Method and device for controlling the movement of biomacromolecules in solution

Abstract

The present invention discloses a microfluidic system and method for controlling the movement of biomacromolecules, such as nucleic acids, in a fluid. The system of the present invention includes a fluid channel having an inlet and multiple outlets; one or more ultra-high frequency acoustic wave resonators, each of which can generate bulk acoustic waves with a frequency of approximately 0.5-50 GHz in the fluid channel; by adjusting the shape and orientation of the bulk acoustic wave generating region of the ultra-high frequency bulk acoustic wave resonator, biomacromolecules, such as nucleic acids, can move to a specified position and direction upon entering a vortex channel induced by the bulk acoustic wave in the solution. The system and method of the present invention can control and separate biomacromolecules, such as nucleic acids, in a solution to obtain a specified nucleic acid, or obtain a purified solution after the nucleic acid is separated.

Description

[0001] This application claims priority to the following Chinese patent applications: Application No. 202010546759.5, filed on June 15, 2020, entitled “Method and Apparatus for Controlling the Movement of Biomacromolecules in Solution”; and Application No. 202011465865.7, filed on December 13, 2020, entitled “Method and Apparatus for Controlling the Movement of Nucleic Acids in Solution”. The entire contents of each of these applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to the fields of biological research methodology and medical devices. Specifically, the present invention relates to a microfluidic system for separating and analyzing biological macromolecules such as nucleic acids and a method for separating and analyzing biological macromolecules such as nucleic acids using the system. Background Art

[0003] Microfluidics systems are widely used to separate nucleic acids from samples. Most reported methods rely on the biospecificity of nucleic acids to distinguish and separate them. However, these methods suffer from significant costs and low throughput.

[0004] Therefore, there is an urgent need for a system and method to separate biomacromolecule particles in a solution and obtain the desired biomacromolecule particles. Summary of the Invention

[0005] The present invention discovers for the first time that ultrahigh frequency bulk acoustic waves can be used to effectively manipulate the movement position and direction of flexible particles such as nucleic acids, proteins, polysaccharides and other biomacromolecules in a solution in a microfluidic system, thereby providing a method and system for separating and obtaining target biomacromolecule particles.

[0006] The present invention provides a method for controlling the movement of target nucleic acids, proteins, polysaccharides and other biomacromolecules (especially nucleic acids) in a solution, comprising:

[0007] (1) allowing a solution containing a target biomacromolecule to flow through a microfluidic device, the device comprising:

[0008] a fluid channel having an inlet and an outlet;

[0009] One or more ultra-high frequency bulk acoustic wave resonators, which are disposed 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 about 0.5-50 GHz in the fluid channel that propagate toward the wall on the opposite side of the fluid channel;

[0010] (2) the UHF resonator emits a bulk acoustic wave that is transmitted toward the wall on the opposite side of the fluid channel, generating a vortex channel in the solution that is defined by the boundary of the bulk acoustic wave generating region of the UHF resonator;

[0011] (3) The target biomacromolecules in the solution enter the vortex channel, move along the vortex channel, and leave the vortex channel at a set position.

[0012] In one aspect of the present invention, the biomacromolecule in the method is a nucleic acid. 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, 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 products of amplification reactions using artificial or natural DNA or RNA as templates. Nucleic acids can be double-stranded or single-stranded, circular or linear. Samples that can be used to detect target nucleic acids include the following: samples from cell cultures, eukaryotic microorganisms, or diagnostic samples such as body fluids, body fluid sediments, gastric lavage specimens, fine needle aspirates, biopsy samples, tissue samples, cancer cells, cells from a patient, cells from a tissue, or in vitro cultured cells from an individual to be tested and / or treated for a disease or infection, or forensic samples. Non-limiting examples of body fluid samples include whole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph fluid, serum, plasma, urine, chyle, feces, ejaculate, sputum, nipple aspirate, saliva, cotton swab samples, wash or lavage fluid and / or wipe samples. The method of the present invention is particularly suitable for separating nucleic acids (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) with a length of ≥100 bp, or ≥300 bp, preferably ≥1 kbp, more preferably ≥10 kbp, for example ≥50 kbp.

[0013] The ultra-high frequency bulk acoustic wave resonator in the present invention refers to a resonator capable of generating 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, for example, a thin film bulk acoustic wave resonator or a solid-state assembly resonator.

[0014] In the method of the present invention, an ultrahigh frequency resonator emits an ultrahigh frequency bulk acoustic wave (UHF) toward the wall on the opposite side of the fluid channel (e.g., the top of the channel). The volume force generated by the attenuation of the sound wave in the fluid causes an acoustic jet to appear in the solution flowing through, resulting in the generation of a localized three-dimensional vortex in the liquid in the microchannel. Because the vortex is generated by the volume force caused by the attenuation of the sound wave, the central axis of the vortex is above the boundary of the bulk acoustic wave. The continuous vortex caused by the ultrahigh frequency bulk acoustic wave forms an acoustic fluid vortex channel. The shape of the vortex channel is substantially the same as the shape of the bulk acoustic wave generation region and is located above the boundary of the bulk acoustic wave generation region. That is, the shape and position of the vortex channel are defined by the boundary of the bulk acoustic wave generation region of the ultrahigh frequency resonator.

[0015] In one aspect of the present invention, the fluid channel of the microfluidic device has an outflow channel for the biological macromolecules such as nucleic acids or proteins whose movement is controlled, which can be called a particle outflow channel. In another aspect of the present invention, the fluid channel also has other outflow channels, such as an outflow channel for a solution that removes or contains less of the biological macromolecules whose movement is controlled, which can be called a solution outflow channel. The width ratio of the openings of the particle outflow channel and the solution outflow channel can be set to about 1:1-1:20, preferably about 1:2-1:15, for example, about 1:4-1:10. In one aspect of the present invention, the position where the biological macromolecules leave the vortex channel is close to the opening of the particle outflow channel.

[0016] In step (3) of the above method, the shape and position of the bulk acoustic wave action area of ​​the ultrahigh frequency bulk acoustic wave resonator can be adjusted so that the biomacromolecules such as nucleic acids or proteins that are controlled to move in the solution enter the vortex channel and move along the vortex channel, and leave the vortex channel at a set position. As a result, the biomacromolecules leave the bulk acoustic wave action area at a specified position and direction and enter the desired outflow channel, for example, the particle outflow channel. The set position for leaving the vortex channel is called the release point, that is, the position where the biomacromolecules leave the bulk acoustic wave action area. The solution without the biomacromolecules that are controlled to move maintains the inflow direction and enters the aforementioned solution outflow channel.

[0017] Since one of the important factors for biomacromolecules such as nucleic acids or proteins to escape from the vortex channel is the influence of laminar flow along the direction of the fluid channel, the release point is usually located in the downstream area of ​​the vortex channel. In another aspect of the present invention, the release point is usually located at the location where the vortex channel turns or changes in curvature, that is, above the location where the boundary of the bulk acoustic wave generation area turns or changes in curvature, that is, there is a turning or curvature change at the boundary of the bulk acoustic wave generation area corresponding to the release point. Without being bound by relevant theories, the applicant believes that the reason for this phenomenon is that at the turning point or corner of the vortex channel, the vortex direction and the direction of the acoustic radiation force suddenly change. Among the biomacromolecules such as nucleic acids or proteins that enter the vortex channel, particles that meet the appropriate conditions (such as the appropriate size) have already been focused at the vortex center. Under the action of the acoustic radiation force, they can change their movement direction with the vortex channel and quickly refocus to the center of the vortex channel after the turn; while particles that do not meet the conditions (such as having a smaller size) will be more affected by the jump in the laminar drag direction and leave the vortex channel.

[0018] In one aspect of the present invention, the method provided herein is suitable for processing liquid samples containing a large number of biomacromolecules, such as nucleic acids or proteins; the large number of biomacromolecules can continuously enter and move along the vortex channel and exit the vortex channel at a predetermined location, thereby achieving rapid and high-throughput processing. In one aspect of the present invention, the method provided herein is suitable for processing samples containing a large number of biomacromolecules, such as whole blood or blood fractions.

