A cellular biomechanical method for differentiating pyroptosis and apoptosis based on dual-resonance piezoelectric technology

Abstract

This invention discloses a cellular mechanics method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology. The invention utilizes a quartz crystal microbalance to monitor in real-time the changes in frequency and resistance of HeLa and HUVECs cell populations, which exhibit no significant cell-cell interactions, adhering to the surface of AT-cut and BT-cut dual-cut quartz crystal chips, during the entire process of pyroptosis and apoptosis. This allows for the measurement of the real-time changes in surface stress exerted on the quartz crystal by the cell populations during pyroptosis and apoptosis, establishing a method for identifying pyroptosis and apoptosis based on differences in cellular mechanical responses.

Description

Technical Field

[0001] This invention relates to a method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology. It is a novel cellular mechanics method for identifying pyroptosis and apoptosis by real-time and dynamic monitoring of the mechanical response changes of HeLa and HUVECs cell populations that adhere to the surface of AT-cut and BT-cut double-cut quartz crystals without obvious cell-cell interactions during pyroptosis and apoptosis. Background Technology

[0002] Cell death, as the endpoint of cellular life and an important protective mechanism in organisms, participates in many life activities. Programmed cell death (PCD) is a common form of cell death regulated by genetics. Apoptosis, as a programmed cell death mechanism, is closely related to the occurrence and development of many diseases, including cardiovascular diseases such as heart failure, atherosclerosis, and cancer. However, currently, cancer cells are increasingly escaping drug-induced apoptosis, leading to poor responses to tumor intervention therapies. Pyroptosis offers a potential solution to these poor responses and shows the potential to bypass apoptosis and activate tumor-specific immune responses. Furthermore, pyroptosis is also a trigger for cardiovascular diseases, playing a crucial role in their pathogenesis. However, the biological functions and responses induced by pyroptosis and apoptosis are distinctly different and influence different disease progressions. Therefore, using appropriate methods to differentiate between pyroptosis and apoptosis is indispensable for drug efficacy evaluation and new drug development.

[0003] Currently, techniques for differentiating pyroptosis and apoptosis primarily rely on differences in morphology, activation pathways, and biochemical products. While optical detection methods based on morphological differences are simple and direct, their identification criteria are relatively subjective and can only observe cell death in a very small area at any given time, making quantitative measurement impossible. Detection methods based on differences in biochemical products and activation pathways offer the advantage of quantitative or semi-quantitative measurement. Examples include detecting the expression of key proteins involved in apoptosis and pyroptosis using Western blotting, ELISA, and immunofluorescence, or detecting gene markers using DNA gel electrophoresis, RT-qPCR, and TUNEL techniques. These methods provide more accurate results, but they are endpoint-based and prone to false positives. Flow cytometry, currently the most widely used method for detecting different cell death pathways, is expensive and difficult to operate, requiring specialized training. Furthermore, flow cytometry detection is not real-time. Therefore, a technique that simultaneously offers real-time, non-destructive, highly sensitive, and easy-to-operate identification of cell death pathways is urgently needed in this field.

[0004] In recent years, cell mechanics techniques have been applied to the detection of cell death mechanisms, such as atomic force microscopy (AFM) and traction force microscopy (AFM). However, these techniques are still based on endpoint testing, which cannot dynamically and in real-time acquire the mechanical information of cells during the death process, nor can they avoid interference with the natural physiological state of cells during the measurement process, affecting the accuracy and effectiveness of the detection. In addition, these techniques are mostly used for the detection of single-cell death mechanisms. This technology provides a cell mechanics method for identifying cell population death mechanisms that can be measured in real-time without affecting the physiological state of cells. It identifies pyroptosis and apoptosis by the changes in surface stress exerted by cells on a quartz crystal, and establishes a cell mechanics model for identifying apoptosis and pyroptosis in a real-time and non-destructive manner. This advantage is of great help in the fields of new drug development and drug efficacy evaluation. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a cellular mechanical method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0007] The cellular mechanical method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology includes the following steps:

[0008] (1) Place AT-cut and BT-cut quartz crystals in a detection cell. The AT-cut and BT-cut quartz crystals have the same fundamental frequency, surface morphology and consistent surface-adhesive molecular modification.

