Cell mechanical property measuring system and method based on ultrasonic radiation force

Abstract

The invention relates to a cell mechanical property measuring system and method based on ultrasonic radiation force, and the system comprises an objective table; the surface acoustic wave chip comprises a working area; the fixing unit is used for fixing the surface acoustic wave chip on an objective table and fixing a living superficial tissue on a working area of the surface acoustic wave chip; the signal generation unit is in signal connection with the surface acoustic wave chip and is used for enabling a working area of the surface acoustic wave chip to generate surface acoustic waves; and the image acquisition unit is opposite to the working area and is used for acquiring an image. Through the arrangement, non-contact control and mechanical characterization of cells in microvessels of superficial tissue of a living body are realized.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a cell mechanical property measurement system and method based on ultrasonic radiation force. Background Art

[0002] Cell mechanics is the study of the mechanical properties of cells, such as elasticity, viscoelasticity, and hardness, as well as the response of cells under mechanical forces. The main focus is on the mechanical properties of red blood cells, particularly their deformability and Young's modulus.

[0003] Existing methods for measuring the hardness and softness of red blood cells mainly include atomic force microscopy (AFM), microfluidics, optical tweezers, flow cytometry, etc. Both AFM and microfluidics require the extraction of cell samples from the organism, which is somewhat invasive. Then, the mechanical properties of the cells are measured in vitro, which makes it impossible to achieve in situ and continuous monitoring. Moreover, the in vitro measurement results are difficult to accurately reflect the true mechanical behavior of the cells in vivo. In addition, AFM requires the probe to directly contact the cell surface, which may cause mechanical damage to the cells, affecting the accuracy of the measurement results, and can only measure the local mechanical properties of the cells. Optical tweezers have a thermal effect that affects the activity of cells, and the equipment purchase and operation costs of flow cytometry are relatively high.

[0004] Therefore, the existing technology lacks a low-cost, contactless system and method for in situ and continuous monitoring of cell mechanical properties. Summary of the Invention

[0005] In order to solve the above problems, the present invention proposes a cell mechanical properties measurement system and method based on ultrasonic radiation force.

[0006] The technical solution adopted by the present invention is a cell mechanical property measurement system based on ultrasonic radiation force, comprising:

[0007] stage;

[0008] A surface acoustic wave chip including a working area;

[0009] a fixing unit, which is used to fix the surface acoustic wave chip on the stage and fix the superficial tissue of the living body on the working area of ​​the surface acoustic wave chip;

[0010] a signal generating unit, whose signal is connected to the surface acoustic wave chip and is used to generate surface acoustic waves in the working area of ​​the surface acoustic wave chip;

[0011] An image acquisition unit is configured to acquire images of the working area.

[0012] Preferably, the surface acoustic wave chip comprises:

[0013] A piezoelectric substrate, which is a 128° Y-cut lithium niobate single crystal, the piezoelectric substrate comprising a structural region and the working region adjacently arranged;

[0014] An interdigital transducer is disposed in the structural region of the piezoelectric substrate, and an electrode lead extends from one end of the interdigital transducer away from the working region.

[0015] Preferably, the number N of interdigital electrode pairs of the IDT is 40-60.

[0016] Preferably, the period length M and wavelength λ of the IDT are both 180μm-200μm, the acoustic aperture W is 10λ-100λ, and the thickness h is 180nm-220nm; the width d1 of the interdigital electrode fingers and the spacing d2 between adjacent fingers are both M / 4.

[0017] Preferably, the length and width of the working area are both between 8 mm and 12 mm.

[0018] Preferably, the output frequency of the signal generating unit is 15 MHz to 25 MHz, the amplitude is 200 Vpp to 700 mVpp, and the period is 180 ms to 220 ms.

[0019] Preferably, a constant temperature insulation layer is provided on the loading platform.

[0020] Preferably, the image acquisition unit includes:

[0021] a microscope for magnifying and imaging the superficial living tissue on the working area;

[0022] A high-speed camera that captures and records images within the microscope's field of view.