[0019] The above method provided by the present invention can be used to obtain or purify desired biomacromolecules in a sample. In one aspect of the present invention, the method can be used to enrich desired biomacromolecules.

[0020] The method provided by the present invention can also be used to remove unwanted biomacromolecules in a sample to obtain a purified solution.

[0021] In one aspect of the present invention, the method further comprises regulating the biomacromolecule particles entering the vortex channel by adjusting the power of the bulk acoustic wave and / or the speed at which the solution flows through the bulk acoustic wave region. Biomacromolecules that do not enter the vortex channel, or that enter the vortex channel but exit the vortex channel before reaching a designated release point, pass through the bulk acoustic wave region and exit in the direction in which the sample entered the fluid channel.

[0022] In one aspect of the present invention, the boundary lines of the bulk acoustic wave generating region of the UHF resonator in the above method (i.e., the shape of the corresponding vortex channel) are configured to facilitate the migration of the target biomacromolecule along the vortex channel to the release point. This prevents the target biomacromolecule from escaping the vortex channel and not leaving the vortex channel at the desired release point.

[0023] In another aspect of the present invention, the target biomacromolecule is maintained in the vortex channel and moves to the release point by adjusting the boundary shape of the bulk acoustic wave generation area of ​​the ultrahigh frequency resonator. As previously mentioned, the presence of a turning point or curvature change in the boundary lines of the bulk acoustic wave generation area may increase the probability of the biomacromolecule escaping the vortex channel. Therefore, the biomacromolecule can be maintained in the vortex channel and moved by reducing the turning point or curvature change in the boundary lines of the bulk acoustic wave generation area, that is, reducing the biomacromolecules that escaping the vortex channel and improving the efficiency of separation.

[0024] In another aspect of the present invention, the angle between the boundary line of the UHF resonator's BAW generation region and the fluid channel is adjusted to maintain the biomacromolecules within the vortex channel as they move toward the release point. The inventors unexpectedly discovered that a smaller angle between the boundary line of the BAW generation region and the fluid channel facilitates the biomacromolecules' movement within the vortex channel, thereby reducing the risk of biomacromolecules escaping the vortex channel and improving separation efficiency.

[0025] In another aspect of the present invention, the bulk acoustic wave action area of ​​the ultrahigh frequency bulk acoustic wave resonator in the microfluidic system has a focusing area and a screening area. The focusing area is located upstream of the bulk acoustic wave action area (i.e., close to the sample inflow direction, far from the release point), and the screening area is located downstream of the bulk acoustic wave action area (i.e., close to the sample outflow direction, close to the release point or including the release point). The setting of the bulk acoustic wave action area in the focusing area is more suitable for keeping the biomacromolecule particles moving in the vortex channel relative to the setting of the screening area: the biomacromolecules in the vortex channel of the focusing area move in the same or similar direction as the laminar flow direction, and the vortex drag force they are subjected to is relatively small, making it easier for the biomacromolecules to enter and remain in the vortex channel; in the downstream screening area, the biomacromolecules focused to the center of the vortex can be moved more stably in the vortex channel than the unfocused biomacromolecules. In another aspect of the present invention, the angle between the boundary line of the bulk acoustic wave action area in the focusing area and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area in the screening area and the fluid channel. For example, the boundary of the body acoustic wave action area in the focusing zone is basically consistent with or basically consistent with the direction of the fluid channel (for example, the angle is less than 10°). The vortex drag force on the particles in the vortex channel in this area basically does not change the motion state of the biomacromolecules along the laminar flow direction, but only causes the biomacromolecules to migrate laterally to the center of the vortex, thereby achieving the focusing of the biomacromolecules; the boundary of the body acoustic wave action area in the screening zone has a larger angle with the fluid channel, guiding the movement direction of the biomacromolecules to deviate from the direction of the fluid channel and transfer to the designated release point. In the screening zone, the biomacromolecules focused to the center of the vortex can be moved more stably in the vortex channel than the unfocused biomacromolecules.

[0026] In the present invention, the microfluidic device generally includes a power regulating device, which regulates the power of the bulk acoustic wave generated by the ultrahigh frequency resonator.

[0027] In the present invention, 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.

[0028] In one aspect of the present invention, the output power of the power regulating device is about 20-5000 mW, preferably 50-2000 mW, and more preferably 100-1500 mW.

[0029] In one aspect of the present invention, the flow rate regulating device 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 0.5-3 mm / s.

[0030] In one aspect of the present invention, the flow rate regulating device can adjust the speed of the solution flowing through the bulk acoustic wave region to about 0.01-100 μL / min, preferably about 0.1-50 μL / min, more preferably about 0.5-30 μL / min.

[0031] In one aspect of the present invention, the height of the fluid channel in the bulk acoustic wave action area of ​​the microfluidic device is about 5-200 μm, preferably about 25-100 μm, more preferably about 30-80 μm, for example about 40-60 μm.

[0032] In one aspect of the present invention, the height of the fluid channel in the bulk acoustic wave action area of ​​the microfluidic device is about 10-45 μm, preferably about 15-30 μm, more preferably about 18-25 μm, for example about 20 μm or less than about 20 μm.

[0033] 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 .

[0034] In one aspect of the present invention, the side length of the BAW generating region of the UHF BAW resonator is about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm. That is, the side length of the corresponding eddy current channel (the distance from the upstream end closest to the sample to the downstream release point) is about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm.

[0035] 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. Based on the sheath flow effect, the auxiliary solution can be used to control the flow direction and range of the sample liquid in the microchannel, so that the sample liquid fully flows through the bulk acoustic wave generation area of ​​the ultrahigh frequency bulk acoustic wave resonator. For example, the flow direction and range of the sample liquid in the microchannel can be controlled by controlling the flow rate and inflow area of ​​the auxiliary solution.

[0036] In one aspect of the present invention, the microfluidic system in the aforementioned method includes multiple UHF resonators having identical BAW generation regions. For example, the BAW generation regions of the multiple UHF resonators have identical release points, such as, for example, identical BAW generation regions. This allows for the collection and reorientation of biomacromolecules, such as nucleic acids or proteins, that have undesirably escaped from the BAW generation region of an upstream UHF resonator, thereby increasing separation efficiency.

[0037] In one aspect of the present invention, the microfluidic system described in the aforementioned method divides the fluid channel into different regions, and ultrahigh frequency resonators for separating different biomacromolecules are positioned in different regions. For example, the ultrahigh frequency resonators for separating different nucleic acids or proteins can have acoustic wave generating regions of different shapes, or apply bulk acoustic waves of different powers, or have different flow rates. Thus, different nucleic acids or proteins in a solution can be separated and directed into different flow channels or outlets.

[0038] In one aspect of the present invention, the aforementioned method can be used to separate (or isolate) biological macromolecules such as nucleic acids or proteins of different types or properties (eg, different sizes or densities, etc.).

[0039] In another aspect of the present invention, different biological macromolecules such as nucleic acids or proteins can be selectively separated (or separated) by one or any combination of the following methods:

[0040] (a) regulating the power of the bulk acoustic wave;

[0041] (b) regulating the time of generating body sound waves;

[0042] (c) Adjust the velocity of the solution flowing through the bulk acoustic wave region.

[0043] The present invention also provides a microfluidic device for controlling the movement of flexible particles, such as nucleic acids, proteins, or polysaccharides, in a solution. The microfluidic device can be used to obtain or purify desired biomacromolecules. The microfluidic device provided herein is used to process biomacromolecules and therefore has configurations or materials suitable for processing bioactive substances. For example, the inner surface of the flow channel can be made of a biocompatible material. In another example, the device has a design to prevent cross-contamination, particularly contamination that could lead to amplification.

