Method and device for separating cells or microvesicles

Abstract

The present invention discloses a microfluidic system and method for separating target cells or vesicles in a fluid. The system of the present invention includes a fluid channel having an inlet and an outlet; one or more ultra-high frequency acoustic wave resonators, and 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; a power regulator, which adjusts the power of the bulk acoustic wave generated by the ultra-high frequency resonator; and a flow rate regulating device, which adjusts the speed at which the solution flows through the bulk acoustic wave area. The power of the generated bulk acoustic wave is adjusted by the power regulator and / or the speed at which the solution flows through the bulk acoustic wave area is adjusted by the flow rate regulating device, so that the cells or vesicles stay in the area affected by the bulk acoustic wave. The system and method of the present invention can capture and release cells or vesicles in the solution, and further process and analyze the obtained cells or vesicles.

Description

[0001] This application claims priority to the following Chinese patent application: Application number 201910512145.2, filed on June 13, 2019, entitled “Method and device for separating cells or microvesicles”, 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 cells or microvesicles and a method for separating and analyzing cells or microvesicles using the system. Background Art

[0003] The manipulation of biological particles, especially cells or microvesicles, is a basic biotechnology and also a cornerstone technology for cutting-edge research. There are many methods available to manipulate biological particles, such as cells or microvesicles, including electrical methods, acoustic methods, optical methods, magnetic methods, chemical methods, fluid manipulation methods, etc. Each method has a specific scope of application and is only effective for specific particle properties. For example, the manipulation of surface acoustic waves and dielectrophoresis is related to the radius of the particle, so it is better for micron-sized particles and has poorer discrimination for nanoscale particles; methods such as electrophoresis can only work when the particles are charged, so the discrimination between uncharged or similarly charged substances is poor. Optical manipulation methods have high precision, but will generate more heat during the manipulation process, which will have a greater impact on the activity of biological molecules, and the optical system is more complex and difficult to integrate with microfluidic devices. Each method has certain limitations when used alone, so in most cases, the combination of different methods will make the sample differentiation and manipulation effect better.

[0004] Single cell manipulation is an area that has received increasing attention in recent years. The manipulation of single cells or microvesicles is very important for studies such as single cell analysis, drug development, organ chips, and cell-cell interactions. Among them, studies based on cell-to-cell specificity (such as studies on cell morphology, surface adhesion, migration speed, protein expression, and gene expression of single cells) are extremely dependent on single cell manipulation technology. However, traditional methods of extracting single cells are complicated, time-consuming, laborious, and inefficient.

[0005] Acoustofluidic technologies, such as surface acoustic waves and acoustophoresis based on standing waves in fluids, have been used to separate, extract and manipulate micronized particles and cells. However, the devices used in these existing technologies are large and difficult to miniaturize.

[0006] Therefore, there is an urgent need for a system and method to capture cells or microvesicles, especially single cells or vesicles, so as to facilitate further processing and analysis of the captured single cells or vesicles. Summary of the invention

[0007] The present invention first discovered that ultra-high frequency bulk acoustic waves can be used to effectively manipulate and separate flexible particles such as cells in a solution in a microfluidic system, thereby providing a method and system for separating and "capturing" target cells. The method and system of the present invention can also be used to accurately control the type and number of cells captured, and is particularly suitable for obtaining a single or limited number of cells.

[0008] Specifically, the present invention provides a method for separating flexible particles such as cells or vesicles from a solution, comprising the following steps:

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

[0010] a fluid channel having an inlet and an outlet;

[0011] 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 about 0.5-50 GHz in the fluid channel and propagate to the wall on the opposite side of the fluid channel;

[0012] (2) the ultra-high frequency resonator emits a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and a vortex is generated in the solution in the area affected by the bulk acoustic wave;

[0013] (3) adjusting the power of the bulk acoustic wave (for example, by a power regulator) and / or adjusting the speed at which the solution flows through the area affected by the bulk acoustic wave (for example, by a flow rate regulating device) so that the flexible particles stay in the area affected by the bulk acoustic wave, i.e., do not move forward relative to the fluid channel.

[0014] In one aspect of the present invention, the method further comprises the step of making the resting flexible particles leave the resting position, that is, "releasing" the flexible particles. For example, the captured flexible particles can be released into a desired channel or container.

[0015] The UHF BAW resonator in the present invention refers to a resonator capable of generating sound waves with a frequency exceeding 0.5 GHz (preferably exceeding 1 GHz), for example, a frequency of 0.5-50 GHz. The UHF BAW resonator may be a thin film BAW resonator or a solid-state assembly resonator.

[0016] The microfluidic device generally includes a power regulating device, which regulates the power of the bulk acoustic wave generated by the ultrahigh frequency resonator.

[0017] 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.

[0018] Flexible microparticles refer to nano or micron particles with deformation properties. Flexible particles can be artificial or natural, and the particles are usually micelles with a membrane structure, especially micelles with a lipid bilayer or a lipid bilayer. The flexible particles involved in the present invention usually have a diameter of about 0.01-30um, preferably 0.5-25um, and more preferably 0.8-20um. In one aspect of the present invention, the flexible particles are naturally occurring particles, such as cells or cell vesicles released by cells into the external environment, including exosomes, microvesicles, vesicles, membrane vesicles, blisters, bubbles, prostatic bodies, microparticles, intraluminal vesicles, endosomal-like vesicles or exocytic vesicles. These cell-related vesicles are vesicle-like bodies with a double-layer membrane structure that fall off the cell membrane or are secreted by cells. The vesicles usually have a diameter of about 30-1000nm, for example, a diameter of about 800-1000nm.

[0019] In one aspect of the present invention, the cell comprises a cell cluster. The cell cluster is usually composed of several, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, cells. In one aspect of the present invention, the vesicle comprises a vesicle population. The vesicle population is usually composed of several, such as 2-50 vesicles.

[0020] In one aspect of the present invention, the diameter of the cell or vesicle is about 0.8-30 um, such as 1-25 um, and also such as 5-20 um.

[0021] In one aspect of the present invention, the output power of the power regulating device is about 0.5-800 mW, preferably 0.5-500 mW, and more preferably 0.5-350 mW.

[0022] 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 0.5-2.5 mm / s.

[0023] 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, and more preferably about 0.5-20 μL / min.

[0024] In one aspect of the present invention, the height of the fluid channel is about 1.5-10 times the diameter of the cell or vesicle, preferably about 1.5-6 times the diameter, and more preferably about 2-4 times the diameter.

[0025] In one aspect of the present invention, the height of the fluid channel of the microfluidic device is about 25-200 μm, preferably about 25-100 μm, more preferably about 30-80 μm, such as about 40-60 μm.

[0026] 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 .

[0027] 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 can be a liquid such as a buffer. The auxiliary solution can be used to resuspend the "captured" cells or vesicles, or to add reagents for treating the "captured" cells or vesicles, such as fluorescent markers that specifically identify the cells or vesicles or their specific markers. Based on the sheath flow effect, the auxiliary solution can also be used to control the flow direction and range of the sample liquid in the microfluidic channel.

[0028] In one aspect of the present invention, the aforementioned method is used to separate and obtain a single cell or several (eg, 2-100, preferably 2-10) cells, or to separate and obtain a single cell or several (eg, 2-100, preferably 2-10) cell clusters.

[0029] In one aspect of the present invention, the aforementioned method further includes selectively capturing cells or vesicles of different types or different properties (e.g., different sizes or densities, etc.) in the area affected by the bulk acoustic wave. In another aspect of the present invention, it also includes controlling the number of cells or vesicles that stay in the area affected by the bulk acoustic wave. In another aspect of the present invention, the cells or vesicles and their number that stay in the area affected by the bulk acoustic wave can be selected by one of the following methods or any combination thereof:

[0030] (a) regulating the power of the bulk acoustic wave;

[0031] (b) regulating the time of generating body sound waves;

[0032] (c) adjusting the velocity of the solution flowing through the bulk acoustic wave region.

[0033] In one aspect of the present invention, the above method controls the cells or vesicles that stay in the area affected by the bulk acoustic wave to be single or several (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-20). In another aspect, the speed at which the solution flows through the bulk acoustic wave area is adjusted to be about 0.1-10 mm / s, preferably about 0.5-3 mm / s. In another aspect, the power of the bulk acoustic wave is adjusted to be about 0.1-500 mW, preferably about 0.5-200 mW. In another aspect, the speed at which the solution flows through the bulk acoustic wave area is adjusted to be about 0.1-10 mm / s, preferably about 0.5-3 mm / , and the power of the bulk acoustic wave is adjusted to be about 0.1-500 mW, preferably about 0.5-200 mW.

[0034] In one aspect of the present invention, the aforementioned method further comprises allowing the retained cells or vesicles to leave the retention position, i.e., releasing the cells or vesicles. In another aspect of the present invention, the cells or vesicles may be released by one or any combination of the following methods:

[0035] (a) Stopping body sound waves;

[0036] (b) reducing the power of the bulk acoustic wave;

[0037] (c) Increase the velocity of the solution flowing through the bulk acoustic wave region.