[0023] The present invention also discloses a method for measuring cell mechanical properties based on ultrasonic radiation force, using the above-mentioned cell mechanical properties measurement system based on ultrasonic radiation force, the method comprises the following steps:

[0024] S100, fixing the surface acoustic wave chip on a stage, and fixing the living superficial tissue on a working area of ​​the surface acoustic wave chip;

[0025] S200, generating surface acoustic waves in the working area of ​​the surface acoustic wave chip by the signal generating unit;

[0026] S300, recording the deformation process of cells in the superficial tissue of the living body by the image acquisition unit;

[0027] S400: Calculating the mechanical properties of the cell based on the deformation process of the cell.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. Compared with traditional in vitro measurement methods, the ultrasonic radiation force-based cell mechanical property measurement system of the present invention can directly and continuously measure the mechanical properties of cells (red blood cells, white blood cells, etc.) in living organisms. This avoids the differences between in vitro and in vivo environments, more realistically reflects the mechanical behavior of cells under physiological conditions, and provides a basis for more accurate biological research. By utilizing the acoustic radiation force generated by surface acoustic waves (SAW) to manipulate cells, the risk of cell damage is reduced, ensuring cell integrity and the accuracy of measurement results.

[0030] 2. Traditional ultrasound has insufficient resolution and manipulation accuracy, making it difficult to achieve precise manipulation of cells at the microscale. This is especially true in superficial tissues (such as thin and delicate structures like the mouse auricle). Penetration depth and focusing accuracy are limited, making it difficult to effectively manipulate cells within microvessels. However, surface acoustic wave (SAW) energy is primarily concentrated within a wavelength depth near the surface of the piezoelectric material. It can effectively penetrate superficial tissues, precisely manipulate and characterize cells within microvessels, overcoming the limitations of traditional ultrasound in superficial tissue applications. It is well-suited for manipulation of microscale objects, offering advantages such as non-contact operation, high manipulation accuracy, and good biocompatibility.

[0031] 3. The integration of AFM, microfluidic chips or traditional ultrasonic technology into a wearable platform and their application to micro-scale manipulation of superficial tissues presents great technical challenges. However, the present invention utilizes a wearable surface acoustic wave chip and effectively couples it to living superficial tissues through a stage and a fixing unit for in vivo experiments. By allowing sound waves to penetrate tissues for manipulation and measurement, non-invasive cell mechanics detection is achieved, greatly improving biocompatibility and application potential. Through wearable design and non-invasive measurement methods, interference with the organism is minimized, cell mechanics research in a more natural physiological state is achieved, and precise manipulation and mechanical characterization of red blood cells in microvessels of superficial tissues are achieved, providing new technical means for biological research and clinical applications. The measurement system also has the advantages of convenience and efficiency, making it suitable for use by a large population. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention is described in detail below with reference to the embodiments and accompanying drawings, in which:

[0033] Figure 1 This is a diagram of the surface acoustic wave chip structure;

[0034] Figure 2 is a schematic diagram of an interdigital transducer;

[0035] Figure 3 It is an experimental flow chart of the present invention;

[0036] Figure 4 This is a flow chart of the in vitro red blood cell extrusion experiment;

[0037] Figure 5 This is the flow chart of the in vivo red blood cell extrusion experiment;

[0038] Figure 6 This is a simulation diagram of red blood cell deformation;

[0039] Figure 7 This is a diagram of the blood vessels of experimental mice;

[0040] Figure 8 This is a microscopic image of the results of an in vitro red blood cell experiment.

[0041] 10. Piezoelectric substrate; 11. Working area; 20. Interdigital transducer; 30. PCB board. DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions, and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar components or components having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0043] The present invention aims to realize an in vivo red blood cell extrusion experiment, and uses ultrasound to measure the red blood cell deformation and calculate its Young's modulus, thereby achieving a non-destructive and painless measurement.

[0044] In one embodiment, a cell mechanical property measurement system based on ultrasonic radiation force includes a stage, a surface acoustic wave chip, a fixing unit, a signal generating unit, and an image acquisition unit. The stage provides a stable placement platform for the surface acoustic wave chip and the superficial tissue of a living body. The fixing unit is used to fix the surface acoustic wave chip on the stage. The surface acoustic wave chip includes a working area 11. The fixing unit is also used to fix the superficial tissue of a living body on the working area 11 of the surface acoustic wave chip. The fixing unit can be a tape, a mechanical clamp, etc. The signal generating unit is connected to the surface acoustic wave chip, and the signal generating unit is used to generate surface acoustic waves in the working area 11 of the surface acoustic wave chip. The signal generating unit may include a signal source and a power amplifier. The signal source is responsible for generating an electrical signal of a specific frequency. The power amplifier amplifies the electrical signal generated by the signal source. The amplified electrical signal is transmitted to the surface acoustic wave chip, thereby generating surface acoustic waves in the working area 11 of the surface acoustic wave chip. The image acquisition unit, which faces the working area 11, captures images and may include a microscope and a high-speed camera. The microscope is used to optically magnify the superficial living tissue on the SAW chip working area 11, clearly displaying microscopic structures such as microvessels and cells within the tissue. The high-speed camera captures and records, in real time, the dynamic deformation images and motion trajectories of cells within the microscope's field of view under the influence of SAW radiation forces.

