may be a thin film bulk acoustic wave resonator or a solid-state assembly type resonator, for example, an acoustic wave resonator in a thickness stretching vibration mode.

[0078] In yet another aspect thereof, the thickness of the piezoelectric layer of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device of the present invention is in the range of 1 nm to 2 um.

[0079] In yet another aspect thereof, the fluid channel inlet of the microfluidic device of the present invention comprises a sample inlet and an auxiliary solution inlet disposed on one side or both sides of the sample inlet.

[0080] In yet another aspect thereof, the fluid channel of the microfluidic device of the present invention has at least two outlets, one of which is used to receive a liquid sample that passes through the bulk acoustic wave area and enters the downstream after the specified flexible particles that are pushed to the top of the fluid channel and stay in the bulk acoustic wave influence area are removed.

[0081] In one aspect of the present invention, the fluid channel of the microfluidic device is divided into different regions, and different flexible particles are arranged in different regions to separate them. For example, different regions have ultra-high frequency resonators for separating different flexible particles, which may have sound wave generating regions of different shapes; or different regions apply body sound waves of different powers, or different regions have different flow velocities, or different regions have different flow channel heights, or a combination thereof.

[0082] The present invention also provides a kit, comprising a microfluidic device as defined above or an ultrahigh frequency bulk acoustic wave resonator as defined therein, and reagents for analyzing cell microvesicles (such as exosomes) or nucleic acids. The analysis includes, but is not limited to: DNA and RNA amplification (such as rapid amplification of cDNA ends (RACE); degenerate oligoprimer PCR; mitochondrial DNA PCR; genomic PCR, digital PCR, RT-PCR, proximity ligation PCR; immuno-PCR); sequencing; immunochemistry; metabolite analysis; enzymatic analysis; reporter gene expression analysis; and hybridization studies.

[0083] Reference numerals

[0084] 100 microfluidic devices

[0085] 101 Fluid Channels

[0086] 200 chip shell

[0087] 201 Coulter Cell Counter

[0088] 202 Ultra-high frequency bulk acoustic wave resonator

[0089] 203 top electrode layer

[0090] 204 piezoelectric layer

[0091] 205 bottom electrode layer

[0092] 206 Sound wave reflection layer Sound impedance layer

[0093] 207 bottom lining

[0094] 208 Top

[0095] 300PCL controller

[0096] 301 High Frequency Signal Generator

[0097] 302 Power Amplifier

[0098] 303 Impedance Meter

[0099] 400 Liquid injection and flow rate regulation device

[0100] 500 sound jets

[0101] 501 Vortex

[0102] 600 Larger Size Particles

[0103] 601 medium size particles

[0104] 602 Smaller size particles BRIEF DESCRIPTION OF THE DRAWINGS

[0105] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0106] Figure 1 A schematic diagram of the structure of a microfluidic device system provided in an embodiment of the present application;

[0107] Figure 2 A schematic diagram of the structure of an ultra-high frequency bulk acoustic wave resonator in a microfluidic device system provided in an embodiment of the present application; wherein (a) represents Figure 1 The microfluidic system shown in FIG. 1 is a top view of the microchannel (left side) and a cross-sectional view of AA (right side); (b) is a top view of the UHF BAW resonator (left side) (the black pentagonal part is the acoustic wave action area of ​​the UHF BAW resonator) and a cross-sectional view of BB (right side); (c) is a top view (left side) and a cross-sectional view (right side) of the microchannel + UHF BAW resonator;

[0108] Figure 3 Showing the effect of flow channel height on the acoustic jet and vortex induced by bulk acoustic waves.

[0109] Figure 4 It is shown that the method and device of the present invention can separate exosomes of different sizes in clinical blood samples as needed.

[0110] Figure 5 It was shown that the methods and apparatus of the present invention can separate nucleic acids of different sizes. DETAILED DESCRIPTION

[0111] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0112] Example 1 Experimental methods and materials

[0113] Microfluidic channel and UHF BAW resonator preparation:

[0114] Microfluidic channels made of polydimethylsiloxane (PDMS) were prepared by soft lithography.

[0115] The BAW resonator device is prepared by chemical vapor deposition, metal sputtering, photolithography and other methods on a silicon-based wafer. The specific method is as follows:

[0116] 1. Use a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:1 to thoroughly clean the surface of the silicon wafer. This method can effectively remove organic and inorganic matter on the silicon wafer.

[0117] 2. On the cleaned silicon wafer, a layer of aluminum nitride film is formed by surface sputtering, and then a layer of silicon dioxide film is deposited by ion-enhanced chemical vapor deposition. Then, the same method is used to alternately deposit aluminum nitride film and silicon dioxide film to form a Bragg acoustic reflection structure in which aluminum nitride and silicon dioxide alternately overlap.

