Acoustofluidic microfluidic chip and system, and use thereof

Abstract

The present invention provides an acoustofluidic microfluidic chip and system, and a use thereof. The acoustofluidic microfluidic chip comprises a microporous array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip which are sequentially stacked in a thickness direction. The acoustofluidic microfluidic chip of the present invention uses acoustofluidics to manipulate micro-objects such as cells or cell secretions, can realize accurate capture, in-situ cell culture, and selective recovery of the micro-objects, has the advantages of simple structure, low cost, high flux, high integration and the like, and has high application value.

Description

Acoustofluidic microfluidic chips and systems and their applications Technical Field

[0001] The present application relates to the fields of life science and medicine. Specifically, the present application relates to an acoustofluidic microfluidic chip and system and its applications. Background Art

[0002] The rapid development of single-cell analysis technology over the past decade is primarily due to the rapid development of single-cell analysis tools. Currently, single-cell analysis technology is relatively mature in the field of single-cell gene sequencing. However, in-depth characterization at the single-cell level, such as the measurement, analysis, and recovery of multiple cytokine secretion and complex intercellular interactions, has lagged behind. This is primarily due to the lack of effective single-cell manipulation technologies and more streamlined digital and automated platforms for parallel screening, isolation, in situ characterization, culture, and efficient recovery of single cells.

[0003] Single-cell analysis technology is currently in its early stages of development and has yet to establish a stable competitive landscape. However, it has garnered significant attention from industry and academia worldwide, with universities, research institutions, and major biotechnology companies already developing their approaches. Most reported methods utilize light-induced dielectrophoresis (ODEP) or dielectrophoresis (DEP) combined with microfluidics to achieve cell manipulation. However, these approaches suffer from significant costs, system complexity, or limited functionality. In comparison, approaches combining acoustofluidics with microfluidics are easier to establish and significantly reduce complexity and cost. However, most reported methods rely on the physical properties and size of microparticles for separation, or on the specificity of microparticles for differentiation and separation. These methods are generally more suitable for screening cell populations but lack the ability to culture, monitor, or selectively recover single cells in situ.

[0004] Therefore, there is an urgent need to propose a cell manipulation system and method that can achieve cell capture, differential screening, in situ culture and efficient recovery to realize high-throughput, low-cost single-cell analysis.

[0005] Summary of the Invention

[0006] The present invention aims to, at least to some extent, address the technical problems existing in the prior art. To this end, the present invention provides an acoustofluidic microfluidic chip and system, a method for manipulating micro-objects, a method for in situ cell culture, and a method for obtaining cell secretions. The acoustofluidic microfluidic chip and system utilize acoustofluidics to manipulate micro-objects, such as cells or cell secretions, enabling precise capture of micro-objects, in situ cell culture, and selective recovery. These chips and systems offer advantages such as a simple structure, low cost, high throughput, and high integration, resulting in high application value.

[0007] In one aspect of the present invention, the present invention proposes an acoustic fluid microfluidic chip. According to an embodiment of the present invention, the acoustic fluid microfluidic chip includes a micropore array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked in sequence along the thickness direction, the surface of the micropore array chip close to the microfluidic channel layer is concave to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating target micro-objects; the microfluidic channel layer includes a fluid channel, a plurality of in situ culture through-holes that penetrate the microfluidic channel layer along the thickness direction and are connected to the fluid channel, and an inlet and an outlet formed on the surface of the fluid channel layer, the fluid channel is formed from the inlet in a direction intersecting with the thickness direction. The micropores are arranged in a one-to-one correspondence with each other, and one end along the thickness direction is connected to the micropores, and the other end corresponds to the bulk acoustic wave resonator and is used to receive the bulk acoustic waves emitted by the bulk acoustic wave resonator. The dimension of the in situ culture through-holes along the direction perpendicular to the thickness is larger than that of the micropores, and the projection of the bulk acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-holes and at least part of the micropores.

[0008] In the acoustofluidic microfluidic chip according to an embodiment of the present invention, the BAW resonators arranged on the BAW resonator array chip can generate BAWs, which exert force on the microfluid in the fluid channel to generate acoustofluidic vortices, thereby realizing the manipulation of micro-objects.

[0009] In situ culture through-holes designed in the acoustofluidic microfluidic chip can be used for in situ cell culture and proliferation. At the same time, compared with open spaces, the combination of in situ culture through-holes and microfluidics can not only ensure the biological culture environment and isolate samples from each other to avoid contamination, but also avoid acoustofluidic signal crosstalk between each micropore or in situ culture through-hole, thereby improving the accuracy of cell manipulation.

[0010] Therefore, the acoustofluidic microfluidic chip according to the embodiment of the present invention can realize the precise capture of micro-objects, in situ cell culture and selective recovery, and has the advantages of simple structure, low cost, high throughput and high integration, and has high application value.

[0011] In another aspect of the present invention, the present invention proposes an acoustic fluid microfluidic system. According to an embodiment of the present invention, the acoustic fluid microfluidic system includes: the acoustic fluid microfluidic chip, the acoustic fluid microfluidic chip includes a micropore array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip stacked in sequence along the thickness direction; the surface of the micropore array chip close to the microfluidic channel layer is concave to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating target micro-objects; the microfluidic channel layer includes a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and connected to the fluid channel, and an inlet and an outlet formed on the surface of the fluid channel layer, the fluid channel is formed from the inlet along the The direction intersecting with the thickness direction extends to the outflow outlet; the multiple in situ culture through-holes form an in situ culture through-hole array; the bulk acoustic wave resonator array chip includes multiple bulk acoustic wave resonators, and the multiple bulk acoustic wave resonators form a bulk acoustic wave resonator array; the in situ culture through-holes are arranged in a one-to-one correspondence with the micropores and one end along the thickness direction is connected to the micropore, and the other end corresponds to the bulk acoustic wave resonator and is used to receive the bulk acoustic wave emitted by the bulk acoustic wave resonator, the dimension of the in situ culture through-hole along the direction perpendicular to the thickness direction is larger than that of the micropore, and the projection of the bulk acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-hole and at least part of the micropore.

[0012] In yet another aspect of the present invention, a method for manipulating a micro-object is provided. According to an embodiment of the present invention, the method includes: an introduction step of injecting a biological sample containing a target micro-object into a fluid channel of an acoustofluidic microfluidic chip through an inlet, the acoustofluidic microfluidic chip including a micropore array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked sequentially along a thickness direction, the microfluidic channel layer forming the fluid channel and the inlet communicating with the fluid channel; a capture step of capturing the target microorganism into a micropore array using the micropore array chip, the micropore array chip including the micropore array communicating with the fluid channel; a discharge step of discharging the biological sample remaining in the fluid channel from the fluid channel; and a manipulation step of releasing the target micro-object from the micropore array chip and activating a bulk acoustic wave resonator located on the bulk acoustic wave resonator array chip, emitting a bulk acoustic wave into an in-situ culture through-hole through the micropore array chip to generate a vortex, thereby manipulating the target micro-object in the in-situ culture through-hole, the in-situ culture through-hole penetrating the microfluidic channel layer along the thickness direction, located between the micropore and the bulk acoustic wave resonator, and communicating with the fluid channel.

[0013] In another aspect of the present invention, a method for in situ screening of cultured cells is proposed. According to an embodiment of the present invention, the method comprises: an introduction step: injecting a biological sample containing target cells into a fluid channel of an acoustic fluidic microfluidic chip through an inlet, wherein the acoustic fluidic microfluidic chip comprises a microporous array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip stacked in sequence from bottom to top along the thickness direction, a fluid channel is formed in the microfluidic channel layer, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and connected to the fluid channel, and an inlet connected to the fluid channel; a capture step: utilizing the microporous array chip to capture the target cells into a microporous array, wherein the microporous array chip comprises the microporous array connected to the fluid channel; a discharge step: discharging the remaining uncaptured biological sample in the fluid channel from the fluid channel; an incubation step: injecting cell culture fluid into the fluid channel The in situ culture through-holes are penetrated into the microfluidic channel layer and enter the in situ culture through-holes, so that the target cells and the cell culture fluid are incubated in the in situ culture through-holes, and the in situ culture through-holes penetrate the microfluidic channel layer along the thickness direction, are located between the micropores and the bulk acoustic wave resonator, and are connected to the fluid channel; a specific binding step: after the incubation is completed, an immune reagent containing a ligand is injected into the fluid channel, so that the immune reagent contacts the incubated target cells, and the ligand can specifically bind to the secretions of the incubated target cells; a detection and screening step: screening the target cells by fluorescence signals, and selectively opening the bulk acoustic wave resonator based on the screening results of the fluorescence signals to empty the unselected in situ culture through-holes in the fluid channel; a culturing step: flipping the acoustofluidic microfluidic chip along the thickness direction, closing the bulk acoustic wave resonator, and in situ culturing the screened target cells.