[0045] During use, the surface acoustic wave chip is first firmly placed on the stage through the fixing unit, and then the living superficial tissue is precisely fixed on the working area of ​​the chip; then the signal generating unit is turned on to establish a signal connection with the surface acoustic wave chip, so that the chip working area generates specific surface acoustic waves, exerting acoustic radiation force on the cells in the tissue to cause them to deform; at the same time, the microscope in the image acquisition unit optically magnifies the working area, and the high-speed camera records the dynamic deformation image and motion trajectory of the cells under the action of the surface acoustic wave in real time. Subsequently, the mechanical properties of the cells, such as deformation ability and Young's modulus, are obtained by analyzing these image data.

[0046] Compared to traditional in vitro measurement methods, the ultrasonic radiation force-based cell mechanical property measurement system in this embodiment can directly and continuously measure the mechanical properties of living cells (red blood cells, white blood cells, etc.) in vivo. This avoids the differences between in vitro and in vivo environments, more realistically reflects the mechanical behavior of cells under physiological conditions, and provides a basis for more accurate biological research. By utilizing the acoustic radiation force generated by surface acoustic waves (SAW) to manipulate cells, the risk of cell damage is reduced, ensuring cell integrity and the accuracy of measurement results.

[0047] Traditional ultrasound has insufficient resolution and manipulation accuracy, making it difficult to achieve precise manipulation of cells at the microscale. This is especially true in superficial tissues (such as thin and delicate structures like the mouse auricle), where penetration depth and focusing accuracy are limited, making it difficult to effectively manipulate cells within microvessels. However, surface acoustic waves (SAWs), whose energy is primarily concentrated within a wavelength depth near the surface of the piezoelectric material, can effectively penetrate superficial tissues, precisely manipulate and characterize cells within microvessels, overcoming the limitations of traditional ultrasound in superficial tissue applications. They are well-suited for manipulation of microscale objects, offering advantages such as non-contact operation, high manipulation accuracy, and good biocompatibility.

[0048] There are great technical challenges in integrating AFM, microfluidic chips or traditional ultrasonic technology into a wearable platform and applying them to microscale manipulation of superficial tissue. However, this embodiment utilizes a wearable surface acoustic wave chip and effectively couples it to the superficial tissue of a living body through a stage and a fixing unit for in vivo experiments. By allowing sound waves to penetrate the tissue for manipulation and measurement, non-invasive cell mechanics detection is achieved, greatly improving biocompatibility and application potential. Through wearable design and non-invasive measurement methods, interference with the organism is minimized, and cell mechanics research in a more natural physiological state is achieved, thereby achieving precise manipulation and mechanical characterization of red blood cells in microvessels of superficial tissues, providing new technical means for biological research and clinical applications. The measurement system also has the advantages of convenience and efficiency, making it suitable for use by a large population.

[0049] In one embodiment, the surface acoustic wave chip includes a piezoelectric substrate 10 and an interdigital transducer 20. Specifically, since the surface acoustic wave chip designed in the present invention needs to utilize the energy of the surface acoustic wave, the piezoelectric material selected must have a high electromechanical coupling coefficient and a low propagation loss. The piezoelectric single crystal material of the surface wave chip used in the present invention is 128°Y lithium niobate, which has a high electromechanical coupling coefficient and the energy loss of the sound wave during propagation is small. Its main parameters are: the electromechanical coupling coefficient is 5.5%, the temperature coefficient is -75×10-6 / ℃, and the surface acoustic wave propagation speed is 3990m / s. The piezoelectric substrate 10 includes a structural area and a working area 11 arranged adjacent to each other, the interdigital transducer 20 is placed in the structural area of ​​the piezoelectric substrate 10, and the electrode lead extends from the end of the interdigital transducer 20 away from the working area 11. The specific structure of the surface acoustic wave chip is as follows: Figure 1 As shown, metal electrode leads extend from the bus bars of the IDT 20 and are connected to pads at the edge of the chip for applying radio frequency (RF) drive signals through external connections (such as gold wire bonding or probe contacts).

