Cell membrane mechanical property measuring method based on nanotube electroosmotic flow

Abstract

The invention provides a cell membrane mechanical property measuring method based on nanotube electroosmotic flow, and belongs to the technical field of nanoelectrodes and cell physiology. An Ag / AgCl electrode is inserted into the tip of the nanotube, the size of the tip of the nanotube is 80-100nm, under the stimulation of an external electric field of 50mV, the tip of the nanotube generates a picoliter electroosmosis fluid to trigger periodic cell membrane vibration, and the distance between the tip of the nanotube and the surface of the membrane is associated with a collected current signal, so that the cell membrane is obtained. The infinitesimal displacement of the membrane is reflected as observable current waveform change, the frequency f of a current signal is obtained through fast Fourier transform, and the frequency f is substituted into a calculation formula of a cell membrane elastic parameter k / m = 4pi2f2 to obtain the cell membrane mechanical property. According to the method, the mechanical property of the cell membrane is directly obtained from the current signal by utilizing the derived calculation formula of the cell membrane elasticity parameter k / m = 4pi2f2, and the method can be widely applied to detection of the mechanical property of the cell membrane in different biological states.

Description

Technical Field

[0001] The invention relates to the technical field of nano-electrodes and cell physiology, and in particular to a method for measuring the mechanical properties of cell membranes based on nanotube electroosmotic flow. Background Art

[0002] Cells are connected to the internal and external environment through the plasma membrane, and the mechanical properties of the cell membrane dominate many biological events. Therefore, the detection of the mechanical properties of the cell membrane can provide a lot of valuable information. Previous attempts, such as atomic force microscopy (AFM) and magnetic / optical tweezers have been successfully used to study the structure and function of the cell membrane (Li M, Liu L, Xi N, et al. Research progress in quantifying the mechanical properties of single living cells using atomic force microscopy [J]. Chinese Science Bulletin, 2014, 59 (31): 4020-4029.). Some studies have reported that cell mechanics and cell stiffness can be measured by detecting tip-sample interactions using atomic force microscopy (Han Y, Wang J, Wang K, et al. Fabrication of atomic force microscope spherical tips and its application in determining the mechanical property of cancer cells [J]. Micro & Nano Letters, 2016, 11 (12): 881-884; Wang X, Liu H, Zhu M, et al. Mechanical stability of the cell nucleus–roles played by the cytoskeleton in nuclear deformation and strain recovery [J]. Journal of Cell Science, 2018, 131 (13): jcs209627.). Although atomic force microscopy is a powerful tool for measuring the mechanical properties of cell membranes with high spatial resolution and sensitivity, it also has some limitations. First, AFM works by directly contacting the cell membrane, which is likely to affect the mechanical state of the cell itself. Second, AFM measurements are usually performed in a controlled environment, which may not fully replicate the dynamic and complex conditions of living cells. Therefore, for characterization of biological samples, especially living cells, applying forces using non-contact techniques is preferable.

[0003] Recognizing the non-contact advantages of magnetic and optical manipulation, researchers have developed magnetic and optical tweezers to study biological forces and movements (Liang Y, Liang G, Xiang Y, et al. Manipulation and Assessment of Human Red Blood Cells with Tunable ``Tug-of-War″Optical Tweezers[J]. Physical Review Applied, 2019, 12(6): 064060; Tajik A, Zhang Y, Wei F, et al. Transcription upregulation via force-induced direct stretching of chromatin[J].Nature Materials,2016,15(12):1287-1296.). However, both techniques have certain drawbacks. While optical tweezers provide high spatial precision, their intense lasers can also cause photodamage to living cell membranes. In addition, optical tweezers are limited in force, which may limit their application in probing rigid membrane structures. Magnetic tweezers can apply large forces without causing photodamage, but their relatively low spatial resolution is not conducive to measuring subtle membrane deformations. Membrane detection techniques, such as optical or magnetic tweezers, have revealed mechanical strength through membrane anchoring modifications, but developing label-free methods to reduce the impact of exogenous interference remains challenging.