[0014] In another aspect, the present invention provides a method for recovering target cells. According to an embodiment of the present invention, the method comprises: culturing the target cells in selected in situ culture wells of the acoustofluidic microfluidic chip using the aforementioned method for in situ screening and culturing cells; activating the bulk acoustic wave resonator to emit bulk acoustic waves into the in situ culture wells through the bulk acoustic wave resonator, generating vortices to capture the cultured target cells; and injecting liquid into the fluid channel and collecting effluent discharged from the fluid channel, wherein the effluent contains the target cells.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] (1) The present invention provides a high-throughput, rapid micro-object manipulation method. In the design of the acoustofluidic microfluidic chip of the present invention, a bulk acoustic wave resonator array is used to realize multi-channel microfluidic parallel manipulation. At the same time, since the in-situ culture through-holes of the bulk acoustic wave resonator array chip correspond one-to-one with the spatial position of the micropores, crosstalk between the acoustofluidic signals is avoided and the mutual isolation of the samples is ensured, thereby improving the accuracy of micro-object manipulation, and is particularly suitable for single cells. In addition, the combination of the in-situ culture through-holes and the bulk acoustic wave resonator array chip realizes the selective manipulation of samples and the in-situ proliferation and culture of target samples.

[0017] (2) The present invention does not require expensive equipment other than a radio frequency signal source, such as a high-power laser or a high numerical aperture (NA) objective lens. The design and processing of the acoustofluidic microfluidic chip can be accomplished based on traditional microfluidic preparation methods, resulting in a simple process and high processing efficiency. The independent switching configuration of the bulk acoustic wave resonator significantly improves the controllability of the sample.

[0018] (3) The present invention integrates multiple functions such as microfluidic manipulation, in situ culture, and selective recovery. Rare cells can be screened through a closed acoustofluidic microfluidic chip, avoiding the problem of sample contamination during the cell transfer process from cell capture-transfer-culture-recovery.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0021] Figure 1 shows a schematic diagram of the structure of an acoustofluidic microfluidic chip, (A) is a diagram of the main structure; (B) is a spatial decomposition diagram;

[0022] Figure 2 shows a schematic diagram of the microwell array chip structure, (A) is the main structure diagram; (B) is an enlarged view of the microwell;

[0023] FIG3 shows a top view of a BAW resonator array chip;

[0024] FIG4 shows a schematic diagram of the structure of the acoustofluidic microfluidic system;

[0025] FIG5 shows a schematic diagram of the structure of a BAW resonator array chip, wherein (A) is an inverted view of the BAW resonator array chip; (B) is an enlarged view of the in situ culture through-hole and BAW resonator;

[0026] FIG6 shows a schematic flow chart of a method for manipulating micro-objects;

[0027] FIG7 shows a schematic diagram of the capture results of CHO green fluorescent cells in the acoustofluidic microfluidic chip;

[0028] Figure 8 shows images of fluorescent cells captured by a bulk acoustic wave resonator. (A) shows the trajectory of a fluorescent cell captured by a bulk acoustic wave resonator. (B) A sequence of images from top to bottom shows the trajectory of a single CHO cell before and after capture along the path planned in Figure 8(C).

[0029] FIG9 shows a single adherent CHO cell being peeled off and transferred by a bulk acoustic wave resonator;

[0030] FIG10 is a diagram of a single adherent CHO cell captured into a well, (A) is a BAW resonator array; (B) is a microwell array; (C) is a diagram of a BAW resonator capturing a CHO cell into a well;

[0031] FIG11 is a diagram showing the effect of BAW operation at different powers on the survival rate of living cells;

[0032] Figure 12 is a microscopic photograph of single cell distribution;

[0033] FIG13 is a schematic diagram showing the principle of a bulk acoustic wave resonator generating vortices to capture secretions;

[0034] FIG14 is a micrograph showing controllable manipulation of a single cell using a bulk acoustic wave resonator;

[0035] FIG15 is a proliferation curve of cell culture.

[0036] Reference numerals:

[0037] 100: micropore array chip; 110: micropore; 200: microfluidic flow channel layer; 210: fluid channel; 220: in situ culture through-hole; 230: inlet; 240: outlet; 300: bulk acoustic wave resonator array chip; 310: bulk acoustic wave resonator; 400: radio frequency signal generating device; 500: microfluidic injection device; 600: control device; 700: recovery device; 800: signal amplification device. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0040] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0041] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0042] In this document, the terms "longitudinal", "transverse", "upper", "lower", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the present invention.

[0043] In this document, unless otherwise specified or limited, terms such as "connected," "connect," and "fixed" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components or interaction between two components, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0044] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0045] The present invention provides an acoustofluidic microfluidic chip, an acoustofluidic microfluidic system, a method for manipulating micro-objects, a method for in situ cell culture, and a method for obtaining cell secretions, which will be described in detail below.

[0046] Acoustofluidic microfluidic chip

[0047] In one aspect of the present invention, an acoustofluidic microfluidic chip is provided. The overall structure of the acoustofluidic cell manipulation chip of the present invention will be described in detail below with reference to FIG1 to FIG3.

[0048] Figure 1 shows the main structure of the acoustofluidic microfluidic chip, and Figure 2 shows a spatially exploded view of the acoustofluidic microfluidic chip. The acoustofluidic microfluidic chip includes a microwell array chip 100, a microfluidic channel layer 200, and a bulk acoustic wave resonator array chip 300, which are stacked sequentially along the thickness direction.

[0049] Microwell array chip

[0050] A plurality of micropores 110 are formed on the surface of the micropore array chip 100 close to the microfluidic channel layer 200 . The plurality of micropores form a micropore array for accommodating target micro-objects.

[0051] Target micro-objects include cells and / or cell secretions. Exemplarily, cell secretions include but are not limited to enzymes, antibodies, cytokines, growth factors, exosomes, etc.

[0052] The plurality of micropores 110 can be arranged along the X and Y directions to form a micropore array. The array arrangement can be linear, circular, rectangular, or spherical, preferably rectangular. For example, the row spacing and column spacing of the micropore array are each independently 100 μm to 1 cm, specifically 100 μm, 300 μm, 500 μm, 1 cm, or a range formed by any two endpoints, preferably 500 μm to 1 cm.

[0053] The micropores 110 in the micropore array are used to accommodate target micro-objects, specifically single or a small number (such as 2-4) cells, thus, can be used not only for single cell analysis, but also for multi-cell interaction analysis. The shape of the micropores can be a circular or polygonal geometric structure, preferably a circular structure. For example, the depth of the micropores 110 is 10 to 100 μm, specifically 10 μm, 20 μm, 50 μm, 80 μm, 100 μm or any two point values ​​as the range value formed by the endpoint value, preferably 10 to 50 μm; the pore size of the micropores 110 is 10 to 75 μm, specifically 10 μm, 25 μm, 50 μm, 70 μm, 75 μm or any two point values ​​as the range value formed by the endpoint value, preferably 10 μm to 25 μm.

[0054] The microwell array can be a conventional microwell array or a microwell array with dielectrophoresis (DEP) function.

[0055] a) Traditional microwell array: A transparent substrate material is provided with a certain number and size of microwells. The following processing methods may be used, but are not limited to: wet oxidation is used to form an oxide layer on the surface of the transparent substrate, SU-8 photoresist is uniformly coated on the surface, the photoresist is exposed to ultraviolet light through a photomask, and then developed and rinsed to ultimately obtain the microwells.