[0050] The interdigital transducer is the most important part of the surface acoustic wave chip and determines the performance of the surface acoustic wave chip. Its main structure is as follows Figure 2As shown, the main parameters include the number of finger pairs N, the acoustic aperture W and the periodic segment length M. In acoustic devices, there is a relationship between the -3dB acoustic bandwidth and the number of finger electrode pairs Δf-3dB / f0=Cm / N, where Cm is a material-related coefficient, which is usually 0.3184 for 128°Y lithium niobate. When the number of finger pairs N is too large, surface acoustic waves are difficult to output and will cause mutual interference. When it is too small, the surface acoustic wave formation is unstable. It is preferred that the number of finger electrode pairs N of the fork finger transducer is 40 to 60. The size of the aperture will also affect the performance of the fork finger transducer. If the aperture is too small, strong diffraction and second-order effects will occur. If the aperture is too large, a large equivalent capacitance will be generated. Therefore, the general design considers the range of 10 to 100 wavelengths. The period length affects the frequency of the IDT. The present invention employs a uniform interdigital design with d1 = d2 = M / 4 and M = λ = v / f0. The period length M and wavelength λ of the IDT are preferably between 180 μm and 200 μm. The interdigital electrode finger width d1 and the spacing d2 between adjacent fingers are both M / 4. The vibration intensity of the excited surface acoustic waves decreases with increasing electrode thickness, and so does the energy. Therefore, the thickness h is preferably between 180 nm and 220 nm.

[0051] In summary, the transducer parameters used in the present invention are more preferably d1 = d2 = 50 μm, M = 200 μm, N = 50 pairs, and the interdigital thickness is more preferably 200 nm.

[0052] Furthermore, the length and width of the working area are both between 8mm and 12mm. This size range can accommodate a certain number of cells, facilitating mechanical property measurements of multiple cells, improving the representativeness and reliability of the measurement data. It is also compatible with the size of common superficial tissue samples in vivo, providing good coverage of cells in the tissue and facilitating in situ cell measurements.

[0053] In one embodiment, the signal generation unit has an output frequency of 15 MHz to 25 MHz. This frequency range matches the physical properties of the piezoelectric substrate (a 128° Y-cut lithium niobate single crystal) and the interdigital transducer in the surface acoustic wave chip, enabling efficient excitation of surface acoustic waves. Within this frequency range, the piezoelectric substrate, leveraging its inherent piezoelectric effect, effectively converts electrical signals into surface acoustic waves, ensuring that the surface acoustic waves have sufficient energy and an appropriate wavelength to generate a suitable acoustic radiation force on the cells. The amplitude is 200 Vpp to 700 mVpp. Within this range, the acoustic radiation force can significantly deform the cells, but not be so strong as to cause irreversible damage, thereby ensuring accurate measurement of the cells' mechanical properties. The period is 180 ms to 220 ms. If the period is too short, the cells may not have enough time to fully deform, resulting in inaccurate measurement results; if the period is too long, measurement efficiency will be reduced. This period range ensures sufficient time for the cells to deform while allowing for multiple measurements to be completed within a certain timeframe, improving measurement efficiency and data reliability.

[0054] In one embodiment, a constant temperature insulation layer is installed on the stage. This layer maintains a stable temperature environment for the cells, preventing temperature fluctuations that could cause a decrease in cell activity or altered physiological functions. This ensures that the measurement results truly reflect the mechanical properties of the cells, improving measurement accuracy. Stable temperature is crucial for experimental reproducibility.

[0055] In one embodiment, a method for measuring cell mechanical properties based on ultrasonic radiation force, using the cell mechanical properties measurement system based on ultrasonic radiation force in the above embodiment, comprises the following steps:

[0056] S100, fixing the surface acoustic wave chip on the stage, and fixing the superficial tissue of the living body on the working area of ​​the surface acoustic wave chip;

[0057] S200, generating a surface acoustic wave in the working area of ​​the surface acoustic wave chip by using a signal generating unit;

[0058] S300, recording the deformation process of cells in the superficial tissue of the living body by an image acquisition unit;

[0059] S400: Calculate the mechanical properties of the cell based on the cell's deformation process.

[0060] This method directly affixes superficial living tissue to the chip's working area for measurement, avoiding the need to extract cell samples from the organism and preserving the cells' true in vivo physiological environment to the greatest extent possible. The image acquisition unit records the cell deformation process in real time at a high frame rate, enabling continuous monitoring of cell mechanical properties. By utilizing the acoustic radiation force of surface acoustic waves acting on cells, this method avoids the potential mechanical damage caused by direct contact of the atomic force microscope probe with the cell surface. Compared to optical tweezers, this method avoids the problem of thermal effects affecting cell viability. Compared to flow cytometry, which requires expensive flow cytometer equipment, this measurement method utilizes relatively simple and low-cost equipment. During operation, this method does not require the large amounts of expensive reagents and consumables typically consumed by flow cytometry. By recording and analyzing the cell deformation process in detail, multiple parameters reflecting the cell's mechanical properties, such as deformability and Young's modulus, can be obtained. These parameters comprehensively describe the cell's mechanical properties from different perspectives, providing a wealth of information for a deeper understanding of its physiological functions and changes.