[0038] In one aspect of the present invention, the method can controllably release the retained cells or vesicles. In another aspect of the present invention, the method can control the types of released cells or vesicles, such as releasing cells or vesicles of different sizes. In another aspect of the present invention, by adjusting the power of the body acoustic wave, the "captured" cells are controlled to be released in order from small to large size, for example, the power of the body acoustic wave decreases by about 5-20%. In another aspect of the present invention, the method can control the number of released cells or vesicles, for example, by adjusting the power of the body acoustic wave and / or the flow rate of the solution, the "captured" cells are controlled to be released in different quantities, thereby controllably obtaining the required number of cells or vesicles, especially single cells or vesicles.

[0039] In one aspect of the present invention, the method further comprises the step of dispersing the clustered cells, for example, by adjusting the power of the bulk acoustic wave generated by the ultra-high frequency resonator to disperse the clustered cells.

[0040] In one aspect of the present invention, the method further comprises increasing the power of the bulk acoustic wave generated by the ultrahigh frequency resonator to destroy the cell membrane or vesicle membrane to release substances in the cell or vesicle, such as proteins or nucleic acids.

[0041] In one aspect of the present invention, the solution in the method is a liquid containing cells or vesicles to be captured, such as body fluid, whole blood, any blood fraction containing cells, fragmented tumor, tumor cell suspension, cell culture or culture supernatant. In another aspect of the present invention, the solution is blood, including whole blood or blood fractions.

[0042] In one aspect of the present invention, the method is used to separate cells or vesicles to be separated from a solution containing other particles or cells. In another aspect of the present invention, the ratio of the content of the cells or vesicles to be separated in the solution to the other particles or cells is less than 1:10. 4 , preferably less than 1:10 5 , more preferably less than 1:10 6 In yet another aspect of the present invention, the content of the cells or vesicles to be separated in the solution is less than 10 / ml or less.

[0043] In one aspect of the present invention, the microfluidic device in the method is configured so that when processing particles or cells containing different particles or cells, the position where the desired cells or vesicles are captured (or referred to as the capture point) is different from the position where other cells or vesicles leave the area of ​​action of the bulk acoustic wave (or referred to as the release point). Preferably, the position where other cells leave the area of ​​action of the bulk acoustic wave is set upstream of the position where the desired cells are captured. In another aspect of the present invention, the area of ​​action of the bulk acoustic wave of the ultrahigh frequency bulk acoustic wave resonator of the microfluidic device in the method is configured so that the position where the desired cells are captured (or referred to as the capture point) is different from the position where other cells leave the area of ​​action of the bulk acoustic wave (or referred to as the release point).

[0044] In the present invention, cells include natural or cultured cells of higher plants or animals (e.g., mammals including humans), as well as unicellular organisms or simple multicellular organisms such as bacteria and fungi. In one aspect of the present invention, the cell is a eukaryotic animal cell, preferably a mammalian cell, and more preferably a human cell. In another aspect of the present invention, the cell is a blood cell, a stem cell, a somatic cell or a tumor cell, such as a circulating tumor cell (CTC). The circulating tumor cell is selected from but not limited to the following epithelial cell cancers: prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, head and neck cancer, pancreatic cancer and lung cancer. In another aspect of the present invention, the cell is a placental trophoblast cell or a nucleated red blood cell, and more preferably, the cell is from the peripheral blood of a pregnant woman.

[0045] In one aspect of the invention, the method further comprises using the isolated cells or vesicles 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.

[0046] In one aspect of the present invention, the method is used for the combination of cells or vesicles and microcapsules, in particular the combination of a single cell or vesicle and a single microcapsule. In one aspect of the present invention, it includes allowing a single or several cells or vesicles to stay in the acoustic wave influence area of ​​an ultra-high frequency resonator body, and also includes allowing a single or several microcapsules to stay in the acoustic wave influence area of ​​the same ultra-high frequency resonator body, and then the single or several cells or vesicles are combined with a single microcapsule, for example, the combination of the single or several cells or vesicles and a single microcapsule is formed at a capture point. The method also includes changing the parameters of the microfluidic device so that the combined microcapsule-cell / vesicle is released.

[0047] Microcapsules are devices comprising multiple compartments, which are widely used in different molecular biology applications, including but not limited to: nucleic acid sequencing, protein sequencing, nucleic acid quantification, sequencing optimization, detection of gene expression, quantification of gene expression, and single-cell analysis of genomic markers or expressed markers. The partitions of the microcapsules may contain one or more reagents (e.g., enzymes, unique identifiers such as barcodes, antibodies, etc.). The materials used for the microcapsules, especially the materials of the shells of the microcapsules, can enable the microcapsules to rupture under the applied stimulus. The reagents encapsulated in the microcapsules can be various molecules, chemicals, particles and elements suitable for the sample preparation reaction of the analyte. For example, the microcapsules used in the sample preparation reaction for DNA sequencing of the target may contain one or more of the following reagents: enzymes, restriction enzymes, ligases, polymerases, fluorophores, oligonucleotide barcodes, buffers, etc. In another example, the microcapsules used in the sample preparation reaction for single-cell analysis may contain: lysis buffer, detergent, fluorophore, oligonucleotide barcode, ligase, protease, heat-activatable protease, protease or nuclease inhibitor, buffer, enzyme, antibody, nanoparticles, etc. Microcapsules are usually encapsulated and prepared by emulsifiers or droplet-based microfluidics. The emulsion or droplet contains a water-in-oil formulation or a water-in-oil-in-water (W / 0 / W) double emulsion formulation, or the emulsion or droplet includes a polymer such as agarose or PEG. Microcapsules generally have a size from 10nm to 100μm. Preferably, in the method of the present invention, the microcapsule has a size from 1μm to 100μm.

[0048] In one aspect of the present invention, the method comprises the following steps:

[0049] (a) adjusting the parameters of the UHF BAW resonator, such as adjusting the BAW power and / or flow rate, so that a single or several cells or vesicles stay in the BAW action area of ​​the UHF BAW resonator;

[0050] (b) injecting gel microspheres from the sample inlet, and adjusting the parameters of the ultra-high frequency bulk acoustic wave resonator, such as by adjusting the bulk acoustic wave power and flow rate, so that a single or several microcapsules stay in the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator while keeping the cells or vesicles retained in step (a) from being released;

[0051] (c) The microcapsule and the retained cells or vesicles are combined in the bulk acoustic wave action area of ​​the UHF BAW resonator. The combination of the single or several cells or vesicles and a single microcapsule is usually formed at the capture point in the bulk acoustic wave action area of ​​the UHF BAW resonator.

[0052] In one aspect of the present invention, the method further comprises the following steps:

[0053] (d) By changing the parameters of the microfluidic device, such as by reducing the BAW power or stopping the BAW, the combined microcapsule-cell / vesicle is released.

[0054] The present invention also provides a kit comprising a microcapsule and an ultrahigh frequency bulk acoustic wave resonator as defined above. The kit can be used to combine the microcapsule and the cell or vesicle (especially a single cell or vesicle), and to perform one or more of the following analyses on the cell or vesicle (especially a single cell or vesicle): DNA and RNA amplification (e.g., rapid amplification of cDNA ends (RACE); degenerate oligo-primed 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. The kit may also contain reagents for the analysis.

[0055] The present invention also provides a microfluidic device for separating desired flexible particles from a solution.

[0056] In one aspect of the present invention, a microfluidic device for separating cells or vesicles is provided, comprising:

[0057] a fluid channel having an inlet and an outlet;

[0058] 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 about 0.5-50 GHz in the fluid channel and propagate to the wall on the opposite side of the fluid channel;

[0059] A power regulating device for regulating the power of the bulk acoustic wave generated by the ultrahigh frequency resonator;

[0060] a flow rate regulating device, which regulates the speed at which the solution flows through the bulk acoustic wave region,

[0061] The ultra-high frequency resonator can emit bulk acoustic waves toward the wall on the opposite side of the fluid channel, causing the solution flowing through the bulk acoustic wave region to generate a vortex. The power of the bulk acoustic wave is adjusted by the power regulator and / or the speed of the solution flowing through the bulk acoustic wave region is adjusted by the flow rate regulating device, so that the cells or vesicles stay in the bulk acoustic wave region.

[0062] The microfluidic device provided by the present invention is used to process biologically active cells or molecules, and therefore has a configuration or material for processing biologically active substances. For example, the inner surface of the flow channel can be made of a biocompatible material. For another example, it has a design to prevent cross contamination, especially to prevent contamination amplification.

[0063] In yet another aspect of the present invention, the output power of the power regulating device of the microfluidic device is about 0.5-800 mW, preferably 0.5-500 mW, and more preferably 0.5-350 mW.

[0064] In yet another aspect of the present invention, the flow rate regulating device of the microfluidic 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 0.5-2.5 mm / s.

[0065] In yet another aspect of the present invention, the flow rate regulating device of the microfluidic 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, and more preferably about 0.5-20 μL / min.

[0066] In yet another aspect of the present invention, the height of the fluid channel of the microfluidic device is about 1.5-10 times the diameter of the desired cell or vesicle, preferably about 1.5-6 times the diameter, and more preferably about 2-4 times the diameter.

[0067] In yet another aspect of the present invention, the height of the fluid channel of the microfluidic device is about 25-200 μm, preferably about 25-100 μm, more preferably about 30-80 μm, such as about 40-60 μm.

