Surface acoustic wave micro-fluidic chip and cell compression ratio measuring method

Abstract

The invention discloses a surface acoustic wave micro-fluidic chip and a cell compression ratio measuring method, and belongs to the technical field of micro-fluidic analysis. The at least two interdigital transducers are oppositely arranged and are parallel to each other; the micro-channel is fixed on the surface of the piezoelectric substrate and is positioned between the two interdigital transducers; the finger strips of the two interdigital transducers and the central axis of the micro-channel are obliquely arranged at a certain included angle; the surface acoustic wave generated by the interdigital transducer is propagated to the fluid in the micro-channel along the piezoelectric substrate to form a surface acoustic wave standing wave field; the surface acoustic wave standing wave field and the flow field in the micro-channel change the movement mode of the cell by adjusting the Stokes drag force and the acoustic radiation force borne by the cell so as to measure the cell compression ratio. Aiming at the limitation of an inclination angle surface acoustic wave standing wave micro-fluidic chip in measurement of mechanical properties of cells in the prior art, accurate regulation and control are realized.

Description

Technical Field

[0001] The present application relates to the technical field of microfluidic analysis, and more specifically, to a surface acoustic wave microfluidic chip and a method for measuring cell compression ratio. Background Art

[0002] In recent years, with the continuous progress of micro-nano processing technology, the microfluidic technology relying on this has also achieved extensive development. Microfluidic technology is a technology for manipulating trace fluids and substances in fluids, with advantages such as high throughput, precise manipulation, and sample conservation, and plays a huge role in the field of biological sample analysis. In the field of cell mechanical property measurement, multiple technical routes have been developed. The passive fluid microfluidic method can measure the mechanical properties of cells by photographing the extrusion process of cells passing through a narrow microchannel. Although this method has achieved high-throughput single-cell measurement and made up for the deficiencies of the traditional atomic force microscope measurement with low throughput and large cell damage, there are still problems such as easy blockage of the microchannel and inability to measure stably for a long time. The microfluidic cell mechanical typing based on optics, electricity, and magnetism can avoid the blockage of the microchannel, but often requires additional labeling and modification of cells, and it is difficult to improve the throughput. The surface acoustic wave microfluidic chip based on acoustic excitation has been widely applied in the fields of cell sorting, enrichment, arrangement, etc. due to its good biocompatibility, label-free, flexible and adjustable characteristics. Among them, the tilted angle surface acoustic wave standing wave microfluidic chip has been widely used in the sorting of microscale particles and cells due to its high acoustic energy density and unique manipulation ability for continuously flowing particles in the microchannel.

[0003] The working principle of the inclined angle surface acoustic wave standing wave microfluidic chip is to use two oppositely placed parallel interdigital transducers to excite surface acoustic waves propagating in opposite directions on a piezoelectric substrate. These waves leak into the fluid in the microchannel region to form a standing wave sound field, and acoustic radiation force is applied to the microspheres in the fluid. Due to the different mechanical properties of the microspheres (such as size, density, compressibility), the acoustic radiation forces they receive under the action of the standing wave sound field will be different. As the fluid flows, the microspheres form different flow trajectories in the cavity and finally produce different lateral offsets at the outlet, achieving separation. This principle enables this type of chip to have the potential to measure the mechanical properties of cells through trajectory measurement without observing cell deformation. In such chips, microspheres exhibit two typical motion modes according to different mechanical properties: the locked mode (the microspheres move along the nodal line of the standing wave sound field) and the drift mode (the microspheres fluctuate back and forth between the fluid streamline and the nodal line of the standing wave sound field). The lateral offsets of microspheres in the two motion modes are significantly different, while the lateral offsets of microspheres in the same motion mode are small. Therefore, traditional designs can often only achieve binary separation of a single mechanical property threshold of microspheres, making it mainly applied to the sorting of tumor cells and difficult to perform more refined measurement of cell mechanical properties. Aiming at the limitation of the traditional inclined angle surface acoustic wave standing wave microfluidic chip in the precise measurement of cell mechanical properties, existing solutions include: adjusting the chip driving power to observe the conversion of cell motion modes and extracting the critical driving power to measure the compressibility; or changing the included angle between the flow field and the sound field by setting a multi-segment line microchannel and observing the cell wall collision points to measure the compressibility. However, these solutions still have problems of low measurement accuracy and difficulty in achieving efficient single-cell measurement.

[0004] Chinese Patent Application, Application No. CN202410774164.3, Publication Date October 1, 2024, discloses a manufacturing method of a surface acoustic wave microfluidic chip with a uniform near field, belonging to the field of microfluidic technology. This application aims at the problem of uneven transverse distribution of the sound field amplitude excited by the straight interdigital transducer in the existing surface acoustic wave microfluidic chip, and proposes a manufacturing method of a surface acoustic wave microfluidic chip with a uniform transverse sound field distribution. Specifically, first, a model for calculating the sound field distribution of the aperture tapered interdigital transducer is constructed; further, the sound field distributions when the transducer finger bars have different weightings are calculated; furthermore, an optimization algorithm is applied to obtain the weighting method that makes the sound field distribution in the near field region uniform; finally, a microfluidic channel is fabricated and installed in the near field region of the interdigital transducer. However, this solution uses a traditional microfluidic channel structure with a constant cross-section. In measurement applications, it is necessary to adjust the driving power or change the flow rate multiple times to observe cell movement, and the measurement efficiency needs to be further improved. Summary of the Invention

[0005] 1. Technical Problems to be Solved

[0006] In view of the limitations of the existing angled surface acoustic wave standing wave microfluidic chips in cell mechanical property measurement, the present application provides a surface acoustic wave microfluidic chip and a method for measuring cell compressibility. Through the synergistic effect of the surface acoustic wave standing wave field and the flow field, combined with the measurement method using standard microspheres as a reference, precise control of the acoustic radiation force and Stokes drag force is achieved.