[0004] Invention patent CN 112014429 B discloses a cell membrane vibration detection method based on ultra-micro electroosmotic flow regulation, which uses the cell membrane vibration frequency of quartz nanopores to detect cell membrane surface vibrations. Electroosmotic flow drives cell membrane vibrations, making it easier to understand cell membrane biophysics at the level of a single cell. However, this method only preliminarily reveals that cell membrane vibrations can be detected through electroosmotic flow regulation, and does not provide specific data analysis of the captured signals. Summary of the invention

[0005] The purpose of the present invention is to provide a method for measuring the mechanical properties of cell membranes based on nanotube electroosmotic flow and its application. The method controls the size of nanotubes and the applied voltage to generate picoliter electroosmotic flow at the nanotube mouth, thereby capturing the tiny deformation of the cell membrane and using the derived cell membrane elastic parameter k / m=4π 2 f 2 The calculation formula directly and effectively derives the mechanical properties of cell membranes from a two-dimensional perspective.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a method for measuring the mechanical properties of cell membranes based on nanotube electroosmosis. The method comprises constructing a single-cell sensing platform, inserting an Ag / AgCl electrode at the tip of a nanotube, wherein the tip size of the nanotube is 80-100 nm, and under the stimulation of an external electric field of 50 mV, the tip of the nanotube generates picoliters of electroosmotic fluid, triggering periodic cell membrane vibration, and linking the distance between the tip of the nanotube and the membrane surface with the collected current signal. The tiny displacement of the membrane is reflected as an observable change in the current waveform, and the frequency f of the current signal is obtained by fast Fourier transform, and is substituted into the cell membrane elastic parameter k / m=4π 2 f 2 The calculation formula for the mechanical properties of cell membrane is obtained.

[0007] As a preferred solution, the tip size of the nanotube is 100 nm.

[0008] As a preferred solution, when the nanotube tip moves from the outer surface of the cell membrane to the inner surface of the cell membrane, under a continuous external potential of 50 mV, the current trajectory shows different stages: before the nanotube tip contacts the cell membrane, the current does not change; after the nanotube tip moves downward, the current suddenly decreases, indicating that the nanotube tip is in contact with the cell membrane; stable and regular periodic oscillations are observed from the current time trajectory; as the contact between the nanotube tip and the cell membrane deepens, the waveform of the oscillation signal changes in the middle of the waveform; when the nanotube tip penetrates the cell membrane, the regular periodic current signal disappears.

[0009] As a preferred solution, the current wave can be decomposed into two sine waves of different frequencies, with different elastic parameters k / m in the vertical direction and the horizontal direction, and the cell membrane has mechanical strength in both the vertical direction and the horizontal direction.

[0010] As a preferred approach, the tip of the nanopipette is placed near the edge of a selected cell, picoliter-scale electroosmotic measurement signals are recorded, the tip is moved to collect signals at multiple locations around the cell membrane, the tip is moved to the center of the cell, vibration information from different regions of the cell surface is collected, and measurement data of the mechanical polarity of the cell membrane are obtained.

[0011] As a preferred embodiment, the mechanical properties of the cell membrane are heterogeneous. At the outer edge of the cell, the elastic parameter k / m value measured at a point with a larger curvature is higher than the k / m value measured at a point with a smaller curvature, and the k / m value of the cytoplasm in the cell body is higher than that of the extracellular part.

[0012] The invention also discloses the application of the cell membrane mechanical property measurement method based on nanotube electroosmotic flow in screening pathological cells and analyzing the mechanical changes of cell membrane after drug treatment.

[0013] In order to achieve picoliter electroosmotic measurement (PEOM), a typical conical 80-100 nm glass nanotube was made by pulling a quartz capillary. COMSOL Multiphysics was used for numerical simulation to show the electric field distribution and fluid dynamics inside the nanotube under applied bias voltage. By simulating the potential and voltage distribution inside the nanotube, it can be calculated that when the applied voltage is 50 mV, the liquid flow rate at the tip of the nanotube is 3×10 -14 The force generated by cell membrane deformation is about piconewton level. By controlling the size of the nanotubes and the applied voltage, the nanotube mouth generates picoliter electroosmotic flow, which can capture the tiny deformation of the cell membrane.