[0068] In another aspect of the present invention, the area of ​​the BAW generation region of the high-frequency BAW resonator of the microfluidic device is about 500-200000 μm 2 , preferably about 5000-50000 μm 2 , most preferably about 10000-25000 μm 2 .

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

[0070] In yet another aspect of the present invention, the microfluidic device is configured to capture desired cells or vesicles at a position different from the position where other cells or vesicles in the solution leave the area where the bulk acoustic wave acts.

[0071] In yet another aspect of the present invention, the BAW action region of the UHF BAW resonator of the microfluidic device is arranged in such a way that the position where the desired cells are captured is different from the position where other cells leave the BAW action region. In yet another aspect of the present invention, the position where other cells leave the BAW action region is arranged upstream of the position where the desired cells are captured.

[0072] In yet another aspect of the present invention, the ultra-high frequency bulk acoustic wave resonator of the microfluidic device may be a thin film bulk acoustic wave resonator or a solid-state assembly type resonator, for example, an acoustic wave resonator in a thickness stretching vibration mode.

[0073] In yet another aspect of the present invention, the thickness of the piezoelectric layer of the ultra-high frequency bulk acoustic wave resonator of the microfluidic device is in the range of 1 nm to 2 um.

[0074] Reference numerals

[0075] 100 microfluidic device 101 fluid channel

[0076] 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 207 bottom lining layer 208 top

[0077] 300 PCL controller 301 high frequency signal generator 302 power amplifier 303 impedance meter

[0078] 400 Liquid injection and flow rate regulation device

[0079] 500 Acoustic Jet 501 Vortex

[0080] 600 Larger size particles 601 Medium size particles 602 Smaller size particles BRIEF DESCRIPTION OF THE DRAWINGS

[0081] 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.

[0082] Figure 1 A schematic diagram of the structure of a microfluidic device system provided in an embodiment of the present application;

[0083] 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;

[0084] Figure 3 It shows that Hela cells are captured in a microfluidic device system provided in an embodiment of the present application. Figure 3 (a) The motion trajectory of a single Hela cell in a vortex channel formed by the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator of the microfluidic device system (the trajectory of a single cell at different times is superimposed); Figure 3 (b) Schematic diagram showing the movement trajectory of Hela cells (superposition of the movement trajectories of multiple cells) and analysis diagram; Figure 3 (c) is the time and speed analysis diagram of Hela cells.

[0085] Figure 4 The movement of cells in a vortex channel of a microfluidic device system provided in an embodiment of the present application is shown. Figure 4 (a) Image showing cell movement in a vortex; Figure 4 (b) shows cell sedimentation after stopping BSA; Figure 4 (c) shows the presence of multiple cells in the vortex tunnel.

[0086] Figure 5 It is shown that the cell clusters can be controllably decomposed and released in a microfluidic device system provided in an embodiment of the present application. Figure 5(a) shows that cell clusters are captured and broken down into single cells in the acoustic vortex tunnel of the present invention under the condition of a bulk acoustic wave power of 66 mV. Figure 5 (b) shows that when the power of the BAW emitted by the UHF BAW resonator exceeds a certain level, the cells are destroyed by the vortex. Figure 5 (c) Fluorescence image showing cell clusters captured in the acoustic vortex tunnel of the present invention. Figure 5 (d) Fluorescence image showing the decomposition of cell clusters into single cells and the release of single cells in the acoustic vortex tunnel of the present invention (power 15 mV, flow rate 2 μL / min).

[0087] Figure 6 It is shown that a microfluidic device system provided in an embodiment of the present application is capable of separating and capturing Hela cells from a blood mixture containing Hela cells and blood cells. Figure 6 (a) shows that HeLa cells are trapped at the edge of the insonation zone of the high-frequency bulk acoustic wave resonator, while white blood cells and red blood cells flow through the insonation zone. Figure 6 (b) is a graph showing the relationship between the cell capture efficiency and different BAW powers and sample solution flow rates.

[0088] Figure 7 It shows that a microfluidic device system provided in an embodiment of the present application is capable of separating and capturing different cells from a blood mixture. Figure 7 (a) shows that HeLa cells were trapped at the edge of the insonation zone of a high-frequency bulk acoustic wave resonator and remained active for 560 s. Figure 7 (b) shows that the device and method of the present invention can capture leukocytes and maintain their activity. Figure 7 (c) shows that the device and method of the present invention can capture Hela cells, white blood cells and red blood cells respectively.

[0089] Figure 8 Displays the temperature distribution around the UHF BAW resonator. The image was processed using Matlab software.

[0090] Fig. 9 It shows that a microfluidic device system provided in an embodiment of the present application is capable of separating and capturing Hela cells from a whole blood mixture containing Hela cells. Fig. 9 (c) and (d) show the photos of Hela cells being captured and blood cells being released under two microfluidic settings. Fig. 9 (a) and (b) are Fig. 9 Analysis and schematic diagram of (c) and (d). Fig. 9 (e) Shows the upstream and downstream areas of the body acoustic wave action area ( Fig. 9 (c) and (d) (color lines) of blood cell distribution.

[0091] Fig.10 It is shown that a microfluidic device system provided in an embodiment of the present application is capable of capturing circulating tumor cells from blood samples of liver cancer patients.

[0092] Fig.10 (a-1) and (a-2) show the flow conditions of the liquid in the flow channel when bulk acoustic waves and vortices are generated by applying and discharging electricity, respectively. Fig.10 (a-3) shows captured single cells stained green by Calcein-A. Fig.10 (a-4) and Fig.10 (a-5) are the fluorescence and bright field images, respectively. Fig.10 (b) shows the staining of captured cells. Calcein-A is stained green, while the red fluorescence of anti-EpCAM label represents the presence of cancer marker EpCAM in captured cells.

[0093] Fig.11 It is shown that under the condition of constant flow rate, the number of captured cells is linearly related to the power applied to the UHF BAW resonator.

[0094] Fig.12 It is shown that a microfluidic device system provided in an embodiment of the present application combines a single cell or several cells with microcapsules one-to-one. DETAILED DESCRIPTION

[0095] 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.

[0096] Example 1 Experimental methods and materials

[0097] Microfluidic channel and UHF BAW resonator preparation:

[0098] Microfluidic channels made of polydimethylsiloxane (PDMS) were prepared by soft lithography.

[0099] 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:

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Instruments and Materials

[0108] High frequency signal generator: (MXG Analog Signal Generator, Agilent, N5181A 100kHz-3GHz

[0109] Power Amplifier: Mini-Circuits, with 35dBm enhancement of the original RF source power

[0110] Syringe pump: New Era Pump Systems, Inc., NE-1000

[0111] cell:

[0112] Hela cell line: Guangzhou Geneo Biotechnology Co., Ltd., ATCC#CCL2

[0113] 3T3 cells: Guangzhou Geneo Biotechnology Co., Ltd., ATCC#CRL-1658

[0114] Cell culture:

[0115] 293T cells were cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100 U / ml penicillin (Thermo) and 100 ug / ml streptomycin (Thermo). The cell density was 1 x 10 5 / mL~2x 10 6 / mL. When conducting microfluidic experiments, it can be diluted to 1x10 5 / mL for the experiment. PBS buffer (Gibco).

[0116] Cell or tumor markers or stains:

[0117] Calcein-AM (Beijing Solebow Technology Co., Ltd., China)

[0118] Anti-EpCAM (Biolegend, USA)

[0119] 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA)

[0120] Example 2

[0121] 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, in particular flexible particles with a diameter of about 0.8-30um. The flexible particles can be artificial or natural, and the particles are usually micelles with a membrane structure, in particular micelles with a lipid bilayer or a lipid-like bilayer. The flexible particles involved in the present invention usually have a diameter of about 0.8-30um. In one aspect of the present invention, the flexible particles are naturally occurring particles, such as cells or vesicles released by cells into the extracellular environment.

[0122] The method and device of the present invention can be used to separate and capture flexible particles in a solution, such as separating and capturing target cells in blood, or separating and capturing vesicles in plasma.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In one aspect of the present invention, the height of the fluid channel of the microfluidic device is about 20-200 μm, preferably about 20-100 μm, more preferably about 30-80 μm, for example about 40-60 μm. In one aspect of the present invention, the microfluidic device is used to capture cells, and the height of the fluid channel is generally about 1.5-10 times the diameter of the target cell, preferably about 1.5-6 times the diameter, more preferably about 2-4 times.

[0127] 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.

[0128] 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.

[0129] like Figure 1As 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 blood, tissue fluid, extracellular fluid, lymph, cerebrospinal fluid, aqueous humor, urine, sweat, etc.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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).

[0134] 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.

[0135] 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.

[0136] 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.