[0118] 3. On the Bragg reflection layer structure, a 600nm molybdenum film is sputtered as the bottom electrode. Then, standard photolithography techniques, including coating, exposure, and development, are used to photolithograph the molybdenum electrode film, and then etching is performed to form a bottom electrode with a target pattern.

[0119] 4. Sputter another layer of aluminum nitride film on the molybdenum electrode as a piezoelectric layer. Use dry etching to define the pattern of the aluminum nitride film.

[0120] 5. Use negative photoresist to transfer the pattern on the mask, and then sputter a 50nm thick titanium-tungsten alloy layer, which can increase the adhesion of the gold electrode as an adhesion layer. Then use the evaporation method to grow a 300nm thick gold film on the upper electrode. Finally, use acetone to remove the gold film around the target pattern to form a gold electrode with the target pattern.

[0121] Finally, the BAW resonator device is bonded and integrated with the PDMS microchannel chip. The BAW resonator device is set in the middle of the channel.

[0122] The BAW resonator device is connected to a network analyzer using a standard SMA interface, and the resonance peak is found by testing the spectrum, so that the frequency of the BAW wave emitted by the BAW resonator device in the microchannel can be measured.

[0123] Instruments and Materials

[0124] High frequency signal generator: (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz

[0125] Power Amplifier: Mini-Circuits, with 35dBm enhancement of the original RF source power

[0126] Syringe pump: New Era Pump Systems, Inc., NE-1000

[0127] Example 2

[0128] In the specific implementation process of this embodiment, a microfluidic device is provided, which can be used to separate and capture flexible particles in a solution. The flexible particles can be artificial or natural. The flexible particles can be biological macromolecules such as nucleic acids. The flexible particles can also be micelles with a membrane structure, in particular micelles with a lipid bilayer or a lipid-like bilayer. In one aspect of the present invention, the flexible particles are naturally occurring particles, such as cell microvesicles released by cells into the extracellular environment. Cell microvesicles include exosomes, vesicles, membrane vesicles, blisters, bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytic vesicles, etc.

[0129] The method and device of the present invention can be used to separate and capture flexible particles in a solution, for example, to separate and obtain target vesicles in blood.

[0130] like Figure 1 As shown, the microfluidic device 100 includes a fluid channel 101, an ultra-high frequency bulk acoustic wave resonator 202, a bulk acoustic wave driving and power regulating device, and a liquid injection and flow rate regulating device 400.

[0131] The microfluidic device provided by the present invention can exist alone or as part of a microfluidic system, for example, in the form of a detachable chip. Microfluidic systems or devices can be used to contain and transport fluid materials such as liquids, and the flow channel size is in the micrometer or even nanometer level. Typical microfluidic systems and devices usually include structures and functional units of millimeter or smaller size.

[0132] The fluid channel of the microfluidic device, or microchannel, is generally closed except for the opening for fluid to enter and flow out. The cross section of the fluid channel usually has a size of 0.1-500 μm, which can be various shapes, including ellipse, rectangle, square, triangle, circle, etc. The fluid channel can be prepared with various known micro-preparation techniques, and its materials include but are not limited to silica, silicon, quartz, glass or polymeric materials (such as PDMS, plastics, etc.). The channel can be coated with a coating. The coating can change the characteristics of the channel and can be patterned. For example, the coating can be hydrophilic, hydrophobic, magnetic, conductive, or biologically functionalized.

[0133] In one aspect of the present invention, the height of the fluid channel of the microfluidic device is about 5-60 um, preferably about 8-45 um, and more preferably about 10-30 um.

[0134] In one aspect of the present invention, the microfluidic device is used to capture vesicles including exosomes, and the height of the fluid channel thereof is generally about 10-60 um, preferably about 8-45 um, and more preferably about 10-20 um.

[0135] In one aspect of the present invention, the width of the fluid channel of the microfluidic device is about 50-1000 μm, preferably about 100-500 μm, and more preferably about 150-300 μm.

[0136] The microchannel 100 in this embodiment has an inlet and an outlet for fluid to enter and exit. The inlet is connected to a liquid injection device for receiving liquid injection. The inlet of this embodiment includes a sample inlet 101 and a buffer inlet 102. Among them, the buffer inlet is two inlets arranged on both sides of the sample inlet, which intersect and communicate with the sample inlet. The microchannel inlet is arranged through the above-mentioned three-phase flow mode (sample flow in the middle, buffer flow on both sides), which is conducive to passive focusing of the sample introduced into the middle sample inlet.