[0061] The present invention utilizes photolithography technology to manufacture surface acoustic wave chips and microcavities, and the chips can generate surface acoustic wave traveling waves with a frequency of 20MHz. Figure 3 As shown, in vitro experiments were first performed to verify the feasibility of the chip, and then the chip was used for in vivo experiments.

[0062] The specific technical steps are as follows:

[0063] Step 1: Preparation of surface acoustic wave chip.

[0064] It mainly includes the following steps:

[0065] (1) Cleaning and surface treatment of the piezoelectric substrate 10, which is usually achieved by chemical cleaning or plasma cleaning.

[0066] (2) Spin coating of photoresist: A layer of photosensitive chemical, i.e., photoresist, is evenly coated on the surface of the piezoelectric substrate 10. The coating method is usually spin coating, that is, the photoresist is dropped onto the rotating piezoelectric substrate 10 and formed into a uniform thin layer using centrifugal force. The spin coating is first performed at 500 revolutions within 10 seconds, and then at 3000 revolutions within 30 seconds.

[0067] (3) Soft baking: After coating is completed, soft baking (pre-baking) is required, usually on a hot plate at 95°C for 15 minutes. The purpose of this step is to remove the solvent in the photoresist, enhance its adhesion, and prepare for the next exposure step.

[0068] (4) Exposure: Align the pre-treated piezoelectric substrate 10 with the mask and then irradiate with ultraviolet light. The pattern on the mask will block part of the light, thereby forming a pattern on the photoresist. The exposure dose is selected as 180mJ / cm 2The exposed portion of the photoresist becomes soluble. The mask and the IDT 20 have the same shape.

[0069] (5) Post-baking: Post-baking is usually performed to further cure the photoresist, enhance its chemical and heat resistance, and prepare for subsequent etching or metal deposition steps.

[0070] (6) Development: Place the exposed piezoelectric substrate 10 in a developer for 1-3 minutes to remove the soluble photoresist. Rinse and soak with water, then rinse with isopropyl alcohol and blow dry with nitrogen. The pattern on the mask is then transferred to the piezoelectric substrate 10.

[0071] (7) Inspection: After the entire lithography process is completed, an inspection is required to confirm the accuracy and integrity of the pattern. This is usually done using a microscope or other inspection equipment.

[0072] (8) Sputtering: Before sputtering, the piezoelectric substrate 10 must be thoroughly cleaned to remove dust, grease, and other contaminants from the surface. This is typically accomplished by chemical cleaning and plasma cleaning. The cleaned piezoelectric substrate 10 and target are loaded into the sputtering equipment. An inert gas, such as argon, is introduced into the vacuum environment.

[0073] (9) Etching: Etching is performed using a dry etching method.

[0074] (10) Removing photoresist: After the electrode manufacturing process is completed, the remaining photoresist needs to be removed.

[0075] (11) Testing and packaging: The finished SAW chip needs to undergo performance testing to confirm that its characteristics such as frequency response and sensitivity meet the requirements. It is then packaged to protect the chip and facilitate integration with electronic devices.

[0076] (12) Connection: Fix the surface acoustic wave chip that has passed the test on the PCB board 30 with UV curing glue. The size of the PCB board is slightly larger than the chip, about 2cm×2.5cm. Connect one end of the chip to the positive electrode on the PCB and the other end to the negative electrode on the PCB.

[0077] Step 2: Microcavity design.

[0078] In order to better simulate the in vivo vascular environment, the microcavity is designed to be approximately 5-10um, which is equivalent to the diameter of the blood vessel. Since surface acoustic waves will attenuate during transmission, the construction width on both sides of the microcavity is controlled at 2mm. Soft lithography technology is used to construct the microcavity to form a channel mold. Polydimethylsiloxane (PDMS) is selected as the microfluidic channel material to ensure optimal biocompatibility. After it solidifies, the PDMS structure is carefully peeled off from the mold. Finally, the PDMS microfluidic channel is bonded to the previously manufactured SAW device to complete the assembly of the core chip.

[0079] Microcavity preparation.