[0009] (2) After cells are added and the cells are stably adhered to the quartz crystal interface (12 hours), lipopolysaccharide (LPS) and cholera toxin subunit B (CTB) are added to the detection cell to induce pyroptosis, and tumor necrosis factor-α (TNF-α) and cycloheximide (CHX) are added to induce apoptosis. The frequency and resistance changes of AT-cut and BT-cut quartz crystals are monitored in real time throughout the process. Preferably, 40 μL of LPS and 20 μL of Cholera toxin B subunit are added to the detection cell to induce pyroptosis, or 40 μL of TNF-α and 20 μL of cycloheximide are added to induce apoptosis.

[0010] (3) The surface stress (ΔS) generated by the cells on the quartz crystal is determined by the following formula:

[0011]

[0012] Among them, K AT =2.75×10 -12 cm 2 dyn -1 K BT = -2.65 × 10 -12 cm 2 dyn -1 The stress coefficients of AT-cut and BT-cut quartz crystals are respectively; t q AT and t q BT These are the thicknesses of the quartz crystal, expressed in cm; f0 AT =f0 BT =9MHz is the original resonant frequency of the quartz crystal resonator; Δf t AT and Δf t BT It is the frequency shift after subtracting the corresponding f0 from the new frequency shift caused by the surface stress of the resonator at time t.

[0013] (4) The storage modulus (G′) and loss modulus (G″) of the cell are determined using the following formulas:

[0014]

[0015] Where Γ is the half bandwidth, ρ c For cell density, based on the assumption that it is consistent with the density of pure water: ρ 水 =1.00g / cm 3 Z q Z represents the acoustic impedance of the quartz crystal. q AT =8.84*10 5 g / cm·s,Z q BT =1.35*10 6 g / cm·s,ρ c =0.9933g / cm 3 The half-bandwidth Γ and the dynamic resistance R have the following relationship: ΔΓ=ΔR / 4πL q L q The inductance of a quartz crystal in a cell culture medium;

[0016] (5) Identify pyroptosis and apoptosis based on the difference in ΔS changes during pyroptosis and apoptosis, and simultaneously obtain the changes in cell viscoelasticity during pyroptosis and apoptosis.

[0017] The present invention will be further described below:

[0018] This invention utilizes both AT-cut and BT-cut dual-type quartz crystal chips to monitor the dynamic changes in surface stress (ΔS) and cell population storage modulus (G′) and loss modulus (G″) exerted on the quartz crystal during pyroptosis and apoptosis in HeLa and HUVECs cells in real time and over a long period without affecting cell function. The difference in surface stress (ΔS) exerted by cells on the quartz crystal is used to differentiate between pyroptosis and apoptosis. The AT-cut and BT-cut quartz crystal resonators have the same stress coefficient, but with opposite signs. The stress changes exerted by cells on the quartz crystal can be calculated based on the different frequency shifts of the two resonators at the same interface. Therefore, the fundamental premise of this technology is to ensure that the surface mass changes, solution viscosity, and electrode roughness of the AT-cut and BT-cut quartz crystals are consistent during the monitoring process. Based on these necessary and sufficient conditions, the magnitude and direction of the stress are calculated according to the different responses generated by transverse stress on the AT-cut and BT-cut quartz crystals.

[0019] This invention employs a 9MHz quartz crystal double-cut resonator with a 100nm gold plating, a compatible Teflon detection cell, and a four-channel quartz crystal microbalance network analyzer 250B to dynamically monitor changes in the mechanical response of cells during pyroptosis and apoptosis. The quartz crystal resonator chip used for measurement has a diameter of 12.5mm, and the gold electrode plating has a diameter of 5mm.