[0044] In one aspect of the present invention, the aforementioned method is used to separate nucleic acids. In another aspect of the present invention, the nucleic acid molecule is 100-10,000 bases or base pairs in length, preferably 200-2,000 bases or base pairs, more preferably 300-1,000 bases or base pairs, for example, about 500 bases or base pairs. In another aspect of the present invention, the height of the fluid channel of the microfluidic device is about 10-45 μm, preferably about 15-30 μm, more preferably about 18-25 μm, for example, about 20 μm or less. In another aspect of the present invention, the solution containing the biomacromolecule particles contains a nucleic acid agglutinating agent; in another aspect of the present invention, the solution containing the biomacromolecule particles further contains a buffered saline solution, particularly a buffered saline solution of a relatively high concentration. In the present invention, the addition of a nucleic acid agglutinating agent to the solution containing the biomacromolecule particles facilitates the separation of smaller nucleic acids, such as nucleic acids with a molecular length of 100-2,000 bases or base pairs.

[0045] In the present invention, nucleic acid chains are condensed in a solution containing a nucleic acid condensing agent. The nucleic acid condensing agent that can be used in the method of the present invention includes various nucleic acid condensing agents known in the art.

[0046] In one aspect of the present invention, the coagulant can be a nonionic polymer, such as a polyethylene glycol polymer. In a specific example, the molecular weight of the polyethylene glycol polymer is in the range of 1,000 to 100,000, such as in the range of 2,000 to 20,000, between 5,000 and 15,000, or between 8,000 and 12,000. The nonionic polymer can be present in the solution at a concentration in the range of 0.1 wt% to 25.0 wt%, such as in the range of 0.5 wt% to 15.0 wt%, in the range of 0.5 wt% to 10 wt%, or in the range of 2.5 wt% to 7.5 wt%.

[0047] In one aspect of the present invention, the coagulant can be a concentrated alkali metal salt or alkaline earth metal salt, such as a halide salt. For example, the coagulant can include magnesium chloride at a concentration in the range of 1 mM to 1 M, such as 30 mM to 1 M, 50 mM to 1 M, or 50 mM to 800 mM.

[0048] In one aspect of the present invention, the coagulant is a metal complex. The metal complex may have a 3+ charge and may be provided to the solution as a salt. For example, the metal complex salt may be a halide salt, such as a chloride salt or an iodide salt. Specifically, the metal complex includes cobalt, for example, to form a cobalt organic complex, such as a cobalt-ammine complex. In one example, the metal complex may include hexaammine cobalt. In another example, the metal complex includes tris(ethylenediamine)cobalt. In another example, the metal complex includes 1,3,6,8,10,13,16,19-octaazabicyclo-[6.6.6]eicosanecobalt.

[0049] In the present invention, the solution containing the biomacromolecule particles further contains a buffered saline solution, such as a phosphate-buffered saline solution. The solution may include salts such as alkali metal halides, such as sodium or potassium halides, sodium or potassium phosphates, and polysorbates. In the solution containing the biomacromolecule particles, the salts are present at high concentrations, for example, in an amount ranging from 100 mM to 5000 mM, such as from 150 mM to 3000 mM or from 500 mM to 2500 mM. In one example, sodium chloride or potassium chloride may be present in a concentration ranging from 0.5 M to 2 M, such as from 0.8 M to 1.8 M or from 0.8 M to 1.6 M.

[0050] In one aspect of the present invention, the solution containing biomacromolecule particles comprises a polyethylene glycol polymer and an alkali metal halide salt, such as a sodium halide salt or a potassium halide salt. In another aspect of the present invention, the polyethylene glycol polymer is present in an amount of about 0.1 wt% to 25.0 wt%, preferably about 1.0 wt% to 10.0 wt%, and the alkali metal halide salt is present in an amount of about 100 mM to 5000 mM, preferably about 500-2000 mM.

[0051] In one aspect of the present invention, a microfluidic device for separating biological macromolecules such as nucleic acids or proteins is provided, comprising:

[0052] a fluid channel having an inlet and an outlet;

[0053] One or more ultra-high frequency bulk acoustic wave resonators, which are disposed 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 about 0.5-50 GHz in the fluid channel that propagate toward the wall on the opposite side of the fluid channel;

[0054] a power regulating device for regulating the power of the bulk acoustic wave generated by the ultrahigh frequency resonator;

[0055] A flow rate regulating device, which regulates the speed at which the solution flows through the bulk acoustic wave region,

[0056] The ultra-high frequency resonator can emit bulk acoustic waves toward the wall on the opposite side of the fluid channel, generating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generation area of ​​the ultra-high frequency resonator. Biomacromolecules such as nucleic acids in the solution enter the vortex channel and move along the vortex channel, and leave the vortex channel at a set position, which is called a release point.

[0057] In one aspect of the present invention, there is a turning point or curvature change in the bulk acoustic wave generating region corresponding to the release point.

[0058] In one aspect of the present invention, the boundary lines of the bulk acoustic wave generating area of ​​the ultrahigh frequency resonator are set to be suitable for biological macromolecules such as nucleic acids to be retained in the vortex channel and move along the vortex channel to the release point.

[0059] In one aspect of the present invention, the boundary line of the bulk acoustic wave generating region of the ultrahigh frequency resonator is shaped so that biomacromolecules, such as nucleic acids, are retained in the vortex channel and move to the release point, for example by reducing the occurrence of inflections or changes in curvature in the boundary line of the bulk acoustic wave generating region.

[0060] In one aspect of the present invention, the angle between the boundary line of the bulk acoustic wave generating region of the ultrahigh frequency resonator and the fluid channel is such that a biomacromolecule, such as a nucleic acid, is retained in the vortex channel and moves to a release point. For example, the angle between the boundary line of the bulk acoustic wave generating region and the fluid channel is relatively small.

[0061] In one aspect of the present invention, the bulk acoustic wave action region of the ultrahigh frequency bulk acoustic wave resonator has a focusing region and a screening region, wherein the focusing region is located upstream of the bulk acoustic wave action region, and the screening region is located downstream of the bulk acoustic wave action region, wherein the arrangement of the bulk acoustic wave action region of the focusing region is more suitable for keeping biological macromolecules such as nucleic acids moving in a vortex channel relative to the arrangement of the screening region. In one aspect of the present invention, the angle between the boundary line of the bulk acoustic wave action region of the focusing region and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action region of the screening region and the fluid channel. In one aspect of the present invention, the velocity of the liquid flowing through the bulk acoustic wave action region of the focusing region is controlled to be less than the velocity of the liquid flowing through the bulk acoustic wave action region of the screening region.

[0062] In one aspect of the present invention, the output power of the power regulating device is about 20-5000 mW, preferably 50-2000 mW, and more preferably 100-1500 mW.

[0063] In one aspect of the present invention, the flow rate regulating device 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 0.5-3 mm / s.

[0064] In one aspect of the present invention, the flow rate regulating device 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, more preferably about 0.5-30 μL / min.

[0065] In one aspect of the present invention, the height of the fluid channel of the microfluidic device is about 5-200 μm, preferably about 25-100 μm, more preferably about 30-80 μm, for example about 40-60 μm.

[0066] In one aspect of the present invention, the height of the fluid channel in the bulk acoustic wave action area of ​​the microfluidic device is about 10-45 μm, preferably about 15-30 μm, more preferably about 18-25 μm, for example about 20 μm or less than about 20 μm.

[0067] 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 .

[0068] In one aspect of the present invention, the side length of the BAW generating region of the UHF BAW resonator is about 30-500 μm, preferably about 40-300 μm, and most preferably about 50-200 μm.

[0069] In one aspect of the present invention, the inlet of the fluid channel includes a sample inlet and an auxiliary solution inlet disposed on one side or both sides of the sample inlet.

[0070] In one aspect of the present invention, the fluid channel has at least two outflow channels, one of which is the outflow channel for the controlled movement of biological macromolecules such as nucleic acids or proteins, called a particle outflow channel; the other is the outflow channel for removing the solution of the controlled movement of biological macromolecules such as nucleic acids or proteins, which can be called a solution outflow channel.

[0071] In one aspect of the present invention, a plurality of ultra-high frequency resonators having the same bulk acoustic wave generating region are provided.

