Cell drug screening device and method based on microstructure-induced shear acoustic streaming

Abstract

The invention discloses a cell drug screening device and method based on microstructure induced shear acoustic streaming. The micro-structure array processed and manufactured on the acoustic flow driving chip is utilized, so that the high-frequency acoustic surface wave and the micro-structure unit generate acoustic-solid coupling and oscillation, an acoustic streaming effect is generated through excitation in the micro-channel cavity, a stable acoustic flow field is formed, the acoustic flow field generates micro-acoustic streaming shearing, and the acoustic flow effect is improved. The biological cells in the acoustic flow field are subjected to controllable shearing force to change the permeability of cell membranes, so that the medicine is accelerated to enter the cells, and the medicine screening of the cells is promoted; the sizes, the shapes and the periods of the microstructure units are different, the microstructure units respectively and correspondingly excite micro-acoustic streams with different flow speeds, corresponding shearing forces are different, and then different stimulation effects are brought to cell membranes; screening the cell drugs to obtain the specific effect of the drugs on the cells.

Description

Technical Field

[0001] The invention belongs to the field of analytical instruments, and in particular relates to a cell drug screening device and method based on microstructure-induced shear acoustic flow. Background Art

[0002] Rapid drug screening is of great significance in individualized treatment, saving time and resources, accelerating new drug discovery and development, supporting drug combination therapy, and exploring new therapeutic approaches. It plays a vital role in improving the effect and quality of clinical treatment and promoting the progress of medical research. However, due to the inherent resistance of cell membranes to chemotherapeutic drugs, it is difficult for drug molecules to penetrate cells. The traditional in vitro drug screening process is time-consuming and expensive. It generally consumes a large number of samples and experimental animals, and has high requirements on the operating skills of technicians. It is difficult to carry out effective and economical screening of a certain number of samples in a short period of time.

[0003] In order to overcome the limitations of passive intracellular drug delivery strategies, a variety of techniques for changing cell membrane permeability have been developed, such as electroporation, photoevaporation, sonoevaporation, and microinjection. However, although these methods ensure the effective delivery of extracellular substances into cells, they also cause damage to cells and may induce cell apoptosis. In addition, cell apoptosis can also affect drug screening results. Therefore, non-invasive intracellular drug delivery strategies are essential for achieving rapid drug screening.

[0004] Several studies on high-frequency sound-wave-assisted drug delivery systems have been demonstrated in a variety of application areas, and these systems enable non-invasive processing of biological samples. Similar to the acoustic pressure-induced shear stress that can change the permeability of cell membranes, acoustic fluidization can also apply shear stress. However, developing a high-throughput single-cell drug screening method with the help of acoustic fluidization, especially using biologically friendly high-frequency sound waves, is a challenging task. Summary of the invention

[0005] In view of the problems existing in the above prior art, the present invention proposes a cell drug screening device and method based on microstructure induced shear acoustic flow, which can well change the permeability of the cell membrane surface and promote the cell's absorption of drugs.

[0006] One object of the present invention is to provide a cell drug screening device based on microstructure-induced shear acoustic streaming.

[0007] The cell drug screening device based on microstructure induced shear acoustic flow of the present invention comprises: a microscopic observation device, a central processing unit, a power amplifier, a signal generator, a microfluidic cavity module and an acoustic flow driving chip; wherein the acoustic flow driving chip is placed on the sample stage of the microscopic observation device, and the microfluidic cavity module is located under the acoustic flow driving chip; the acoustic flow driving chip is connected to the power amplifier, the power amplifier is connected to the signal generator, and the microscopic observation device and the signal generator are connected to the central processing unit;

[0008] The acoustic flow driving chip includes a substrate, an interdigital transducer and a microstructure metamaterial; the interdigital transducer and the microstructure metamaterial are respectively arranged on the substrate, and the distance between the interdigital transducer and the microstructure metamaterial is not greater than 10 mm; the microstructure metamaterial includes a plurality of different microstructure arrays, each microstructure array includes a plurality of microstructure units of the same shape and size arranged in a two-dimensional array, and the microstructure unit is a protruding structure formed on the substrate, and has a set regular shape and size; the size and position of each different microstructure array are independent of each other, and the size, shape or period of the microstructure unit of each different microstructure array is different; the unit size, shape and period of different microstructure arrays correspond to the micro-acoustic flow with different flow rates, and the corresponding shear force is different as the speed of the micro-acoustic flow is different, thereby bringing different stimulation effects to the cell membrane;