[0020] Specifically, the cellular mechanical method for distinguishing pyroptosis and apoptosis based on dual-resonance piezoelectric technology is as follows:

[0021] AT-cut and BT-cut quartz crystals, having the same frequency, surface state, and / or modified with the same surface adhesion molecules, are placed in a detection cell. Test cells are added to the culture dish or detection cell, and the cell traction force is determined using the following formula:

[0022]

[0023] In the formula, K AT =2.75×10 -12 cm 2 dyn -1 K BT = -2.65 × 10 -12 cm 2 dyn -1 The stress coefficients of AT-cut and BT-cut quartz crystals are respectively; t q AT and t q BT These are the thicknesses of the quartz crystal, expressed in cm; f0 AT =f0 BT =9MHz is the original resonant frequency of the quartz crystal resonator; Δft AT and Δf t BT It is the frequency shift after subtracting the corresponding f0 from the new frequency shift caused by the surface stress of the resonator at time t.

[0024] Substituting the above values ​​into formula (1), it can be simplified to:

[0025] ΔS=380.8Δf AT -582.2Δf BT (2)

[0026] According to formula (2), the unit of ΔS is dyne / cm. ΔS>0 corresponds to the cell group adhering to the surface of the quartz crystal being in a state of tensile stress. That is, the cells exert a contractile traction force on the bottom matrix through the adhesion spot complex during the spreading and adhesion process. This process causes the quartz crystal to be subjected to compressive stress, and ΔS is positive. When ΔS<0, the cell group adhering to the surface of the quartz crystal is in a state of compressive stress. That is, the protruding force in the opposite direction to the contractile traction force is dominant, causing the quartz crystal to be subjected to tensile stress, and ΔS is negative.

[0027] Furthermore, since the operating frequency of quartz crystals is in the megahertz range, the attenuation length of its thickness shear wave is much smaller than that of a cell, and a living cell can be considered as a semi-infinite viscoelastic load. Therefore, the viscoelastic modulus of a cell can be determined using the following formula:

[0028]

[0029] In the formula, Z q AT =8.84*10 5 g / cm·s,Z q BT =1.35*10 6 g / cm·s represents the acoustic impedance of AT-cut and BT-cut quartz crystals, respectively; ρ c =0.9933g / cm 3 Let L be the cell density, assuming the cell density is the same as the density of pure water; q f0 represents the dynamic inductance of the quartz crystal being measured. AT =f0 BT =9MHz is the original resonant frequency of the quartz crystal resonator. Δf and ΔR are the frequency shift and dynamic resistance caused by the uniform viscoelastic layer on the surface of the quartz crystal relative to air, respectively.

[0030] The frequency shift and resistance at 9MHz AT and BT cuts caused by the culture medium are known to be:

[0031]

[0032] The frequency shift caused by transverse stress in AT and BT shears is:

[0033]

[0034] Therefore, by subtracting the frequency shift caused by transverse stress from the total frequency shift, we can obtain the frequency shift caused by cellular viscoelasticity as follows:

[0035]

[0036] In summary, by subtracting the frequency shift and dynamic resistance caused by the culture medium from the frequency shift and dynamic resistance caused by cell viscoelasticity, we can obtain the corrected frequency shift (Δf) and dynamic resistance (ΔR):

[0037]

[0038]

[0039] Formula (9-12) and L AT =10.7*10 -3 H, L BT =40.1*10 -3 Substituting H into formulas (3) and (4), we can calculate:

[0040] G′(AT)=0.5297ΔR 2 -0.009576Δf 2 (13)

[0041] G"(AT)=-0.1425ΔfΔR (14)

[0042] G′(BT)=-0.0879ΔR 2 -0.02233Δf 2 (15)

[0043] G″(BT)=-0.08868ΔfΔR (16)

[0044] The method of this invention allows for real-time, simultaneous, quantitative, and continuous measurement of the forces generated by cells and their viscoelastic responses during pyroptosis and apoptosis. Pyroptosis and apoptosis can be distinguished based on the differences in the cell's response to surface stress applied to a quartz crystal. When cells are stably adhered to the surface of a quartz crystal, the cells exert compressive stress on the crystal under the dominance of traction force, at which point ΔS is positive. When pyroptosis occurs, the compressive stress on the quartz crystal transforms into tensile stress, meaning the cell exerts a protruding force in the opposite direction to the traction force, and ΔS changes from positive to negative. Conversely, when apoptosis occurs, the compressive stress on the quartz crystal further increases, but the cell still exerts a dominant traction force, and the direction of the force applied to the quartz crystal remains unchanged, so ΔS remains positive. Attached Figure Description