[0137] like Figure 1 As shown, the microfluidic device of this embodiment includes a liquid injection and flow rate regulating device 400, which is used to control the liquid injection and the flow rate of the liquid. The liquid can be a liquid containing a sample. For example, the sample is a liquid containing cells to be captured. The sample can include body fluid, whole blood, any blood fraction containing cells, fragmented tumors, tumor cell suspensions, cell cultures or culture supernatants, etc. The liquid can be various body fluids, including tissue fluid, extracellular fluid, lymph fluid, cerebrospinal fluid, aqueous humor, urine, sweat, etc.

[0138] The flow rate of the injected liquid can be controlled by an external pressure source, an internal pressure source, electronic dynamics or magnetic field dynamics. The external pressure source and the internal pressure source can be pumps, such as peristaltic pumps, syringe pumps or pneumatic pumps. In this embodiment, a syringe pump finely adjusted by a computer is used to control the flow rate of the liquid injection.

[0139] In the present invention, the flow rate of the liquid ranges from about 0.1-10 mm / s, preferably about 0.3-5 mm / s, and more preferably about 0.5-2.5 mm / s. In another aspect of the present invention, the flow rate of the liquid ranges from about 0.1-100 μL / min, preferably about 0.1-50 μL / min, and more preferably about 0.5-20 μL / min.

[0140] The channel may be a single channel, or a plurality of channels arranged in parallel or in other forms and having a common output and input, wherein the inflow and outflow of the fluid in each channel and the flow rate thereof may be controlled jointly or independently as required.

[0141] The microfluidic device of the present invention has one or more ultra-high frequency bulk acoustic wave resonators 200, which are arranged on a wall of a fluid channel (usually arranged at the bottom of the flow channel). The ultra-high frequency bulk acoustic wave resonator can generate a bulk acoustic wave with a frequency of about 0.5-50 GHz in the fluid channel and propagate to the wall on the opposite side of the fluid channel (usually the top of the flow channel).

[0142] The ultra-high frequency bulk acoustic wave resonator that can be used in the present invention can be a thin film bulk acoustic wave resonator or a solid-state assembly type resonator, for example, a thickness stretching vibration mode acoustic wave resonator.

[0143] like Figure 1 As shown, the microfluidic device of this embodiment has a plurality of ultra-high frequency bulk acoustic wave resonators 202 arranged at the bottom of the flow channel.

[0144] The ultra-high frequency bulk acoustic wave resonator is a bulk acoustic wave generating component that can generate a bulk acoustic wave in the fluid channel that propagates toward a wall on the opposite side of the fluid channel.

[0145] like Figure 2 As shown in the cross-sectional view on the right side of (b), the UHF BAW resonator includes an acoustic wave reflection layer 206, a bottom electrode layer 205, a piezoelectric layer 204 and a top electrode layer 203 arranged in order from bottom to top. The overlapping area of ​​the bottom electrode layer, the piezoelectric layer, the top electrode layer and the acoustic wave reflection layer constitutes a BAW generation area. Figure 2 (b) As shown in the top view on the left, the top surface of the UHF BAW resonator is arranged on the wall of the fluid channel, generating a BAW propagating perpendicular to the wall toward the opposite wall; generally speaking, the area formed by the top surface of the UHF BAW resonator is the BAW generating area, also referred to as the BAW area or BAW action area in this article. In one aspect of the present invention, the area of ​​the BAW action area is about 500-200000 μm 2 , preferably about 5000-50000 μm 2 , most preferably about 10000-25000 μm 2 .like Figure 2 The bulk acoustic wave active region of the embodiment shown is a pentagon with a side length of about 120 μm.

[0146] In the present invention, the shape of the acoustic wave action area includes at least but is not limited to one of the following: a circle, an ellipse, a semicircle, a parabola, a polygon with an acute angle or an obtuse angle at the vertex, a polygon with a vertex replaced by an arc, a polygon with a vertex of an acute angle, a semicircle or a parabola, or a square array or annular array of the same shape arranged repeatedly. The present application provides the acoustic wave action area of ​​the above shape, but the acoustic wave action area of ​​any other shape is also within the protection scope of the present application.

[0147] like Figure 2 As shown in the cross-sectional view on the right, the fluid channel of this embodiment may have a plurality of UHF BAW resonators. In one aspect of the invention, they are arranged in a straight line in the same direction as the direction of fluid movement.

[0148] The ultra-high frequency bulk acoustic wave resonator used in the present invention is a thickness stretching vibration mode, in which the piezoelectric material thin film layer is grown in the vertical direction and excited by coupling the vertical electric field through the d33 piezoelectric coefficient. The ultra-high frequency bulk acoustic wave resonator used in the present invention can generate a localized acoustic flow at the interface between the device and the liquid without the help of a coupling medium or structure.