[0014] Before the tip of the nanotube approaches the cell membrane, the open-circuit current of the nanotube at 50 mV is ~400 pA. The cations in the diffusion layer near the inner wall of the nanotube are regarded as a cylindrical sheath. Under the action of the external electric field, the solvated cations close to the sliding surface are forced to migrate from the anode to the cathode, thereby inducing electroosmosis inside the nanotube. The external factors affecting the directional movement of ions are observed from the current feedback.

[0015] Fourier transform is used to describe the picoliter electroosmotic measurement sensing event, explaining the different current waveforms that appear at different stages of the interaction between the nanotube tip and the cell membrane. The power carried by the wave per unit frequency is used to analyze the discrete periodic signal oscillation, and the distance between the nanotube tip and the membrane surface is converted into a current signal. The tiny displacement of the membrane is reflected as an observable change in the current waveform.

[0016] In order to explain the different current waveforms appearing at different stages of the interaction between the nanotube tip and the cell membrane, Fourier transform was used to quantitatively describe the PEOM sensing event, and the acquired data were fast Fourier transformed to obtain frequency domain information. The typical current-time (It) trajectory of the entire transmembrane process of the nanotube tip was recorded at +50mV, including approach, contact (consisting of three stages from the outer surface of the cell membrane to the inner surface of the cell membrane) and penetration.

[0017] In order to quantitatively describe the PEOM sensing events, the concept of power spectral density (PSD), which is the power carried by the wave per unit frequency, is introduced to analyze discrete periodic signals. The current signal was selected for quantitative study, and the peaks were evenly distributed in time and amplitude, which can be attributed to the stable, consistent and regular interaction between the nanotube tip and the cell membrane at this stage. The oscillatory current signal consists of lower and higher frequencies, which can be explained by the membrane elasticity in the vertical direction affected by the strength of the cytoskeleton and the local membrane elasticity in the horizontal direction affected by the membrane fluidity. Therefore, the PEOM strategy represents a label-free method to reveal the mechanical properties of living cell membranes from a two-dimensional perspective.

[0018] In order to deeply understand the mechanical significance of the two characteristic peaks in the spectrum, we started from the basic dynamic principles and focused on the relationship between the movement of the cell membrane and the current signal measured by PEOM. The proposed PEOM method can convert the distance between the nanotube tip and the membrane surface into a current signal, so the tiny displacement of the membrane can be reflected as an observable change in the current waveform.

[0019] According to the typical current-time (It) trajectory of the oscillating current signal, the trajectory is periodic, smooth, and exhibits the characteristics of a sine function. The first-order differential result of the trace shows that the rate of change fits the sine function well. From the corresponding FFT results, it can be seen that the current wave can be decomposed into two sine waves with different frequencies. Therefore, a combined model of two orthogonal spring oscillators is used to describe the plasma membrane vibration. Considering the periodic motion of the mass point on the cell membrane patch when the nanotube tip hits it, the cell membrane patch moves periodically in the vertical direction, which can be simulated by an ideal spring oscillator model, and the stiffness coefficient of the spring represents the bending modulus of the membrane. By superimposing the two sine functions obtained from the spring oscillator model, we found that the superimposed function fits the original It trajectory well, which confirms the validity of the analytical model.