[0009] The microfluidic cavity module includes a liquid inlet, a liquid outlet and a microfluidic cavity; the surface of the microfluidic cavity is an opening, the interior is a cavity, and the liquid inlet and the liquid outlet are respectively arranged on two opposite sides of the microfluidic cavity;

[0010] The surfaces of the microstructure metamaterial of the acoustic fluidic drive chip and the microfluidic cavity of the microfluidic cavity module are processed to be hydrophilic; the acoustic fluidic drive chip is placed upside down on the microfluidic cavity module, the microstructure metamaterial of the acoustic fluidic drive chip faces the microfluidic cavity of the microfluidic cavity module, and the microfluidic cavity module and the acoustic fluidic drive chip are bonded to form an integral body; an adhesion layer is prepared at the bottom of the microfluidic cavity so that cells can adhere to the bottom of the microfluidic cavity; the required cell suspension is introduced into the microfluidic cavity and allowed to stand to ensure that the cells are uniformly attached to the bottom of the microfluidic cavity, then the drug is introduced into the microfluidic cavity, and finally a fluorescent probe for marking the drug or the cell is injected; the acoustic fluidic drive chip and the microfluidic cavity module bonded to form an integral body with the drug introduced are placed on the stage of a microscopic observation device, and are located within the imaging field of view of the microscopic observation device;

[0011] The central processing unit sends a digital signal to the signal generator, which converts the digital signal into an electrical signal and transmits it to the power amplifier; the power amplifier converts the received electrical signal command into an amplified power signal, which is input into the interdigital transducer of the acoustic flow driving chip, stimulating the interdigital transducer to generate high-frequency surface acoustic waves; the high-frequency surface acoustic waves and the microstructure metamaterial undergo acoustic-solid coupling, causing oscillations at the boundaries of the microstructure units, and this oscillation drives the culture fluid in the flow field located in the microfluidic cavity to produce a stable form of acoustic flow field, which generates micro-acoustic flow shear, and the biological cells located in the acoustic flow field are subjected to controllable shear forces; different microstructure arrays can stimulate different flows. The change of micro-acoustic flow shear affects the permeability of drug molecules in the cell membrane. The greater the flow rate, the greater the shear force, thereby increasing the stimulation to the cell membrane and the deeper the drug penetrates into the cell. Different microstructure arrays correspond to different microfluidic cavity areas, so the degree of drug penetration into cells in different microfluidic cavity areas is also different, thereby screening cell drugs. Fluorescent probes that mark drugs or cells are used to observe the process of drug penetration and cell response. Microscopic observation devices are used to collect images of cells in real time, and the images are analyzed by a central processing unit to obtain the specific effects of drugs on cells.

[0012] According to the required micro-acoustic flow velocity and the fluid mechanics equations, the finite element method is used to perform parametric scanning on the unit size, shape and period to obtain the unit size, shape and period of the microstructure array. According to the shear force formula: F s =ηAdv / dy, and the cell surface is regarded as a fixed boundary. As the speed of the microacoustic flow increases, the corresponding shear force will also increase, thereby bringing different stimulation effects on the cell membrane.

[0013] The frequency of the high-frequency surface acoustic wave generated by the exciting interdigital transducer is 5 to 20 MHz.

[0014] The analysis includes drug permeability, cell membrane changes, cell morphology changes and cell survival rate, thereby obtaining the specific effects of the drug on cells.

[0015] The microscopic observation device includes a fluorescence microscope and a charge coupled device (CCD) camera; wherein, the CCD camera is arranged behind the eyepiece of the fluorescence microscope, and the CCD camera is connected to the central processing unit; the microfluidic cavity module and the acoustic flow driving chip are placed on the sample stage of the fluorescence microscope, and the microfluidic cavity is located in the imaging field of the fluorescence microscope; the fluorescence microscope collects the fluorescence generated by biological cells after being stimulated by acoustic flow shear and treated with drugs, and generates fluorescence information, and the CCD camera converts the fluorescence information into electrical signals and inputs them into the central processing unit, and the fluorescence expression state of the cells is displayed through the central processing unit.

[0016] The substrate material is lithium niobate.

[0017] The flow rate of the cell culture fluid injected into the microchannel cavity is 0.5-10 μl / min, the height in the microchannel cavity is 300-1000 μm, the length and width are 5-20 mm, and the material is polydimethylsiloxane (PDMS).

[0018] The finger width of the interdigital transducer on the acoustic flow driving chip is 50-200 μm, the index is 20-50 pairs, the center frequency is 5-20 MHz, and the applied power is 100-1000 mW.

[0019] The microstructure units of the microstructure array are columnar, the planar shape of the columns is rectangular, triangular or cross-shaped, the planar size is 50-100 μm, the height is 100-200 μm, and the period of the microstructure units is 100-500 μm.