[0137] like Figure 2As 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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 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 (for example, 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 continuous vortex caused by the ultra-high frequency body acoustic wave forms an acoustic fluid vortex channel. Since the vortex is generated by the body force caused by the attenuation of the sound wave, the central axis of the vortex is above the boundary of the body acoustic wave action. Therefore, the shape of the vortex channel is basically the same as the shape of the body acoustic wave action area, and is located above the boundary of the body acoustic wave action area. The acoustic fluid vortex is caused by the nonlinearity of the propagation of the sound wave in the liquid medium. The strength of the amplitude of the sound wave directly determines the intensity of the acoustic fluid vortex. The amplitude of the ultra-ultrasonic device, that is, the amplitude of the sound wave, can be adjusted by adjusting the applied power, thereby controlling the flow rate of the acoustic fluid vortex. The forces on the particles in the vortex (including larger particles 600, medium 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. When the forces on the particles in the fluid reach a certain balance, the particles stop at the relevant position of the vortex channel and do not move relative to the flow channel. Since the magnitude of the fluid drag force is positively related to the particle, such as the particle diameter, and the magnitude of the acoustic radiation force is positively related to the square of the particle size. As the particles increase in size, the forces they receive will change from being dominated by fluid drag to being dominated by acoustic radiation force, and the acoustic radiation force will push the particles to the center of the vortex. Larger particles are subject to greater acoustic radiation force 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 to the downstream of the body sound wave action area under the action of the lateral drag force generated by the laminar flow.

[0142] The fluid drag force and acoustic radiation force caused by sound waves on particles entering the vortex channel can be calculated according to certain formulas, but the inertial lift force generated by the acoustic fluid vortex can hardly be calculated using simple physical principles and formulas, especially in the case of fluids containing complex components. Compared with the two-dimensional particle capture in the prior art, the method and device of the present invention involve deformable cells or vesicles, whose force conditions and movement trajectories in the acoustic fluid vortex and the channel formed by it are more complicated, because the interaction between each vortex and the migration of particles between vortices have an impact on the capture in the fluid, especially when there are a large number of cells or vesicles in the fluid, because the cells or vesicles interact and influence each other due to collisions and other phenomena, the force and movement in the vortex are different from the force and movement calculated and simulated theoretically, and the movement mode and trajectory in the vortex channel cannot be predicted based on theoretical calculations and simulations.

[0143] The applicant unexpectedly discovered through experiments that in the method and device of the present invention, when cells or vesicles in the solution pass through the acoustic fluid vortex channel area caused by ultra-high frequency body acoustic waves, under the conditions of suitable flow rate and body acoustic wave power, they reach a stable state at a certain position (some) of the vortex channel without any relative movement to the flow channel, that is, they are captured (the position where the cell or vesicle reaches a stable state can be called the capture position); while other cells or vesicles in the solution that do not meet the stable state (for example, cells or vesicles with smaller volume) do not enter the vortex channel or leave the body acoustic wave action area at a certain position (some) of the vortex channel under the lateral drag force generated by the laminar flow, and move downstream, that is, they are released; the position where the cell or vesicle leaves the vortex channel is also called the release point. In the vortex channel, due to the action of the laminar flow, the cells or vesicles at the center of the vortex usually move to the area where the vortex channel is located downstream of the fluid channel, that is, the capture position is usually in the area where the vortex channel is located downstream of the fluid channel.

[0144] The inventors also unexpectedly found that the position where the cells or vesicles leave the vortex channel is usually at the jump point of the vortex and the acoustic radiation force. Under the condition that the body acoustic wave power that generates the vortex, the solution flow rate, the shape and size of the fluid channel remain unchanged, the position where the cells or vesicles leave the vortex channel is usually at the place where the vortex channel turns or turns, that is, above the position where the boundary of the corresponding body acoustic wave action area turns or turns. Without being bound by relevant theories, the applicant believes that the reason for this phenomenon is that at the turning point or turning point of the vortex channel, the vortex direction and the direction of the acoustic radiation force suddenly change. Among the cells or vesicles that enter the vortex channel, the particles that meet the appropriate conditions (such as the appropriate size) have been focused to the center of the vortex. Under the action of the acoustic radiation force, they can change the direction of movement with the vortex channel and quickly refocus to the center of the vortex channel after the turn; while the particles that do not meet the conditions (such as having a smaller size) will be more affected by the jump in the laminar drag direction, and thus leave the vortex channel.

[0145] In one preferred embodiment of the present invention, the microfluidic device is configured so that when processing cells or vesicles containing different cells or vesicles, the position where the target cells or vesicles are captured (or called the capture point) is different from the position where the non-target cells or vesicles leave the area where the body acoustic wave acts (or called the release point).

[0146] In one preferred embodiment of the present invention, the BAW action area of ​​the UHF BAW resonator of the microfluidic device is set in such a way that when processing cells or vesicles of different sizes, the position where the target cells or vesicles are captured (or called the capture point) is different from the position where the non-target cells or vesicles leave the BAW action area (or called the release point). For example, when the BAW action area of ​​the UHF BAW resonator is a pentagon, it is set so that one of the corners faces the inflow direction of the channel liquid, and is placed symmetrically along the center line of the flow channel: at this time, the non-target cells or vesicles leave the BAW action area, that is, the release point, at both ends of the downstream edge line along the fluid direction; and the position where the target cells or vesicles are captured, that is, the capture point is in the middle of the downstream edge line along the fluid direction; the two do not overlap. As a result, the non-target cells have the least effect on the stability of the captured cells. In another preferred embodiment of the present invention, the position where the target cells or vesicles are captured (or called the capture point) is set upstream of the non-target cells or vesicles leaving the BAW action area (or called the release point).

[0147] The configuration of the microfluidic device capable of achieving different capture points and release points can be obtained through theoretical calculation or actual experiment.

[0148] For example, when screening circulating tumor cells from the blood, due to the extremely high cell density in the blood, if the release site coincides with the capture site, all cells will pass through the capture site, which will lead to strong cell-to-cell interactions, and circulating tumor cells at the capture site may fall off due to the collision of a large number of red blood cells and white blood cells. In one embodiment of the present invention, the capture point is separated from the release point, for example, the release point is set before the capture site, and non-target red blood cells and white blood cells will be taken away by the laminar flow without passing through the capture site, and only the target circulating tumor cells can reach the capture position.

[0149] Thus, the inventors of the present applicant have discovered and provided a method for more effectively isolating target cells or vesicles in a sample, in particular, a method for isolating target cells or vesicles with extremely small numbers or extremely low proportions from a sample containing other cells or vesicles.

[0150] 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 0.5GHz-50GHz, preferably more than 1GHz-about 10GHz.

[0151] 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.

[0152] 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 0.5-800mW, preferably 0.5-500mW, and more preferably 0.5-350mW. Since the thin film bulk acoustic wave resonator has a 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.

[0153] 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.

[0154] 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 cells and vesicles.

[0155] Example 3 Isolation of cells in buffer

[0156] Hela cells were dissolved in DMEM culture medium to prepare samples (cell content was about 1*10 5 / mL). In addition, Hela cells were labeled with Calcein-AM to observe their movement pathway. The sample was injected into the microfluidic channel from the sample inlet.

[0157] The height of the microchannel is 50 μm. The frequency of the ultra-high frequency bulk acoustic wave resonator is 1.83 GHz, and the output power is 30 mW. The sample input flow rate is controlled to be 1 μL / min, that is, about 0.67 mm / s.

[0158] The results are as follows Figure 3 shown. Figure 3 (a) shows the motion trajectory of a single Hela cell in a vortex channel formed by the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator of the microfluidic device system. It is a superposition of multiple images of the same single cell at different times to show its motion trajectory. Figure 3 (b) Schematic diagram of the movement trajectories of different individual cells: the upper figure is the top view, and the lower figure is the side view. Figure 3 (c) is the time and speed analysis diagram of Hela cells.

[0159] In the microfluidic device of the present invention, the bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator causes the fluid flowing through to generate a vortex, and an acoustic fluid vortex channel or a vortex tunnel is formed along the boundary of the acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator. When passing through the bulk acoustic wave action area, the cell enters the vortex tunnel, and under the conditions of suitable flow velocity and bulk acoustic wave power, after reaching a stable state at a certain position (positions) of the vortex channel, it does not produce movement relative to the flow channel, that is, it is captured (the position where the cell or vesicle reaches a stable state can be called the capture position). Due to the interaction between the vortex and the laminar flow, the cell at the capture position is subjected to very weak lateral flow force, so it no longer moves forward (horizontally), that is, it is captured. Moreover, the captured cells float above the bottom of the microfluidic channel and have no contact with the microfluidic channel.

[0160] Figure 3 The image sequence in (a) shows and demonstrates the process of single HeLa cell capture. The capture process can be divided into three stages occurring in regions abc, as Figure 3 (a) and Figure 3 (c) As shown. In region a, the cells move at a uniform speed in the lateral flow. In region b, the direction of cell movement is changed by the vortex array, and the cells enter the vortex tunnel generated by the acoustic wave while being focused on the 3-D axis and moving along a fixed path. Cells with different initial positions have very different speeds when entering the vortex array. Due to the difference in relative direction with the vortex, cells with initial positions in the lower layer of the microchannel accelerate, while cells with initial positions in the upper layer slow down. In region c, the speed of cell movement gradually slows down. Finally, the cells are trapped at a fixed point where the drag force and the acoustic radiation force balance each other. Since the trajectory of the cells is adjusted in the vortex tunnel, the process of cell deceleration and capture has good stability, and cells entering different initial positions have good consistency in deceleration and capture.