[0045] Figure 1 The curves show the adhesion of 2000 HeLa cells to AT-cut and BT-cut quartz crystal chips, the dynamic QCM response during LPS and CTB pyroptosis induction, and the changes in cell stress and viscoelasticity. (A) Cell adhesion on AT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by LPS and CTB; (B) Cell adhesion on BT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by LPS and CTB; (C) HeLa cell adhesion and stress changes applied to the quartz crystal during LPS and CTB induction; (D) Western blotting detection of pyroptosis of HeLa cells induced by LPS and CTB; (E) Cell adhesion on AT-cut quartz crystal chips and storage modulus changes induced by LPS and CTB; (F) Cell adhesion on BT-cut quartz crystal chips and storage modulus changes induced by LPS and CTB; (G) Cell adhesion on AT-cut quartz crystal chips and loss modulus changes induced by LPS and CTB; (H) Cell adhesion on BT-cut quartz crystal chips and loss modulus changes induced by LPS and CTB.

[0046] Figure 2 The curves show the adhesion of 2000 HeLa cells to AT-cut and BT-cut quartz crystal chips, their dynamic QCM response during TNF and CHX apoptosis induction, and the changes in cell stress and viscoelasticity in response to the quartz crystals. (A) Cell adhesion on AT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by TNF and CHX; (B) Cell adhesion on BT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by TNF and CHX; (C) Stress changes applied to quartz crystals during TNF and CHX-induced apoptosis; (D) Western blotting detection of HeLa cell apoptosis induced by TNF and CHX; (E) Cell adhesion on AT-cut quartz crystal chips and storage modulus changes induced by TNF and CHX; (F) Cell adhesion on BT-cut quartz crystal chips and storage modulus changes induced by TNF and CHX; (G) Cell adhesion on AT-cut quartz crystal chips and loss modulus changes induced by TNF and CHX; (H) Cell adhesion on BT-cut quartz crystal chips and loss modulus changes induced by TNF and CHX.

[0047] Figure 3The curves show the adhesion of 2000 HUVECs cells to AT-cut and BT-cut quartz crystal chips, the dynamic QCM response during LPS and CTB pyroptosis induction, and the changes in cell stress and viscoelasticity. (A) Cell adhesion on AT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by LPS and CTB; (B) Cell adhesion on BT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by LPS and CTB; (C) HUVECs cell adhesion and stress changes applied to the quartz crystal during LPS and CTB induction; (D) Western blotting detection of HeLa cell pyroptosis induced by LPS and CTB; (E) Cell adhesion on AT-cut quartz crystal chips and storage modulus changes induced by LPS and CTB; (F) Cell adhesion on BT-cut quartz crystal chips and storage modulus changes induced by LPS and CTB; (G) Cell adhesion on AT-cut quartz crystal chips and loss modulus changes induced by LPS and CTB; (H) Cell adhesion on BT-cut quartz crystal chips and loss modulus changes induced by LPS and CTB.

[0048] Figure 4 The curves show the adhesion of 2000 HUVECs cells to AT-cut and BT-cut quartz crystal chips, their dynamic QCM response during TNF and CHX apoptosis induction, and the changes in cell stress and viscoelasticity in response to the quartz crystals. (A) Cell adhesion on AT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by TNF and CHX; (B) Cell adhesion on BT-cut quartz crystal chips and frequency shift and dynamic resistance changes induced by TNF and CHX; (C) Stress changes applied to quartz crystals during TNF and CHX-induced apoptosis; (D) Western blotting detection of HUVECs cell apoptosis induced by TNF and CHX; (E) Cell adhesion on AT-cut quartz crystal chips and storage modulus changes induced by TNF and CHX; (F) Cell adhesion on BT-cut quartz crystal chips and storage modulus changes induced by TNF and CHX; (G) Cell adhesion on AT-cut quartz crystal chips and loss modulus changes induced by TNF and CHX; (H) Cell adhesion on BT-cut quartz crystal chips and loss modulus changes induced by TNF and CHX. Detailed Implementation