[0020] Based on the kinematic equation of the cell membrane, the corresponding force conditions and mechanical parameters were further studied through mathematical analysis. Assuming that the impact force F generated by the electroosmotic flow on the cell membrane is proportional to the distance x from the nanotube tip to the cell membrane, it can be deduced that:

[0021] F=-kx (1)

[0022] Where k is the stiffness coefficient. According to Newton's second law:

[0023]

[0024] Combining equation (1) and equation (2):

[0025]

[0026] make Then (3) can be simplified as:

[0027]

[0028] So the equation of motion of the particle is:

[0029]

[0030] And because:

[0031] ω=2πf (6)

[0032] Then we can get:

[0033] k / m=4π 2 f 2 (7)

[0034] Where A is the amplitude, ω is the angular frequency, φ is the phase, and f is the frequency. The elastic parameter k / m can be calculated from the motion equations in the vertical and horizontal directions, where k is the stiffness coefficient and m is the mass. The results show that the cell membrane has mechanical strength in both the vertical and horizontal directions, which may be due to the support of the cytoskeleton and the fluidity of phospholipids.

[0035] To further explore the changes in the entire oscillatory system, we analyzed multiple representative waveforms corresponding to cells under different conditions. We observed two characteristic frequencies in the frequency domain, indicating two different membrane vibration modes. By statistically analyzing the amplitudes of these two characteristic frequencies, we found significant differences between different conditions. Next, the linear discriminant analysis (LDA) machine learning method was used to classify the three current signals. The results showed that at a 95% confidence level, LDA could well divide the signals into three clusters. These results indicate that PEOM can be effectively used to analyze the mechanical changes of cell membranes after drug treatment.

[0036] The advantages of the present invention are that it proposes to use picoliter electroosmotic pressure (PEOM) to measure the mechanical properties of cell membranes. The ultra-sensitive oscillating current signal generated by the nanotube tip during the transmembrane process reflects the mechanical interaction between the cell membrane and the tip. The derived cell membrane elasticity parameter k / m=4π 2 f 2 The calculation formula directly and effectively derives the mechanical properties of the cell membrane from a two-dimensional perspective. By controlling the size of the nanotubes and the applied voltage, a picoliter electroosmotic flow is generated at the nanotube mouth, which enables the capture of tiny deformations of the cell membrane. Periodic cell membrane vibration modes are observed from the current traces. These vibration modes carry information about the mechanical properties of the membrane to indicate its biological state. In addition, we have demonstrated the exciting potential of PEOM for label-free observation of changes in membrane mechanical properties in different biological processes, including cytoskeletal changes, membrane tension changes, and mechanical polarization. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Nanotube-based PEOM sensing platform.

[0038] Figure 2 Quantitative analysis of current traces obtained by PEOM.

[0039] Figure 3 Simulation of the motion of a membrane in two orthogonal vibration modes.

[0040] Figure 4 PEOM was used to explore the effects of external stimuli on the mechanical properties of cell membranes.

[0041] Figure 5 Heterogeneity of cell membrane mechanical properties. DETAILED DESCRIPTION

[0042] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0043] Example 1.

[0044] 1. Construction of a single-cell sensing platform. Consistent with commonly used sensing platforms, the sensing platform generally includes a micromanipulation system to control the movement of nanotubes, a probe connected to a current amplifier and a digital-to-analog converter to record weak electrical signals, a microscope to observe cells and nanotubes, a working electrode, a reference electrode, and a Faraday cage to shield noise.

[0045] 2. Preparation of nanoelectrodes. Usually, quartz glass tubes are used as raw materials, and laser needle pullers are used to prepare nanotubes. A typical glass nanoelectrode for PEOM experiments was prepared by inserting an Ag / AgCl electrode at the tip of a glass nanotube. First, the silver wire was polished with sandpaper to remove the surface oxide layer. The silver wire was then immersed in a saturated sodium hypochlorite solution for 30 minutes. After rinsing with deionized water several times, a dense silver chloride film was visible on the surface of the silver wire, indicating the formation of an Ag / AgCl electrode. Nanopipette was prepared using a P-2000 laser aspirator. The program settings for obtaining 100nm nanopipette are as follows:

[0046] Line 1:Heat 700,Fil 4,Vel 32,Del 132,Pul 70;

[0047] Line 2:Heat 750,Fil 4,Vel 16,Del 140,Pull 140.