[0149] like Figure 1 As shown in the right figure, the ultra-high frequency resonator emits a body acoustic wave that is transmitted to the wall on the opposite side of the fluid channel (e.g., the top of the flow channel). The body force generated by the attenuation of the sound wave in the fluid causes an acoustic jet 500 to appear in the solution flowing through, resulting in a local three-dimensional vortex 501 in the liquid in the microchannel. The forces on the particles (including larger-sized particles 600, medium-sized particles 601, and smaller-sized particles 602) in the area affected by the body acoustic wave include the fluid drag force (Stokes drag force) generated by the vortex, the inertial lift force (inertial lift force) generated by the laminar flow, and the acoustic radiation force (acoustic radiation force) caused by the attenuation of the sound wave.

[0150] In the method of the present invention, the ultra-high frequency resonator emits a body acoustic wave transmitted to the wall on the opposite side of the fluid channel (for example, the top of the flow channel), and the body force generated by the attenuation of the sound wave in the fluid causes an acoustic jet to appear in the solution flowing through, and the fluid with the same flux moves downward around it to form a vortex. In the method of the present invention, when the solution containing flexible particles is transmitted to the body acoustic wave influence area (there is an acoustic jet and a vortex or vortex channel) on the wall on the opposite side of the fluid channel through the ultra-high frequency resonator, the forces on the flexible particles include the fluid drag force (Stokes drag force) generated by the vortex, the acoustic radiation force (acoustic radiation force) caused by the attenuation of the sound wave, and the inertial lift force (inertial lift force) generated by the laminar flow. Different flexible particles are affected differently by the fluid drag force and acoustic radiation force. The fluid drag is proportional to the particle radius; the acoustic radiation force is proportional to the cube / quadratic power of the particle radius (depending on the particle size and the wavelength of the sound wave). Therefore, as the particle size decreases, the acoustic radiation force will decay faster than the drag force. The movement trajectory of larger particles is mainly controlled by the acoustic radiation force after entering the vortex. In the area dominated by the acoustic radiation force, under the action of the upward acoustic radiation force, the particles move to the top of the flow channel. At the top of the flow channel, the flexible particles are subject to the inertial drag force generated by the laminar flow of the solution, and are also subject to resistance caused by the friction and adhesion between the particles and the top due to the pressure of the acoustic radiation force. When the resistance is greater than the inertial drag force, the flexible particles stay at the top of the flow channel and do not flow downstream with the liquid. When smaller particles enter the vortex, the acoustic radiation force is not enough to push them away from the vortex movement trajectory. Therefore, their movement trajectory is dominated by the fluid drag force, and they move with the vortex. They can also leave the body acoustic wave area under the inertial drag force generated by the laminar flow of the solution and flow downstream with the liquid.

[0151] The applicant unexpectedly discovered through experiments that in the method and apparatus of the present invention, when the vesicles in the solution pass through the acoustic fluid region caused by ultra-high frequency bulk acoustic waves, the flexible particles that are pushed to the top of the flow channel and stay ("blocked") are related to factors such as the bulk acoustic wave power (related to the amplitude and intensity of the sound wave), the distance between the ultra-high frequency resonator and the top of the flow channel, and the speed at which the solution flows through the bulk acoustic wave region.

[0152] Without being bound by relevant theories, the applicant believes that when the power approaches zero, neither the acoustic radiation force nor the vortex is sufficient to act on the particles, so the phenomenon is dominated by the laminar drag force. As the power increases, the force of the vortex is not enough to change the particle motion dominated by the acoustic radiation force, so the particles begin to be pushed to the top of the flow channel. As the power continues to increase, the acoustic fluid is strong enough that the acoustic radiation force cannot push the particles to the top, but can only push the particles to the center of the vortex, so that the particles enter the vortex tunnel and move along the tunnel. As the power increases, the size of particles that can hit the opposite side of the flow channel is decreasing, and the size of particles that can be captured by the vortex is also decreasing. Within a certain parameter range, at the same power, the size of particles that can be pushed to the opposite side of the flow channel is smaller than that captured by the vortex.

[0153] Microvesicles of different sizes, especially those in the size range of 20-1000 nm, can be distinguished and separated by a suitable combination of bulk acoustic wave power (related to the acoustic wave amplitude and intensity), the distance between the ultrahigh frequency resonator and the top of the flow channel, and the speed at which the solution flows through the bulk acoustic wave region.

[0154] Thus, the inventors of the present applicant have discovered and provided a method for more effectively isolating microvesicles of target cells.