[0056] b) DEP micropore array chip: The purpose of the DEP electrode is to assist cell entry through dielectrophoresis force, thereby improving the cell entry rate. The DEP micropore array chip includes:

[0057] (I) Substrate: Specifically, the substrate material can be transparent materials such as glass, PDMS, and rubber.

[0058] (II) DEP electrode layer, the dielectrophoresis electrode layer is arranged on the substrate. The electrode material can be a conductive material such as gold (Au) or indium tin oxide (ITO). The dielectrophoresis electrode layer contains a dielectrophoresis electrode, and the number of dielectrophoresis electrodes can be one, that is, all cells are controlled by one dielectrophoresis electrode; or it can be multiple, and multiple dielectrophoresis electrodes form a dielectrophoresis electrode array, that is, one or more cells are controlled by multiple dielectrophoresis electrodes, wherein the dielectrophoresis electrodes in the dielectrophoresis electrode array preferably correspond one to one to the micropores in the micropore array, thereby achieving precise control of specific cells entering the pores. Specifically, the electrode pattern can be transferred to a transparent substrate coated with an electrode coating by photolithography to form a DEP electrode array.

[0059] (III) Microwell array: The microwell array is disposed on the dielectrophoresis electrode. Specifically, a photoresist (e.g., SU-8) can be coated on the surface of a dielectrophoresis electrode array chip. A chromium photomask patterned with the microwell array is aligned with the dielectrophoresis electrode array. The photoresist is exposed to ultraviolet light through the photomask, followed by development and rinsing, ultimately yielding a microwell array chip.

[0060] When the DEP switch is turned on, cells introduced into the fluid channel are actively captured into microwells equipped with interdigitated electrodes through the DEP force. Excess biological sample / not captured by the DEP is drained from the fluid channel through the microfluidics and removed from the microfluidic device through the outflow hole. When the DEP switch is turned off, the captured biological sample is released into the microwell array, achieving cell distribution and improving cell penetration rate. In some embodiments, each dielectrophoresis electrode is equipped with an independent switch, allowing for independent control.

[0061] Microfluidic channel layer

[0062] The microfluidic channel layer 200 includes a fluid channel 210, a plurality of in situ culture through-holes 220 that penetrate the microfluidic channel layer 200 along the thickness direction and are connected to the fluid channel 210, and an inlet 230 and an outlet 240 formed on the surface of the fluid channel layer 200. The fluid channel 210 extends from the inlet 230 to the outlet 240 along a direction intersecting the thickness direction; the plurality of in situ culture through-holes 220 form an in situ culture through-hole array.

[0063] In situ culture through-holes 220 are arranged in a one-to-one correspondence with microwells 110. One end along the thickness direction is connected to microwell 110, and the other end corresponds to BAW resonator 310 and is used to receive BAW waves emitted by the BAW resonator. The in situ culture through-holes not only facilitate in situ cell culture but also provide an independent reaction environment, ensuring mutual isolation of samples, preventing crosstalk between acoustic and fluidic signals, and improving the precision of micro-object manipulation. In some embodiments, the microwell array and the in situ culture through-hole array have the same periodic distribution.

[0064] The dimension of the in situ culture through-hole 220 perpendicular to the thickness direction is larger than that of the micro-hole 110. Therefore, on the one hand, the range of the bulk acoustic wave generated is large, which helps to better act on the micro-objects in the micro-hole; on the other hand, it also helps to provide a suitable environment for cell capture and in situ culture in the in situ culture through-hole.

[0065] The row spacing and column spacing of the in situ culture through-hole array are independently 100 μm to 1 cm, specifically 100 μm, 300 μm, 500 μm, 1 cm, or a range formed by any two point values ​​as endpoint values, preferably 500 μm to 1 cm; the height of the in situ culture through-hole 220 is 10 to 5000 μm, specifically 10 μm, 50 μm, 100 μm, 500 μm, 1000 μm, 1500 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, or a range formed by any two point values ​​as endpoint values, preferably 50 to 2000 μm; the volume of the in situ culture through-hole 220 is 8×10 4 ~10×10 8 μm 3 , specifically 8×10 4 μm 3 , 1×10 5 μm 3 , 1×10 6 μm 3 , 5×10 6 μm 3 , 7×10 6 μm 3 , 1×10 7 μm 3 , 1×10 8 μm 3, 10×10 8 μm 3 Or any two point values ​​as the range of endpoints, preferably 7×10 6 ~4×10 8 μm 3 .

[0066] The in situ culture through-holes 220 are not limited in shape and can be cylindrical, cubic, irregular, etc., preferably cylindrical, with a diameter of 100 to 500 μm, specifically 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, or a range formed by any two point values ​​as endpoints. The in situ culture unit can be prepared and processed using any microfluidic chip preparation method, and the materials can be selected from but not limited to PDMS, rubber, glass, PMMA, photoresist (SU-8 light), etc. In this embodiment, the in situ culture through-hole array is obtained by exposing the photoresist to SU-8 light using a photomask.

[0067] The fluid channel 210 extends perpendicular to the thickness direction and is formed by being recessed from one side of the microfluidic channel layer 200 near the microwell array. Specifically, the microfluidic channel layer has one or more fluid channels, which can be arranged horizontally or vertically. For example, the number of fluid channels can range from 1 to 10,000 or more, so that one to 10,000 or more samples can be loaded simultaneously.

[0068] The cross-sectional width of the fluid channel 210 perpendicular to the thickness direction is 10 to 250 μm, specifically 10 μm, 100 μm, 150 μm, 200 μm, 250 μm, or a range formed by any two endpoints. The concave depth of the fluid channel is 10 to 250 μm, specifically 10 μm, 30 μm, 50 μm, 60 μm, 80 μm, 100 μm, 150 μm, 200 μm, 250 μm, or a range formed by any two endpoints. The inlet 230 and outlet 240 are respectively exposed on both sides of the microfluidic channel layer 200 perpendicular to the thickness direction. Thus, the fluid channel provides a channel for the inflow and outflow of solutions and samples, as well as a selective sample recovery channel. The shape of the fluid channel is preferably rectangular, circular, elliptical, etc.

[0069] Bulk acoustic wave resonator array chip

[0070] The BAW resonator array chip 300 includes a plurality of BAW resonators 310, which form a BAW resonator array. Specifically, the plurality of BAW resonators are laid flat on a single crystal silicon wafer substrate along the X and Y directions to form a BAW resonator array, and a radio frequency signal source can provide BAW signals to the BAW resonators. Each BAW resonator can be provided with an independent switch, and micro-objects can be selectively captured by individually controlling one or more target units. The microfluidic channel layer 200 can be integrally formed with the BAW resonator array chip 300.

[0071] In the present invention, the BAW resonators in the BAW resonator array can be distributed in a one-to-one correspondence with the microwells, or one microwell can correspond to multiple BAW resonators, or multiple microwells can correspond to multiple BAW resonators. A one-to-one correspondence between the microwells and BAW resonators in the microwell array is preferred. In some embodiments, the BAW resonators 310 can correspond one-to-one with the in situ culture through-holes 220, i.e., one BAW resonator 310 corresponds to one in situ culture through-hole 220; or one BAW resonator 310 can correspond to multiple in situ culture through-holes 220. This can be flexibly selected based on actual conditions, with a one-to-one correspondence being preferred, thereby enabling precise manipulation. Preferably, the microwells 110, the in situ culture through-holes 220, and the BAW resonators 310 correspond one-to-one, and the microwell array, the in situ culture through-hole array, and the BAW resonator array have consistent periodic distribution. This allows for isolation between samples, provides independent reaction / culture environments, avoids crosstalk between acoustic-fluidic signals, and facilitates micro-object manipulation and cell culture.

[0072] The projection of the BAW resonator 310 along the thickness direction covers at least a portion of the in situ culture through-hole 220 and at least a portion of the microwell 110. Thus, on the one hand, the BAW generates a wide range of sound waves, which helps to better act on the micro-objects in the microwell; on the other hand, it also provides a suitable environment for in situ cell culture in the in situ culture through-hole.