[0048] 3. Measurement of the mechanical properties of cell membranes. The prepared nanopipette is fixed by a bracket and connected to the front stage of the current amplifier with a reference electrode to form a current loop. At the same time, the head stage is mounted on a micromanipulator arm, and the microcontrol system can be manually adjusted to achieve nanoscale movement of the nanopipette. All devices are placed on the moving platform of an inverted microscope, which makes it possible to accurately position the tip of the nanopipette near the surface of a single living cell. While applying a 50mV bias voltage, the tip of the nanopipette gradually approaches the cell membrane. The entire process is recorded by bright field imaging and It curve monitoring.

[0049] 4. Measurement of cell membrane mechanical polarity. First, place the tip of the nanopipette near the edge of a selected cell and record the PEOM signal for one minute. Then move the tip to collect signals at multiple locations around the cell membrane. Then, move the tip to the center of the cell to collect vibration information from different areas of the cell surface.

[0050] PEOM is based on the traditional patch clamp procedure. Figure 1 As shown in a, an Ag / AgCl electrode is inserted into the nanotube as a working electrode. Another Ag / AgCl electrode is immersed in the bath as a reference electrode. Under external electric field stimulation, the tip of the nanotube produces picoliters of electroosmotic fluid, which can trigger periodic cell membrane vibrations. The periodic motion of the cell membrane can be observed from the current trajectory, thereby revealing the mechanical properties of the membrane and its biological state. Subsequently, a micromanipulator is used to precisely move the nanotube tip, allowing the tip to move vertically from the outer surface of the cell membrane to the inner surface of the cell membrane in the z direction. Nanoscale motion ( Figure 1 b). The process was monitored in real time by recording the current in PBS (pH 7.4) buffer. Before approaching the cell membrane, the open circuit current of the nanotube was measured to be ~400pA at 50mV. According to the double layer theory, the cations in the diffusion layer near the inner wall of the nanotube can be regarded as a cylindrical sheath. Under the action of the applied electric field, the solvated cations close to the sliding surface are forced to migrate from the anode to the cathode, thereby inducing electroosmosis inside the nanotube. This directional movement of ions is the source of the current of PEOM, which shows that external factors affecting the directional movement of ions, such as the periodic movement of the cell membrane, can be observed from the current feedback.

[0051] like Figure 1 As shown in Figure c, when the nanotube tip moves from the outer surface of the cell membrane to the inner surface of the cell membrane, under a continuous external potential of +50 mV, the current trajectory can be divided into three different stages. From the typical current-time (It) trajectory, no events were observed initially before the nanotube tip contacted the cell membrane. After the nanotube tip moved downward, the current suddenly decreased, indicating that the nanotube tip was in contact with the cell membrane. At this time, stable and regular periodic oscillations were observed from the It trace, and a downward spike appeared (baseline was ~0.4 nA), confirming that the vibrational movement of the membrane was triggered by the electroosmotic flow of the nanotube tip ( Figure 1 c, stage 1). As the nanotube tip contacts the cell membrane more deeply, the waveform of the oscillating signal changes in the middle of the waveform, changing the baseline to ∼0.2 nA ( Figure 1 c, stage 2). In the next stage, the waveform of the oscillating signal changes again, and as the baseline moves to ∼0.0 nA, an upward spike appears ( Figure 1c, stage 3). It is noteworthy that the regular periodic current signal disappears when the nanotube tip penetrates the cell membrane. This indicates that the oscillation phenomenon is detected only when the nanotube tip contacts the cell membrane. The ultrasensitive oscillating current signal generated by the nanotube tip during the transmembrane process can accurately reflect the mechanical interaction between the cell membrane and the tip, providing excellent resolution for real-time detection of the mechanical properties of the cell membrane without labeling.