[0155] In the present invention, the frequency of the thin film bulk acoustic wave resonator is mainly determined by the thickness and material of the piezoelectric layer. The thickness of the piezoelectric layer of the thin film bulk acoustic wave resonator used in the present invention ranges from 1nm to 2um. The frequency of the ultra-high frequency bulk acoustic wave resonator of the present invention is about 0.5-50GHz, preferably about 1-10GHz.

[0156] The BAW generated by the UHF BAW resonator is driven by a signal from a high frequency signal generator. The pulse voltage signal driving the resonator can be driven with pulse width modulation, which can generate any desired waveform, such as a sine wave, square wave, sawtooth wave or triangle wave. The pulse voltage signal can also have an AM or FM start / stop capability to start or eliminate the BAW.

[0157] The microfluidic device of the present invention also includes a power regulating device, which regulates the power of the bulk acoustic wave generated by the ultra-high frequency resonator. In this embodiment, the power regulating device is a power amplifier with a power regulating function. In one aspect of the present invention, the output power of the power regulating device is about 0.5-2000mW, preferably about 5-1500mW, more preferably about 15-900mW, for example, 70-300mW. Since the thin film bulk acoustic wave resonator has high energy conversion efficiency and basically no loss, the output power of the power regulating device can be basically regarded as the output power of the thin film bulk acoustic wave resonator to generate bulk acoustic waves in the fluid. In the microfluidic device of the present invention, the power regulating device can be connected to a high-frequency signal generator. The output circuit of the power amplifier is respectively connected to the bottom electrode, piezoelectric layer, and top electrode of the ultra-high frequency bulk acoustic wave resonator.

[0158] The microfluidic device of the present invention may also include a detection device for detecting signals of characteristics of cells in the sample or the markers they carry. These characteristics may include physical properties such as molecular size, molecular weight, molecular magnetic moment, refractive index, electrical conductivity, charge, absorbance, fluorescence, polarity, etc. For example, the detection device includes a detection electrical detection device, such as a Coulter counter, for cell counting. The detection device may also be a photodetector, which includes an illumination source and an optical detection component for detecting physical parameters such as charge, absorbance, fluorescence, polarity, etc.

[0159] In such Figure 1 and Figure 2 The microfluidic device of the present invention shown in the figure has a Coulter counter (its device basis is an impedance meter 303), which is arranged at a specified distance from the intersection of the sample inlet and the buffer inlet in the microchannel, and is arranged at a specified distance from the outlet of the microchannel in the microchannel. The Coulter counter is a sensor that uses the electrical properties of cells to achieve cell counting and detection. Structurally, the Coulter counter is composed of multiple electrode strips, mostly two or three electrodes. Its working principle is that when cells pass through the electrodes, they will replace the same volume of electrolyte, causing the dielectric impedance between the electrodes to change, and a transient potential pulse will be generated, which can be detected by an external impedance meter. Thus, cell counting can be achieved. At the same time, because its processing technology is compatible with the processing technology of bulk acoustic wave devices (ultra-high frequency bulk acoustic wave resonators), it can be integrated on the same substrate to achieve detection of vesicles, etc.

[0160] The microfluidic device provided by the present invention can also be used to capture / separate nucleic acids. The microfluidic device and method provided by the present invention are particularly suitable for separating short-chain nucleic acids. For example, the method of the present invention is suitable for separating short-chain nucleic acids with a length of ≤1500bp, preferably ≤500bp, more preferably ≤200bp, for example ≤100bp. In this aspect of the present invention, the height of the fluid channel of the microfluidic device is generally about 5-25um, preferably about 6-25um, and more preferably about 7-20um.

[0161] Example 3 Effect of flow channel height on acoustic jet and vortex caused by body acoustic wave

[0162] The acoustic jets and / or vortices generated by the ultra-high frequency bulk acoustic wave resonator used in the present invention in the microchannels at different heights are observed. When the microchannel of the present invention is used to separate cell microvesicles (20-800nm) in the size range of exosomes (about 20-250nm), the suitable height range is below 60 microns, such as 60 microns, 40 microns, and 20 microns.

[0163] Among them, hollow glass microspheres (with a density close to that of water) are added to the fluid cavity, and the liquid flow velocity distribution is characterized by the particle motion trajectory.

[0164] The results are as follows Figure 3 As shown in the figure, the microchannel sizes in the upper, middle and lower figures decrease in sequence.