[0073] When bulk acoustic waves propagate through a fluid, the acoustic field exerts a non-zero time-averaged force on the fluid medium along the direction of the acoustic wave propagation, thereby inducing a non-periodic flow of the liquid, namely acoustic streaming, which manifests itself as micro-vortices (i.e., bulk acoustic wave vortices). When a micro-object is near an acoustic vortex, it is acted upon by a variety of forces, including acoustic radiation and Stokes drag, causing it to ultimately balance at a potential energy point close to the fluid vortex, thereby achieving micro-object capture, which can include operations such as peeling, clamping, and movement.

[0074] The BAW resonator 310 can be arranged inside the in-situ culture through-hole 220, or outside the in-situ culture through-hole 220 and near the opening of the in-situ culture through-hole 220, so that the in-situ culture through-hole 220 receives the BAW emitted by the BAW resonator, that is, the micro-object capture potential well is located inside the in-situ culture through-hole 220, so as to achieve manipulation. In some embodiments, the BAW resonator array and the in-situ culture through-hole array are formed in a chimeric and sealed manner, and the BAW resonator 310 is located inside the in-situ culture through-hole 220. In this way, the BAW emitted by the BAW resonator can better act on the micro-objects in the micropore 110. In other embodiments, each BAW resonator 310 in the BAW resonator array is provided with an independent switch. Thus, independent manipulation is facilitated.

[0075] The projection of the BAW resonator 310 along the thickness direction covers at least part of the in situ culture through-hole 220 and at least part of the microwell 110. This helps to receive the BAW emitted by the BAW resonator in the in situ culture through-hole, thereby better affecting the cells in the microwell.

[0076] The row spacing and column spacing of the BAW resonator array are independently 100 μm to 1 cm, specifically 100 μm, 200 μm, 300 μm, 500 μm, 1000 μm, 1 cm, or a range formed by any two point values ​​as endpoint values, preferably 500 μm to 1 cm; each BAW resonator 310 can form a BAW generation area facing the in situ culture through hole 220, and the area of ​​the BAW generation area is not less than 300 μm 2 , specifically 300μm 2 , 1000μm 2 , 5000μm 2 , 10000μm 2 , 20000μm 2 , 30000μm 2 Or any two point values ​​as the range value formed by the endpoint value, preferably 300 to 30000 μm 2 , the power is 50~500mw.

[0077] The resonant frequency of the BAW resonator is not less than 1 GHz, and can be 1 GHz, 10 GHz, 20 GHz, 30 GHz, 40 GHz, 50 GHz, or a range formed by any two endpoints, preferably 1 to 10 GHz. This allows for better effects on cells in the microwells.

[0078] Acoustofluidic microfluidic system

[0079] In another aspect of the present invention, an acoustofluidic microfluidic system is proposed. The structure of the acoustofluidic microfluidic system will be described in detail below with reference to FIG. 4 .

[0080] The acoustofluidic microfluidic system includes an acoustofluidic microfluidic chip, which includes a micropore array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip stacked in sequence along the thickness direction; the surface of the micropore array chip close to the microfluidic channel layer is recessed to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating target micro-objects; the microfluidic channel layer includes a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and connected to the fluid channel, and an inlet and an outlet formed on the surface of the fluid channel layer, and the fluid channel is connected to the microfluidic channel layer along the thickness direction. The direction intersecting the thickness direction extends to the outflow outlet; a plurality of in situ culture through-holes form an in situ culture through-hole array; the bulk acoustic wave resonator array chip includes a plurality of bulk acoustic wave resonators, and the plurality of bulk acoustic wave resonators form a bulk acoustic wave resonator array; the in situ culture through-holes and the micropores are arranged in a one-to-one correspondence and one end along the thickness direction is connected to the micropore, and the other end corresponds to the bulk acoustic wave resonator and is used to receive the bulk acoustic wave emitted by the bulk acoustic wave resonator, the size of the in situ culture through-hole along the direction perpendicular to the thickness direction is larger than the micropore, and the projection of the bulk acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-hole and at least part of the micropore.

[0081] The acoustofluidic microfluidic system further includes: a radio frequency signal generating device 400 , a microfluidic injection device 500 and a control device 600 .

[0082] The radio frequency signal generating device 400 is connected to the bulk acoustic wave resonator 310 and is used to drive the bulk acoustic wave resonator 310 to emit bulk acoustic waves.

[0083] The microfluidic injection device 500 is used to inject a liquid containing target micro-objects into the fluid channel 210 through the inlet 230. One microfluidic injection device 500 can correspond to one fluid channel 210, thereby allowing different liquids to be injected into different fluid channels or changing different injection conditions, such as flow rate, according to different needs. One microfluidic injection device 500 can also correspond to multiple fluid channels 210 to achieve high-throughput operation. For example, the microfluidic injection device 500 can be a microfluidic pump.

[0084] The control device 600 is connected to the BAW resonator 310, the RF signal generator 400, and the microfluid injection device 500. Thus, the control device 600 can adjust the BAW resonator 310 on / off, the RF signal's operating frequency and output power, and the microfluid flow rate. For example, the control device 600 can be a computer.

[0085] The microwell array chip 100 comprises a substrate; a dielectrophoresis electrode layer disposed on the substrate and containing dielectrophoresis electrodes; and a microwell array disposed on the dielectrophoresis electrode layer. A control device 600 is connected to the dielectrophoresis electrodes. Thus, when the dielectrophoresis electrodes are switched on, cells introduced into the fluid channel are actively captured into microwells equipped with interdigitated electrodes by dielectrophoretic forces. Excess biological sample / biological material not captured by dielectrophoresis is drained from the fluid channel by microfluidics and removed from the microfluidic device through the outflow port. When the dielectrophoresis electrodes are switched off, the captured biological sample is released into the microwell array, achieving cell distribution.

[0086] The fluid channel 210 extends perpendicular to the thickness of the microfluidic channel layer 200. The inlet 230 and outlet 240 are respectively exposed on either side of the microfluidic channel layer 200 perpendicular to the thickness. The acoustofluidic microfluidic system may further include a recovery device 700 for collecting liquid flowing out of the outlet 240. For example, the recovery device 700 may be a variety of sample collection devices, such as a culture dish, a centrifuge tube, or a well plate.

[0087] The acoustofluidic cell microfluidic system may further include a signal amplifying device 800, which is connected to the RF signal generating device 400 and the BAW resonator 310, respectively, so as to provide a BAW signal with a frequency range of not less than 1 GHz to the BAW resonator.

[0088] For ease of understanding, the following describes in detail the method for using the acoustofluidic microfluidic system of the present invention, taking in situ cell culture as an example:

[0089] Using a microfluidic injection device, a biological sample solution, such as cells, is introduced into the fluid channel through the inlet. Gravity settles the biological sample, which is then randomly distributed across the surface of the microwell array chip. When the DEP switch is turned on, the DEP force actively captures the cells introduced into the fluid channel into microwells equipped with interdigitated electrodes. Excess biological sample / not captured by DEP is expelled from the fluid channel by microfluid injection and removed through the outlet. When the DEP switch is turned off, the captured biological sample is released into the microwell array, achieving cell distribution.

[0090] The RF signal generator and signal amplifier are turned on to provide a BAW signal to the BAW resonator, generating a BAW vortex within the in-situ culture through-hole with a frequency ranging from gigahertz to tens of gigahertz, facing the microwell. This high-speed acoustic flow captures the micro-objects in the microwell array chip. Because the in-situ culture through-hole is larger than the microwell, the acoustofluidic microfluidic chip is flipped so that the in-situ culture through-hole is positioned below the microwell (as shown in Figure 5). At this point, the BAW resonator is turned off, releasing the captured biological sample from the vortex and allowing it to fall by gravity into the in-situ culture through-hole, enabling in-situ culture and long-term monitoring.

[0091] Each BAW resonator on the BAW resonator array chip has its own switch setting, allowing the selective capture of micro-objects by individually controlling one or more target units. Once a target unit is selected, only the switch of the target BAW resonator is turned on, meaning only the selected micro-objects can be captured by the BAW resonator from the micropore array, while those not captured remain in the micropores of the micropore array. By introducing a microfluid at a certain rate, laminar flow forces the target micro-objects out of the fluid channel, where they are finally introduced through the outlet into a designated recovery device, achieving the recovery purpose.

[0092] The features and advantages described above for the acoustofluidic microfluidic chip are also applicable to the acoustofluidic cell manipulation system and will not be repeated here.