[0049] The quartz crystal resonator used in this invention is a 9MHz AT-cut or BT-cut quartz crystal with a diameter of 12.5mm. The gold electrode coating thickness is 100nm (5mm in diameter), and a 20nm chromium plating layer serves as an adhesion layer between the gold electrode coating and the quartz crystal matrix. This patent is based on dual-resonator piezoelectric technology. By real-time monitoring of the frequency shift caused by HeLa and HUVECs cells adhering to the quartz crystal, the dynamic changes in the stress (ΔS) exerted by HeLa and HUVECs cells on the quartz crystal are quantitatively measured. The positive and negative values ​​of ΔS represent different stresses exerted by the cell population as a whole on the quartz crystal. ΔS>0 corresponds to the cell population adhering to the quartz crystal surface being in a state of tensile stress, occurring during the process of cell spreading and adhering on the substrate. The cells exert a contractile traction force on the quartz crystal through the adhesion focal complex, causing the quartz crystal to experience compressive stress. Conversely, ΔS<0 corresponds to the resultant force exerted by the cell population on the quartz crystal being in the opposite direction to the contractile traction force, indicating that the protruding force dominates, and the cell population is in a state of compressive stress, causing the quartz crystal to experience tensile stress.

[0050] The specific steps of the cellular mechanical method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology are as follows:

[0051] (1) Place the gold electrode portion of the AT-cut and BT-cut quartz crystal in 1 drop of 80℃ Piranha solution (a 1:3 mixture of 30% H2O2 and 98% H2SO4) for 30 seconds, then rinse with Milli-Q water and ethanol, and then dry with ultra-high purity N2. Repeat this process three times.

[0052] (2) Four independent quartz crystal resonators from the same batch were assembled one by one into a Teflon detection cell, with two silicon thin-layer O-rings on each side of the vertical direction of the detection cell. The Piranha-treated gold electrode surface was exposed to an anhydrous ethanol solution containing 20 mM 3-mercaptopropionic acid (MPA) and 1 mM triethylene glycol mono-11-mercaptoundecyl ether (TGME) and left to stand at room temperature in the dark for 16 hours to form a self-assembled monolayer.

[0053] (3) Remove the solution and immerse the surface of the self-assembled monolayer gold electrode in 150 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 30 mM N-hydroxysuccinmide (NHS) in phosphate buffer (PBS, pH=5.5) and let it stand at room temperature for 30 minutes.

[0054] (4) Remove the solution and expose the interface to 20 μg / mL fibronectin (FN) prepared with PBS (pH=8.2) and let it stand at room temperature in the dark for 6 hours.

[0055] (5) After 6 hours, remove the solution, add 2000 HeLa or HUVECs cells to the detection pool, connect the detection pool to the 250B-2 quartz crystal network analyzer, and start data monitoring.

[0056] (6) After HeLa or HUVECs cells have stably adhered to the gold electrode surface (more than 10 hours after cell addition), pyroptosis or apoptosis drugs are added for induction.

[0057] (7) Pyroptosis experimental group: 20 μL of cell culture medium was taken and 20 μL of LPS (Conc. 1 μg / ml) was added for induction and monitoring. To improve the pyroptosis induction efficiency, 40 μL of culture medium was taken after 6 hours and 20 μL of LPS (Conc. 1 μg / ml) and 20 μL of CTB (Conc. 10 μg / ml) were added together for pyroptosis induction.

[0058] (8) In the apoptosis experimental group, 60 μL of cell culture medium was taken out and 40 μL of TNF-α (Conc. 20 ng / ml) and 20 μL of CHX (Conc. 10 μg / ml) were added to induce apoptosis.

[0059] QCM signal monitoring was conducted continuously from cell culture to drug induction, with frequency shift and dynamic resistance data continuously acquired for over 30 hours at a sampling rate of one set per second. Throughout the entire detection process, the quartz crystal resonator was placed in a constant-temperature cell culture incubator at 37°C and 5% CO2.