[0165] The high-speed camera was used to shoot at a speed of 5000fps and the images were stitched together through 100 frames. Each line segment in the figure represents the trajectory of the particle. Since the time of 100 frames is the same (20ms), the length of the line segment represents the distance the particle moves during this time period. The longer the line segment, the faster the particle moves. It can be seen that under the same power, as the height decreases, the movement speed of the particles in the vortex increases. Since the drag force of the fluid is proportional to the flow rate, when the flow channel height is lower, the vortex will have a stronger fluid drag force. On the other hand, as the height decreases, the center of the vortex will also be closer to the UHF resonator, which means that as the vortex enters the upper part of the UHF resonator, the particle trajectory will be closer to the surface, and the particle will be subject to a greater acoustic radiation force and change its trajectory to enter the center of the vortex. It can be seen that lowering the height of the flow channel can increase the vortex fluid velocity and thus increase the drag force while keeping the power condition unchanged.

[0166] Example 4 Isolation of exosomes from plasma samples

[0167] According to the method described in Examples 1 and 2, prepare and set Figure 4 (a) Microfluidic channel and UHF resonator shown. Figure 4(a) and (b) are top views of the microfluidic channel. The top is the channel inlet, and the arrow on the right indicates the direction of liquid flow. The surface of the ultrahigh frequency resonator (i.e., the area where the bulk acoustic wave occurs, shown as a five-pointed star in the figure) is located on one side of the channel (the left side in the figure). The flow channel inlet of the microfluidic channel includes two solution inlets. The left inlet is fed with plasma samples obtained from volunteers, which are centrifuged at high speed to remove blood cells and some vesicles. The plasma sample is stained with Calcein-AM. The PBS solution is passed through the right side, and the dotted line indicates the separation of the PBS solution and the plasma sample flow. By adjusting the flow rate of the PBS solution and the focusing effect of the sheath flow, the plasma sample flow is allowed to fully pass through the bulk acoustic wave generation area of ​​the ultrahigh frequency resonator. The two downstream outlets are the waste liquid outlet and the outlet (exosome outlet).

[0168] like Figure 4 (b) When the UHF resonator starts working, under the action of acoustic radiation force, larger vesicles are pushed to the flow channel surface above the UHF resonator when passing through the BAW region and are captured, while smaller exosomes escape the capture of the BAW region and enter the downstream, and are collected and detected through the exosome outlet.

[0169] Figure 4 (c), (d) and (e) are the results of exosome screening of plasma.

[0170] The first sample was 10-fold diluted plasma. Figure 4 (c) and Figure 4 (d) as shown. Figure 4 (c) shows that there are two sizes of vesicles before separation, which are manifested as two peaks. Through the method and system of the present invention, the vesicles with a size of about 137 nm on the right side of Figure (c) are removed from the plasma sample, and the vesicles with a size of about 45 nm on the left side of Figure (c) are retained.

[0171] Experimental parameters: flow rate Vpbs=Vplasma is 2μL / min, equivalent to V is 1.07mm / s, the power applied by the ultrahigh frequency resonator is 209Mw, and the flow channel height is 20um.

[0172] The second sample was undiluted plasma. Figure 4 As shown in (e), the vesicles with a size of about 144 nm on the right side of the figure were removed from the plasma stock sample, while the vesicles with a size of about 107 nm on the left side of the figure were retained.

[0173] Experimental parameters: flow rate Vpbs is 2μL / min, Vplasma is 3μL / min, the power applied by the ultra-high frequency resonator is 660Mw, and the flow channel height is 20um.

[0174] It can be seen that through the microfluidic device of the present invention, vesicles of different sizes, including exosomes, can be separated as needed by adjusting the power and flow rate of the bulk acoustic waves generated by the ultrahigh frequency resonator.

[0175] Example 5 Isolation of Nucleic Acids

[0176] According to the method described in Examples 1 and 2, prepare and set Figure 5 (a) Microfluidic channel and UHF resonator shown on the left. Figure 5 This is a top view. Figure 5 (a) In the leftmost image above, the upper part is the inlet of the flow channel, which is used to input PBS solutions containing nucleic acid fragments of different sizes. The surface of the ultra-high frequency resonator, that is, the area where the bulk acoustic wave is generated, is shown as a five-pointed star in the figure. The height of the microchannel is about 7 microns.

[0177] The nucleic acid sample is a double-stranded nucleic acid obtained by PCR amplification reaction. The nucleic acid with the accurate number of nucleotides can be obtained by selecting (synthesizing) appropriate primers according to the sequence of the DNA template and then amplifying. The nucleic acid is stained and quantified using the Qubit sDNA HS kit, dissolved in PBS solution, and adjusted to about 85ng / μl.