[0093] method

[0094] In another aspect, the present invention provides a method for manipulating a biological sample. Referring to FIG6 , the method includes: an introduction step ( S100 ), a capture step ( S200 ), a discharge step ( S300 ), and a manipulation step ( S400 ). Each step is described in detail below.

[0095] S100 introduction steps

[0096] In this step, a biological sample containing target micro-objects is injected into the fluid channel of an acoustofluidic microfluidic chip through an inlet. The acoustofluidic microfluidic chip comprises a microwell array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked sequentially along the thickness direction. The microfluidic channel layer forms a fluid channel and an inlet connected to the fluid channel. The features and advantages described above for the acoustofluidic microfluidic chip also apply to this step and will not be repeated here.

[0097] S200 capture step

[0098] In this step, the target microorganisms are captured in a micropore array using a micropore array chip, which includes a micropore array connected to a fluid channel.

[0099] The microwell array chip includes: a substrate; a dielectrophoretic electrode layer disposed on the substrate and containing dielectrophoretic electrodes; and a microwell array disposed on the dielectrophoretic electrode layer. The capture step includes activating the dielectrophoretic electrodes to capture target micro-objects into the microwells; and the manipulation step includes deactivating the dielectrophoretic electrodes and emitting bulk acoustic waves into the in-situ culture through-holes via a bulk acoustic wave resonator to generate eddy currents for manipulating the target micro-objects within the in-situ culture through-holes. The features and advantages previously described for the microwell array chip also apply to this microwell array chip and will not be further elaborated here.

[0100] When the DEP switch is turned on, cells introduced into the fluidic channel are actively captured into microwells equipped with interdigitated electrodes through DEP force. Excess biological sample / biosample not captured by DEP is expelled from the fluidic channel through microfluidics. When DEP is turned off, the captured biosample is released into the microwell array, achieving cell distribution and helping to improve cell penetration rate.

[0101] S300 discharge step

[0102] In this step, the biological sample remaining in the fluid channel is discharged from the fluid channel.

[0103] S400 operation steps

[0104] In this step, the micropore array chip releases the target micro-object and activates the bulk acoustic wave resonator located on the bulk acoustic wave resonator array chip, and emits bulk acoustic waves into the in situ culture through-hole through the bulk acoustic wave resonator to generate eddy currents, so as to manipulate the target micro-object in the in situ culture through-hole. The in situ culture through-hole passes through the microfluidic flow channel layer along the thickness direction, is located between the micropore and the bulk acoustic wave resonator, and is connected to the fluid channel.

[0105] In some embodiments, the resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz, for example, 1 to 50 GHz; the flow rate of the biological sample containing the target micro-object in the fluid channel is 0.1 to 12.5 μL / min, specifically 0.1 μL / min, 1 μL / min, 3 μL / min, 5 μL / min, 10 μL / min, 12.5 μL / min or a range value formed by any two point values ​​as endpoint values; the biological sample containing the target micro-object is a single cell suspension.

[0106] In another aspect of the present invention, the present invention provides a method for in situ screening of cultured cells, the method comprising:

[0107] Introducing step: injecting a biological sample containing target cells into a fluid channel of an acoustofluidic microfluidic chip through an inlet, wherein the acoustofluidic microfluidic chip includes a micropore array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked sequentially from bottom to top along a thickness direction, a fluid channel formed in the microfluidic channel layer, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along a thickness direction and communicating with the fluid channel, and an inlet communicating with the fluid channel;

[0108] Capturing step: capturing target cells into a micropore array using a micropore array chip, wherein the micropore array chip includes a micropore array connected to a fluid channel;

[0109] Expelling step: Expelling the remaining uncaptured biological sample from the fluid channel;

[0110] Incubation step: injecting a cell culture fluid into the fluid channel and into the in situ culture through-hole, so that the target cells and the cell culture fluid are incubated in the in situ culture through-hole, wherein the in situ culture through-hole penetrates the microfluidic channel layer along the thickness direction, is located between the micropore and the bulk acoustic wave resonator, and is connected to the fluid channel;

[0111] Specific binding step: After the incubation is completed, an immune reagent containing a ligand is injected into the fluid channel so that the immune reagent contacts the incubated target cells. The ligand can specifically bind to the secretions of the incubated target cells.

[0112] Detection and screening step: screening target cells by fluorescence signals, and selectively opening the BAW resonator based on the screening results of the fluorescence signals to empty unselected in situ culture through-holes in the fluid channel;

[0113] Cultivation step: turning the acoustofluidic microfluidic chip over along the thickness direction, closing the bulk acoustic wave resonator and culturing the screened target cells in situ.

[0114] Compared with the in situ culture through-holes in the in situ culture through-hole array, the micropores in the micropore array chip are relatively small, which is not conducive to cell culture. Therefore, a bulk acoustic wave resonator is used to capture cells from the micropores and mix them with the cell culture medium. The acoustofluidic microfluidic chip is then inverted for culture, which provides a large culture space and better cell culture effects, such as good cell activity and abundant cell secretions.

[0115] When performing fluorescence detection, the fluorescence microscope can collect fluorescence signals from the side of the microwell array chip 100 away from the microfluidic channel layer 200 through the microwells 110 and the in-situ culture through-holes 220 in sequence.

[0116] The micropore array chip includes: a substrate; a dielectrophoresis electrode layer, which is arranged on the substrate and contains dielectrophoresis electrodes; and a micropore array, which is arranged on the dielectrophoresis electrode layer; the discharge step includes: opening the dielectrophoresis electrode and / or emitting bulk acoustic waves into the in-situ culture through-holes through a bulk acoustic wave resonator, discharging uncaptured biological samples from the fluid channel, and closing the dielectrophoresis electrode / or bulk acoustic wave resonator.

[0117] When the DEP switch is turned on, cells introduced into the fluidic channel are actively captured into microwells equipped with interdigitated electrodes through the DEP force. Excess biological sample / uncaptured by the DEP is expelled from the fluidic channel through the microfluidics. When the DEP switch is turned off, the captured biological sample is released into the microwell array, achieving cell distribution and helping to improve cell penetration rate. Next, a bulk acoustic wave resonator is used to emit bulk acoustic waves into the in situ culture through-holes, generating eddy currents that can be used to manipulate the target micro-object within the in situ culture through-holes.

[0118] The resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz; the flow rate of the biological sample containing cells in the fluid channel is 0.1 to 12.5 μL / min; and the biological sample containing cells is a single cell suspension.

[0119] The discharge step includes injecting liquid into the fluid channel and collecting the effluent discharged from the fluid channel.

[0120] The detection and screening steps include: turning on the bulk acoustic wave resonator corresponding to the unselected in situ culture through-hole to generate vortex in the unselected in situ culture through-hole, injecting liquid into the fluid channel and using the liquid to empty the fluid in the unselected in situ culture through-hole.

[0121] Before flipping the acoustofluidic microfluidic chip along the thickness direction, the culturing step further includes: turning on the bulk acoustic wave resonator corresponding to the selected in-situ culture through-hole to generate vortex to capture the target cells into the in-situ culture through-hole.

[0122] The specific binding step comprises: turning on the bulk acoustic wave resonator to generate vortex in the in situ culture through-hole to enhance the contact between the immune reagent and the incubated target cells.

[0123] As used herein, the term "ligand" refers to any molecule that can specifically bind to a target cell secretion, including but not limited to an antibody or binding protein.

[0124] The ligand is pre-coated on the surface of a solid phase carrier; the solid phase carrier includes microbeads, which are at least one of polystyrene beads, Luminex™ beads, magnetic beads, and microrods; the diameter of the microbeads is 200 nm to 25 μm, specifically 200 nm, 500 nm, 1 μm, 5 μm, 8 μm, 25 μm, or a range formed by any two point values ​​as endpoints; the resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz; the flow rate of the biological sample containing the target cells in the fluid channel is 0.1 to 12.5 μL / min; and the biological sample containing the target cells is a single-cell suspension.