[0007] 2. Technical Solution

[0008] The objectives of the present application are achieved through the following technical solutions.

[0009] One aspect of the present application provides a surface acoustic wave microfluidic chip, comprising: a piezoelectric substrate; at least two interdigital transducers, which are attached to the piezoelectric substrate by sputtering, and the interdigital transducers are arranged facing each other and parallel to each other; a microchannel, which is bonded to the piezoelectric substrate and is located between the two interdigital transducers; the finger bars of the two interdigital transducers are placed obliquely at a certain angle with respect to the central axis of the microchannel; the microchannel is divided into an inlet region for introducing a cell suspension, a pre-focusing region, a flow rate changing region, and an outlet region; wherein, the inlet region has three inlets, the middle inlet is for introducing the measurement sample suspension, and the two side inlets are for introducing fluids with the same carrier liquid composition as the measurement sample suspension; the surface acoustic wave generated by the interdigital transducers propagates along the piezoelectric substrate and leaks into the fluid in the microchannel to form a surface acoustic wave standing wave field; the surface acoustic wave standing wave field and the flow field in the microchannel change the motion mode of the cells by adjusting the Stokes drag force and acoustic radiation force acting on the cells, so as to measure the cell compressibility; the motion modes include a drift mode and a locking mode. The piezoelectric ceramics such as lead titanate, piezoelectric crystals such as lithium niobate, piezoelectric thin films such as zinc oxide, and solid materials such as quartz glass, and the interdigital transducers are prepared by chemical or physical methods.

[0010] Furthermore, the cross-sectional area of the microchannel increases along the flow rate direction.

[0011] Furthermore, the microchannel also includes a pre-focusing region and a flow rate changing region; the pre-focusing region uses the sheath flow pre-focusing method to ensure that the measurement sample is located at a specified lateral position in the microchannel before entering the flow rate changing region, and the lateral line width of the measurement sample aggregation is less than 10 μm; the flow rate changing region includes a surface acoustic wave standing wave action region, and it is required that when the standing wave field is inclined, the flow rate changing region completely covers the surface acoustic wave standing wave action region; a single outlet is used at the outlet to ensure the collection of all measurement samples.

[0012] The cross-section of the flow velocity change region is a polygon. In this application, the polygon refers to the cross-sectional shape of the microchannel, including but not limited to geometric shapes such as rectangles, triangles, trapezoids, etc. Preferably, the polygon is a rectangle, and the cross-section of the internal channel of the microchannel is a rectangle that gradually increases along the flow direction; the flow velocity change region includes the region where the surface acoustic wave standing wave acts. It is required that when the standing wave field is inclined, the flow velocity change region completely covers the region where the surface acoustic wave standing wave acts; a single outlet is used at the outlet to ensure that all measurement samples are collected.

[0013] Furthermore, the microchannel is made of a biocompatible material.

[0014] Furthermore, the biocompatible material includes glass, polydimethylsiloxane, and polymethyl methacrylate.

[0015] Furthermore, the interdigital transducer includes: the finger widths and spacings of two interdigital transducers are equal, and the finger width and spacing are 1 / N of the surface acoustic wave wavelength; preferably, N is taken as 4, and the value range of N is from 2 to 6.

[0016] The inclination angle of the interdigital transducer with respect to the central axis of the microchannel is θ, the operating frequency of the interdigital transducer is f0, and the substrate surface acoustic wave wavelength can be determined by the operating frequency f0 of the interdigital transducer and the surface wave sound velocity c of the substrate s Calculated as: λ0 = c s / f0. The width of the operating region of the interdigital transducer is between 3000μm and 10000μm as required, so that the sample to be measured can undergo a motion mode conversion at the used flow velocity without settling due to the decrease in flow velocity.

[0017] This application uses a microchannel with a variable cross-section, so that the flow velocity along the central axis of the microchannel gradually decreases in the flow direction. The relevant parameters of the microchannel are determined by the measurement range, measurement accuracy, and measurement flux. The flow velocity change rate of the central axis of the microchannel is determined according to the measurement accuracy, and the boundary function of the xy plane of the microchannel is determined according to the measurement mechanical property range and the input flow rate. The following elaborates on the specific design process: First, design for the input flow rate Q of the microchannel and the input sound pressure amplitude p of the standing wave sound field generated by the interdigital transducer in the microchannel fluid domain. To ensure wide applicability, generally a larger input flow rate Q and a smaller input sound pressure amplitude p in are selected. The height of the microchannel is h. The mechanical properties of the microspheres to be measured are characterized by the mechanical property coefficient Ψ in , written as Ψ s = a s φ, which is the coupling quantity of the microsphere radius a and the microsphere acoustic contrast factor φ. Among them, the acoustic contrast factor is 2 f1 and f2 are the acoustic monopole scattering coefficient and acoustic dipole scattering coefficient of the microsphere, respectively, written as f1 = (K0 - K ) / K0, p ),

[0018] K p is the microsphere compression ratio, K0 is the fluid compression ratio, ρ p is the microsphere density, and ρ0 is the fluid density. The measurement range of the microsphere mechanical property coefficient to be measured is between Ψ l ~Ψ h . The required measurement accuracy is characterized by the fact that when the mechanical property coefficient changes by ΔΨ(x), the microspheres will be locked at different nodes of the standing wave sound field.