[0178] like Figure 5 (a) Display system settings and fluorescence observation phenomena: the left picture is the bright field, and the right picture is the fluorescence signal observation picture. Figure 5 (b) is the control, where only Qubit dye was added to PBS. Figure 5 (c)- Figure 5 (g) A PBS solution containing nucleic acids of different sizes was passed through. Figure 5 (b)- Figure 5 The left figure in (g) shows the phenomenon when the UHF resonator generates bulk acoustic waves after power (2100 mW) is applied, and the right figure shows the phenomenon when the UHF resonator stops generating bulk acoustic waves and a PBS solution is input.

[0179] As shown in the figure, when the UHF resonator generates BAW after applying power (2100mW), under the action of acoustic radiation force, nucleic acids of 76bp, 151bp, 200bp, 500bp, and 1000bp are pushed to the surface of the flow channel above the UHF resonator and captured when passing through the BAW area. When the UHF resonator stops generating BAW, the captured nucleic acids fall off and flow in the direction of the liquid flow; the dotted circle indicates the nucleic acids that move after falling off.

[0180] The results showed that nucleic acids ranging from about 50 bp to 1 kbp could be captured and released by the microfluidic device of the present invention.

[0181] It can be seen that the microfluidic device and method provided by the present invention can capture nucleic acids of different sizes as needed by adjusting the power and flow rate of the bulk acoustic wave generated by the ultra-high frequency resonator, and then release them into the solution to achieve the purpose of separating or purifying nucleic acids. Without being limited by this theory, the applicant believes that when the microfluidic device provided by the present invention is used to capture nucleic acids, especially small molecule nucleic acids, in the area where the bulk acoustic wave acts, the nucleic acids are mainly affected by the acoustic radiation force caused by the attenuation of the acoustic wave according to the height of the flow channel and the frequency of the bulk acoustic wave.

[0182] The microfluidic device and method provided by the present invention can also release nucleic acids of different sizes one by one in order from small to large after capturing nucleic acids of different sizes by adjusting the power and flow rate of the bulk acoustic waves generated by the ultrahigh frequency resonator, thereby achieving the purpose of further separation.

[0183] In summary, the microfluidic device and method for separating flexible particles provided in the present application can achieve selective capture and controllable release of flexible particles of different sizes, including cell microvesicles or nucleic acids, thereby obtaining or purifying flexible particles for further analysis.

[0184] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating flexible particles, characterized in that: include: (1) allowing a solution sample containing flexible particles to flow through a microfluidic device, the device comprising: a fluid channel having an inlet and an outlet; One or more ultra-high frequency bulk acoustic wave resonators are arranged at the bottom of the fluid channel, and the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel; (2) the ultra-high frequency bulk acoustic wave resonator emits a bulk acoustic wave that is transmitted toward the top of the fluid channel; (3) adjusting the distance from the ultrahigh frequency bulk acoustic wave resonator to the top of the flow channel and / or adjusting the power of the bulk acoustic wave and / or adjusting the speed at which the solution flows through the area affected by the bulk acoustic wave, so that the specified flexible particles are pushed to the top of the fluid channel and stay in the area affected by the bulk acoustic wave.

2. The method according to claim 1, characterized in that It also includes: (4) Obtain liquid samples entering the downstream of the bulk acoustic wave region.

3. The method according to claim 1, characterized in that The diameter of the flexible particles is 0.2-1 μm.

4. The method according to claim 1, characterized in that The flexible particles are naturally occurring particles.

5. The method according to claim 1, characterized in that: The flexible particles include: vesicles, blisters, or bubbles.

6. The method according to claim 5, characterized in that The vesicles include: exosomes, microvesicles, prostatic bodies, intraluminal vesicles, or endosomal-like vesicles.

7. The method according to claim 1, characterized in that The distance from the ultra-high frequency bulk acoustic wave resonator to the top of the flow channel is adjusted to 8-60 μm.

8. The method according to claim 1, characterized in that The power of the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator is adjusted to 0.5-2000mW.

9. The method according to claim 1, characterized in that: wherein the speed at which the solution flows through the bulk acoustic wave region is adjusted to 0.1-10 mm / s; or The speed at which the solution flows through the bulk acoustic wave region is adjusted to 0.1-100 μL / min.

10. The method according to claim 1, characterized in that The area of ​​the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is 500-200000 μm 2 .

11. The method according to claim 1, characterized in that: The inlet includes a sample inlet and an auxiliary solution inlet arranged on one side or both sides of the sample inlet.

12. The method according to any one of claims 1 to 11, characterized in that wherein the solution sample contains different flexible particles, wherein the different flexible particles include flexible particles having different diameters; The method includes controlling the flexible particles that are pushed to the top of the fluid channel and stay there, and obtaining the flexible particles that are not pushed to the top of the fluid channel and stay there downstream of the area affected by the bulk acoustic wave.