[0125] The micropore array chip includes: a substrate; a dielectrophoresis electrode layer, which is arranged on the substrate and contains dielectrophoresis electrodes; and a micropore array, which is arranged on the dielectrophoresis electrode layer. The capture step includes: turning on the dielectrophoresis electrode and / or emitting bulk acoustic waves into the in-situ culture through-holes through a bulk acoustic wave resonator, discharging uncaptured biological samples from the fluid channel, and closing the dielectrophoresis electrode and / or the bulk acoustic wave resonator.

[0126] In another aspect of the present invention, the present invention provides a method for recovering target cells, the method comprising:

[0127] culturing the target cells in the selected in situ culture through-holes of the acoustofluidic microfluidic chip using the aforementioned method for in situ screening and culturing cells;

[0128] Turning on the BAW resonator to emit BAW into the in-situ culture through-hole through the BAW resonator, thereby generating eddy currents to capture the cultured target cells;

[0129] A liquid is injected into the fluid channel and an effluent discharged from the fluid channel is collected, wherein the effluent contains the target cells.

[0130] It should be noted that the features and advantages described above for the acoustofluidic microfluidic chip are also applicable to this method and will not be repeated here.

[0131] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0132] Example 1

[0133] In this embodiment, an acoustofluidic microfluidic system is provided, which can be used to achieve operations such as capture, in situ cultivation, and selective recovery of micro-objects in a solution, as shown in Figures 1 to 5.

[0134] 1. Structure

[0135] The acoustofluidic microfluidic system includes an acoustofluidic microfluidic chip, a radio frequency signal generator, a signal amplifier, a microfluidic injection device (microfluidic pump), a recovery device, and a computer control system. One end of the fluid channel is connected to the microfluidic pump, and the other end is connected to the recovery device.

[0136] The acoustofluidic microfluidic chip comprises a micropore array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip which are sequentially stacked along the thickness direction.

[0137] The microfluidic channel layer has a groove structure on one side close to the micropore array. The microfluidic channel layer with the groove structure is embedded and sealed with the micropore array chip to form a fluid channel.

[0138] The substrate of the microwell array chip can be configured with dielectrophoresis (DEP) electrodes, where the electrodes are distributed near the microwell array and correspond one-to-one to the microwells; the DEP electrode array is controlled by a separate switch. When the DEP switch is turned on, the dielectrophoretic force captures the biological sample in the microwell, and when the switch is closed, the sample is released.

[0139] The micropores in the micropore array, the in situ culture through-holes in the in situ culture through-hole array, and the BAW resonators in the BAW resonator array are in one-to-one correspondence and have the same periodic distribution. The resonators are set at the bottom of the BAW resonator array chip, and the BAW generation area is located below the BAW resonator, with an active area of ​​1000μm 2 above.

[0140] Structural parameters: The row and column spacing of the microwell array are independently 500 μm, the microwells are circular, 20 μm deep, and 25 μm in diameter. The row and column spacing of the UHF BAW resonator array are independently 500 μm. The row and column spacing of the in situ culture through-hole array are independently 500 μm, the height of the in situ culture through-holes is 1000 μm, the in situ culture through-holes are cylindrical, 200 μm in diameter, and the volume of the in situ culture through-holes is 1×10 7 μm 3 The cross-sectional width of the fluid channel perpendicular to the thickness direction is 30 μm, and the concave depth of the fluid channel is 100 μm.

[0141] 2. Preparation method

[0142] (1) Microwell array chip

[0143] Use an N-type silicon wafer, clean the wafer, and use wet oxidation to form an oxide film on the surface of the silicon wafer; evenly apply SU-8 photoresist on the oxidized silicon surface, place the micropore array mask on the photoresist surface, and use ultraviolet exposure technology to etch the micropore array; pour liquid PDMS (PDMS and curing agent 10:1) into the newly prepared template, wait for it to solidify and then demold it to obtain a micropore array chip.

[0144] DEP microwell array chip: The purpose of the DEP electrode is to assist single cells in entering the pores through dielectrophoretic force and improve the single cell penetration rate. The DEP microwell array chip consists of a two-layer structure: (I) DEP electrode layer, which uses photolithography to transfer the electrode pattern to a transparent substrate coated with an electrode coating to form a DEP electrode array. The substrate layer can be a transparent material such as glass, PDMS, or rubber. The electrode material can be indium tin oxide (ITO) or a metal electrode, such as one or more of the following metals: indium, tin, and zinc. In some methods, the metal electrode is formed by solidifying liquid metal. The metal electrode is an alloy having a melting point below 200°C, preferably 40°C to 80°C, so that the alloy is in a liquid state during the electrode preparation process and then introduced into the engraved electrode flow channel to form a metal electrode after cooling; (II) a single-cell microwell array layer is coated on the surface of the DEP electrode array chip using photoresist (SU-8), a chromium photomask with a microwell array pattern is aligned with the DEP electrode array, and the photoresist is exposed to ultraviolet light through the photomask, followed by development and rinsing to finally obtain a single-cell microwell array chip.

[0145] (2) Bulk Acoustic Wave Resonator Array Chip

[0146] The BAW resonator array chip includes multiple BAW resonators, which include a bottom electrode layer, a piezoelectric layer, and a top electrode layer from bottom to top. The overlapping area of ​​the bottom electrode layer, the piezoelectric layer, the top electrode layer, and the acoustic wave reflecting part constitutes a BAW generating area. A Bragg reflector layer is provided on one side of the BAW resonator. The substrate layer is located below the Bragg reflector layer and is used to support the BAW generating component. Among them, the Bragg reflector layer is an acoustic impedance layer, which is composed of a low acoustic impedance layer and a high acoustic impedance layer stacked alternately. The high acoustic impedance layer and the low acoustic impedance layer can be composed of metals such as silicon, silicon dioxide, aluminum nitride, molybdenum, and materials such as parylene with different acoustic impedances. The bottom electrode layer and the top electrode layer can be composed of metals such as gold, aluminum, molybdenum, iron, titanium, copper, and alloys. The thickness of the bottom electrode layer is 800 angstroms, and the thickness of the top electrode is 2000 angstroms; the piezoelectric layer can be composed of piezoelectric materials such as aluminum nitride, zinc oxide, lead zirconate titanate, lithium niobate, etc.; the thickness of the piezoelectric layer is 100100000 angstroms.

[0147] A plurality of BAW resonators are laid out on a single crystal silicon wafer substrate along the X and Y directions to form a BAW resonator array.

[0148] (3) Microfluidic channel layer

[0149] A photoresist (SU-8) is coated on the surface of a BAW resonator array chip. A chromium photomask with a patterned microwell array is aligned with the BAW resonator array. The photoresist is exposed to ultraviolet light through the photomask, followed by development and rinsing, ultimately yielding the microwell array and thus the in situ culture unit. The BAW resonator is positioned within the microwell array, so the micro-object capture potential well is located within the in situ culture through-hole. The sidewalls of the in situ culture through-hole have a groove structure that forms a fluid channel when bonded to the microwell array chip. The inlet and outlet are exposed on either side of the microfluidic channel layer, perpendicular to the thickness.

[0150] (4) The bulk acoustic wave resonator array chip, the microfluidic channel layer, and the micropore array chip are sealed and bonded in a top-down order. The bonding can be carried out by ultraviolet irradiation, vacuum plasma, and curing adhesive bonding, and finally an acoustic fluidic microfluidic chip with the functions of micro-object manipulation, in situ cultivation, and selective recovery is obtained.

[0151] Example 2

[0152] According to the same experimental setup as in Example 1, a concentration of 1×10 5 A CHO green fluorescent cell solution sample with a concentration of cells / mL was injected into the acoustofluidic microfluidic chip through the inlet, and the flow rate was controlled at 1 to 3 μL / min.

[0153] Cells are randomly distributed across the surface of the microwell array chip by gravity sedimentation. When the DEP switch is turned on, cells introduced into the fluidic channel are actively captured by DEP force into microwells equipped with interdigitated electrodes. Excess cell solution / cells not captured by DEP are drained from the fluidic channel through the outflow holes. When DEP is turned off, the captured cells are released into the microwell array, achieving single-cell distribution. Figure 7 shows a single-cell penetration rate of 93%.

[0154] Example 3

[0155] According to the same experimental device and access parameters as in Example 2, CHO cells were individually distributed in the microwell array chip, and the bulk acoustic wave resonator was turned on with an operating frequency of 2 GHz and a power of 300 mW.