[0060] Dynamic changes in cellular traction forces during pyroptosis and apoptosis

[0061] This invention demonstrates, as Figure 1 , 2The QCM response of AT-cut and BT-cut chips with 2000 HeLa cells adhering to the surface of FN-modified quartz crystals is shown, including the changes in frequency shift Δf and dynamic resistance ΔR over time, as well as the changes in cell-induced stress (ΔS), cell storage modulus (G′), and loss modulus (G″) on the quartz crystal. These parameters are analyzed using Western spectroscopy. Blot analysis of the cleavage of the pyroptosis executive protein GSDMD and the expression of its cleavage products (N-terminal and C-terminal proteins) verified the feasibility of this pyroptosis induction mechanism. During cell adhesion, as HeLa cells spread on the gold electrode and integrins and other focal adhesion complexes gradually formed, the cells exerted a gradually increasing compressive stress (ΔS) on the quartz crystal, stabilizing at nearly 20,000 dyne / cm. At this stage, cellular traction dominated, and the cells exerted compressive stress on the underlying hard matrix through this traction, thereby regulating cell adhesion. As the focal adhesion complex matured, the compressive stress exerted by the cells on the quartz crystal gradually stabilized, a process consistent with the stabilization time of ΔS. Subsequently, pyroptosis induction using LPS showed a decreasing frequency and increasing resistance in the QCM response, with ΔS decreasing from a positive to a negative value (see [reference needed]). Figure 1 -C. To improve pyroptosis induction efficiency, LPS and CTB were added 6 hours later to induce pyroptosis again. Under the combined action of LPS and CTB, the frequency further decreased and the resistivity further increased. Simultaneously, ΔS continued to decrease in negative values. After pyroptosis induction in HeLa cells, the stress exerted by the cells on the quartz crystal changed from positive to negative, indicating that pyroptosis altered the direction of the resultant force exerted by the HeLa cell population on the basal matrix, becoming a mode dominated by the direction of the protruding force. Further analysis of the changes in viscoelastic response during pyroptosis, such as... Figure 1 As shown in E, F, G, and H, the storage modulus (G′) and loss modulus (G″) show an increasing trend in the early stages of cell adhesion, and then gradually stabilize as the focal adhesion complex matures. After the first pyroptosis induction, both G′ and G″ show significant increases, with G′ nearly doubling and G″ increasing by approximately 3000 Pascal. After LPS and CTB co-induction, the storage and loss moduli show even more significant increases, with the storage modulus increasing nearly threefold (AT cut to 30,000 Pascal; BT cut to 25,000 Pascal), while the loss modulus also increased by approximately 25,000 Pascal at AT cut and 20,000 Pascal at BT cut. However, the mechanical response of HeLa cells during apoptosis is completely opposite to that during pyroptosis (see...). Figure 2 After detecting the cleavage of the apoptosis-executing protein Caspase3 by Western blotting and verifying the occurrence of TNF and CHX-induced apoptosis ( Figure 2-D), we monitored the mechanical response of apoptosis. The frequency and resistance responses during cell adhesion were consistent with those of the pyroptosis group. However, after apoptosis induction with the addition of TNF and CHX, a slight increase in frequency and a decrease in resistance were observed on both AT and BT cuts, which was the opposite of the pyroptosis response. In addition, the compressive stress on the quartz crystal increased after apoptosis, but ΔS remained positive. Further comparison of the viscoelastic modulus changes between apoptosis and pyroptosis revealed that after apoptosis induction, a significant decrease in both storage modulus and loss modulus was observed on both AT and BT cuts. Specifically, the storage modulus eventually decreased to approximately 3000 pascal on AT cuts and approximately 5000 pascal on BT cuts; while the loss modulus eventually decreased to approximately 46,000 pascal on AT cuts and approximately 49,000 pascal on BT cuts.

[0062] In summary, the pyroptosis mechanical response of HeLa cells is manifested as follows: (1) changing the direction of the total stress exerted by the cell population on the quartz crystal. (2) increasing the cell storage modulus and loss modulus. The apoptosis mechanical response of HeLa cells is manifested as follows: (1) increasing the compressive stress exerted by the cell population on the quartz crystal. (2) decreasing the cell storage modulus and loss modulus.