[0080] The preparation of microcavities is similar to that of chips, and the specific steps are as follows:

[0081] First, the silicon wafer surface is cleaned to remove contaminants. The wafer is placed on a homogenizer and SU-83025 photoresist is poured onto it. A spin coater is used to evenly distribute the photoresist. A soft bake is performed by baking the wafer on a hot plate at 95°C for 15 minutes. Next, the designed mask and wafer are placed, and the exposure is aligned using a photolithography machine. A post-exposure bake is then performed, with the wafer baked at 80°C for 1 minute and then at 95°C for 8 minutes. After cooling, it is placed in a developer solution, soaked in water, and then washed with isopropyl alcohol. A hard bake is performed by baking on a hot plate at 95°C for 15 minutes, followed by an oxygen plasma treatment for 2 minutes to remove the photoresist. Finally, a stepper is used to measure the thickness. For the fabrication of the polydimethylsiloxane (PDMS) channels, the base and curing agent are mixed in a 10:1 weight ratio, and a vacuum pump is used to remove air bubbles. The PDMS is poured into a silicon wafer mold and cured at 80°C in an incubator for 1 hour. A 0.75 mm diameter punch is used to drill holes to create the inlet and outlet ports. The prefabricated PDMS microporous structure and glass substrate were treated with air plasma for 5 minutes to create hydroxyl functional groups on the surface. The treated surfaces were then permanently bonded together. The bonded microchannels were then placed on a PDMS film with alignment marks and heated in an oven for curing, completing the microcavity fabrication. Finally, the prepared cavity was bonded onto a previously fabricated surface acoustic wave chip to complete the core chip assembly.

[0082] Step 3: Design of in vitro experimental platform.

[0083] The prefabricated PDMS cavity with a width of 10 μm and the designed surface acoustic wave chip were treated in an air plasma cleaner to generate hydroxyl functional groups on the surface. The treated surfaces were then permanently bonded together, and the treated microcavity was then firmly bonded to the PDMS.

[0084] Specific in vitro experimental steps include Figure 4As shown, the output of the signal source is connected to the input of a power amplifier, and the output of the power amplifier is connected to a surface acoustic wave chip. The surface acoustic wave chip is mounted on a microscope for easy observation. The flow rate of the liquid in the microcavity is controlled by a microfluidic pump to simulate the flow rate of blood. The signal generator is turned on, and the waveform is set to a pulse wave, the signal frequency is set to 20MHz, the amplitude is set to 200-700mVpp, and the period is 200ms. When the acoustic field is applied, red blood cells are affected by the acoustic radiation force and are pushed against the cavity wall and squeezed. The force applied to the red blood cells is controlled by adjusting the signal amplitude. At lower flow rates, red blood cells are easily captured and can undergo corresponding deformation. The change in the relative aspect ratio of the red blood cells before and after deformation is recorded. Cell squeezing experiments are conducted at different power levels. The higher the power, the greater the cell deformation. The cell deformation process is recorded.

[0085] Step 4: AFM measurement of the Young's modulus of red blood cells in vitro.

[0086] Atomic force microscopy (AFM) is the gold standard for measuring Young's modulus of cells. This paper uses measurements of the Young's modulus of red blood cells to support the acoustic method. Atomic force microscopy (AFM) is the recognized gold standard for measuring the Young's modulus of single cells. This paper uses AFM to directly measure the Young's modulus of red blood cells in vitro. The results obtained will serve as a reference for verifying and calibrating the accuracy of the Young's modulus values ​​calculated using the acoustic method.

[0087] The AFM measurement process primarily involves the following steps: First, a fresh red blood cell sample is prepared and fixed to a suitable substrate using a cell adhesion reagent to ensure cell stability during the measurement. The sample is then placed under the atomic force microscope, and an AFM probe with an appropriate elastic modulus is selected. The probe's elastic modulus must be precisely calibrated before the experiment.

[0088] During measurement, the AFM system controls the probe to gradually approach the RBC surface with nanometer-level precision. When the probe tip contacts the cell surface, the probe bends due to the cell's elastic resistance. The AFM system accurately records the probe deflection and applied force. As the probe penetrates further into the cell, the applied force gradually increases, forming the loading portion of the force-indentation curve. Subsequently, as the probe begins to withdraw, the applied force decreases with indentation depth, forming the unloading portion of the force-indentation curve. The loading curve is typically selected for analysis. Using the Hertz model in contact mechanics, a nonlinear fit is performed on the loading portion of the force-indentation curve to extract the RBC's Young's modulus. The Hertz model describes the force-indentation relationship when a spherical indenter presses into an elastic half-space. By fitting experimental data, the Young's modulus, which is related to cell elasticity, can be calculated. It is important to note that the RBC's Young's modulus may not be constant but rather depends on indentation depth. Typically, at low indentation depths, it primarily reflects the elasticity of the cell membrane. As indentation depth increases, the contribution of the cytoskeleton and cytoplasm increases, and the Young's modulus also changes accordingly. Therefore, when analyzing AFM data, it is necessary to consider the effect of indentation depth on Young's modulus and make a comprehensive interpretation based on the characteristics of the force-indentation curve.