[0156] Figure 8A shows the trajectory of fluorescent cells captured by a BAW resonator. When the BAW resonator operates in a liquid environment, it generates micro-vortices (green fluorescence clearly indicates the cell's trajectory within the micro-vortices). The device's ultra-high frequency enables acoustic streaming velocities of up to m / s, enabling the high-speed acoustic streaming to detach, capture, and displace microparticles.

[0157] The image sequence from top to bottom in FIG8B shows the motion trajectory of a single CHO cell before and after being captured along the path planned in FIG6C .

[0158] FIG9 shows a single adherent CHO cell being peeled off and transferred by the acoustofluidic resonator according to an embodiment of the present invention.

[0159] As shown in FIG10 , a BAW resonator traps a single CHO cell into a well.

[0160] Figure 11 shows the effect of BAW operation at different power levels on the viability of living cells. The results show that, at the same exposure time, increasing power levels do not significantly alter the cell viability of the microvortices generated by BAW. This demonstrates that manipulation of biological samples using BAW is virtually safe and non-destructive.

[0161] Example 4: Detection of Antibody Secretion by Single B Cells Based on Acoustofluidic Microfluidic Chip

[0162] This example exemplifies the process of detecting single B cell secretions based on the principle of acoustofluidic microfluidic chip. The reagents used are shown in the following table.

[0163] 1. Obtaining Single B Cell Distribution Arrays

[0164] A single B cell was captured and manipulated into a microwell by an acoustofluidic microfluidic chip connected to an automatic displacement module (the combined effect of the DEP force and fluid force generated by the microfluidic chip was used to achieve single cell entry into the well to form a single cell array), resulting in a distributed microwell array of single B cells.

[0165] The separation parameters are set as follows:

[0166] The resonator frequency is 1-50 GHz, and the power is 50-500 mW. In this embodiment, the optimal parameters are set to 1.5 GHz-2.5 GHz, and the power is 50-300 mW. As shown in FIG12 , the single-cell distribution microwell array is obtained, and one cell to be tested is distributed in each microwell.

[0167] 2. B cell activation

[0168] incubating the B cells in the microwell array under conditions favorable for antibody expression;

[0169] The incubation conditions include: culture medium RPMI 1640 + 10% FBS + IL-2 (50 ng / ml) + 50 μM β-mercaptoethanol, adding the corresponding immune antigen to activate B cells, and incubating in a constant temperature of 37°C and 5% carbon dioxide incubator for 12-24 hours to achieve an activated state.

[0170] 3. Rotation and floating treatment

[0171] A bulk acoustic wave resonator is used to capture microbeads coupled with a primary affinity reagent (biotin-modified anti-IgG secondary antibody coupled with streptavidin PS microbeads, 5 μM in diameter) and introduced into the microwells individually or in multiples. The bulk acoustic wave resonator harmonic parameters are adjusted to keep the microbeads stable in the vortex to capture antibody proteins secreted by B cells.

[0172] The BAW resonator processing parameters are set as follows:

[0173] The resonator frequency is 1.5 GHz and the power is 300 mW.

[0174] 4. Second affinity reagent (fluorescently labeled secondary antibody / fluorescently labeled antigen) binds to the secretion

[0175] Add fluorescently labeled antigens or fluorescently labeled secondary antibodies (the fluorescent group can be Alexa Fluo.488, 594, 647 or Cy3, Cy5, etc., diluted with PBS, the ratio is 1:50-1:1000), and bind to the microspheres that capture the cell-secreted antibodies.

[0176] 5. Secreted Protein Characterization

[0177] Wash with DPBS to remove unbound fluorescently labeled secondary antibodies or antigens. Use fluorescence microscopy to image and measure fluorescence intensity to characterize the amount of cell-secreted protein. Turn on the BAW resonator to aggregate the antibody proteins secreted by the cells, as shown in Figure 13. The vortex generated by the BAW resonator can aggregate particles in the culture medium.

[0178] The BAW resonator parameters are set as follows:

[0179] The resonator frequency is 2 GHz and the power is 200 mW.

[0180] 6. Target cell recovery

[0181] Cells with high fluorescence intensity were screened, their coordinates marked, cultured in situ, and recovered after single clones formed (Figure 14). High-performing single clones were recovered and transferred to 96-well plates for further mass expansion. Cell proliferation was observed over seven days of continuous expansion, as shown in Figure 15. This demonstrates that the acoustofluidic microfluidic chip of the present invention can manipulate micro-objects, screen and culture cells in situ, and recover target cells, without affecting cell viability.

[0182] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An acoustic fluid microfluidic chip, characterized in that: The acoustofluidic microfluidic chip comprises a micropore array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip stacked in sequence along the thickness direction. The surface of the micropore array chip close to the microfluidic channel layer is concave to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating target micro-objects; The microfluidic channel layer comprises a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and communicating with the fluid channel, and an inlet and an outlet formed on a surface of the fluid channel layer, wherein the fluid channel extends from the inlet to the outlet along a direction intersecting the thickness direction; the plurality of in situ culture through-holes form an in situ culture through-hole array; The BAW resonator array chip includes a plurality of BAW resonators, and the plurality of BAW resonators form a BAW resonator array; The in situ culture through-holes are arranged in a one-to-one correspondence with the micropores, and one end along the thickness direction is connected to the micropores, and the other end corresponds to the bulk acoustic wave resonator and is used to receive the bulk acoustic waves emitted by the bulk acoustic wave resonator. The dimension of the in situ culture through-holes along the direction perpendicular to the thickness is larger than that of the micropores, and the projection of the bulk acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-holes and at least part of the micropores.

2. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The target micro-objects include cells and / or cell secretions.

3. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The row spacing and column spacing of the microwell array are independently 100 μm to 1 cm; The depth of the micropores is 10 to 100 μm; The pore diameter of the micropores is 10 to 75 μm.

4. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The microwell array chip comprises: substrate; a dielectrophoretic electrode layer, the dielectrophoretic electrode layer being disposed on the substrate and comprising dielectrophoretic electrodes; and The micropore array is arranged on the dielectrophoresis electrode layer.

5. The acoustofluidic microfluidic chip according to claim 4, characterized in that: The dielectrophoretic electrode layer includes a plurality of dielectrophoretic electrodes, and the plurality of dielectrophoretic electrodes form a dielectrophoretic electrode array; The dielectrophoresis electrodes in the dielectrophoresis electrode array correspond one to one with the micropores in the micropore array; Each of the dielectrophoresis electrodes is provided with an independent switch.

6. The acoustofluidic microfluidic chip according to claim 4, characterized in that: The bulk acoustic wave resonator array and the in-situ culture through-hole array are embedded and sealed, and the bulk acoustic wave resonators are located in the in-situ culture through-holes.

7. The acoustofluidic microfluidic chip according to claim 1, characterized in that: Each BAW resonator in the BAW resonator array is provided with an independent switch; The row spacing and column spacing of the BAW resonator array are independently 100 μm to 1 cm; Each of the BAW resonators can form a BAW generating region facing the in-situ culture through hole, and the area of ​​the BAW generating region is not less than 300 μm 2 .

8. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The row spacing and column spacing of the in situ culture through-hole array are independently 100 μm to 1 cm; The height of the in situ culture through-hole is 10 to 5000 μm; The in situ culture through-hole is cylindrical and has a diameter of 100 to 500 μm; The volume of the in situ culture through-hole is 8×10 4 ~10×10 8 μm 3 .

9. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The micropores, in-situ culture through-holes and bulk acoustic wave resonators correspond to each other one by one, and the micropore array, the in-situ culture through-hole array and the bulk acoustic wave resonator array have consistent periodic distribution.

10. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The fluid channel extends perpendicular to the thickness direction and is formed by being recessed from one side of the microfluidic channel layer close to the microwell array; The cross-sectional width of the fluid channel perpendicular to the thickness direction is 10 to 250 μm, and the concave depth of the fluid channel is 10 to 250 μm; The inlet and the outlet are respectively arranged on two sides of the microfluidic channel layer perpendicular to the thickness direction.

11. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz.