[0019] Next, analyze the position of the microsphere mode conversion in the microchannel. In the inclined angle surface acoustic wave standing wave microfluidic chip, when the incident sound pressure amplitude and the microsphere mechanical properties are fixed, the critical velocity for the microsphere motion mode conversion is v0 = 2k y E ac Ψ s / (3ηsinθ). When the fluid flow velocity is greater than ν0, the motion mode of the microsphere is a drift mode, that is, the microsphere oscillates forward between the node line of the standing wave sound field and the fluid streamline; when the fluid flow velocity is less than ν0, the motion mode of the microsphere is a locked mode, that is, the microsphere does not follow the streamline but moves along the node line of the standing wave sound field.

[0020] In the critical velocity formula, k y is the surface acoustic wave number and can be written as k y = 2π / λ0. is the sound energy density, ρ0 and c0 are the density of the fluid and the sound speed in the fluid respectively. In specific experimental operations, E ac is proportional to the driving power P of the signal generator and can also be written as E ac = αP, where α is the conversion coefficient of the driving power and the sound energy density and can be obtained through the sound pressure calibration of the surface acoustic wave microfluidic chip. η is the viscosity of the fluid. Furthermore, because the measurement range of the particle mechanical property coefficient is Ψ l ~Ψ h , it is necessary to make the particles with mechanical properties of Ψ l and Ψ h both be able to undergo motion mode conversion in the variable cross-section microchannel. Therefore, substituting Ψ l , Ψ h into the critical velocity formula for particle motion mode conversion, the minimum value ν min of the critical velocity is obtained as ν y = 2k ac E l Ψ max / (3ηsinθ) and the maximum value ν y of the critical velocity is ν ac = 2k h / (3ηsinθ). Considering the design of locking particles with a mechanical property coefficient interval of ΔΨ(χ) on different nodal lines, according to the formula, the minimum required flow velocity change between two nodes is Δν = 2k y E ac ΔΨ(χ) / (3ηsinθ), and the distance between adjacent nodes is Δχ = λ0 / (2sinθ). Based on this, the flow velocity distribution on the central axis of the microchannel can be calculated.

[0021] Set the flow velocity on the central axis of the microchannel in the acoustic field action area as ν f (χ) = ∫mdχ = M(χ) + C, where the change rate m of the flow velocity can be determined by the required measurement accuracy ΔΨ(χ): m = 4k y E ac ΔΨ(χ) / (3ηλ0), and the integral constant C can be determined by the measurement range Ψ l 、Ψ h : C = k y E ac (Ψ l + Ψ h ) / (3ηsinθ) - M(O). According to this flow velocity change curve, based on the flow rate Q, the function of the channel width can be calculated: w(χ) = Q / [2Hν f (x)], where H is the channel height.

[0022] Furthermore, according to the measurement range, calculate the length I of the acoustic surface wave standing wave action area I = 2×MAX(lh, l l ), where I h satisfies ν max = M(I h ) + C, and l l satisfies ν min = M(l l ) + C. Based on this, calculate the interdigital aperture W = I / cosθ, and the length L of the flow velocity change area. L needs to be slightly larger than the length I of the acoustic surface wave standing wave action area to ensure the stable attenuation of the flow velocity. The acoustic surface wave standing wave action area is located in the middle of the flow velocity change area, and thus the detailed dimensions of the variable cross-section microchannel are determined.

[0023] Furthermore, the chip also includes: a drive circuit, electrically connected to the interdigital transducer, to drive the interdigital transducer to generate acoustic surface waves; the drive circuit includes a signal source and a power amplifier circuit.

[0024] Furthermore, the piezoelectric substrate is a piezoelectric ceramic, a piezoelectric crystal, or a solid material with a piezoelectric thin film attached to its surface;

[0025] Another aspect of the present application also provides a method for measuring the cell compression ratio based on a surface acoustic wave microfluidic chip, which is characterized by including: numerically simulating the flow field distribution and acoustic field distribution of the surface acoustic wave microfluidic chip, calculating the movement trajectories of standard microspheres with different mechanical property coefficients under different incident acoustic pressure amplitudes and different input flow rates, and taking it as a standard microsphere movement trajectory table; injecting standard microspheres with known compression ratios and densities into the inlet area of the microchannel, and making the standard microspheres enter the flow rate change area through sheath flow pre-focusing to obtain the movement trajectories of the standard microspheres under different driving powers and flow rate conditions; comparing the standard microsphere movement trajectory table with the recorded movement trajectories of the standard microspheres to establish the conversion relationship between the driving power and the acoustic pressure amplitude; obtaining the movement trajectories of the cells in the cell suspension to be measured and preprocessing the movement trajectories of the cells; comparing the preprocessed movement trajectories of the cells with the standard microsphere movement trajectory table to obtain the microsphere trajectory that best matches the movement trajectories of the cells and the corresponding mechanical property coefficients; wherein, by simulating the physical field in the surface acoustic wave microfluidic chip, changing the input flow rate and the incident acoustic pressure amplitude, and changing the mechanical property coefficient Ψ of the microspheres s , a movement trajectory table of microspheres with different mechanical properties under different input flow rates and incident acoustic pressure amplitudes is obtained. Microspheres with known sizes, densities, and compression ratios are used to observe the movement trajectories of the microspheres in the flow rate change area under different flow rates and driving powers. Compare the movement trajectories of the microspheres obtained in the experiment with the movement trajectory table of microspheres with the same mechanical properties and the same flow rate established by simulation, and find the trajectory with the smallest mean square error, and take the acoustic pressure amplitude corresponding to the trajectory as the incident acoustic pressure amplitude corresponding to the current driving power. After obtaining multiple sets of driving powers and the corresponding incident acoustic pressure amplitudes, calculate the relationship curve between the two through polynomial fitting Confirm the conversion coefficient α between the driving power and the acoustic energy density to complete the acoustic pressure calibration of the surface acoustic wave microfluidic chip. Perform trajectory tracking on the sequential images to obtain the movement trajectories of single cells. Compare the movement trajectories of the obtained single cells with the microsphere movement trajectory table obtained by simulation, fix the input flow rate and the input acoustic pressure amplitude corresponding to the calibrated driving power, find the simulation microsphere movement trajectory with the smallest mean square error, and extract its mechanical property coefficient Ψ s .