[0020] Another object of the present invention is to provide a cell drug screening method based on microstructure-induced shear acoustic streaming.

[0021] The cell drug screening method based on microstructure-induced shear acoustic streaming of the present invention comprises the following steps:

[0022] 1) Designing the unit size, shape and period of the microstructure array according to the required flow rate of the microacoustic flow;

[0023] 2) treating the surfaces of the microstructure metamaterial of the relative acoustic flow driving chip and the microfluidic cavity of the microfluidic cavity module to make them hydrophilic;

[0024] 3) placing the acoustic fluidic driving chip upside down on the microfluidic cavity module, with the microstructure metamaterial of the acoustic fluidic driving chip facing the microfluidic cavity of the microfluidic cavity module, and bonding the microfluidic cavity module and the acoustic fluidic driving chip to form a whole;

[0025] 4) preparing an adhesion layer at the bottom of the microfluidic cavity so that the cells can adhere to the bottom of the microfluidic cavity;

[0026] 5) Pass the required cell suspension into the microfluidic cavity and let it stand to ensure that the cells are evenly attached to the bottom of the microfluidic cavity.

[0027] The drug is then passed into the microfluidic cavity, and finally a fluorescent probe that labels the drug or cell is injected;

[0028] 6) placing the integrated acoustic flow driving chip and microfluidic cavity module into which the drug is introduced on the stage of the microscopic observation device, within the imaging field of the microscopic observation device;

[0029] 7) The central processing unit sends a digital signal to the signal generator, which converts the digital signal into an electrical signal and transmits it to the power amplifier; the power amplifier converts the received electrical signal command into an amplified power signal, which is input into the interdigital transducer of the acoustic flow driving chip, and stimulates the interdigital transducer to generate a high-frequency surface acoustic wave;

[0030] 8) High-frequency surface acoustic waves and microstructure metamaterials undergo acoustic-solid coupling, causing oscillations at the boundaries of the microstructure units. This oscillation drives the culture fluid in the flow field within the microchannel cavity to produce a stable acoustic flow field, which generates micro-acoustic flow shear, and biological cells in the acoustic flow field are subjected to controllable shear forces.

[0031] 9) Different microstructure arrays can stimulate microacoustic flow at different flow rates, thus exerting different shear forces on cells. Changes in microacoustic flow shear affect the permeability of drug molecules in cell membranes. The greater the flow rate, the greater the shear force, thereby increasing the stimulation to the cell membrane and the deeper the drug penetrates into the cell. Different microstructure arrays correspond to different microfluidic cavity areas, so the degree of drug penetration into cells in different microfluidic cavity areas is also different, thereby screening cell drugs.

[0032] 10) Use fluorescent probes to label drugs or cells to observe the process of drug penetration and cell response; use a microscopic observation device to collect images of cells in real time, and analyze the images through a central processing unit to obtain the specific effects of the drugs on the cells.

[0033] Wherein, in step 1), according to the required flow rate of the micro-acoustic flow and based on the fluid mechanics equations, the unit size, shape and period are parametrically scanned using the finite element method to obtain the unit size, shape and period of the microstructure array.

[0034] In step 2), plasma is used to treat the surface of the microstructure metamaterial of the acoustic flow driving chip and the surface of the microfluidic cavity of the microfluidic cavity module, so that functional groups are formed on the surface to facilitate bonding and increase its hydrophilicity.

[0035] In step 4), preparing the adhesion layer includes the following steps: after bonding is completed, a coating material is introduced into the microfluidic cavity and incubated for 1 to 2 hours, and then the excess coating material is washed away with a buffer to obtain an adhesion layer. The coating material is polylysine, collagen or fibronectin. The buffer is phosphate buffered saline (PBS).

[0036] In step 7), according to the shear force F s Formula: F s=ηAdv / dy, η is the viscosity of the fluid, v is the velocity of the acoustic flow, A is the surface area of ​​the cell membrane, dv / dy is the rate of change of the acoustic flow velocity in the y direction; and the cell surface is regarded as a fixed boundary. As the velocity of the micro-acoustic flow increases, the corresponding shear force will also increase, thereby increasing the stimulation effect on the cell membrane.

[0037] In step 10), the analysis contents include drug permeability, changes in cell membrane, changes in cell morphology and cell survival rate, so as to obtain the specific effect of the drug on the cells.