[0161] Figure 3 (b) Schematic diagram further illustrates the movement of cells in the flow channel and under the action of bulk acoustic waves. Figure 3 (b) is a schematic diagram of the superposition of the movement trajectories of different individual cells. Figure 3 The upper figure of (b) is a top view, and the lower figure is a side view. From the top view, it can be seen that the cell trajectories are only distributed at the boundary of the UHF BAW resonator's acoustic wave action area. Each single cell finally stays at the capture point downstream of the boundary of the UHF BAW resonator's acoustic wave action area. From the side view, it can be seen that the cells are floating above the bottom of the channel and have no contact with the bottom.

[0162] Example 4 Suspension and sedimentation of cells in microfluidic channels

[0163] In the prior art acoustic flow vortex method, the movement of particles or cells in the z-axis of the microchannel (i.e., the vertical direction of the flow channel) is ignored because the in-plane acoustic flow is the main vortex. However, the trajectory of the z-axis is very important for single-cell manipulation and high-precision sensing. Due to the unique TE vibration mode of the ultra-high frequency body acoustic wave adopted by the present invention, the dominant vortex in the microfluidic system of the present invention is out-of-plane. In order to better study the vortex tunnel generated by the ultra-high frequency body acoustic wave in the microfluidic system of the present invention, Hela cells labeled with Calcein-AM were used, and a confocal microscope (Leica, Germany) was used to observe and measure the movement of cells on the z-axis. In order to capture the particle trajectory in the vortex tunnel, the xzt mode was used. In this mode, the shooting speed can reach 37 frames per second.

[0164] The same experimental settings as in Example 3 were used, but the input flow rate of the sample containing Hela cells was reduced to 0.1 μL / min. After the sample containing cells and the buffer solution were passed into the microfluidic channel, the signal generator was turned on, and the cells were captured in the area of ​​acoustic wave action. Then the pressures of the inlet and outlet were adjusted to be the same, and the fluid stagnated. The movement of the cells in the vortex was observed. Then the signal generator was turned off, and the movement of the cells was observed.

[0165] The results are as follows Figure 4 shown. Figure 4 (a) is an image of cell movement in a vortex, which is a composite stack image (6 images, 27ms apart), where the red dot shows the center of the particle in each frame, the green dotted circle indicates the range of particle movement and the red arrow indicates the direction of particle movement. When the signal generator is turned on, the bulk acoustic wave is generated, forming a vortex and vortex tunnel in the flow channel, and the particles are relatively "static" suspended at a distance from the chip surface. Figure 4 As shown in (b), when the power is turned off, the bulk acoustic wave disappears and the particles settle to the chip surface under the combined action of gravity and buoyancy. The results show that the particles are indeed trapped in the vortex tunnel.

[0166] Figure 4 (c) shows that Calcein-AM-labeled HeLa cells are trapped in a vortex tunnel that has the same shape as the boundary of the insonation region of an UHF BAW resonator.

[0167] For cell manipulation, cells in contact with the device may be killed by the negative effects triggered by the device, thereby deteriorating biocompatibility. In addition, cell-cell adhesion and cell-device surface adhesion caused by contact are also a serious problem, especially for single cell manipulation. The method provided by the present invention can avoid the problem of cell-cell or cell-device surface contact and adhesion in cell capture and separation operations.

[0168] Example 5 Cell disruption

[0169] like Figure 5 As shown in (b), the inventors found in the experiment that the excitation power of the ultra-high frequency bulk acoustic wave resonator in the microfluidic system was increased to more than 800mW, for example, at 1300mW, under the combined action of the shear force of the high-speed vortex and the volume force generated by the velocity gradient, the cell membrane of the cell will be destroyed, the cell will be lysed, and the substances in the cell, such as proteins and nucleic acids, will be released (as shown in the red circle in the figure), and enter the downstream of the system with the liquid flow, which can be further studied. The power that causes cell or vesicle destruction is related to the volume of the cell or vesicle. Since cell fragmentation is mainly affected by the squeezing of the acoustic radiation force, and the acoustic radiation force is generally proportional to the cube of the volume of the cell or vesicle, the excitation power of the ultra-high frequency bulk acoustic wave resonator that causes the destruction and lysis of cells or vesicles with a smaller volume is higher.

[0170] In cell research, in order to obtain information inside the cell, such as DNA, RNA, and protein, the cell destroys the cell membrane, including the cell membrane and the nuclear membrane, so that the substances wrapped in the membrane can be released for downstream analysis. For example, in DNA sequencing, it is necessary to destroy the cell membrane with a lysis solution to obtain DNA and then participate in the polymerase chain reaction; the protein blotting method commonly used in protein detection also needs to obtain protein from the cell. At present, the commonly used cell lysis method is mainly chemical, and different lysis solutions are added to achieve cell lysis. Compared with the physical lysis method, the chemical method may affect downstream analysis because it will introduce exogenous agents. Among the physical methods, there are ultrasonic lysis, thermal lysis, laser lysis and other methods. At present, the only method that can achieve accurate lysis of a small number of cells is laser lysis. However, due to the limitations of the laser and the optical path, laser lysis is costly and has poor compatibility with microfluidic chips.

[0171] By using the method and system of the present invention, single-cell lysis can be achieved with high precision to obtain unique property information of individual cells.

[0172] Example 6 Separation of different cells in a diluted blood sample

[0173] The forces that affect the movement of cells in the acoustic vortex of the present invention, including the fluid drag force (Stokes drag force), the inertial lift force (inertial lift force) generated by the acoustic fluid vortex and the acoustic radiation force (acoustic radiation force) caused by the sound wave, are closely related to the size of the cells. It is necessary to study whether the method of the present invention can be used to separate cells of different sizes in a solution, especially in blood, and the accuracy of the separation.

[0174] Hela cells were dissolved in DMEM culture medium and mixed with whole blood diluted 200 times to prepare the test sample (the Hela cell content was adjusted to about 1x10 5 / mL). Hela cells were labeled with Calcein-AM (green). Blood was stained with DAPI, so that white blood cells appeared blue under the microscope.

[0175] The sample is injected into the microchannel from the sample inlet. And the PBS buffer is injected into the microchannel from the buffer inlets on both sides. The injected sample is passively focused by three-phase flow, so that the sample can flow better through the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator.

[0176] Figure 6 It is shown that Hela cells in a blood mixture containing Hela cells and blood cells are selectively captured by the device and method of the present invention.

[0177] Specifically, Figure 6 (a) shows that Hela cells (dyed green, indicated by the solid circle in the figure) stay in the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator, while white blood cells (dyed blue, indicated by the dotted circle in the figure) and red blood cells continue to move along the direction of fluid movement. Figure 6 (a) The rightmost image is an enlarged view of the left image. As can be seen from the image, the capture of three Hela cells (indicated by arrows) is very stable, even if other cells in the blood (white blood cells and red blood cells) try to enter and occupy this position.

[0178] In addition, if Figure 6 As shown in (b), the cell capture efficiency is related to different BAW powers and sample solution flow rates. In this experiment, the capture efficiency is defined as the number of cells captured in the acoustic action area of ​​a single UHF BAW resonator when 10 target cells are input. Within a certain range, the cell capture efficiency increases with the applied power and decreases with the increase in the lateral flow rate. The reason is that the increase in lateral flow will induce stronger shear force, which will cause cell escape. The increased power produces higher-speed vortices, which can offset the negative impact of the lateral flow on the BAW vortex tunnel. In order to obtain a stable capture area with high resolution, the applied power and the lateral flow rate of the liquid can be balanced.

[0179] By using the microfluidic system and method of the present invention, target cells can be extracted from a mixed sample (such as blood) into another solution (such as a buffer or a fluorescent dye). Figure 7As shown in (a), by adjusting the power and flow rate, Hela cells (dyed green) stay in the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator, while white blood cells (dyed blue) and red blood cells continue to move in the direction of fluid movement, leaving the microfluidic device; then, while maintaining the power and flow rate, the blood containing cells is stopped and PBS liquid is input, and the Hela cells still stay in the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator, while the solution is converted from blood to PBS; then the power is lowered, and the Hela cells are separated from the vortex and flow with the PBS liquid to the next structure of the microfluidic channel, that is, the process of capturing Hela cells from blood to PBS buffer is completed. Similarly, Hela cells can be transferred from blood or PBS buffer to other solutions, such as Trypan dye solution.

[0180] Moreover, the device and method of the present invention can capture cells to meet the requirements of single cell separation. Under the conditions of the method and device of the present invention, that is, when the height of the microchannel is about 1.5-6 times the diameter of the target cell, and more preferably about 2-4 times, the generated vortex channel is most suitable for holding one cell within the vertical height range. In this way, the cells captured in the acoustic wave action area of ​​each high-frequency body acoustic wave resonator are arranged side by side along the edge of the acoustic wave action area, and basically no oscillation occurs, reducing the damage caused by contact and collision between cells. And it is easy to carry out the next step of the single cell separation operation by changing the vortex formation conditions to make the cells leave the vortex tunnel.

[0181] Experiments have shown that the method and device of the present invention can distinguish and separate cells of different sizes, and the resolution can achieve separation of blood cells and other cells in the blood (such as circulating tumor cells such as Hela cells).