13. The method according to claim 12, characterized in that The control is performed by one or any combination of the following methods: (a) adjusting the distance between the UHF BAW resonator and the top of the flow channel; (b) regulating the power of the bulk acoustic wave; (c) adjusting the velocity of the solution flowing through the bulk acoustic wave region.

14. The method according to claim 12, characterized in that Exosomes are obtained downstream of the area affected by the body acoustic wave, and the diameter of the exosomes is 20-300nm.

15. The method according to claim 12, characterized in that The distance between the ultra-high frequency bulk acoustic wave resonator and the top of the flow channel is 15 μm to 25 μm, and the power of the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator is adjusted to 600-1500 mW.

16. A microfluidic device for separating flexible particles, characterized in that: include: a fluid channel having an inlet and an outlet; One or more ultra-high frequency bulk acoustic wave resonators, which are arranged on a wall of the fluid channel, and the ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of 0.5-50 GHz in the fluid channel and propagate toward the top of the fluid channel; A power regulating device for regulating the power of the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator; A flow rate regulating device, which regulates the speed at which the solution flows through the bulk acoustic wave region, The ultra-high frequency bulk acoustic wave resonator can emit bulk acoustic waves toward the top of the fluid channel, so that the solution flowing through the bulk acoustic wave area generates an acoustic jet. The microfluidic device is configured to adjust the distance from the ultra-high frequency bulk acoustic wave resonator to the top of the flow channel and / or adjust the power of the bulk acoustic wave through the power adjustment device and / or adjust the speed of the solution flowing through the bulk acoustic wave influence area through the flow rate adjustment device, so that the specified flexible particles are pushed to the top of the fluid channel and stay in the bulk acoustic wave influence area.

17. The microfluidic device according to claim 16, characterized in that: The diameter of the flexible particles is 0.2-1 μm.

18. The microfluidic device according to claim 16, characterized in that: The flexible particles are naturally occurring particles.

19. The microfluidic device according to claim 16, characterized in that: The flexible particles include: vesicles, blisters, or bubbles.

20. The microfluidic device according to claim 19, characterized in that The vesicles include: exosomes, microvesicles, prostatic bodies, intraluminal vesicles, or endosomal-like vesicles.

21. The microfluidic device according to claim 16, characterized in that: The distance between the ultra-high frequency bulk acoustic wave resonator and the top of the flow channel is 8-80 μm.

22. The microfluidic device according to claim 16, characterized in that The output power of the power regulating device is 0.5-2000mW.

23. The microfluidic device according to claim 16, characterized in that The flow rate regulating device can adjust the speed of the solution flowing through the bulk acoustic wave region to 0.1-10 mm / s.

24. The microfluidic device according to claim 16, characterized in that The flow rate regulating device can adjust the speed of the solution flowing through the bulk acoustic wave region to 0.1-100 μL / min.

25. The microfluidic device according to claim 16, characterized in that The area of ​​the bulk acoustic wave generation region of the ultra-high frequency bulk acoustic wave resonator is 500-200000 μm 2 .

26. The microfluidic device according to claim 16, characterized in that The inlet includes a sample inlet and an auxiliary solution inlet arranged on one side or both sides of the sample inlet.

27. The microfluidic device according to claim 16, characterized in that The ultra-high frequency bulk acoustic wave resonator is a thin film bulk acoustic wave resonator or a solid-state assembly type resonator.

28. The microfluidic device according to claim 16, characterized in that The thickness of the piezoelectric layer of the ultra-high frequency bulk acoustic wave resonator of the device is in the range of 1 nm to 2 μm.

29. The microfluidic device according to claim 16, characterized in that The fluid channel has at least two outlets, one of which is used to receive a liquid sample that is pushed to the top of the fluid channel and stays in the area affected by the bulk acoustic wave, allowing the designated flexible particles to pass through the bulk acoustic wave area and enter the downstream.

30. The microfluidic device according to claim 16, characterized in that A plurality of ultra-high frequency bulk acoustic wave resonators having the same bulk acoustic wave generating area are arranged.

31. The microfluidic device according to claim 16, characterized in that The fluid channel is divided into different regions, and ultrahigh frequency bulk acoustic wave resonators for separating different cells or vesicles are arranged in different regions. The ultra-high frequency bulk acoustic wave resonator for separating different cells or vesicles has acoustic wave generating areas of different shapes, Alternatively, the ultra-high frequency bulk acoustic wave resonator for separating different cells or vesicles applies bulk acoustic waves of different powers, Alternatively, the different regions have different flow rates.

32. A kit, characterized in that It includes a microfluidic device for separating flexible particles as described in any one of claims 16 to 31, and reagents for analyzing microvesicles.

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