[0072] In one aspect of the present invention, the fluid channel is divided into different regions, and ultrahigh frequency resonators for separating different nucleic acids, proteins, or other biomacromolecules are disposed in different regions. For example, the ultrahigh frequency resonators for separating different nucleic acids, proteins, or other biomacromolecules may have acoustic wave generating regions of different shapes, or may apply bulk acoustic waves of different powers, or have different flow rates.

[0073] In one aspect of the present invention, the ultrahigh frequency bulk acoustic wave resonator is a thin film bulk acoustic wave resonator or a solid-state assembly type resonator, for example, a thickness stretching vibration mode acoustic wave resonator.

[0074] In one aspect of the present invention, 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 um.

[0075] In one aspect of the present invention, the device is a device for analyzing nucleic acids, such as a device for sequencing.

[0076] Reference numerals

[0077] 100 microfluidic device 101 fluid channel

[0078] 200 chip housing 201 Coulter cell counter 202 UHF BAW resonator 203 top electrode layer 204 piezoelectric layer 205 bottom electrode layer 206 acoustic wave reflection layer acoustic impedance layer

[0079] 300PCL controller 301 high frequency signal generator 302 power amplifier 303 impedance meter

[0080] 400 Liquid injection and flow rate regulation device

[0081] 500 Acoustic Jet 501 Vortex

[0082] 600 Larger size particles 601 Medium size particles 602 Smaller size particles BRIEF DESCRIPTION OF THE DRAWINGS

[0083] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to 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 any creative labor.

[0084] Figure 1 A schematic structural diagram of a microfluidic device system provided in an embodiment of the present application;

[0085] Figure 2A 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 (a) A top view (left) and a cross-sectional view (right) of the microfluidic system shown in FIG. 1 ; (b) A top view (left) of the UHF BAW resonator (the black pentagonal portion represents the acoustic wave generation region of the UHF BAW resonator) and a cross-sectional view (right) of the microfluidic system shown in FIG. 2 ; (c) A top view (left) and a cross-sectional view (right) of the microfluidic channel + UHF BAW resonator.

[0086] FIG3 shows an embodiment of a microfluidic device system provided in an example of the present application for controlling and separating nucleic acids in a solution.

[0087] Figure 4 Another embodiment of the present application is shown of a microfluidic device system for controlling and separating nucleic acids in a solution.

[0088] Figure 5 Another embodiment of the present application is shown of a microfluidic device system for controlling and separating nucleic acids in a solution.

[0089] Figure 6 An embodiment of the present application shows an implementation method of a microfluidic device system for controlling and separating nucleic acids in a solution containing a nucleic acid agglutinating agent. DETAILED DESCRIPTION

[0090] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only 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 making any creative efforts are within the scope of protection of the present invention.

[0091] Example 1 Experimental methods and materials

[0092] Microfluidic channel and ultrahigh frequency bulk acoustic wave resonator preparation:

[0093] Microfluidic channels made of polydimethylsiloxane (PDMS) were fabricated by soft lithography.

[0094] The BAW resonator device is fabricated on a silicon-based wafer by chemical vapor deposition, metal sputtering, photolithography, and other methods. The specific methods are as follows:

[0095] 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.

[0096] 2. On a cleaned silicon wafer, a thin film of aluminum nitride is formed by sputtering, followed by a thin film of silicon dioxide using ion-enhanced chemical vapor deposition. Subsequently, the same method is used to alternately deposit aluminum nitride and silicon dioxide films, forming a Bragg acoustic reflection structure with alternating layers of aluminum nitride and silicon dioxide.

[0097] 3. A 600nm thick molybdenum film is sputtered onto the Bragg reflector structure as the bottom electrode. Standard photolithography techniques, including resist coating, exposure, and development, are then used to photolithograph the molybdenum electrode film. This is then etched to form the bottom electrode with the target pattern.

[0098] 4. Sputter an aluminum nitride film on the molybdenum electrode as a piezoelectric layer. Use dry etching to define the pattern of the aluminum nitride film.

[0099] 5. Use negative photoresist to transfer the pattern on the mask. Then, sputter a 50nm thick layer of titanium-tungsten alloy to serve as an adhesion layer to enhance the adhesion of the gold electrode. Next, use evaporation to grow a 300nm thick gold film as the top electrode. Finally, use acetone to remove the gold film around the target pattern, forming a gold electrode with the target pattern.

[0100] Finally, the BAW resonator device was bonded and integrated with the PDMS microchannel chip, and the BAW resonator device was set in the middle of the channel.

[0101] The BAW resonator device is connected to a network analyzer using a standard SMA interface. The resonance peak is found by testing the spectrum, and the frequency of the BAW generated by the BAW resonator device can be measured.

[0102] Instruments and materials

[0103] High frequency signal generator: (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz

[0104] Power amplifier: Mini-Circuits, with 35 dBm enhancement

[0105] Syringe pump: New Era Pump Systems, Inc., NE-1000

[0106] Example 2

[0107] In the specific implementation process of this embodiment, a microfluidic device is provided, which can be used to separate and capture biological macromolecular particles such as nucleic acids or proteins in a solution, especially nucleic acids.

[0108] 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 .

[0109] The microfluidic device provided by the present invention may exist independently, or may be part of a microfluidic system, for example, in the form of a detachable chip.

[0110] The fluid channel of described microfluidic device, or be called microfluidic channel, is except the opening for fluid to enter and flow out, generally is closed.The cross section of fluid channel has the size of 0.1-500 μ m usually, and it can be various shapes, including ellipse, rectangle, square, triangle, circle etc. Fluid channel can be prepared with various known micro-preparation technologies, and its material includes but is not limited to silica, silicon, quartz, glass or polymeric material (such as PDMS, plastic etc.). The channel can be coated with coating. Coating can change the characteristic of channel, and can be patterned. For example, coating can be hydrophilic, hydrophobic, magnetic, conductive, or biological functionalization.

[0111] In one aspect of the present invention, the height of the fluid channel of the microfluidic device is about 5-200 μm, preferably about 25-100 μm, more preferably about 30-80 μm, for example about 40-60 μm.

[0112] In one aspect of the present invention, when used to separate smaller nucleic acids, the height of the fluid channel of the microfluidic device is about 10-45 μm, preferably about 15-30 μm, more preferably about 18-25 μm, for example about 20 μm or less.

[0113] 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.

[0114] The microchannel 100 in this embodiment has an inlet and an outlet for fluid entry 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 in 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 sample inlet in the middle.

[0115] like Figure 1As shown, the microfluidic device of this embodiment includes a liquid injection and flow rate regulating device 400 for controlling liquid injection and liquid flow rate. The liquid can be a liquid containing a sample. For example, the sample is a liquid containing biomacromolecules to be captured. The sample can include body fluids, 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 blood, tissue fluid, extracellular fluid, lymph, cerebrospinal fluid, aqueous humor, urine, sweat, etc.

[0116] 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 and internal pressure sources can be pumps, such as peristaltic pumps, syringe pumps, or pneumatic pumps. In this embodiment, a syringe pump finely tuned by a computer is used to control the flow rate of the injected liquid.

[0117] In the present invention, the flow rate of the liquid is in the range of about 0.1-10 mm / s, preferably about 0.3-5 mm / s, and more preferably about 0.5-3 mm / s. In another aspect of the present invention, the flow rate of the liquid is in the range of about 0.1-100 μL / min, preferably about 0.1-50 μL / min, and more preferably about 0.5-30 μL / min.

[0118] The channel can be a single channel, or a plurality of channels arranged in parallel or in other forms and having a common output and input, wherein the outflow, inflow and flow rate of the fluid in each channel can be controlled jointly or independently as needed.

[0119] The microfluidic device of the present invention has one or more ultra-high frequency bulk acoustic wave resonators 202 disposed on a wall of a fluid channel (typically at the bottom of the channel). The ultra-high frequency bulk acoustic wave resonators can generate bulk acoustic waves with a frequency of approximately 0.5-50 GHz in the fluid channel that propagate toward the opposite wall of the fluid channel (typically the top of the channel).

[0120] 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.