[0182] In addition, the size of the captured cells can be selected by adjusting the strength of the acoustic streaming and the lateral flow velocity. White blood cells and red blood cells can also be selectively captured from blood samples, and the results are shown in Figure 7 (b) and Figure 7 (c) Figure 7 In (b), the blood sample was stained with DAPI so that in the fluorescent view, the white blood cells (WBC) were stained blue. Figure 7 (b) shows that when the power is adjusted to 13.2 mV, single leukocytes are captured and good cell activity is maintained. Figure 7 (c) shows that different cells are captured at different powers. For example, at a power of 6.6 mV, both white blood cells and red blood cells are not captured; at a power of 13.2 mV, white blood cells are captured, but red blood cells are not captured; at a power of 20.9 mV, both white blood cells and red blood cells are captured.

[0183] The inventors used software (COMSOL, USA) to build a model to analyze the separation process of the above three types of cells. The resolution of cell separation in the simulation is much worse than the effect observed in the actual experiment. This may be because factors such as the interaction and deformation between cells are not reflected in the simulation model.

[0184] Example 7: Maintenance of Activity of Captured Cells

[0185] In addition, the retention of cell activity is of great significance for cell research. However, most of the existing cell separation methods will have adverse effects on cell activity or even kill cells. In existing cell manipulation technologies, factors that affect cells include mechanical shear force, Joule heat or electron field. When the sound waves generated by the ultra-high frequency bulk acoustic wave resonator are absorbed into the liquid, the mechanical energy is converted into kinetic energy of the fluid and heat. Thermal effects may lead to biological effects, which may cause conformational changes, protein misfolding, entanglement and / or non-specific aggregation of proteins, etc.

[0186] However, the methods and devices of the present invention are able to maintain the full viability of captured cells.

[0187] In this experiment, trypan blue (an important stain for selectively marking dead tissue or cells) was injected into the captured cells as a probe for active cells.

[0188] Experiments and results Figure 7 (a) As shown. HeLa cells were captured according to the above method and conditions. Then their activity was detected with trypan blue. The experiment proved that during the viability test, the cells were exchanged between buffer and trypan blue solution, repeated 3 times. At 560 seconds, the cells were still active: the cell membrane still remained selectively permeable to trypan blue.

[0189] In addition, in the experimental setup described above, Rhodamine B (a temperature-dependent fluorescent dye) was added to the solution and the temperature distribution around the UHF BAW resonator was measured at different applied powers. Figure 8 As shown, it is shown that the thermal effect of the resonator is very weak, especially under conditions of selective cell capture, such as when the applied power is less than 100 mW.

[0190] Example 8 Separation and capture of different cells in whole blood

[0191] Blood is the most primitive and most suitable environment for cells. In the blood environment, cells have the best vitality and complete functions, which is important for biological research, such as cell metabolism and proteomics. For these reasons, the separation of CTCs from whole blood is an important requirement for research, but also a technical challenge.

[0192] However, whole blood is a more challenging sample than diluted blood samples. From the perspective of physical parameters, whole blood is more viscous and turbid, which has serious negative effects on cell manipulation. Physical fields, such as dielectric fields, magnetic fields, and fluid dynamic fields, are chaotic in blood samples. In addition, the high density of cells, especially red blood cells (10 9 / mL), which would cause strong interactions between cells, thus changing the trajectory of the specimen and affecting the stability of cell capture.

[0193] The inventors have demonstrated that the apparatus and methods of the present invention are capable of isolating and capturing target cells in whole blood.

[0194] Hela cells were dissolved in DMEM culture medium and mixed with whole blood to prepare test samples (the Hela cell content was adjusted to about 1x10 5 / mL).

[0195] The inlet of the microfluidic system is designed as a three-phase flow, including a central sample inlet and buffer inlets on both sides: the sample is injected into the microfluidic channel from the central sample inlet; and the PBS buffer is injected into the microfluidic channel from the buffer inlets on both sides. As a result, the PBS buffer added on both sides can be used as a clamping sheath flow, limiting the lateral range of the sample fluid and ensuring that all samples will pass above the ultra-high frequency bulk acoustic wave resonator located in the center of the flow channel. The injected sample is passively focused by the three-phase flow method, so that the sample can better flow through the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator.

[0196] In order to better separate the target cells and increase the separation efficiency, the microfluidic device of the present invention is optimized. For example, the setting of the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator is adjusted to separate the capture point of the target cells and the release point of the non-target cells.

[0197] The BAW action area of ​​the UHF BAW resonator of this embodiment is set in such a way that when processing cells of different sizes, the position where the target cells are captured (or called the capture point) is different from the position where the non-target cells leave the BAW action area (or called the release point). For example, when the BAW action area of ​​the UHF BAW resonator is a pentagon, it is set so that one of the corners faces the inflow direction of the channel liquid, and it is placed symmetrically along the center line of the flow channel: at this time, the position where the non-target cells leave the BAW action area, that is, the release point, is located at both ends of the downstream sideline along the fluid direction; and the position where the target cells are captured, that is, the capture point, is in the middle of the downstream sideline along the fluid direction; the two do not overlap. As a result, the non-target cells have minimal impact on the stability of the captured cells.

[0198] The results are as follows Fig. 9 As shown, HeLa cells in whole blood containing HeLa cells were selectively captured.

[0199] Fig. 9 (c) and (d) show the photos of Hela cells being captured and blood cells being released under two microfluidic settings. Fig. 9 (a) and (b) are Fig. 9 (c) and (d) are analysis and schematic diagrams. In this embodiment, the BAW action area of ​​the UHF BAW resonator is a pentagon. Fig. 9 In (c), the BAW action area is set to have a protruding corner (a corner of a pentagon) downstream of the liquid inflow direction: at this time, one of the points where the blood cells leave the BAW action area is near the corner of the pentagon; and the location where the Hela cells are captured, i.e., the capture point, is also near the same corner. Therefore, the blood cells have some influence on the stability of the captured Hela cells. Fig. 9 (d) The mid-body acoustic wave action area is adjusted so that one corner faces the direction of liquid inflow in the channel and is placed symmetrically along the center line of the flow channel: at this time, the release points of blood cells are located at both ends of the downstream edge along the flow direction and upstream of the capture point (i.e., the position where the cells pass first); while the capture point of Hela cells is in the middle of the downstream edge along the flow direction; the two do not overlap. Therefore, the blood cells have the least impact on the stability of the captured Hela cells. Fig. 9 (e) Shows the upstream and downstream areas of the body acoustic wave action area ( Fig. 9 The green and blue curves indicate that blood cells are released at two symmetrical release points on average without passing through the capture point. Fig. 9 (f-1) shows the passive focusing of the injected sample using a three-phase flow input method, that is, the sample is injected into the microchannel from the middle sample inlet, and the PBS buffer is injected into the microchannel from the buffer inlets on both sides, so that the sample can better flow through the acoustic wave action area of ​​the high-frequency bulk acoustic wave resonator. Fig. 9 (f-2) shows the flow of blood cells when the power is turned on and bulk acoustic waves are generated in the microchannel. Fig. 9 (f-3)-(f-5) show that CTCs are selectively captured: the composite stack image sequence shows the trajectories of captured CTCs. Fig. 9 (f-6) Results showing selective capture of CTCs: The merged image shows that the device of the present invention stably captures three CTCs while releasing blood cells.

[0200] In a more preferred method of the present invention, in order to reduce the negative impact caused by cell-cell interaction, the microfluidic device, such as the body acoustic wave action area of ​​the ultra-high frequency body acoustic wave resonator, is adjusted to distinguish the capture area of ​​the target cell and the release point of the non-target cell, which helps to capture the target cell. In the case of separating the added Hela cells from the diluted blood sample, since the blood cell density is relatively low, the negative impact caused by the interaction between the captured cells (Hela cells) and the released cells (blood cells) is acceptable. However, in the case of separation from undiluted whole blood, the high viscosity of the blood limits the velocity gradient of the fluid, and the extremely high cell density triggers collisions and friction between cells. The captured Hela cells may deviate from the original trajectory or capture point under the interaction of other cells. By adjusting the body acoustic wave action area of ​​the ultra-high frequency body acoustic wave resonator of the microfluidic device, the capture point is separated from the release point, especially the release point is placed before the capture point (upstream), and the non-target cells will be taken away by the laminar flow without passing through the capture point, and only the target cells can reach the capture position.

[0201] Example 9 Capture of CTCs in Patient Blood Samples

[0202] Circulating tumor cells, or CTCs, are tumor cells found in the circulation of cancer patients. The term generally does not include blood tumor cells. In the patient's circulation, CTCs are present in very small amounts, with less than 1 CTC per milliliter of blood. Circulating tumor cells are of great clinical significance for the diagnosis of cancer. Due to the small number of CTCs and the presence of a large number of cells with similar physical properties in the blood, detecting CTCs in the blood or isolating CTCs from the blood is a major challenge.

[0203] This application demonstrates that the methods and devices of the present invention can separate and capture CTCs from the blood of liver cancer patients.