[0063] Specifically, the differences in the mechanical responses of pyroptosis and apoptosis are mainly reflected in the following aspects: after pyroptosis, Δf shows a continuous decrease, while after apoptosis, Δf slightly increases; simultaneously, ΔR increases after pyroptosis and decreases after apoptosis. The difference in Δf response during the two death processes leads to differences in the stress (ΔS) exerted by the cells on the quartz crystal. During pyroptosis, the cell volume expands, and the pyroptotic cell group as a whole generates a protruding force pointing towards the periphery of the quartz crystal, placing the quartz crystal in a state of tensile stress, corresponding to a negative ΔS value; while during apoptosis, the destruction of microtubules in the cell's internal skeletal structure causes the apoptotic cells to exert greater compressive stress on the underlying matrix, corresponding to an increased ΔS value. Furthermore, this invention also provides real-time changes in the viscoelastic responses of HeLa cells during pyroptosis and apoptosis. During pyroptosis, both the storage modulus and loss modulus of the cells increase significantly, while after apoptosis, both storage modulus and loss modulus decrease significantly.

[0064] Similarly, the stress responses of pyroptosis and apoptosis were consistently validated in HUVECs cell lines.

[0065] Following pyroptosis in HUVECs, ΔS decreased from positive to negative. Conversely, ΔS showed an increasing trend after apoptosis. However, the viscoelastic response of HUVECs induced by TNF and CHX differed from that induced by HeLa. Both storage and loss moduli increased after HUVEC apoptosis, indicating inconsistent viscoelastic responses induced by HUVECs and HeLa at the same concentration, suggesting a significant difference in viscoelastic responses between normal and cancer cells undergoing apoptosis.

[0066] In summary, this invention patent proposes a novel cellular mechanics method for identifying pyroptosis and apoptosis by observing the stress response of cells to quartz crystals. Furthermore, this technology can also provide real-time viscoelastic changes in different cell lines during pyroptosis and apoptosis.

Claims

1. A cellular mechanical method for distinguishing pyroptosis and apoptosis based on dual-resonance piezoelectric technology, characterized in that, The method includes the following steps: (1) Place AT-cut quartz crystals and BT-cut quartz crystals in a detection cell. The AT-cut quartz crystals and BT-cut quartz crystals have the same fundamental frequency, surface morphology and consistent surface-adhesive molecular modification. (2) Add cells. After the cells are stably adhered to the quartz crystal interface, lipopolysaccharide and cholera toxin B subunit are added to the detection cell to induce cell pyroptosis. Tumor necrosis factor α and cyclohexylimide are added to induce cell apoptosis. The frequency and resistance changes of AT-cut and BT-cut quartz crystals are monitored in real time throughout the process. (3) The surface stress (ΔS) generated by the cells on the quartz crystal is determined by the following formula: Among them, K AT =2.75×10 -12 cm 2 dyn -1 K BT = -2.65 × 10 -12 cm 2 dyn -1 The stress coefficients of AT-cut and BT-cut quartz crystals are respectively; t q AT and t q BT These are the thicknesses of the quartz crystal, expressed in cm; Δf t AT and △f t BT It is the frequency shift after subtracting the corresponding f0 from the new frequency shift caused by the surface stress of the resonator at time t. (4) The storage modulus (G′) and loss modulus (G″) of the cell are determined using the following formulas: Where Γ is the half bandwidth, ρ c For cell density, based on the assumption that it is consistent with the density of pure water: ρ 水 =1.00g / cm 3 Z q ρ is the acoustic impedance of the quartz crystal. c =0.9933g / cm 3 The half-bandwidth Γ and the dynamic resistance R have the following relationship: ΔΓ=ΔR / 4πL q L q Δf is the inductance of the quartz crystal in the cell culture medium; Δf is the frequency shift caused by the uniform viscoelastic layer on the surface of the quartz crystal relative to air. (5) Identify pyroptosis and apoptosis based on the difference in ΔS changes during pyroptosis and apoptosis, and simultaneously obtain the changes in cell viscoelasticity during pyroptosis and apoptosis.

2. The cellular mechanical method for identifying pyroptosis and apoptosis based on dual-resonance piezoelectric technology as described in claim 1, characterized in that, In step (2), 40 μL of lipopolysaccharide and 20 μL of cholera toxin B subunit are added to the detection pool to induce pyroptosis, and 40 μL of tumor necrosis factor α and 20 μL of cyclohexylimide are added to induce apoptosis.

Images