[0038] Advantages of the present invention:

[0039] (1) The present invention utilizes a microstructure array fabricated on an acoustic flow drive chip, and each microstructure unit is bonded to a substrate, so that high-frequency surface acoustic waves and microstructure units generate acoustic-solid coupling and oscillate, thereby exciting an acoustic flow effect in the microfluidic cavity to form a stable acoustic fluid vortex. The cell sheets are arranged near the microfluidic vortex, and ultimately the shearing effect of the acoustic flow on the cell membrane sheet will change the permeability of the cell membrane, thereby accelerating the entry of drugs into the cell and promoting drug screening of the cell.

[0040] (2) The microstructure unit is optimized by COMSOL finite element simulation software, and the required acoustic flow field is excited by matching the excitation frequency, microstructure unit structure and size, and cavity boundary conditions.

[0041] It can selectively regulate the surface acoustic wave excitation power and generate acoustic flow vortices of different flow rates to stimulate different fluid shear forces; it can selectively arrange microstructures of different spacing and forms in the same cavity to generate different fluid shear forces at different positions.

[0042] In general, in order to generate appropriate acoustic flow vortices, the periodic spacing of the microstructure unit is generally set to 2 to 5 times the unit size. The height of the microfluidic cavity is mainly matched with the height of the microstructure unit and the thickness of the cell sheet. In order to better operate and ensure that the cell sheet adheres and grows under the microstructure array, the height of the microfluidic cavity is generally set to 2 to 5 times the height of the microstructure unit. The width of the microfluidic cavity is mainly matched with the width of the microstructure array. In order to ensure the closedness of the environment inside the microfluidic cavity, the microfluidic width is usually set slightly larger than the width of the microstructure array. In order to reduce the loss of surface acoustic waves during propagation, the thickness of the inner wall of the microfluidic cavity is optimally the width of one microstructure unit, and the distance from one side of the inner wall of the microfluidic cavity to the other side of the microstructure array is optimally the width of 2 to 3 microstructure units. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1A schematic diagram of an embodiment of a cell drug screening device based on microstructure-induced shear acoustic streaming according to the present invention;

[0044] Figure 2 The flowchart of the method for preparing the surface acoustic wave chip of the present invention;

[0045] Figure 3 The simulation effect diagram of microstructure-induced shear acoustic streaming obtained by an embodiment of the cell drug screening device based on microstructure-induced shear acoustic streaming of the present invention, wherein (a) is a schematic diagram of the shear acoustic streaming generated by the microstructure, and (b) is a schematic diagram of the acoustic streaming driving chip;

[0046] Figure 4 This is a schematic diagram of a cell drug screening device based on microstructure-induced shear acoustic fluidics according to an embodiment of the present invention, in which four different microstructure arrays are simultaneously provided on a single acoustic fluidic driving chip. DETAILED DESCRIPTION

[0047] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0048] In this embodiment, the cell attributes are MCF7 breast cancer cells, and the size is 10 μm to 30 μm.

[0049] like Figure 1 As shown, the cell drug screening device based on microstructure induced shear acoustic flow of this embodiment includes: a microscopic observation device, a central processing unit, a power amplifier, a signal generator, a microfluidic cavity module and an acoustic flow driving chip; wherein the acoustic flow driving chip is placed on the sample stage of the microscopic observation device, and the microfluidic cavity module is located under the acoustic flow driving chip; the acoustic flow driving chip is connected to the power amplifier, the power amplifier is connected to the signal generator, and the microscopic observation device and the signal generator are connected to the central processing unit;

[0050] The acoustic flow driving chip includes a substrate, an interdigital transducer and a microstructure metamaterial; the interdigital transducer and the microstructure metamaterial are respectively arranged on the substrate, and the distance between the interdigital transducer and the microstructure metamaterial is not greater than 10 mm; the microstructure metamaterial includes a plurality of different microstructure arrays, each microstructure array includes a plurality of microstructure units of the same shape and size arranged in a two-dimensional array, and the microstructure unit is a protruding structure formed on the substrate, and has a set regular shape and size; the size and position of each different microstructure array are independent of each other, and the size, shape or period of the microstructure unit of each different microstructure array is different; the unit size, shape and period of different microstructure arrays correspond to the micro-acoustic flow with different flow rates, and the corresponding shear force is different as the speed of the micro-acoustic flow is different, thereby bringing different stimulation effects to the cell membrane;

[0051] The microfluidic cavity module includes a liquid inlet, a liquid outlet and a microfluidic cavity; the surface of the microfluidic cavity is an opening, the interior is a cavity, and the liquid inlet and the liquid outlet are respectively arranged on two opposite sides of the microfluidic cavity;

[0052] The microscopic observation device includes a fluorescence microscope and a CCD camera; wherein, the CCD camera is arranged behind the eyepiece of the fluorescence microscope, and the CCD camera is connected to the central processing unit; the microfluidic cavity module and the acoustic flow driving chip are placed on the sample stage of the fluorescence microscope, and the microfluidic cavity is located in the imaging field of the fluorescence microscope; the fluorescence microscope collects the fluorescence generated by biological cells after being stimulated by acoustic flow shear and treated with drugs, and generates fluorescence information, and the CCD camera converts the fluorescence information into electrical signals and inputs them into the central processing unit, and the fluorescence expression state of the cells is displayed through the central processing unit.