[0121] 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.

[0122] The UHF BAW resonator is a BAW-generating component capable of generating BAWs within the fluid channel that propagate toward the opposite wall of the fluid channel. The UHF resonator can emit BAWs toward the opposite wall of the fluid channel, generating a vortex channel in the solution defined by the boundaries of the UHF resonator's BAW-generating region. Biomacromolecules in the solution enter and migrate along the vortex channel, exiting the vortex channel at a predetermined location, referred to as a release point.

[0123] like Figure 2 As shown in the cross-sectional view on the right side of (b), the UHF BAW resonator 202 includes an acoustic wave reflecting 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 reflecting layer constitutes the BAW generating 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 perpendicularly 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 herein as the BAW area or BAW action area. In one aspect of the present invention, the area of ​​the BAW action area is about 500-200,000 μm 2 , preferably about 5000-50000 μm 2 , most preferably about 10000-25000 μm 2 .like Figure 2 The illustrated BAW action zone of this embodiment is pentagonal, with a side length of approximately 120 μm. The continuous vortices generated by the ultrahigh-frequency BAW in the solution form an acoustofluidic vortex channel. Because vortices are generated by volume forces induced by acoustic wave attenuation, and the central axis of the vortex is above the BAW action boundary, the shape of the vortex channel is essentially the same as that of the BAW action zone, located above the boundary of the BAW action zone.

[0124] In the present invention, the shape of the bulk 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 or obtuse vertices, a polygon with vertices replaced by arcs, a polygon with vertices that are any combination of acute angles, semicircles or parabolas, or a square or circular array of the same shape that is repeatedly arranged. Other acoustic wave action areas of any shape are also within the scope of protection of this application. In the present invention, a preferred shape of the bulk acoustic wave generating area of ​​the ultra-high frequency resonator is spindle-shaped.

[0125] In one aspect of the present invention, the boundary lines of the bulk acoustic wave generating region of the ultrahigh frequency resonator in the above method (i.e., the shape of the corresponding vortex channel) are configured to facilitate the migration of biomacromolecule particles, such as nucleic acids or proteins, along the vortex channel to a release point. This prevents biomacromolecule particles from escaping the vortex channel without leaving the vortex channel at the desired release point.

[0126] In another aspect of the present invention, the shape of the boundary lines of the bulk acoustic wave generation area of ​​the ultrahigh frequency resonator is adjusted so that biomacromolecule particles such as nucleic acids or proteins are kept in the vortex channel and move to the release point. As mentioned above, the presence of a turning point or curvature change in the boundary lines of the bulk acoustic wave generation area may increase the probability of biomacromolecule particles escaping from the vortex channel. Therefore, the biomacromolecule particles can be kept in the vortex channel and moved by reducing the turning point or curvature change in the boundary lines of the bulk acoustic wave generation area, that is, reducing the biomacromolecule particles that escaping from the vortex channel, thereby improving the efficiency of separation.

[0127] In another aspect of the present invention, the angle between the boundary line of the UHF resonator's BAW generation region and the fluid channel is adjusted to maintain biomacromolecule particles within the vortex channel as they move toward the release point. The inventors unexpectedly discovered that a smaller angle between the boundary line of the BAW generation region and the fluid channel facilitates the retention of biomacromolecule particles within the vortex channel, thereby reducing the risk of biomacromolecule particles escaping the vortex channel and improving separation efficiency.

[0128] In another aspect of the present invention, the bulk acoustic wave action area of ​​the ultrahigh frequency bulk acoustic wave resonator in the microfluidic system has a focusing area and a screening area. The focusing area is located upstream of the bulk acoustic wave action area (i.e., close to the sample inflow direction, far from the release point), and the screening area is located downstream of the bulk acoustic wave action area (i.e., close to the sample outflow direction, close to the release point or including the release point). The setting of the bulk acoustic wave action area in the focusing area is more suitable for keeping the biomacromolecule particles moving in the vortex channel relative to the setting of the screening area: the biomacromolecule particles in the vortex channel of the focusing area move in the same or similar direction as the laminar flow direction, and the vortex drag force they are subjected to is relatively small, making it easier for the biomacromolecule particles to enter and remain in the vortex channel; in the downstream screening area, the biomacromolecule particles focused to the center of the vortex can be moved more stably in the vortex channel than the unfocused biomacromolecule particles. In another aspect of the present invention, the angle between the boundary line of the bulk acoustic wave action area in the focusing area and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area in the screening area and the fluid channel. For example, the boundary of the bulk acoustic wave action area in the focusing zone is substantially aligned with or substantially aligned with the direction of the fluid channel (e.g., an angle less than 10°). The vortex drag force on the biomacromolecule particles in the vortex channel in this zone does not substantially change the motion state of the biomacromolecule particles along the laminar flow direction, but only causes the biomacromolecule particles to migrate laterally to the vortex center, thereby focusing the biomacromolecule particles. The boundary of the bulk acoustic wave action area in the screening zone is at a larger angle to the fluid channel, guiding the biomacromolecule particles to deviate from the fluid channel direction and transfer to a designated release point. In the screening zone, the biomacromolecule particles focused at the vortex center can be moved more stably in the vortex channel than unfocused biomacromolecule particles. In another aspect of the present invention, the liquid flow velocity in the bulk acoustic wave action area of ​​the focusing zone is controlled to be less than the liquid flow velocity in the bulk acoustic wave action area of ​​the screening zone.

[0129] like Figure 2 As shown in the cross-sectional view on the right, the fluid channel of this embodiment may have multiple UHF BAW resonators. In one aspect of the present invention, they are arranged in a straight line in the same direction as the direction of fluid movement.

[0130] The ultra-high frequency bulk acoustic wave resonator employed in this invention operates in a thickness stretching vibration mode. The piezoelectric material thin film is grown vertically and excited by coupling to a vertical electric field through the d33 piezoelectric coefficient. This ultra-high frequency bulk acoustic wave resonator can generate localized acoustic streaming at the interface between the device and the liquid, without the need for a coupling medium or structure.

[0131] The ultra-high frequency bulk acoustic wave resonator used in the present invention generates an ultra-high frequency bulk acoustic wave that substantially does not generate a standing wave in the solution. Figure 1As shown in the figure on the right, the UHF resonator emits a bulk acoustic wave (BAW) toward the opposite wall of the fluid channel (e.g., the top of the channel). The volume force generated by the attenuation of the sound wave in the fluid causes an acoustic jet 500 to appear in the solution flowing through it, resulting in a localized, three-dimensional vortex 501 in the liquid in the microchannel. The continuous vortices caused by the UHF BAW form an acoustofluidic vortex channel. Because the vortex is generated by the volume force caused by the attenuation of the sound wave, the central axis of the vortex is above the boundary of the BAW action area. Therefore, the shape of the vortex channel is essentially the same as the shape of the BAW action area, located above the boundary of the BAW action area. Acoustofluidic vortices are caused by the nonlinearity of sound wave propagation in the liquid medium. The strength of the sound wave amplitude directly determines the intensity of the acoustofluidic vortex. By adjusting the applied power, the amplitude of the ultra-ultrasonic device, that is, the amplitude of the sound wave, can be controlled, thereby controlling the flow rate of the acoustofluidic vortex. The forces acting on the particles in the vortex (including larger particles 600, medium-sized particles 601, and smaller particles 602) 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 sound waves. Since the magnitude of the fluid drag force is positively correlated with the particle, such as the particle diameter, and the magnitude of the acoustic radiation force is positively correlated with the square of the particle size. As the particles grow larger, the forces they are subjected to will change from being dominated by fluid drag to being dominated by the acoustic radiation force, which pushes the particles toward the center of the vortex. Larger particles are subject to greater acoustic radiation forces and move to the center of the vortex; while smaller particles rotate on the periphery under the action of the vortex drag force, and further move downstream to the area where the body sound waves act under the action of the lateral drag force generated by the laminar flow.