[0204] 1 ml blood samples from three patients with stage IV liver cancer were injected into the microchannel from the sample inlet. PBS buffer and staining solutions containing Calcein-AM and anti-EpCAM antibodies labeled with red fluorescence were injected into the microchannel from the inlets on both sides. The injected sample was passively focused by three-phase flow, so that the sample could flow better through the acoustic action area of ​​the high-frequency bulk acoustic wave resonator. The central sample flow rate was controlled to be about 1 μL / min, i.e., about 0.67 mm / s, while the PBS flow rate was about 5 μL / min.

[0205] The results are as follows Fig.10 shown. Fig.10(a-1) shows the distribution of fluid in the microchannel when the power is not turned on: PBS liquid flows into the channel from the top left channel, the blood sample enters from the top right channel, and the dye solution is input into the bottom right channel; under the clamping action of the PBS liquid flow and the dye solution, the blood sample flows through the bulk acoustic wave signal generating area of ​​the pentagonal high-frequency bulk acoustic wave resonator set near the right side of the flow channel. Fig.10 (a-2) shows that when power is applied, the power is adjusted to 30mW, the high-frequency BAW resonator generates a vortex in the microchannel, the blood cells pass through the BAW signal generation area, and the target cells are captured; then the blood sample and PBS buffer inlet are closed, the dye solution occupies the BAW signal generation area, and the cells are dyed. After dyeing, the dye inlet is closed and PBS buffer is injected to wash the remaining blood cells and dye. Fig.10 (a3-5) shows CTCs captured in the body acoustic wave signal generation area and blood cells that are not captured (passing through the body acoustic wave signal generation area). Fig.10 (b) shows the staining of a captured single target cell, which is stained green by Calcein-AM, indicating that the captured cell remains active, while the red fluorescence of the anti-EpCAM label represents the presence of the cancer marker EpCAM in the captured cell.

[0206] The 3T3 cell line (3T3 is a cell line with low EpCAM expression) was also selected as a negative control. Fig.10 Same as in (a), the results are shown in Fig.10 (c). Fig.10 The results in (c) show that both CTC and 3T3 were stained with Calcein-AM (green), CTC was stained with anti-EpCAM antibody, and 3T3 was not stained red because EpCAM was hardly expressed thereon. The results prove that the captured single cells or cell clusters are cancer cells.

[0207] Experiments have shown that the method of the present invention can capture CTCs in patient whole blood samples and perform in situ staining. Compared with staining in a centrifuge tube by centrifugation and / or magnetic beads, the in situ staining analysis method provided by the present invention has great advantages, including no need for repeated centrifugation for washing and changing dyes or solutions.

[0208] Example 10 Controlled Release of Cells

[0209] The inventors of the present application also unexpectedly discovered that in the method and apparatus of the present invention, within a certain range, the number of cells captured in the vortex tunnel generated by the bulk acoustic waves of the ultra-high frequency bulk acoustic wave resonator is linearly related to the power applied to the ultra-high frequency bulk acoustic wave resonator, thereby providing controllable release of cells, especially a few or single cells.

[0210] Prepare a Hela cell sample, labeling, and a microchannel according to the method of Example 3. Inject the sample into the microchannel from the sample inlet and generate a vortex tunnel by the bulk acoustic wave of an ultra-high frequency bulk acoustic wave resonator.

[0211] As Fig.11 shown in (a), the number of cells captured in the vortex or vortex tunnel generated by the bulk acoustic wave of the ultra-high frequency bulk acoustic wave resonator is affected by the flow rate of the cells and the power applied to the ultra-high frequency bulk acoustic wave resonator.

[0212] The inventors unexpectedly found that there is a linear relationship within a local range among the number of captured cells (N), the applied power (P), and the lateral fluid velocity (V). The inventors found through experiments that when V = 0.33 mm / s, N = 0.4*P; when V = 0.67 mm / s, N = 0.28*P; when V = 1.34 mm / s, N = 0.18*P; when V = 2 mm / s, N = 0.15*P. Where the coefficient k ≈ 0.47*exp(-V / 0.83)+0.14. This formula is applicable when the cell-cell interaction is less and weaker, and when there is no adhesion and aggregation between cells to form cell clusters.

[0213] The inventors found through experiments that the preferred conditions are: when V < 1 mm / s, P < 100 mW; when 1 mm / s < V < 3 mm / s, P < 300 mW. For Hela cells, on the premise of ensuring the accuracy of sieving, the lateral flow rate is most preferably about 0.5 - 1 mm / s, and the applied power is recommended to be 5 - 20 mW. At this time, the number of cells captured by a single device is between 1 and 6. If more cells are needed, it can be achieved by arranging the devices in an array.

[0214] The following table shows the results of the capture numbers:

[0215]

[0216] Therefore, the present application also provides a method for controllable cell capture and release, in which the number of captured cells can be controlled, and the number of released cells can be controlled, so as to control the cells to be released one by one, that is, to obtain single cells. Fig.11Taking (b) as an example, by controlling the gradient to reduce the power and controlling the vortex intensity, the captured cells can be released in sequence. For example, for cells with a diameter of 15-20 μm, the power to capture multiple cells is about 35 mW at a lateral flow rate of 0.67 min / s. Among them, the power is preferably less than 100 mW. Higher power may cause cell-to-cell adhesion, which is not conducive to single-cell operation. Cell capture is performed at an input power of 30-35 mW. When the target number of cells is reached, the power is gradually reduced. For example, each reduced gradient is 5-20% of the total power, such as about 10%. The smaller the gradient, the more precise the control, but the longer the cells are captured, the more likely cell-to-cell adhesion will occur.

[0217] like Fig.11 As shown in (b), when the capture power is about 33Mw, when two cells are captured, the single release power is about 13mW and the complete release power is about 0.1mW; when three cells are captured, the release power of the first cell is about 20mW, the release power of the second cell is about 16.5mW, and the release power of the third cell is about 0.1mW; when four cells are captured, the release power of the first cell is about 21mW, the release power of the second cell is about 17mW, the release power of the third cell is about 6.6mW, and the release power of the fourth cell is about 0.1mW.

[0218] It can be seen. In the system and method of the present invention, the number of cells captured in the vortex can be adjusted by regulating the applied power. The captured cells are arranged along the boundary of the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator. Under the action of the lateral fluid, the cells tend to move downstream along the boundary of the device, so when the capture conditions change, the first cells to fall off are the cells at the downstream boundary. By gradually reducing the power, the ability of the vortex to capture cells gradually decreases. When it is reduced to the point where it is unable to capture all the current cells, the cells closest to the downstream will leave the vortex, and the other captured cells will move downstream one cell at a time to fill the space left vacant by the released cells. Therefore, the controlled release of the captured cells can be achieved by gradually reducing the applied power. It is worth emphasizing that this release can reach the single-cell level, that is, the captured cells can be released one by one in sequence.

[0219] Example 11 Combination of cells and gel microspheres

[0220] Since it was found that in the system and method of the present invention, the relationship between the increase in applied power and the number of captured cells or other flexible particles of similar properties is linear, based on this, the number of cells and / or other particles captured in the vortex can be adjusted by regulating the applied power, and the controlled release of the captured cells or other particles can be achieved by gradiently reducing the applied power.

[0221] In this experiment, a method was tested to capture single cells and assemble them into individual gel microspheres for further analysis such as sequencing of the single cells.

[0222] The method of this embodiment includes the following steps:

[0223] (1) injecting a cell sample to be processed from a sample inlet (the cell content is diluted to a level suitable for the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator to capture a single to about ten cells), and adjusting the bulk acoustic wave power and flow rate of the ultra-high frequency bulk acoustic wave resonator so that a single cell is captured in the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator;

[0224] (2) injecting gel microspheres from the sample inlet, adjusting the BAW power and flow rate of the UHF BAW resonator, and allowing a single gel microsphere to enter the BAW action area of ​​the UHF BAW resonator and be captured while keeping the single cell captured in step (1) from being released, so that the gel microsphere and the captured cell are paired one-to-one;

[0225] (3) By controlling the gradient to reduce the power and control the vortex intensity, the paired gel microsphere-cells are released.

[0226] In single-cell studies such as single-cell sequencing, it is necessary to assemble gel microspheres (which are wrapped with primers, lysate, etc.) with single cells. The current assembly methods are all based on the random method of Poisson distribution, which requires a large number of gel microspheres and cells with extremely low concentrations to ensure that each cell can be wrapped in the same drop with a single gel microsphere to complete the subsequent reaction. Such technology will lead to a large amount of waste of gel microspheres. More importantly, the assembly process is random and uncontrollable, and it is impossible to perform preliminary screening of the required cells, nor can it be guaranteed that a certain specified cell will be wrapped by the gel microspheres. Utilizing the single-cell capture ability of the acoustic fluid vortex, the inventors capture the single cell in the acoustic fluid system provided by the present invention, and then introduce a single gel microsphere. In the acoustic fluid tunnel, the cell and the microsphere can complete the preliminary assembly. In this way, it can be determined that the single cell that needs to be studied is obtained, and it is also guaranteed that this cell can be assembled with the gel microsphere.