[0053] like Figure 2 As shown, the method for preparing the acoustic flow driving chip of this embodiment includes the following steps:

[0054] (1) obtaining a lithium niobate substrate, the characteristics of which include: a 3-inch wafer, a thickness of 500 μm, a cutting direction of Y-128°, and double-sided polishing;

[0055] (2) Spin coating a red red RJZ304 photoresist on the top of the lithium niobate substrate, with a low speed of 450 r / min, a spinning time of 9 s, and a high speed of 3000 r / min, and a spinning time of 60 s; using ultraviolet lithography, the substrate is subjected to ultraviolet exposure and development based on interdigital transducer parameters, and excess developer is removed and blown dry;

[0056] (3) sputtering 5 nm Cr and 50 nm Au on the substrate by magnetron sputtering, and then using a stripping solution to ultrasonically clean and strip the excess photoresist to obtain an interdigital transducer;

[0057] (4) Spin coating MicroChem SU-8 2075 photoresist on the top of the obtained substrate, with a low speed of 500 r / min, a spinning time of 5 to 10 s, and a high speed of 1300 to 2500 r / min, and a spinning time of 30 s; using ultraviolet lithography, the substrate is subjected to ultraviolet exposure and development based on the parameters of the microstructure array, and the excess developer is removed and blown dry;

[0058] (5) Finally, an acoustic streaming drive chip consisting of an interdigital transducer and a microstructured metamaterial on a lithium niobate substrate was obtained.

[0059] The cell drug screening method based on microstructure-induced shear acoustic streaming of this embodiment comprises the following steps:

[0060] 1) According to the required flow rate of the micro-acoustic flow, the unit size, shape and period are parametrically scanned using the finite element method according to the fluid mechanics equations; in this embodiment, the schematic diagram of the shear acoustic flow generated by the microstructure is shown in Figure 3 (a) as shown; Figure 3 As shown in (b), the width of the electrodes of the IDT is 100 μm, the spacing is 100 μm, and there are 20 pairs;

[0062] like Figure 4 As shown, in this embodiment, the parameters of the four microstructure arrays are: the shape of the microstructure unit is a rectangular column, the plane size is 50μm×50μm, the height is 150μm, and the periodic spacing of the microstructure unit in each direction is

[0063] 100μm×200μm; the shape of the microstructure unit is a triangular prism with a side length of 50μm and a height of 150μm. The periodic spacing of the microstructure unit in each direction is 200μm×200μm; the shape of the microstructure unit is a triangular prism with a side length of 70μm and a height of

[0064] 150μm, period is 150 microns; the shape of the microstructure unit is a rectangular column, the plane size is 30μm×100μm, high

[0065] 150μm, and the periodic spacing of the microstructure units in each direction is 100μm×200μm; to generate acoustic streams with different flow rates.

[0066] 2) using oxygen plasma to treat the surface of the microstructure metamaterial of the acoustic flow drive chip and the surface of the microfluidic cavity of the microfluidic cavity module, so that oxygen-containing functional groups are formed on the surface to facilitate bonding and increase its hydrophilicity;

[0067] 3) The acoustic fluidic driving chip is placed upside down on the microfluidic cavity module, the surface of the microstructure metamaterial of the acoustic fluidic driving chip is facing the microfluidic cavity of the microfluidic cavity module, and the microfluidic cavity module and the acoustic fluidic driving chip are bonded into one; the dimensions of the inside of the microfluidic cavity are: height 600 μm, length and width 10 mm;

[0068] 4) introducing polylysine into the microfluidic cavity and incubating for 1 to 2 hours, and then washing with phosphate buffered saline (PBS) to remove excess polylysine to obtain an adhesion layer, so that the cells can adhere to the bottom of the microfluidic cavity;

[0069] 5) The cell properties are MCF7 breast cancer cells, the size is 10μm to 30μm, and the flow rate of the sample is

[0070] 0.5~10μl / min; pass the required cell suspension into the microfluidic cavity, gently shake or rotate the cavity, and then

[0071] The cells were placed in an incubator at 37°C for 1 to 4 hours to ensure that the cells were evenly attached to the bottom of the microfluidic cavity. The drugs were then introduced into the microfluidic cavity, and finally the fluorescent probes for labeling drugs or cells were injected.