[0089] Step 5: Design of in vivo experimental platform.

[0090] Mice aged 4-10 months and diabetic model mice were anesthetized separately and connected to a life support system to maintain their normal heartbeat and breathing. The mice were placed on an inverted microscope stage with a 37°C warming pad. The prepared SAW chip (without integrated PDMS channels) was placed on the microscope stage. The mouse's ear was carefully flattened and fixed in the central working area of ​​the chip (i.e., the blank area to the right of the IDT), ensuring good contact between the ear skin and the chip surface. A small amount of coupling agent such as ultrasound gel or saline can be applied to enhance sound wave transmission. In order to prevent slight movements during the experiment, the surface acoustic wave chip was fixed with tape, and the ears of anesthetized normal mice and diabetic mice were placed in the middle of the chip. The positions of the mice and the chip were fixed to prevent relative movement during the experiment. Adjust the focal plane of the microscope to clearly observe the capillaries and blood flow in the ear tissue, such as Figure 7 As shown in FIG. A signal source and a power amplifier are connected to the SAW chip. A high-speed camera system is used to record blood flow images of the selected capillary segment at a high frame rate, for example, 2000 frames per second.

[0091] During the experiment, if Figure 5As shown, the signal source is turned on, the frequency set to 20 MHz, and the power is gradually increased to excite the SAW chip. The surface acoustic waves are coupled through the thin layer of ear tissue into the capillary region. Under a microscope, real-time observation and recording is performed to determine whether the acoustic radiation force on red blood cells flowing through this region causes changes in velocity, trajectory deviation, or morphological changes (extrusion deformation). The deformation of the red blood cells is recorded at different power levels, and image sequences are acquired for subsequent analysis.

[0092] Step 6: Calculate the red blood cell deformation and Young's modulus.

[0093] Based on the data and images collected during the in vitro experiments, red blood cell deformation was analyzed and the Young's modulus of red blood cells was calculated using a formula. Furthermore, the Young's modulus of cells measured using AFM was statistically analyzed and compared with the acoustic method used in this study.

[0094] Definition of red blood cell shape - relative aspect ratio change rate: ΔAR = (AR after -AR before ) / AR before , where AR = major axis / minor axis. Young's modulus definition: Describes the strain response of a material to a uniaxial stress in the direction of that stress, within the range of Hooke's law. E = stress / strain (unit: Pa), where stress is the acoustic radiation force per unit area of ​​the cell, and strain represents the relative deformation of the cell under the action of the force.

[0095] Based on the erythrocyte deformation data collected in the in vivo experiment, the erythrocyte deformation amounts of normal mice and diabetic mice were counted.

[0096] The cell mechanical property measurement system based on ultrasonic radiation force of the present invention has realized for the first time the non-contact manipulation and mechanical characterization of red blood cells in microvessels of superficial tissues of living beings. The wearability of the surface acoustic wave chip enables it to be attached to a living body for in-situ and continuous monitoring; in vivo application has broken through the limitations of the in vitro application of traditional acoustofluidic technology and directly realized the manipulation of microvascular red blood cells in superficial tissues such as the mouse auricle; the application of surface acoustic wave (SAW) technology has realized non-contact cell squeezing and measurement, reducing cell damage; experiments have verified the platform's ability to distinguish the mechanical properties of red blood cells in different mice. In addition, the cell mechanical property measurement method based on ultrasonic radiation force proposed in the present invention provides a new non-contact measurement method for cell mechanics research.

[0097] In a more specific embodiment, the present invention has been verified through experiments, simulations, and use, and has achieved positive results, fully demonstrating its feasibility and effectiveness. The specific results are as follows:

[0098] Feasibility verification: In vitro experiments successfully demonstrated the feasibility of using SAW-driven microfluidic chips to squeeze and deform red blood cells. Figure 8 The comparative experiments clearly showed that red blood cells underwent significant deformation when ultrasound was turned on.