[0227] Hela cell samples, markers and microfluidics were prepared according to the method of Example 3. The gel microspheres used in the experiment were Barcode Gel beads from 10X genomics, which are standard microspheres for single-cell sequencing. The experiment and results are shown in Fig.12As shown. The applied power is 26.3 mW and the flow rate is 1 μL / min. The inlet of the microfluidic system can be connected to a buffer solution (PBS), a HeLa cell solution or a Barcode gel bead sample respectively. The parameters of the system are adjusted so that a single cell and a single barcode gel bead can enter the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator and be captured. The process of this method includes: first adding a buffer solution; when the buffer solution has drained the bubbles in the flow channel, only opening the cell flow channel and placing the Hela cell. After the bulk acoustic wave area of ​​the ultra-high frequency bulk acoustic wave resonator captures a Hela cell (such as Fig.12 a), close the cell flow channel, open the Barcode gel beads flow channel, and put a Barcode gel bead (as shown in Fig.12 b). A single cell and a single Barcode gel bead are captured in the same BAW region of an ultra-high frequency BAW resonator (as shown in Fig.12 c). Then, the buffer flow channel is opened and the applied power is turned off to release the paired cells and gel microspheres (as shown in Fig.12 d).

[0228] In summary, the microparticle capture system and method provided in the present application can achieve selective and specific capture and controllable release of cells or vesicles of different sizes, and further perform single cell or vesicle analysis.

[0229] 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 isolating cells or vesicles, characterized in that include: (1) allowing a solution containing cells or vesicles 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 and propagate to the wall on the opposite side of the fluid channel; (2) the ultra-high frequency bulk acoustic wave resonator emits a bulk acoustic wave that is transmitted to the wall on the opposite side of the fluid channel, and a vortex is generated in the solution in the area affected by the bulk acoustic wave; (3) By adjusting the power of the bulk acoustic wave and / or adjusting the speed of the solution flowing through the bulk acoustic wave influence area, the cells or vesicles are allowed to stay in the bulk acoustic wave influence area, i.e., be captured; other cells or vesicles that are not captured do not enter the vortex channel or leave the bulk acoustic wave action area at a certain position in the vortex channel, wherein the position of leaving the vortex channel is called the release point, and the release point corresponds to the position above the turning point or corner of the boundary of the bulk acoustic wave action area.

2. 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 0.5-800mW.

3. The method according to claim 1, characterized in that The speed of the solution flowing through the bulk acoustic wave region is adjusted to 0.1-10 mm / s.

4. The method according to claim 1, characterized in that The speed at which the solution flows through the bulk acoustic wave region is adjusted to 0.1-100 μL / min.

5. The method according to claim 1, characterized in that The diameter of the cells or vesicles is 0.8-30 μm.

6. The method according to claim 1, characterized in that The height of the fluid channel is 1.5-10 times the diameter of the cell or vesicle.

7. The method according to claim 1, characterized in that The height of the fluid channel of the microfluidic device is 25-200 μm.

8. 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 .

9. 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.

10. The method according to claim 1, characterized in that It is used to separate single or several cells or cell clusters.

11. The method according to claim 1, characterized in that: This includes controlling the cells or vesicles and / or their number staying in the area affected by the body acoustic wave, which is achieved by one or any combination of the following methods: (a) regulating the power of the bulk acoustic wave; (b) regulating the time of generating body sound waves; (c) adjusting the velocity of the solution flowing through the bulk acoustic wave region.

12. The method according to claim 11, characterized in that The speed at which the solution flows through the bulk acoustic wave region is 0.1-10 mm / s.

13. The method according to claim 11, characterized in that The power of the body sound wave is 0.1-500mW.

14. The method according to claim 11, characterized in that The speed of the solution flowing through the bulk acoustic wave region is 0.1-10 mm / s, and the power of the bulk acoustic wave is 0.1-500 mW.

15. The method according to claim 1, characterized in that It also includes making the retained cells or vesicles leave the retention position, which is achieved by one or any combination of the following methods: (a) Stopping body sound waves; (b) reducing the power of the bulk acoustic wave; (c) Increase the velocity of the solution flowing through the bulk acoustic wave region.

16. The method according to claim 15, characterized in that The method controls the type or quantity of cells or vesicles leaving the retention position, including: releasing and obtaining cells or vesicles of different sizes, or releasing and obtaining cells or vesicles of a desired quantity.

17. The method according to claim 1, characterized in that The method also includes adjusting the power of the body acoustic wave to disperse the clustered cells.

18. The method according to claim 1, characterized in that This also includes increasing the power of the bulk acoustic wave to destroy the cell membrane or vesicle membrane.

19. The method according to claim 1, characterized in that The solution is a liquid containing cells or vesicles to be captured, and the liquid includes: body fluid, tumor cell suspension, cell culture or culture supernatant.

20. The method according to claim 1, characterized in that Wherein the solution is blood, the blood includes whole blood or a blood fraction.

21. The method according to claim 1, characterized in that It is used to separate cells or vesicles to be separated from a solution containing other cells or vesicles, wherein the content of the cells or vesicles to be separated in the solution is less than 10 / ml.

22. The method according to claim 21, characterized in that The ratio of the content of the cells or vesicles to be separated in the solution to the content of the other cells or vesicles is less than 1:

10. 4 .

23. The method according to claim 1 or 21, characterized in that The microfluidic device is configured to capture the desired cell or vesicle at a position different from the position where other cells or vesicles in the solution leave the area where the bulk acoustic wave acts; The position where the microfluidic device is set to capture the desired cells or vesicles is called the capture point, and the position where other cells or vesicles in the solution leave the area where the bulk acoustic wave acts is called the release point.

24. The method according to claim 23, characterized in that The BAW action area of ​​the ultra-high frequency BAW resonator of the device is arranged in such a way that the position where the desired cells or vesicles are captured is different from the position where other cells leave the BAW action area, wherein the position where other cells or vesicles leave the BAW action area is arranged upstream of the position where the desired cells or vesicles are captured.

25. The method according to claim 1, characterized in that The cell is a eukaryotic cell.

26. The method according to claim 1, characterized in that The cells are blood cells, stem cells, somatic cells or tumor cells.

27. The method according to claim 26, characterized in that The tumor cells include circulating tumor cells, wherein the circulating tumor cells are selected from the group consisting of an epithelial cell cancer: prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, head and neck cancer, pancreatic cancer, or lung cancer.

28. The method according to claim 1, characterized in that This also includes using the isolated cells for one or more of the following analyses: DNA and RNA amplification; sequencing; immunochemistry; metabolite analysis; enzymatic analysis; reporter gene expression analysis; and hybridization studies.

29. The method according to claim 1, characterized in that It also includes allowing a single cell or vesicle to stay in the area affected by the bulk acoustic wave, and combining the single cell or vesicle with a single microcapsule.

30. The method according to claim 29, characterized in that These include the following steps: (a) adjusting the BAW power and / or flow rate of the UHF BAW resonator so that a single or several cells or vesicles stay in the BAW action area of ​​the UHF BAW resonator; (b) injecting gel microspheres from the sample inlet, and adjusting the bulk acoustic wave power and / or flow rate of the ultra-high frequency bulk acoustic wave resonator under the condition that the cells or vesicles retained in step (a) are not released, so that a single or several microcapsules remain in the bulk acoustic wave action area of ​​the ultra-high frequency bulk acoustic wave resonator; (c) the microcapsules are assembled with the retained cells or vesicles, (d) The assembled microcapsules-cells / vesicles are released by reducing the BAW power or stopping the BAW.

31. A microfluidic device for separating cells or vesicles, 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 to 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 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 that are transmitted to the wall on the opposite side of the fluid channel, so that the solution flowing through the bulk acoustic wave area generates a vortex, and the power of the bulk acoustic wave is adjusted by the power adjustment device and / or the speed of the solution flowing through the bulk acoustic wave area is adjusted by the flow rate adjustment device, so that cells or vesicles stay in the bulk acoustic wave area, that is, are captured; other cells or vesicles that are not captured do not enter the vortex channel or leave the bulk acoustic wave action area at a certain position in the vortex channel, wherein the position of leaving the vortex channel is called a release point, and the release point corresponds to the position above the turning point or corner of the boundary of the bulk acoustic wave action area.

32. The microfluidic device according to claim 31, characterized in that The output power of the power regulating device is 0.5-800mW.

33. The microfluidic device according to claim 31, 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.

34. The microfluidic device according to claim 31, 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.

35. The microfluidic device according to claim 31, characterized in that The height of the fluid channel is 1.5-10 times the diameter of the cells or vesicles to be captured.

36. The microfluidic device according to claim 31, characterized in that The height of the fluid channel of the microfluidic device is 25-200 μm.

37. The microfluidic device according to claim 31, 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 .

38. The microfluidic device according to claim 31, 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.

39. The microfluidic device according to claim 31, characterized in that The microfluidic device is configured to capture the desired cells or vesicles at a position different from the position where other cells or vesicles in the solution leave the area where the bulk acoustic wave acts.

40. The microfluidic device according to claim 39, characterized in that The BAW action area of ​​the ultra-high frequency BAW resonator of the microfluidic device is arranged in such a way that the position of the cells required for capture is different from the position where other cells leave the BAW action area.

41. The microfluidic device according to claim 40, characterized in that The location where other cells leave the bulk insonation zone is positioned upstream of the location where the desired cells are captured.

42. The microfluidic device according to claim 31, 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.

43. The microfluidic device according to claim 31, 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-2 μm.

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