[0072] 6) placing the integrated acoustic flow driving chip and microfluidic cavity module injected with drugs on the stage of the microscopic observation device, within the imaging field of the microscopic observation device;

[0073] 7) The central processing unit sends a digital signal to the signal generator, and the signal generator converts the digital signal into an electrical signal and transmits it to the power amplifier; the power amplifier converts the received electrical signal command into an amplified power signal, applies a 15MHz, 500mW driving signal to the interdigital transducer of the acoustic flow driving chip, and excites the interdigital transducer to generate a high-frequency surface acoustic wave; the high-frequency surface acoustic wave generated by the interdigital transducer has a high frequency range, so the wavelength generated on the substrate is usually within a hundred microns;

[0074] 8) High-frequency surface acoustic waves and microstructure metamaterials undergo acoustic-solid coupling, causing oscillations at the boundaries of the microstructure units. This oscillation drives the culture fluid in the flow field within the microchannel cavity to produce a stable acoustic flow field, which generates micro-acoustic flow shear. The biological cells in the acoustic flow field are subjected to controllable shear forces. The shear force generated by the acoustic flow is explained by Newton's law of viscosity. When there is a velocity difference between adjacent layers of the fluid, shear forces will be generated inside the fluid.

[0075] According to the shear force formula: F s =ηAdv / dy, and the cell surface velocity is 0 as a fixed boundary. As the velocity of the micro-acoustic flow increases, the corresponding shear force will also increase, thereby increasing the stimulation effect on the cell membrane;

[0076] 9) Different microstructure arrays can stimulate microacoustic flow at different flow rates, thus exerting different shear forces on cells. Changes in microacoustic flow shear affect the permeability of drug molecules in cell membranes. The greater the flow rate, the greater the shear force, thereby increasing the stimulation to the cell membrane and the deeper the drug penetrates into the cell. Different microstructure arrays correspond to different microfluidic cavity areas, so the degree of drug penetration into cells in different microfluidic cavity areas is also different, thereby screening cell drugs.

[0077] 10) Use fluorescent probes to label drugs or cells to observe the process of drug penetration and cell response; use a microscopic observation device to collect fluorescent images of cells in real time, and analyze the images through a central processing unit. The analysis content includes drug penetration rate, cell membrane changes, cell morphology changes and cell survival rate, so as to derive the specific effect of the drug on the cells.

[0078] Among them, in step 1), the acoustic flow caused by the vibration of the fluid-solid coupling boundary is mainly considered. In order to increase the vibration amplitude of the fluid-solid coupling boundary, in the early stage of simulation, the microstructure unit is regarded as a one-dimensional cantilever beam, and the relationship between the size of the one-dimensional cantilever beam and the resonant frequency is used to preliminarily determine the structural size range that matches the frequency:

[0079]

[0080] Where n is the modal order, h is the height of the microstructure, EI is the bending stiffness of the cross-sectional shape of the microstructure, and m is the mass. I is the moment of inertia, which is related to the unit size and shape of the microstructure unit. Different unit sizes and shapes have different moments of inertia. The unit size includes the cross-sectional side length and height.

[0081] Furthermore, the finite element method is used to perform parametric scanning on the unit height and shape according to the selected size range to obtain the unit size and shape of the microstructure array that meets the larger vibration amplitude.

[0082] Subsequently, since the magnitude of the acoustic streaming velocity is also related to the period of the unit, an array of periodic models is established in the finite element model, and the amplitude of the microstructure unit is used as a boundary condition to couple with the fluid mechanics equations to solve the acoustic streaming velocity, and a parametric sweep is performed for the periodicity of the unit. Finally, the unit size, shape and period of the final microstructure array are obtained according to the required acoustic streaming velocity.

[0083] Fluid mechanics equations:

[0084]

[0085]

[0086] Among them, ρ is the fluid density, v is the fluid flow rate, p is the fluid pressure, and η is the fluid viscosity.

[0087] Fluid-structure interaction boundary conditions:

[0088]

[0089] σ f n=σ s ·n

[0090] Where w is the amplitude of the solid surface, v b is the fluid velocity at the fluid-solid surface, σ f and σ s are the stresses on the fluid and solid surface, respectively.

[0091] Finally, it should be noted that the purpose of publishing the embodiments is to help further understand the present invention, but those skilled in the art can understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the contents disclosed in the embodiments, and the scope of protection claimed by the present invention shall be subject to the scope defined in the claims.