[0132] The fluid drag force and acoustic radiation force caused by sound waves on particles in the vortex channel after the vortex can be calculated using certain formulas, but the inertial lift force generated by the acoustofluidic vortex is almost impossible to calculate using simple physical principles and formulas, especially in the case of fluids with complex components. Compared with the two-dimensional particle capture methods in the prior art, the methods and devices of the present invention involve nucleic acids. The forces and motion trajectories of nucleic acids in the acoustofluidic vortex and the channel formed by it are more complex. This is because the interactions between the individual vortices and the migration of nucleic acids between vortices affect the capture of nucleic acids in the fluid. The forces and motions of nucleic acids in the vortex differ from those calculated and simulated theoretically, and the motion patterns and trajectories of nucleic acids in the vortex channel cannot be predicted based on theoretical calculations and simulations.

[0133] Through experiments, the applicant unexpectedly discovered that in the method and apparatus of the present invention, when nucleic acids in a solution pass through the acoustofluidic vortex channel region created by ultrahigh-frequency bulk acoustic waves, under conditions of suitable flow rate and bulk acoustic wave power, they will enter and move along the vortex channel. Under the lateral drag force generated by laminar flow, at certain locations in the vortex channel, the nucleic acids will exit the bulk acoustic wave action region and move downstream, i.e., be released. The location where the nucleic acids exit the vortex channel is also referred to as the release point. Because one of the important factors for nucleic acids to escape from the vortex channel is the influence of laminar flow along the fluid channel, the release point is typically located in the downstreammost region of the vortex channel.

[0134] The inventors also unexpectedly discovered that nucleic acids typically exit the vortex channel at the transition point between the vortex and acoustic radiation forces. Under conditions where the bulk acoustic wave power generating the vortex, the solution flow rate, and the shape and dimensions of the fluid channel remain constant, nucleic acids typically exit the vortex channel at a turning point or corner in the vortex channel, i.e., above the turning point or corner at the boundary of the corresponding bulk acoustic wave action area.

[0135] By adjusting the shape and position of the bulk acoustic wave action area of ​​the ultrahigh frequency bulk acoustic wave resonator, the biomacromolecules in the solution enter the vortex channel, move along the vortex channel, and leave the vortex channel at a set position. The nucleic acid thus leaves the bulk acoustic wave action area at a specified position and direction and enters a desired outflow channel, for example, the particle outflow channel. The set position for leaving the vortex channel is called the release point, that is, the position where the nucleic acid leaves the bulk acoustic wave action area. The solution from which the nucleic acid whose movement is controlled is removed maintains the inflow direction and enters the aforementioned solution outflow channel.

[0136] Thus, the inventors of the present applicant have discovered and provided a method for more effectively separating target biomacromolecules such as nucleic acids.

[0137] In the present invention, the frequency of the FBAW resonator is primarily determined by the thickness and material of the piezoelectric layer. The piezoelectric layer thickness of the FBAW resonator employed in the present invention ranges from 1 nm to 2 μm. The frequency of the UHF FBAW resonator of the present invention is approximately 0.5-50 GHz, preferably approximately 1-10 GHz.

[0138] The bulk acoustic waves (BAWs) generated by the ultra-high frequency BAW resonator are driven by a signal from a high-frequency signal generator. The pulsed voltage signal driving the resonator can be driven using pulse-width modulation, which can produce any desired waveform, such as a sine wave, square wave, sawtooth wave, or triangle wave. The pulsed voltage signal can also have amplitude or frequency modulation start / stop capabilities to start or stop the BAW.

[0139] 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 20-5000mW, preferably 50-2000mW, and more preferably 100-1500mW. 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.

[0140] Example 3 Controlling the Movement of Nucleic Acids and Separating and Purifying Nucleic Acids

[0141] According to the method described in Examples 1 and 2, a microfluidic channel and ultra-high frequency resonator system as shown in Figure 3(a) was prepared and arranged. The frequency of the ultra-high frequency bulk acoustic wave resonator is about 1.83 GHz. Figure 3(a) is a top view, and the right side of the figure is the flow channel inlet, which is used to input the PBS solution of the nucleic acid sample. The surface of the ultra-high frequency resonator, that is, the bulk acoustic wave generating area, is shown as a spindle in the figure, and the left tip is the nucleic acid release point. The left side of the microfluidic channel includes three outflow channels, the middle of which is the nucleic acid outflow channel, and the nucleic acid flowing out from the nucleic acid release point at the left tip of the spindle enters the nucleic acid outflow channel. The two channels located above and below the nucleic acid outflow channel are outflow channels for the solution after the target nucleic acid is removed.

[0142] In the microfluidic device of the present invention, the bulk acoustic waves generated by the ultrahigh frequency bulk acoustic wave resonator cause vortices in the fluid flowing through it. Each vortex connects with adjacent vortices, forming an acoustofluidic vortex channel or vortex tunnel along the boundaries of the ultrahigh frequency bulk acoustic wave resonator's acoustic wave action area. The combined effect of multiple vortices and the interaction between vortex flow and laminar flow cause nucleic acid particles that enter the vortex channel to move along the vortex channel, and the nucleic acid particles float above the bottom of the microfluidic channel, with essentially no contact with the microfluidic channel.

[0143] Nucleic acids were stained and quantified using the Qubit sDNA HS Kit and dissolved in PBS. Figure 3(a) shows the system setup and the observations after the fluorescently stained nucleic acid solution was introduced. The top image is the bright field, and the bottom image is the fluorescence signal observation image, showing the uniform distribution of the fluorescently stained nucleic acid solution in the microfluidic channel when the UHF resonator is not operating.

[0144] Figure 3(b) shows the performance of a double-stranded nucleic acid sample of approximately 20k in size in the device shown in Figure 3(a) at different flow rates (0, 0.1, 0.25, 0.5, and 1 μL / min) and BAW operating powers (50-1000 mW). The fluidic channel height is approximately 20 μm.

[0145] The nucleic acid is genomic DNA extracted from sheep whole blood, with a size of about 20 kbp.

[0146] As shown in Figure 3(b), when the nucleic acid solution is continuously input from the flow channel inlet and the UHF resonator continues to work, under the conditions of appropriate flow rate and bulk acoustic wave power, the nucleic acid moves along the edge of the spindle-shaped UHF resonator device to the release point. In the flow channel inlet area, the nucleic acid moves at a uniform speed. When approaching and entering the active area of ​​the spindle-shaped UHF resonator device, the direction of the nucleic acid movement is changed by the spindle-shaped vortex array, and it enters the vortex tunnel generated by the acoustic wave and moves along the spindle edge, eventually leaving the UHF resonator active area at the spindle distal end (i.e., the release point).

[0147] The results demonstrate that the device and method of the present invention have excellent control over the movement of 20 kbp double-stranded DNA. This control capability is positively correlated with the applied power and negatively correlated with the lateral fluid velocity in the flow channel. This experiment, conducted under continuous operation, demonstrates that nucleic acids do not adhere to the UHF resonator device or the flow channel, nor do they aggregate in vortices.

[0148] Judging from the fluorescence signal, at a flow rate of 0.1 μL / min and a power of 1000 mW, all nucleic acids within the working range of the bulk acoustic wave can be continuously enriched, with an enrichment efficiency (i.e., the amount of nucleic acids entering the nucleic acid outflow channel) exceeding 90%. In the vortex channel at the edge of the bulk acoustic wave area, 20 kbp of DNA is concentrated in a vortex tunnel with a diameter of approximately 35 microns. At the release site, after the nucleic acid is released from the vortex, it diffuses into a nucleic acid strip with a width of approximately 125 μm under the combined action of secondary vortices and lateral fluid, flowing downstream.

[0149] Figure 3(c) shows the performance of a double-stranded nucleic acid sample of approximately 20k in size in the device shown in Figure 3(a) at different flow channel heights (50 μm and 20 μm). The flow rate was approximately 0.1 μL / min and the power was approximately 800 mW.