[0099] Numerical simulation verification: Figure 6 Numerical simulations were performed to predict the dynamic behavior of single cells under the action of acoustic radiation forces.

[0100] Effective use of the wearable measurement platform: The experiment successfully integrated an interdigital transducer onto a piezoelectric substrate to create a wearable chip, which was then effectively coupled to the mouse auricle for in vivo experiments, demonstrating the feasibility of the wearable platform in practical operation and use. In vivo experiments successfully used the advanced Segment Anything Model (SAM) to quantitatively analyze cell deformation in complex images, demonstrating the effectiveness of using advanced image processing techniques to assist in analyzing experimental results and improving the accuracy and efficiency of data analysis.

[0101] In this specification, the use of terms such as "Embodiment 1," "this embodiment," and "in one embodiment" indicates that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in the invention or at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example; furthermore, the specific features, structures, materials, or characteristics described may be appropriately combined in any one or more embodiments or examples.

[0102] In the description of this specification, the terms "connect," "install," "fix," "dispose," and "have" are to be understood in a broad sense. For example, "connect" can mean a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in this application based on specific circumstances.

[0103] In the description of this specification, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising the element.

[0104] The above description of the embodiments is to facilitate ordinary technicians in this technical field to understand and apply the technology of this case. People familiar with the technology in this field can obviously make various modifications to these examples easily and apply the general principles described here to other embodiments without having to go through creative work. Therefore, this case is not limited to the above embodiments. Modifications to the following situations should all be within the scope of protection of this case: ① A new technical solution implemented based on the technical solution of the present invention and combined with existing common knowledge, the technical effect produced by the new technical solution does not exceed the technical effect of the present invention; ② The equivalent replacement of some features of the technical solution of the present invention with the known technology, the technical effect produced is the same as the technical effect of the present invention; ③ The technical solution of the present invention is expandable, and the substantive content of the expanded technical solution does not exceed the technical solution of the present invention; ④ The equivalent transformation made by the content of the description and drawings of the present invention is directly or indirectly applied to other related technical fields.

Claims

1. A cell mechanical properties measurement system based on ultrasonic radiation force, characterized in that: include: stage; A surface acoustic wave chip including a working area; a fixing unit, which is used to fix the surface acoustic wave chip on the stage and fix the superficial tissue of the living body on the working area of ​​the surface acoustic wave chip; a signal generating unit, whose signal is connected to the surface acoustic wave chip and is used to generate surface acoustic waves in the working area of ​​the surface acoustic wave chip; An image acquisition unit is configured to acquire images of the working area.

2. The measurement system according to claim 1, characterized in that The surface acoustic wave chip comprises: A piezoelectric substrate, which is a 128° Y-cut lithium niobate single crystal, the piezoelectric substrate comprising a structural region and the working region adjacently arranged; An interdigital transducer is disposed in the structural region of the piezoelectric substrate, and an electrode lead extends from one end of the interdigital transducer away from the working region.

3. The measurement system according to claim 2, characterized in that The number N of interdigital electrode pairs of the IDT is 40-60.

4. The measurement system according to claim 3, characterized in that The period length M and wavelength λ of the IDT are both 180 μm to 200 μm, the acoustic aperture W is 10λ to 100λ, and the thickness h is 180nm to 220nm; the width d1 of the IDT fingers and the spacing d2 between adjacent fingers are both M / 4.

5. The measurement system according to claim 4, characterized in that The length and width of the working area are both between 8 mm and 12 mm.

6. The measurement system according to claim 1, characterized in that The output frequency of the signal generating unit is 15 MHz to 25 MHz, the amplitude is 200 Vpp to 700 mVpp, and the period is 180 ms to 220 ms.

7. The measurement system according to claim 1, characterized in that A constant temperature insulation layer is provided on the loading platform.

8. The measurement system according to claim 1, wherein: The image acquisition unit includes: a microscope for magnifying and imaging the superficial living tissue on the working area; A high-speed camera that captures and records images within the microscope's field of view.

9. A method for measuring cell mechanical properties based on ultrasonic radiation force, using the cell mechanical properties measurement system based on ultrasonic radiation force according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: S100, fixing the surface acoustic wave chip on a stage, and fixing the living superficial tissue on a working area of ​​the surface acoustic wave chip; S200, generating surface acoustic waves in the working area of ​​the surface acoustic wave chip by the signal generating unit; S300, recording the deformation process of cells in the superficial tissue of the living body by the image acquisition unit; S400: Calculating the mechanical properties of the cell based on the deformation process of the cell.

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