Claims

1. A cell drug screening device based on microstructure-induced shear acoustic streaming, characterized in that: The cell drug screening device comprises: a microscopic observation device, a central processing unit, a power amplifier, a signal generator, a microfluidic cavity module and an acoustic flow driving chip; wherein the acoustic flow driving chip is placed on a sample stage of the microscopic observation device, and the microfluidic cavity module is located under the acoustic flow driving chip; the acoustic flow driving chip is connected to the power amplifier, the power amplifier is connected to the signal generator, and the microscopic observation device and the signal generator are connected to the central processing unit; The acoustic flow driving chip includes a substrate, an interdigital transducer and a microstructure metamaterial; the interdigital transducer and the microstructure metamaterial are respectively arranged on the substrate; the microstructure metamaterial includes a plurality of different microstructure arrays, each microstructure array includes a plurality of microstructure units of the same shape and size arranged in a two-dimensional array, the microstructure unit is a protruding structure formed on the substrate, and has a set regular shape and size; the size and position of each different microstructure array are independent of each other, and the size, shape or period of the microstructure unit of each different microstructure array is different; the unit size, shape and period of different microstructure arrays correspond to the micro-acoustic flow with different flow rates, and the corresponding shear force is different as the speed of the micro-acoustic flow is different, thereby bringing different stimulation effects to the cell membrane; The microfluidic cavity module includes a liquid inlet, a liquid outlet and a microfluidic cavity; the surface of the microfluidic cavity is an opening, the interior is a cavity, and the liquid inlet and the liquid outlet are respectively arranged on two opposite sides of the microfluidic cavity; The surfaces of the microstructure metamaterial of the acoustic fluidic drive chip and the microfluidic cavity of the microfluidic cavity module are processed to be hydrophilic; the acoustic fluidic drive chip is placed upside down on the microfluidic cavity module, the microstructure metamaterial of the acoustic fluidic drive chip faces the microfluidic cavity of the microfluidic cavity module, and the microfluidic cavity module and the acoustic fluidic drive chip are bonded to form an integral body; an adhesion layer is prepared at the bottom of the microfluidic cavity so that cells can adhere to the bottom of the microfluidic cavity; the required cell suspension is introduced into the microfluidic cavity and allowed to stand to ensure that the cells are uniformly attached to the bottom of the microfluidic cavity, then the drug is introduced into the microfluidic cavity, and finally a fluorescent probe for marking the drug or the cell is injected; the acoustic fluidic drive chip and the microfluidic cavity module bonded to form an integral body with the drug introduced are placed on the stage of a microscopic observation device, and are located within the imaging field of view of the microscopic observation device; The central processing unit sends a digital signal to the signal generator, which converts the digital signal into an electrical signal and transmits it to the power amplifier; the power amplifier converts the received electrical signal command into an amplified power signal, which is input into the interdigital transducer of the acoustic flow driving chip, stimulating the interdigital transducer to generate high-frequency surface acoustic waves; the high-frequency surface acoustic waves and the microstructure metamaterial undergo acoustic-solid coupling, causing oscillations at the boundaries of the microstructure units, and this oscillation drives the culture fluid in the flow field located in the microfluidic cavity to produce a stable form of acoustic flow field, which generates micro-acoustic flow shear, and the biological cells located in the acoustic flow field are subjected to controllable shear forces; different microstructure arrays can stimulate different flows. The change of micro-acoustic flow shear affects the permeability of drug molecules in the cell membrane. The greater the flow rate, the greater the shear force, thereby increasing the stimulation to the cell membrane and the deeper the drug penetrates into the cell. Different microstructure arrays correspond to different microfluidic cavity areas, so the degree of drug penetration into cells in different microfluidic cavity areas is also different, thereby screening cell drugs. Fluorescent probes that mark drugs or cells are used to observe the process of drug penetration and cell response. Microscopic observation devices are used to collect images of cells in real time, and the images are analyzed by a central processing unit to obtain the specific effects of drugs on cells.

2. The cell drug screening device according to claim 1, characterized in that: The microscopic observation device includes a fluorescence microscope and a charge-coupled device (CCD) camera; wherein the CCD camera is arranged behind the eyepiece of the fluorescence microscope, and the CCD camera is connected to a central processing unit; the microfluidic cavity module and the acoustic flow driving chip are placed on the sample stage of the fluorescence microscope, and the microfluidic cavity is located in the imaging field of the fluorescence microscope; the fluorescence microscope collects the fluorescence generated by biological cells after being stimulated by acoustic flow shear and treated with drugs, and generates fluorescence information, and the CCD camera converts the fluorescence information into electrical signals and inputs them into the central processing unit, and the fluorescence expression state of the cells is displayed through the central processing unit.