[0026] According to the obtained mechanical property coefficients, calculate the equivalent radius of the cells in the cell suspension to be measured; specifically, use sample cells, by changing the input flow rate and the driving power, it is observed that the movement trajectories of a large number of cells change from the drift mode to the locked mode. Under this condition, measure the cell compression ratio, capture the sequential images of the cells moving in the surface acoustic wave standing wave action area through a microscope system equipped with a high-speed camera, correct the scale of the images and select the threshold to obtain the cell area, and calculate the equivalent radius a of the cells through the area c .

[0027] Calculate the compression rate of cells in the cell suspension to be measured according to the equivalent radius; the equivalent radius a of the cells c , the average density ρ of the cells c Substitute into the mechanical property coefficient Ψ of the cells s to calculate the compression rate k of the cells p .

[0028] Among them, the cells in the cell suspension to be measured are under the action of the surface acoustic wave standing wave field and the flow velocity gradient. According to the different compression rates of the cells, they change from the drift mode to the locking mode at different positions, so as to distinguish different cells and measure the cell compression rate.

[0029] 3. Beneficial effects

[0030] Compared with the prior art, the advantages of this application are as follows:

[0031] Based on the traditional equal-section inclined angle surface acoustic wave standing wave microfluidic chip, by designing the change of the microchannel cross-section in the surface acoustic wave standing wave action area, the flow velocity on the central axis of the microchannel gradually decays, and the chip size is designed according to the theoretical formula and the range of the mechanical property coefficients of common cells, so that the chip can measure a large range of cell compression rates with fewer input parameters, and can greatly simplify the complexity of the process for samples with large heterogeneity. Brief description of the drawings

[0032] Figure 1 It is the design of the surface acoustic wave microfluidic chip of this application and the measurement process of the cell compression rate;

[0033] Figure 2 It is the structural schematic diagram of the surface acoustic wave microfluidic chip in the example of this application;

[0034] Figure 3 It is the particle motion trajectories corresponding to different input acoustic pressure amplitudes when the diameter of the polystyrene microsphere is 10 μm and the input flow rate is 0.105 μL / s;

[0035] Figure 4 It is the motion trajectories of microspheres with different compression rates when the diameter of the microsphere is 15 μm, the density is 1050 kg / m3, the input flow rate is 0.105 μL / s, and the input acoustic pressure amplitude is 1.5×105 Pa;

[0036] Figure 5 It is the measurement results of the diameters and compression rates of MDA-MB-231 cells and MCF7 cells.

[0037] Explanation of the reference numerals in the figure:

[0038] 01. Entrance area; 02. Prefocusing area; 03. Flow velocity change area; 04. Exit area; 05. Acoustic surface wave standing wave action area; 06. Interdigital transducer one; 07. Interdigital transducer two; 08. Sheath flow one; 09. Sample flow; 10. Sheath flow two. Detailed implementation mode

[0039] The present application will be described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0040] As Figure 1 and Figure 2 shown, the present application specifically describes an acoustic surface wave microfluidic chip, which includes a piezoelectric substrate, interdigital transducers, a microchannel and a driving circuit. The interdigital transducers are attached to the piezoelectric substrate, and acoustic surface waves are excited by circuit driving. The acoustic surface waves excited by two opposite interdigital transducers propagate relatively on the piezoelectric substrate to form a standing wave sound field in the microchannel, and apply acoustic radiation force to the particles in the microchannel; the microchannel is bonded or adhered to the piezoelectric substrate, and the internal channel of the microchannel is a rectangle with a gradually increasing cross-sectional area, and the specific parameters are obtained through design and calculation; the driving circuit drives the interdigital transducers to work. The measurement sample enters the acoustic surface wave standing wave action area 05 of the acoustic surface wave microfluidic chip in a pre-focused manner using sheath flow one 08 or sheath flow two 10, ensuring that the initial lateral position of the measurement sample in the microchannel is consistent. Under the action of the acoustic surface wave standing wave, measurement samples with different compression ratios will show different movement trajectories and be captured by the wave nodes of the standing wave sound field at different positions in the microchannel. The movement trajectory of the sample is photographed by a microscope imaging system equipped with a high-speed camera.

[0041] The substrate material of the acoustic surface wave microfluidic chip is piezoelectric ceramics, piezoelectric crystals, or any solid with a piezoelectric thin film on its surface. Two opposite interdigital transducers one 06 and interdigital transducer two 07 are fabricated on both sides of the microchannel, and all their finger bars are parallel, with the same width, length, and interval; the generated acoustic surface waves propagate along the substrate and enter the fluid in the microchannel; the two interdigital transducers form an angle θ with the central axis of the microchannel.