[0150] The results show that under the same flow rate and bulk acoustic wave power, the system with a flow channel of 20 microns in height is significantly more effective than the system with a flow channel of 50 microns in height. This proves that reducing the height of the microchannel can improve the effect of the acoustic fluid in capturing small-sized particles. Reducing the flow channel height will increase the velocity gradient of the acoustic fluid vortex, and the gathering of target particles at the center of the vortex is positively correlated with the gradient force of the vortex, thereby improving the capture efficiency of the acoustic fluid. It can be seen from the above figure that in the flow channel with a height of 50μm, the range of the vortex formed at the edge of the bulk acoustic wave is significantly larger than the vortex in the 20μm flow channel.

[0151] Figure 3(d) shows the performance of a double-stranded nucleic acid sample of approximately 5k in size in the device shown in Figure 3(a) at different flow rates (0.1, 0.25, and 0.5 μL / min) and different BAW operating powers (50-1000 mW). The fluid channel height is approximately 20 μm.

[0152] The 5k double-stranded nucleic acid sample is a DNA plasmid, which is a double-stranded circular DNA.

[0153] The results show that the device and method of the present invention have good ability to control the movement of 5 kbp plasmid DNA, and the control ability is positively correlated with the applied power and negatively correlated with the lateral fluid velocity in the flow channel.

[0154] Figure 3(e) shows the performance of circular and linear nucleic acid samples with a size of about 5k in the device shown in Figure 3(a). The height of the fluid channel is about 20 microns.

[0155] The 5k chain nucleic acid is obtained by enzyme-digesting the DNA plasmid of the 5k double-stranded nucleic acid sample in FIG3(d) to form double-stranded chain DNA.

[0156] The results showed that under the same flow rate and bulk acoustic wave power, the system's control over 5k circular nucleic acids was more significant than that over chain nucleic acids of the same size.

[0157] Example 4 Separation of nucleic acids of different sizes in the same system

[0158] According to the method described in Examples 1 and 2, the Figure 4 (a) shows a microfluidic channel and UHF resonator system. The UHF BAW resonator frequency is approximately 1.83 GHz. The fluid channel height is approximately 20 microns. Figure 4(a) is a top view, and the right side of the figure is the flow channel inlet, 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 occurs, is shown as a spindle in the figure, and its left tip is the nucleic acid release point. The left side of the microchannel includes three outflow channels, of which the bottom one is the target nucleic acid outflow channel (in this embodiment, the larger-sized nucleic acid in the mixed nucleic acid sample). The nucleic acid flowing out from the nucleic acid release point at the left tip of the spindle shape enters the nucleic acid outflow channel. The channel above the nucleic acid outflow channel is an outflow channel for non-target nucleic acids (i.e., nucleic acids to be removed). The upper side line of the upstream end of the spindle-shaped ultra-high frequency resonator (the upper right side line of the spindle shape in the figure) is set to be basically parallel to the flow channel, and the nucleic acid release point at the left end is opposite to the lower outlet on the left side of the flow channel.

[0159] The nucleic acid samples used in the experiment were two nucleic acids of different sizes. The large nucleic acid fragment was extracted human genomic DNA, with a size of about 23 kbp, and the small fragment was plasmid DNA, with a size of about 5 kbp.

[0160] Nucleic acids were stained and quantified using the Qubit sDNA HS kit and dissolved in PBS. Figure 4 (a) Bright field image showing the system setup. Figure 4 (b) and (c) are fluorescence signal observation images, showing the phenomena observed after the fluorescent-stained nucleic acid solution is introduced: the upper image is the bright field, and the lower image is the fluorescence signal observation image, showing that the fluorescent-stained nucleic acid solution is evenly distributed in the microchannel.

[0161] like Figure 4 As shown in (a) and (b), a sample liquid flow containing nucleic acid and a PBS liquid flow are respectively introduced upstream of the flow channel. The PBS liquid flow serves as a sheath flow, so that the liquid flow containing nucleic acid flows through the upper edge line of the upstream end of the spindle-shaped ultrahigh frequency resonator (the upper right edge line of the spindle in the figure) in a direction basically parallel to the flow channel.

[0162] Figure 4 (b) shows that when the UHF resonator is not working, a mixed sample of two nucleic acids with sizes of approximately 23 kb (approximately 2.0 ng / μl) and 5 kb (approximately 3.34 ng / μl) flows through the spindle-shaped UHF resonator in a direction basically parallel to the flow channel under the action of the sheath flow of PBS and enters the upper outlet on the left side of the flow channel.

[0163] like Figure 4 As shown in (c), when the UHF resonator is working (1000 mW), the nucleic acid in the mixed nucleic acid sample moves along the edge of the spindle-shaped UHF resonator device to the release point, and enters the lower outlet on the left side of the flow channel after release.

[0164] The nucleic acid at the upper and lower outlets on the left side of the flow channel is detected by gel electrophoresis (eg Figure 4 (d) shows that, compared with the input mixed nucleic acid sample, the nucleic acids collected at the upper outlet on the left side of the flow channel (excluding the nucleic acid outflow channel) have basically no larger nucleic acids (23 kb) and an increased content of smaller nucleic acids (5 kb); the content of larger nucleic acids (23 kb) collected at the lower outlet on the left side of the flow channel (target nucleic acid outflow channel) has increased (enriched).

[0165] Example 5: Purification and Enrichment of Target Nucleic Acids from Degraded Nucleic Acid Samples

[0166] Prepare and set up the microfluidic channel and ultra-high frequency resonator system as described in Example 4. Figure 5 (a) is shown. Figure 5(a) is a bright field image showing the system setup. The right side of the figure is the flow channel inlet, which is used to input the nucleic acid sample solution. The surface of the ultra-high frequency resonator, that is, the area where the bulk acoustic wave is generated, is shown as a spindle in the figure, and its left tip is the nucleic acid release point. The left side of the microchannel includes two outflow channels, of which the lower one is the target nucleic acid outflow channel, and the nucleic acid flowing out from the nucleic acid release point at the left tip of the spindle enters the nucleic acid outflow channel; the lower channel is the solution outflow channel for removing the target nucleic acid.

[0167] Figure 5 (b) is a fluorescence signal observation diagram, showing the phenomena observed when the ultrahigh frequency resonator is working and not working after the fluorescent dye nucleic acid solution is introduced.

[0168] The nucleic acid sample used in the experiment was extracted human genomic DNA, which was about 23 kbp in size. During storage, the sample was degraded to produce small nucleic acid fragments, which showed a large amount of smear in electrophoresis (such as Figure 5 (c) The original solution (control group) is shown.

[0169] Figure 5 (b) shows that when a sample containing nucleic acid is introduced upstream of the flow channel and the UHF resonator is not working, the nucleic acid sample flows through the spindle-shaped UHF resonator and evenly enters the outlet on the left side of the flow channel; when the UHF resonator is working (1000mW), some nucleic acids in the nucleic acid sample move along the edge of the spindle-shaped UHF resonator device to the release point, and after release, enter the lower outlet on the left side of the flow channel.

[0170] By detecting the nucleic acid at the lower outlet on the left side of the flow channel (such as Figure 5 (c) and (d) show that compared with the input mixed nucleic acid sample, the content of the collected target nucleic acid with a larger size (23 kb) is increased (enriched).

[0171] In this embodiment, a two-stage separation strategy is adopted, namely Figure 5The same separation unit is set downstream of the separation unit shown in (a), and the sample of the target nucleic acid outflow channel of the first-stage (upstream) separation unit is passed into the inlet of the second-stage (downstream) separation unit for further separation and enrichment.

[0172] The nucleic acid collected at the lower outlet on the left side of the flow channel of the second-stage separation unit (target nucleic acid outflow channel) is analyzed. Figure 5 (c) is a gel electrophoresis analysis diagram; Figure 5 (d) is analyzed using ImageJ software. As shown in the figure, the content of the 23kb nucleic acid collected at the lower outlet on the left side of the flow channel of the second-stage separation unit (the target nucleic acid outflow channel) is increased, achieving the purpose of purification or enrichment.