3. The cell drug screening device according to claim 1, characterized in that: The microchannel cavity has a height of 300 to 1000 μm, and a length and width of 5 to 20 mm.

4. The cell drug screening device according to claim 1, characterized in that: The microstructure units of the microstructure array are columnar, and the plane shape is rectangular, triangular or cross-shaped, the plane size is 50-100 μm, the height is 100-200 μm, and the period of the microstructure units is 100-500 μm.

5. A screening method for cell drug screening device based on microstructure induced shear acoustic streaming as claimed in claim 1, characterized in that: The screening method comprises the following steps: 1) Designing the unit size, shape and period of the microstructure array according to the required flow rate of the microacoustic flow; 2) treating the surfaces of the microstructure metamaterial of the relative acoustic flow driving chip and the microfluidic cavity of the microfluidic cavity module to make them hydrophilic; 3) placing the acoustic fluidic driving chip upside down on the microfluidic cavity module, with the microstructure metamaterial of the acoustic fluidic driving chip facing the microfluidic cavity of the microfluidic cavity module, and bonding the microfluidic cavity module and the acoustic fluidic driving chip to form a whole; 4) preparing an adhesion layer at the bottom of the microfluidic cavity so that the cells can adhere to the bottom of the microfluidic cavity; 5) Pass the desired cell suspension into the microfluidic cavity and let it stand to ensure that the cells are evenly attached to the bottom of the microfluidic cavity, then pass the drug into the microfluidic cavity, and finally inject the fluorescent probe that marks the drug or cells; 6) placing the integrated acoustic flow driving chip and microfluidic cavity module into which the drug is introduced on the stage of the microscopic observation device, within the imaging field of the microscopic observation device; 7) The central processing unit sends a digital signal to the signal generator, which converts the digital signal into an electrical signal and transmits it to the power amplifier; the power amplifier converts the received electrical signal command into an amplified power signal, which is input into the interdigital transducer of the acoustic flow driving chip, and stimulates the interdigital transducer to generate a high-frequency surface acoustic wave; 8) High-frequency surface acoustic waves and microstructure metamaterials undergo acoustic-solid coupling, causing oscillations at the boundaries of the microstructure units. This oscillation drives the culture fluid in the flow field within the microchannel cavity to produce a stable acoustic flow field, which generates micro-acoustic flow shear, and biological cells in the acoustic flow field are subjected to controllable shear forces. 9) Different microstructure arrays can stimulate microacoustic flow at different flow rates, thus exerting different shear forces on cells. Changes in microacoustic flow shear affect the permeability of drug molecules in cell membranes. The greater the flow rate, the greater the shear force, thereby increasing the stimulation to the cell membrane and the deeper the drug penetrates into the cell. Different microstructure arrays correspond to different microfluidic cavity areas, so the degree of drug penetration into cells in different microfluidic cavity areas is also different, thereby screening cell drugs. 10) Use fluorescent probes to label drugs or cells to observe the process of drug penetration and cell response; use a microscopic observation device to collect images of cells in real time, and analyze the images through a central processing unit to obtain the specific effects of the drugs on the cells.

6. The screening method according to claim 5, characterized in that In step 1), according to the required flow rate of the micro-acoustic flow and the fluid mechanics equations, the unit size, shape and period are parametrically scanned using the finite element method to obtain the unit size, shape and period of the microstructure array.

7. The screening method according to claim 5, characterized in that In step 2), plasma is used to treat the surface of the microstructure metamaterial of the acoustic flow driving chip and the surface of the microfluidic cavity of the microfluidic cavity module, so that functional groups are formed on the surface to facilitate bonding and increase its hydrophilicity.

8. The screening method according to claim 5, characterized in that In step 4), preparing the adhesive layer comprises the following steps: After bonding is completed, the coating material is introduced into the microfluidic cavity and incubated for 1 to 2 hours, and then the excess coating material is washed away with a buffer solution to obtain an adhesion layer.

9. The screening method according to claim 5, characterized in that In step 7), according to the shear force F s Formula: F s =ηAdv / dy, η is the viscosity of the fluid, v is the velocity of the acoustic flow, and the cell surface is regarded as a fixed boundary. As the velocity of the micro-acoustic flow increases, the corresponding shear force will also increase, thereby increasing the stimulation effect on the cell membrane.

10. The screening method according to claim 5, characterized in that In step 10), the analysis contents include drug permeability, changes in cell membrane, changes in cell morphology and cell survival rate, so as to obtain the specific effect of the drug on the cells.

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