[0042] The channel parameters of the acoustic surface wave microfluidic chip are designed according to specific application scenarios and accuracy requirements. The specific steps are as follows: First, select a suitable operating frequency f0 of the interdigital transducer as 19.97 MHz and the angle θ between the central axis of the microchannel and the interdigital transducer as 12°. Select a lithium niobate substrate, and the substrate sound velocity c s is 3994 m / s. The substrate acoustic surface wave wavelength λ0 is calculated according to the formula λ0 = c s / f0 to obtain 200 μm. Confirm that the design is carried out for an input flow rate Q of 0.126 μL / s and an input sound pressure amplitude p in of 1×10 5 Pa. The height H of the microchannel is taken as 60 μm, and the measurement range of the particle mechanical property coefficient Ψl ~Ψ h At 2.57×10 -12 to 7.32×10 -12 m 2 , the required measurement accuracy is characterized by the fact that when the change in the mechanical property coefficient ΔΨ(χ) is 3.72×10 -13 m 2 , the particles will be locked at different nodal lines.

[0043] Calculate the minimum value ν of the critical velocity according to the formula min = 2k ν E ac Ψ I / (3ηsinθ) and the maximum value ν max = 2k ν E ac Ψ h . Where the surface acoustic wave number k ν According to the formula k ν = 2π / λ0, it can be calculated as 3.14×10 4 m -1 . The acoustic energy density can be calculated as 4.48 Pa according to the formula . Since the fluid properties in the microchannel are similar to those of water in the experiment, the density ρ0 and the sound speed c0 should be taken as 998 kg / m3 and 1495 m / s, and the fluid viscosity η is taken as 0.893 mPas. When the tilt angle θ is 12°, sinθ is 0.2079. Substitute Ψ l and

[0044] Ψ h as 2.57×10 -12 m 2 and 7.32×10 -12 m 2 respectively, and the minimum value ν of the critical velocity is obtained as 0.0013 m / s, and the maximum value ν min of the critical velocity is 0.0037 m / s. max

[0045] Furthermore, calculate the velocity change Δν between two adjacent nodes of the standing wave sound field according to ΔΨ(x). In this example, a uniform accuracy ΔΨ is selected, taken as 3.72×10 -13 m 2 , substitute it into the formula Δν = 2k ν E ac ΔΨ / (3ηsinθ), and the calculated velocity change Δν is constant at 1.88×10 -4 m / s. The distance between adjacent nodes of the standing wave sound field can be calculated as 481 μm according to the formula Δx = λ0 / (2sinθ).

[0046] Furthermore, calculate the flow velocity of the microchannel central axis in the SAW standing wave action area 05

[0047] where the change rate of the flow velocity m = Δν / Δx can be determined by the required measurement accuracy ΔΨ: m = 4k ν E ac ΔΨ / (3ηλ0), and the integration constant C can be determined by the measurement range Ψ l 、Ψ h determined, C = k ν E ac (Ψ l +ψ h ) / (3ηsinθ). After calculation, the slope of the flow velocity change curve of the microchannel central axis is v f (x) = 0.3911x + 0.0025. According to this flow velocity change curve, when the flow rate Q is 0.126 μL / s and the microchannel height is 60 μm, the function of the microchannel width can be calculated:

[0048] w(χ) = Q / [2H(mχ + C)] = 1.05×10 -6 / (0.3911x + 0.0025)m.

[0049] Furthermore, calculate the required length l of the SAW standing wave action area 05. In this case, l = (ν max -ν min ) / m is approximately 6000 μm. Based on this, the aperture W of the interdigital transducer is designed to be 6134 μm as W = l / cosθ.

[0050] To ensure that the SAW standing wave action area 05 is completely within the flow velocity change area 03, the length L of the flow velocity change area 03 should be slightly greater than the length of the SAW standing wave action area 05. In this example, it is set to 7000 μm. Based on this, calculate the width w1 of the inlet area 01 of the microchannel flow velocity change area 03 as 2w(L / 2) = 5.42×10 -4 m, and the outlet width w2 = 2w(-L / 2) = 1.9×10 -3 m.

[0051] Specifically, according to the principles of fluid mechanics, when the cross-sectional area of the microchannel increases along the flow direction, the fluid velocity gradually decays according to the continuity equation (Q = vA). Since the Stokes drag force (Fd = 6πηrv) is proportional to the flow velocity, and the acoustic radiation force (Fr) mainly depends on the cell compressibility, density, and acoustic field intensity, this design of the present application creates a gradient distribution of the ratio of the Stokes drag force to the acoustic radiation force in space. When the cells move along the microchannel, at a specific position, when Fr > Fd, the cells change from the drift mode to the locking mode. Cells with different compressibilities have different acoustic radiation force responses, so the mode conversion will occur at different positions. This spatially distributed force balance design enables the system to analyze cells with multiple compressibilities simultaneously in a single experiment, greatly improving the measurement efficiency and range.

[0052] The pre-focusing region 02 of the microchannel of the surface acoustic wave microfluidic chip has three branches converging into the main channel. The functions of the three branches are that the two sides serve as the inlet regions 01 of sheath flow one 08 or sheath flow two 10, and the middle serves as the inlet region 01 of the sample flow 09, realizing the aggregation of the sample on the central axis of the microchannel before the sample enters the flow velocity change region 03. The outlet of the microchannel of the surface acoustic wave microfluidic chip is set as a single outlet, and the width is the width w2 of the acoustic field action region.