[0173] Example 6: Adding a Nucleic Acid Condensing Agent to Assist in the Control and Collection of Smaller Molecular Nucleic Acids

[0174] Prepare and set up the microfluidic channel and ultra-high frequency resonator system as described in Example 4. Figure 6 Figure 6(a) shows a bright field image. The frequency of the UHF BAW resonator is approximately 1.83 GHz. The fluid channel height is approximately 20 microns.

[0175] Figure 6 (a) is a top view. The upper side of the figure shows the flow channel inlet. The surface of the ultrahigh frequency resonator, i.e., the region generating the bulk acoustic wave, is shown as a spindle-shaped structure, with its lower tip being the nucleic acid release point. The lower portion of the microfluidic channel includes a nucleic acid outflow channel. Nucleic acids flowing from the nucleic acid release point at the lower tip of the spindle enter the nucleic acid outflow channel. The channel to the left of the nucleic acid outflow channel is the outflow channel for the solution after the target nucleic acid has been removed.

[0176] Figure 6 (b) shows the fluorescence signal observation. A PBS solution containing a 1 kbp plasmid was used as the nucleic acid sample. The sample was introduced from above the flow channel, with the UHF resonator operating at a frequency of 1600 mW. The images show the observed phenomena with and without the UHF resonator operating after the fluorescent-stained nucleic acid solution was introduced.

[0177] exist Figure 6 In the results shown in (b), no nucleic acid was observed to move along the edge of the spindle-shaped UHF resonator device to the release point.

[0178] PEG (molecular weight 8K) was added to a PBS solution containing a 1 kbp plasmid at a concentration of 0-18 wt %, and NaCl was added at a concentration of 2.5 M in the solution.

[0179] Observe the phenomenon of nucleic acid condensation under a microscope. Figure 6 (c) Figure 6The top image in (c) is a bright-field image of the nucleic acid solution, the middle image is a fluorescence image, and the bottom image is a magnified, localized fluorescence image. (The nucleic acids are fluorescently labeled.) As shown in the figure, no agglomerated nucleic acid particles were observed at a PEG concentration of 0. However, agglomerated nucleic acid particles were observed at PEG concentrations of 12 wt%, 15 wt%, and 18 wt%.

[0180] The nucleic acid solution sample with 18 wt% PEG and 2.5 M NaCl was taken from Figure 6 (a) The flow channel of the system is connected to the top. Figure 6 As shown in (d), at different operating frequencies of the UHF resonator (200-1600 mW), it can be observed that nucleic acids move along the edge of the spindle-shaped UHF resonator device to the release point, and after release, enter the right outlet below the flow channel.

[0181] The nucleic acid is detected at the two outlets below the flow channel. Figure 6 (e) shows gel electrophoresis analysis; (f) shows analysis using ImageJ software. The results show that compared to the input nucleic acid sample, the content of 1 kb nucleic acid in the right outlet below the flow channel is significantly increased, achieving the purpose of purification or enrichment.

[0182] 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 in the scope of protection of the present invention.

Claims

1. A method for controlling the movement of target biomacromolecules in a solution, characterized in that: include: (1) allowing a solution containing biomacromolecule 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, 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 that propagate toward the wall on the opposite side of the fluid channel; (2) the UHF BAW resonator emits a BAW that is transmitted toward the wall on the opposite side of the fluid channel, generating a vortex channel in the solution that is defined by the boundary of the BAW generation region of the UHF BAW resonator; (3) By adjusting the shape and position of the BAW generation region of the UHF BAW resonator, the biomacromolecules in the solution enter the vortex channel and move along the vortex channel, and leave the vortex channel at a set position, which is called the release point. The BAW generation region corresponding to the release point has a turning point or curvature change. The boundary line of the bulk acoustic wave generating area of ​​the ultrahigh frequency bulk acoustic wave resonator is configured to reduce the turning or curvature change in the boundary line of the bulk acoustic wave generating area so as to be suitable for the target biomacromolecule to remain in the vortex channel and move along the vortex channel to the release point; The biomacromolecules entering the vortex channel can be adjusted by adjusting the power of the bulk acoustic wave and / or by adjusting the speed of the solution flowing through the bulk acoustic wave region.

2. The method according to claim 1, characterized in that The biological macromolecule is a nucleic acid, and the length of the nucleic acid molecule is ≥100 bp.

3. The method according to claim 1, characterized in that The bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator has a focusing area and a screening area, wherein the focusing area is located upstream of the bulk acoustic wave action area, and the screening area is located downstream of the bulk acoustic wave action area, wherein the setting of the bulk acoustic wave action area in the focusing area is more suitable for keeping biological macromolecules moving in the vortex channel than the setting of the screening area.

4. The method according to claim 3, characterized in that The angle between the boundary line of the bulk acoustic wave action area of ​​the focusing zone and the fluid channel is smaller than the angle between the boundary line of the bulk acoustic wave action area of ​​the screening zone and the fluid channel; Alternatively, the velocity of the liquid flowing through the bulk acoustic wave action region of the focusing zone is controlled to be lower than the velocity of the liquid flowing through the bulk acoustic wave action region of the screening zone.

5. The method according to claim 1 or 2, characterized in that The power of the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator is 20-5000 mW.

6. The method according to claim 1 or 2, characterized in that The speed of the solution flowing through the bulk acoustic wave region can be adjusted to 0.01-10 mm / s, or The speed at which the solution flows through the bulk acoustic wave region can be adjusted to 0.01-100 μL / min.

7. The method according to claim 1 or 2, characterized in that The height of the fluid channel in the bulk acoustic wave action area is 10-45 μm.

8. The method according to claim 1 or 2, characterized in that The fluid channel of the microfluidic device has an outflow channel for the biomacromolecules whose movement is controlled, and the outflow channel is a particle outflow channel; the fluid channel also has an outflow channel for the solution that removes or contains less of the biomacromolecules whose movement is controlled, and the solution outflow channel is a solution outflow channel.

9. The method according to claim 8, characterized in that The width ratio of the openings of the particle outflow channel and the solution outflow channel is 1:1-1:

20.

10. The method according to claim 1 or 2, characterized in that The solution containing the biomacromolecule particles contains a nucleic acid condensing agent.

11. The method according to claim 10, characterized in that The nucleic acid condensing agent is a nonionic polymer; the content of the nonionic polymer in the solution is 0.1 wt % to 25.0 wt %.

12. The method according to claim 10, characterized in that The nucleic acid condensing agent is a concentrated alkali metal salt or alkaline earth metal salt, and its concentration is in the range of 1 mM to 1 M.

13. The method according to claim 10, characterized in that The solution containing the biomacromolecule particles also includes a buffered saline solution.

14. The method according to claim 13, characterized in that The concentration of the buffered saline solution is in the range of 1 mM to 5000 mM.

15. A microfluidic device for controlling the movement of target biomacromolecules in a solution, 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 that propagate toward the wall on the opposite side of the fluid channel; a power regulating device for regulating the power of the bulk acoustic wave generated by the ultrahigh frequency bulk acoustic wave resonator; a flow rate regulating device for regulating the speed at which the solution flows through the bulk acoustic wave region; the ultrahigh frequency bulk acoustic wave resonator can emit bulk acoustic waves that are transmitted to the wall on the opposite side of the fluid channel, thereby generating a vortex channel in the solution defined by the boundary of the bulk acoustic wave generating region of the ultrahigh frequency bulk acoustic wave resonator; biomacromolecules in the solution enter and move along the vortex channel and leave the vortex channel at a set position, which is called a release point; wherein the bulk acoustic wave generating region corresponding to the release point has a turning point or a curvature change; wherein the boundary line of the bulk acoustic wave generating region of the ultrahigh frequency bulk acoustic wave resonator is configured to reduce the turning point or curvature change in the boundary line of the bulk acoustic wave generating region so as to be suitable for the target biomacromolecule to remain in the vortex channel and move along the vortex channel to the release point; The height of the fluid channel in the bulk acoustic wave action area of ​​the microfluidic device is 10-45 μm. The microfluidic device is a device for analyzing nucleic acids.

16. The microfluidic device according to claim 15, characterized in that The device for analyzing nucleic acids is a device for sequencing.

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