12. The acoustofluidic microfluidic chip according to claim 1, characterized in that: The microfluidic channel layer and the bulk acoustic wave resonator array chip are formed as one body.

13. An acoustofluidic microfluidic system, characterized in that: include: The acoustofluidic microfluidic chip comprises a micropore array chip, a microfluidic channel layer and a bulk acoustic wave resonator array chip which are sequentially stacked in the thickness direction; The surface of the micropore array chip close to the microfluidic channel layer is concave to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating target micro-objects; The microfluidic channel layer comprises a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and communicating with the fluid channel, and an inlet and an outlet formed on a surface of the fluid channel layer, wherein the fluid channel extends from the inlet to the outlet along a direction intersecting the thickness direction; the plurality of in situ culture through-holes form an in situ culture through-hole array; The BAW resonator array chip includes a plurality of BAW resonators, and the plurality of BAW resonators form a BAW resonator array; The in situ culture through-holes are arranged in a one-to-one correspondence with the micropores, and one end along the thickness direction is connected to the micropores, and the other end corresponds to the bulk acoustic wave resonator and is used to receive the bulk acoustic waves emitted by the bulk acoustic wave resonator. The dimension of the in situ culture through-holes along the direction perpendicular to the thickness is larger than that of the micropores, and the projection of the bulk acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-holes and at least part of the micropores.

14. The acoustofluidic microfluidic system according to claim 13, characterized in that: Further including: a radio frequency signal generating device, the radio frequency signal generating device being connected to the BAW resonator and configured to drive the BAW resonator to emit the BAW; a microfluidic injection device, configured to inject a liquid containing the target micro-object into the fluid channel through the inlet; A control device is connected to the bulk acoustic wave resonator, the radio frequency signal generating device and the microfluid injection device respectively.

15. The acoustofluidic microfluidic system according to claim 14, characterized in that: The microwell array chip comprises: substrate; a dielectrophoretic electrode layer, the dielectrophoretic electrode layer being disposed on the substrate and comprising dielectrophoretic electrodes; and The micropore array is provided on the dielectrophoresis electrode layer; The control device is connected to the dielectrophoresis electrode.

16. The acoustofluidic microfluidic system according to claim 14, wherein: The fluid channel extends perpendicular to the thickness direction, and the inlet and outlet are respectively exposed on both sides of the microfluidic channel layer perpendicular to the thickness direction; The acoustofluidic microfluidic system further comprises: a recovery device, the recovery device being used to collect the liquid flowing out of the outflow port; as well as A signal amplifying device is connected to the radio frequency signal generating device and the bulk acoustic wave resonator respectively.

17. A method for manipulating a micro-object, characterized in that: include: Introducing step: injecting a biological sample containing a target micro-object into a fluid channel of an acoustofluidic microfluidic chip through an inlet, wherein the acoustofluidic microfluidic chip comprises a micropore array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked sequentially along a thickness direction, wherein the microfluidic channel layer forms the fluid channel and the inlet connected to the fluid channel; Capturing step: capturing the target microorganism into a micropore array using the micropore array chip, wherein the micropore array chip includes the micropore array connected to the fluid channel; Expelling step: Expelling the remaining biological sample in the fluid channel from the fluid channel; Manipulation step: the micropore array chip releases the target micro-object and activates the BAW resonator located on the BAW resonator array chip, and emits BAW into the in-situ culture through-hole through the BAW resonator to generate vortex, so as to manipulate the target micro-object in the in-situ culture through-hole, wherein the in-situ culture through-hole passes through the microfluidic channel layer along the thickness direction, is located between the micropore and the BAW resonator, and is connected to the fluid channel.

18. The method according to claim 17, characterized in that The resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz; The flow rate of the biological sample containing the target micro-object in the fluid channel is 0.1 to 12.5 μL / min; The biological sample containing the target micro-objects is a single cell suspension.

19. The method according to claim 17, wherein The microwell array chip comprises: substrate; a dielectrophoretic electrode layer, the dielectrophoretic electrode layer being disposed on the substrate and comprising dielectrophoretic electrodes; and The micropore array is provided on the dielectrophoresis electrode layer; The capturing step includes: turning on the dielectrophoresis electrode to capture the target micro-object into the micro-well, The manipulation step includes: closing the dielectrophoresis electrode, emitting bulk acoustic waves into the in-situ culture through-hole through the bulk acoustic wave resonator, generating eddy currents, so as to manipulate the target micro-object in the in-situ culture through-hole.

20. A method for in situ screening of cultured cells, characterized in that: include: Introduction Step: injecting a biological sample containing target cells into a fluid channel of an acoustofluidic microfluidic chip through an inlet, wherein the acoustofluidic microfluidic chip comprises a microwell array chip, a microfluidic channel layer, and a bulk acoustic wave resonator array chip stacked sequentially from bottom to top along a thickness direction, the microfluidic channel layer forming a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and communicating with the fluid channel, and an inlet communicating with the fluid channel; Capturing step: capturing the target cells into a micropore array using the micropore array chip, wherein the micropore array chip includes the micropore array connected to the fluid channel; Expelling step: Expelling the uncaptured biological sample remaining in the fluid channel out of the fluid channel; Incubation step: injecting a cell culture fluid into the fluid channel and into the in situ culture through-hole, so that the target cells and the cell culture fluid are incubated in the in situ culture through-hole, wherein the in situ culture through-hole penetrates the microfluidic channel layer along the thickness direction, is located between the micropore and the bulk acoustic wave resonator, and is connected to the fluid channel; Specific binding step: After the incubation is completed, injecting an immune reagent containing a ligand into the fluid channel so that the immune reagent contacts the incubated target cells, and the ligand can specifically bind to the secretions of the incubated target cells; Detection and screening step: screening target cells by fluorescence signals, and selectively opening the BAW resonator based on the screening results of the fluorescence signals to empty unselected in situ culture through-holes in the fluid channel; Cultivation step: turning the acoustofluidic microfluidic chip over along the thickness direction, closing the bulk acoustic wave resonator and culturing the screened target cells in situ.

21. The method according to claim 20, characterized in that The microwell array chip comprises: substrate; a dielectrophoretic electrode layer, the dielectrophoretic electrode layer being disposed on the substrate and comprising dielectrophoretic electrodes; and The micropore array is provided on the dielectrophoresis electrode layer; The discharge step includes: opening the dielectrophoresis electrode and / or emitting bulk acoustic waves into the in situ culture through-hole through the bulk acoustic wave resonator, discharging uncaptured biological samples from the fluid channel, and closing the dielectrophoresis electrode and / or bulk acoustic wave resonator.

22. The method according to claim 20, characterized in that The resonant frequency of the bulk acoustic wave resonator is not less than 1 GHz; The flow rate of the biological sample containing cells in the fluid channel is 0.1 to 12.5 μL / min; The biological sample containing cells is a single cell suspension; The discharging step includes: Liquid is injected into the fluid channel and the effluent discharged from the fluid channel is collected.

23. The method according to claim 20, characterized in that The detection and screening step includes: turning on the bulk acoustic wave resonator corresponding to the unselected in situ culture through-hole to generate a vortex in the unselected in situ culture through-hole, injecting liquid into the fluid channel and using the liquid to empty the fluid in the unselected in situ culture through-hole.

24. The method according to claim 20, characterized in that Before flipping the acoustofluidic microfluidic chip along the thickness direction, the culturing step further includes: turning on the bulk acoustic wave resonator corresponding to the selected in-situ culture through-hole to generate vortex to capture the target cells into the in-situ culture through-hole.

25. The method according to claim 20, wherein The specific binding step includes: turning on the bulk acoustic wave resonator to generate eddy currents in the in situ culture through-holes to enhance the immunoreactivity of the immune test sample; contact between the agent and the incubated target cells.

26. A method for recovering target cells, characterized in that: include: Cultivating the target cells in the selected in situ culture through-holes of the acoustofluidic microfluidic chip using the method for in situ screening and culturing cells according to any one of claims 20 to 25; Turning on the BAW resonator to emit BAW into the in-situ culture through-hole through the BAW resonator, thereby generating eddy currents to capture the cultured target cells; A liquid is injected into the fluid channel and an effluent discharged from the fluid channel is collected, wherein the effluent contains the target cells.

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