[0053] The method and process for measuring the cell compressibility in this example are described below:

[0054] First, simulate the physical field and the microsphere movement trajectory in the surface acoustic wave microfluidic chip to establish a microsphere movement trajectory table. The steps of numerical simulation for calculating the movement trajectory are as follows. First, construct a two-dimensional model of the xy plane of the microchannel according to the microchannel boundary function, stretch it to the channel height of 60 μm, and construct a three-dimensional model of the microchannel. According to the flow rate ratio of the sample flow 09 to the sheath flow one 08 or the sheath flow two 10 being 1:20, calculate the surface velocity of the three inlet regions 01 according to the total input flow rate Q and the cross-sectional area of the microchannel, and calculate the steady-state three-dimensional flow field in the microchannel through steady-state analysis. After the calculation is completed, extract the flow velocity data of the xy plane at half the height of the microchannel and save it as the internal flow field distribution file of the microchannel corresponding to the input flow rate Q. Import the flow field distribution file into the two-dimensional model of the xy plane of the microchannel. Second, in the two-dimensional model of the microchannel, set the acoustic field incident boundary conditions on both sides of the microchannel, and the incident acoustic pressure amplitude is p in ., and calculate the acoustic field distribution in the xy plane of the microchannel through frequency domain analysis. Third, according to the flow field distribution and the acoustic field distribution of the xy plane at half the height of the microchannel obtained in the first two steps, calculate the Stokes drag force and the acoustic radiation force acting on the microspheres with the mechanical property of Ψ s and define the inlet region 01 and the outlet boundary conditions of the microspheres. At different input flow rates Q, for different input acoustic pressure amplitudes p in and different mechanical properties Ψ sParametric scanning of the microspheres is performed to calculate the microsphere movement trajectories. Finally, according to the corresponding input parameters Q and p in Export, and interpolate the obtained microsphere movement trajectories at intervals of 1 μm from the x coordinate of -3000 μm to 3000 μm, and save them as a microsphere movement trajectory table. To simplify the calculation, tables can be established separately for the sound pressure calibration process and the cell compression rate measurement process.

[0055] Specifically, the present application adopts a measurement method in a continuous flow environment, precisely controls the cell entry position through the sheath flow - 08 or sheath flow two 10 pre - focusing technology, and combines the synergistic effect of the surface acoustic wave standing wave field and the varying flow field to achieve precise tracking of single - cell trajectories. During the measurement process, by comparing the actual movement trajectory of the cell with the pre - established standard microsphere trajectory database, the mechanical characteristic parameters of the cell can be deduced. This dynamic measurement principle avoids the limitations of static measurement or multiple parameter adjustments in traditional methods, and greatly improves the measurement throughput. At the same time, the pre - focusing design ensures that the cells enter the sound field along a predetermined path, enhancing the consistency and accuracy of the measurement.

[0056] First, for the sound pressure calibration process, it is necessary to track the trajectories of polystyrene microspheres with a radius of 5 μm, a density of 1050 kg / m3, and a compressibility of 2.49×10 -10 Pa -1 and polymethyl methacrylate microspheres with a radius of 5 μm, a density of 1150 kg / m3, and a compressibility of 1.7×10 -10 Pa -1 to obtain their particle movement trajectory tables under 13 incident sound pressure amplitudes (0.8×10 5 Pa~2×10 5 Pa, with an interval of 0.1×10 5 Pa) and 3 input flow rates (0.063, 0.105, 0.147 μL / s). As Figure 3 shown, for polystyrene microspheres with a diameter of 10 μm at an input flow rate of 0.105 μL / s, the particle movement trajectories corresponding to different input sound pressure amplitudes are obtained through simulation calculation.

[0057] Secondly, for the cell compression rate measurement process, it is necessary to use 11 microspheres with a radius of 15 μm, a density of 1050 kg / m3, and a compressibility of 3.3~3.4×10 -10 Pa -1 , with an interval of 0.1×10 -10 Pa -1 to obtain their particle movement trajectories under 10 incident sound pressure amplitudes (1.1×10 5 Pa~2×10 5 Pa, with an interval of 0.1×10 5Table of the movement trajectories of microspheres under Pa) and three input flow rates (0.063, 0.105, 0.147 μL / s). As Figure 4 shown, for microspheres with a diameter of 15 μm and a density of 1050 kg / m3, under an input flow rate of 0.105 μL / s and an input acoustic pressure amplitude of 1.5×10 5 Pa, the movement trajectories of microspheres with different compression ratios are obtained through simulation calculations.

[0058] Furthermore, in the measurement, polystyrene and polymethyl methacrylate microspheres are used to calibrate the inclined surface acoustic wave standing wave microfluidic chip. The polystyrene and polymethyl methacrylate microspheres are suspended in a PBS solution containing 0.04% F127 at a concentration of 2 - 5×10 6 particles / mL. The sheath flow on both sides, sheath flow one 08 or sheath flow two 10, also uses a PBS solution containing 0.04% F127 and is introduced into the channel according to a sheath liquid ratio of 20:1. The driving power of the signal generator is changed, the liquid input flow rate is changed, the movement trajectories of the microspheres are observed and photographed by a high-speed camera.

[0059] The steps for tracking the movement trajectories of microspheres based on the sequential images are as follows. First, set the image gray threshold to preliminarily remove the image background to obtain a frame selection area containing the microchannel boundary, measurement samples, and minute impurities. Filter out the microchannel boundary and minute impurities according to the characteristics of the sample frame selection area such as area and roundness, and only frame out the measurement samples as the microspheres to be tracked and locate their coordinates. Second, based on the characteristics of the microsphere position, velocity, etc., associate the microspheres in different frames, and use a suitable linking algorithm to connect the same microspheres in different frames into trajectories. Third, filter the trajectories according to the trajectory characteristics such as the total displacement of the trajectory, the average movement direction change rate of the trajectory, the average movement speed of the trajectory, etc., and filter out incomplete or mis-tracked trajectories. Finally, export the trajectories to obtain the position coordinates and microsphere areas of different microspheres at each moment.

[0060] According to the pre-calibrated fixed points A′(x1′, y1′), B′(x2′, y2′), C′(x3′, y3′) in the microchannel and the corresponding three-point coordinates A(x1, y1), B(x2, y2), C(x3, y3) in the two-dimensional simulation model, determine the transformation matrix between the experimental coordinate system and the simulation coordinate system:

[0061] Correspond the particle movement trajectories obtained in the experiment with the coordinates in the microsphere movement trajectory table through coordinate transformation.

[0062] Where X′ and Y′ are the coordinates of the particle motion trajectory in the experimental coordinate system, and X and Y are the coordinates of the experimental particle motion trajectory after being transformed into the simulation coordinate system. Further, the x-axis of the experimental particle motion trajectory is aligned with the trajectory of the microsphere motion trajectory table through interpolation. The interpolation method is the same as described above, and interpolation is performed at intervals of 1 μm for the x-coordinate ranging from -3000 μm to 3000 μm.

[0063] Calculate the root mean square error in the y-direction between the trajectory in the microsphere motion trajectory table corresponding to different incident sound pressure amplitudes at the same input flow rate and the particle motion trajectory under different driving powers P in the experiment. Where y0 is the y-direction component of the microsphere trajectory in the microsphere motion trajectory table, y1 is the y-direction component of the particle trajectory observed in the experiment, and i represents the point corresponding to the time sequence in the trajectory. Search for the trajectory with the minimum RMSE in the microsphere motion trajectory table and obtain the corresponding incident sound pressure amplitude p. in According to the formula For all the driving powers P of the experimental particle trajectories and their corresponding Perform linear fitting to obtain the conversion coefficient α between the driving power and the sound energy density, and complete the sound pressure calibration of the surface acoustic wave microfluidic chip.

[0064] Further, after the sound pressure calibration of the surface acoustic wave microfluidic chip is completed, inject the cell suspension suspending the sample cells into the microchannel sample inlet area 01. The cell concentration is 3×106 cells / mL, suspended in PBS solution containing 0.04% F127. The sheath flow 08 or sheath flow 10 on both sides also uses PBS solution containing 0.04% F127 and is introduced into the channel according to a sheath flow ratio of 20:1. Change the driving power and the input flow rate, observe the cell motion trajectory and take pictures with a high-speed camera.

[0065] Consistent with the particle motion trajectory tracking processing method with known properties, perform microsphere motion trajectory tracking through the processing of time-sequence images to obtain the motion trajectories of different cells and the area s at each moment. i Transform the cell motion trajectory into the simulation coordinate system through coordinate transformation and interpolation, and keep it aligned with the χ-direction coordinate of the microsphere motion trajectory table.

[0066] Convert the area of each single cell at each moment into the radius of each single cell at each moment through the area formula and calculate the equivalent radius of this cell on average. Where S i is the cell area at each moment, and n is the total number of moments in the trajectory.

[0067] According to the sound pressure calibration result of the surface acoustic wave microfluidic chip, convert the driving power P into its corresponding incident sound pressure amplitude p through the formula in, index to the closest incident sound pressure amplitude in the microsphere motion trajectory table, index to the closest input flow rate in the microsphere motion trajectory table according to the specific input flow rate used in the experimental cell trajectory, find the simulated microsphere motion trajectory with the smallest RMSE under this incident sound pressure amplitude and input flow rate compared with the experimental cell motion trajectory, and find out its mechanical property coefficient Ψ s .

[0068] The measured equivalent radius a of the cell c Substituting into the equation, we can calculate the acoustic contrast factor φ of the cell, and further calculate the compression rate K c .

[0069] According to the above steps, two breast cancer cells, MCF7 and MDA-MB-231, were measured using a variety of input parameters, which are given below according to the signal generator drive power and the total input flow of the surface acoustic wave microfluidic chip. MDA-MB-231 was measured using three sets of input parameters: 1120mW, 0.063μL / s, 1120mW, 0.105μL / s, and 1400mW, 0.105μL / s; MCF7 was measured using four sets of input parameters: 1070mW, 0.063μL / s, 900mW, 0.063μL / s, 979mW, 0.105μL / s, and 1200mW, 0.147μL / s.

[0070] The measurement results are as follows Figure 5 As shown, the MDA-MB-231 cell diameter was 14.35±1.57μm and the compression rate was 3.84±0.16×10 -10 Pa -1 The diameter of MCF7 cells was 19.77±4.39μm, and the compression rate was 4.08±0.14×10 -10 Pa -1 , the two types of cells can be distinguished, and the measurement flux reaches 20 / s, which is much higher than the traditional measurement method of 100 / h. Furthermore, compared with the traditional surface acoustic wave standing wave microfluidic chip with equal cross-sectional tilt angle, which only has two motion modes of drift mode and locking mode, the present application has a third mixed motion mode, which can lock cells with different mechanical properties to different standing wave acoustic field node lines, naturally divide multiple measurement thresholds, and the measurement accuracy is controllable. The present application realizes the conversion of cell motion mode by regulating the competitive relationship between Stokes drag and acoustic radiation force of cells in the surface acoustic wave standing wave action area. The measurement process of the present application is measured in the flowing fluid throughout the whole process, and the cells are tracked as single cells, which greatly improves the flux of single cell compression rate measurement.

[0071] The above has schematically described the present invention and its implementation manners. This description is not restrictive. Without departing from the spirit or basic characteristics of the present application, the present application can be implemented in other specific forms. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Any reference signs in the claims should not limit the claimed claims. Therefore, if those of ordinary skill in the art are inspired by it and, without departing from the gist of the present invention, design similar structural manners and embodiments to the technical solution without creative efforts, they shall fall within the protection scope of this patent. In addition, the term "including" does not exclude other elements or steps, and the word "a" before an element does not exclude including "a plurality of" such elements. The plurality of elements stated in the product claims can also be implemented by one element through software or hardware. The terms "first", "second", etc. are used to denote names and do not denote any particular order.

Claims

1. A surface acoustic wave microfluidic chip, characterized in that Comprising: A piezoelectric substrate; At least two interdigital transducers, attached to the surface of the piezoelectric substrate, with the interdigital transducers arranged facing each other and parallel to each other; A microfluidic channel, fixed to the surface of the piezoelectric substrate and located between the two interdigital transducers; the fingers of the two interdigital transducers are placed obliquely at a certain angle to the central axis of the microfluidic channel; The microfluidic channel is divided into an inlet region, a pre-focusing region, a flow velocity change region, and an outlet region; the inlet region is used to introduce a cell suspension; The surface acoustic wave generated by the interdigital transducer propagates along the piezoelectric substrate into the fluid in the microfluidic channel, forming a surface acoustic wave standing wave field; The surface acoustic wave standing wave field and the flow field in the microfluidic channel change the movement mode of the cells by adjusting the Stokes drag force and acoustic radiation force received by the cells, so as to measure the cell compressibility; the movement mode includes a drift mode and a locking mode.

2. The surface acoustic wave microfluidic chip according to claim 1, wherein: The microfluidic channel further includes a flow velocity change region; The cross-section of the flow velocity change region is polygonal; The cross-sectional area of the flow velocity change region increases along the flow velocity direction.

3. The surface acoustic wave microfluidic chip according to claim 2, wherein: The microfluidic channel further includes a pre-focusing region; The pre-focusing region uses a sheath flow pre-focusing method to converge the cell suspension to a preset position in the microfluidic channel.

4. The surface acoustic wave microfluidic chip according to claim 3, wherein: The microfluidic channel is made of a biocompatible material.

5. The surface acoustic wave microfluidic chip according to claim 4, wherein: The biocompatible material is glass, polydimethylsiloxane or polymethyl methacrylate.

6. The surface acoustic wave microfluidic chip according to claim 4, wherein: The interdigital transducer includes: The finger widths and spacings of the two interdigital transducers are equal, and the finger width and spacing are 1 / N of the surface acoustic wave wavelength.

7. The surface acoustic wave microfluidic chip according to claim 1, wherein: The chip further includes: A drive circuit, electrically connected to the interdigital transducer, driving the interdigital transducer to generate a surface acoustic wave; The drive circuit includes a signal source and a power amplifier circuit.

8. The surface acoustic wave microfluidic chip according to any one of claims 2 to 7, wherein: The piezoelectric substrate is a piezoelectric ceramic, a piezoelectric crystal or a solid material with a piezoelectric thin film attached to its surface.

9. A method for measuring the cell compression ratio based on the surface acoustic wave microfluidic chip according to any one of claims 1 to 8, characterized in that, Comprising: Performing numerical simulation on the flow field distribution and acoustic field distribution of the surface acoustic wave microfluidic chip, calculating the movement trajectories of standard microspheres with different mechanical property coefficients under different incident acoustic pressure amplitudes and different input flow rates, as a standard microsphere movement trajectory table; Injecting standard microspheres with known compressibility and density into the inlet region of the microfluidic channel, and making the standard microspheres enter the flow velocity change region through the sheath flow pre-focusing method to obtain the movement trajectories of the standard microspheres under different driving powers and flow rate conditions; Comparing the standard microsphere movement trajectory table with the recorded movement trajectories of the standard microspheres to establish the conversion relationship between the driving power and the acoustic pressure amplitude; Obtaining the cell movement trajectories of the cell suspension to be measured and preprocessing the cell movement trajectories; Compare the preprocessed cell movement trajectories with the standard microsphere movement trajectory table to obtain the microsphere trajectory that best matches the cell movement trajectory and the corresponding mechanical property coefficients; Calculate the equivalent radius of the cells in the cell suspension to be measured according to the obtained mechanical property coefficients; Calculate the compression ratio of the cells in the cell suspension to be measured based on the equivalent radius; Among them, under the action of the surface acoustic wave standing wave field and the flow velocity gradient, the cells in the cell suspension to be measured change from the drift mode to the locking mode at different positions according to different cell compression ratios, so as to distinguish different cells and measure the cell compression ratio.

10. The method for measuring the cell compression ratio based on the surface acoustic wave microfluidic chip according to claim 9, wherein: The standard microsphere represents a material with a preset material compression ratio and density.

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