[0052] To achieve PEOM, a typical conical glass nanotube was fabricated by pulling a quartz capillary. Scanning electron microscope (SEM) images and bright field microscope images ( Figure 1 d) shows the side view and top view of the nanotube. From the microscope image, it can be seen that the tip of the nanotube is conical and the diameter of the tip hole is about 90nm. From the ionic current rectification (ICR) curve ( Figure 1 e) It can be seen that the ion current of the glass nanotube is slightly negative, and the rectification ratio remains unchanged after 30 minutes of continuous testing ( Figure 1 f), which confirms the stability of the nanotube. Next, we use COMSOL Multiphysics to perform numerical simulations to show the electric field distribution and fluid dynamics inside the nanotube under applied bias voltage. By simulating the potential and voltage distribution inside the nanotube, we can calculate that when the applied voltage is 50mV, the liquid flow rate at the tip of the nanotube is 3×10 -14 L / s.

[0053] To explain the different current waveforms appearing at different stages of the interaction between the nanotube tip and the cell membrane, Fourier transform was used to quantitatively describe the PEOM sensing events. Figure 2 a shows a typical It trace of the entire transmembrane process of the nanotube tip recorded at +50 mV, including approach, contact (consisting of three stages from the outer surface of the cell membrane to the inner surface of the cell membrane), and penetration. The It trace can be divided into five different parts (iv) according to the relative vertical distance between the nanotube tip and the cell membrane. In a single PEOM experiment, the nanotube tip was first immersed in the bath solution without touching the membrane of a single living HeLa cell, and the It trace was observed as a straight line of the pore opening current ( Figure 2 b, region 1). When the nanotube tip approaches and contacts the outer surface of the cell membrane, periodic downward spikes appear, indicating that the nanotube is periodically blocked ( Figure 2 b, region 2). A sine wave with consistent up and down amplitudes was observed on the It trajectory, indicating that the equilibrium position of the nanotube tip is in the middle of the plasma membrane ( Figure 2 b, region 3). As the nanotube tip penetrates deeper, periodic upward spikes are observed on the It trajectory ( Figure 2 b, region 4), which appears to be an upside-down image of the spike in the second part. Finally, after the tip penetrates the cell membrane, the periodic current signal is significantly weakened ( Figure 2 b, area 5).

[0054] In order to quantitatively describe the PEOM sensing event, here we introduce the concept of power spectral density (PSD), that is, the power carried by the wave per unit frequency, to analyze discrete periodic signals. Two characteristic peaks can be observed in the PSD spectrum, located at 50Hz (relatively low frequency fa) and 100Hz (relatively high frequency fb). The comparison of the PSD values ​​of fa and fb in the histogram shows that the PSD value of the third part is significantly higher ( Figure 2 d), compared with the previous Figure 1 b corresponds to the second stage waveform discussed in detail.

[0055] We chose the current signal in the third part for all the following quantitative studies because the peaks are evenly distributed in time and amplitude, which can be attributed to the stable, consistent and regular interaction between the nanotube tip and the cell membrane in this stage. The oscillatory current signal in the third part (stage 2) consists of lower frequency fa and higher frequency fb, which can be explained by the membrane elasticity in the vertical direction affected by the strength of the cytoskeleton and the local membrane elasticity in the horizontal direction affected by the membrane fluidity. Therefore, the PEOM strategy represents a label-free method to reveal the mechanical properties of living cell membranes from a two-dimensional perspective, which is completely different from other methods.

[0056] In order to deeply understand the mechanical significance of the two characteristic peaks in the spectrum, we started from the basic dynamic principles and focused on the relationship between the movement of the cell membrane and the current signal measured by PEOM. As mentioned above, the proposed PEOM method can convert the distance between the nanotube tip and the membrane surface into a current signal, so the tiny displacement of the membrane can be reflected as an observable change in the current waveform.

[0057] Figure 3 a, i show typical it traces of the oscillating current signal recorded in the third part (see Figure 2 ). The trajectory is periodic and smooth, showing the characteristics of a sine function. The first-order differential result of the trace shows that the rate of change fits the sine function well. From the corresponding FFT results ( Figure 3 a, i and ii) we can see that Figure 3 The current waves in a and i can be decomposed into two sine waves of different frequencies. Therefore, a combined model of two orthogonal spring oscillators is used to describe the plasma membrane vibration. Considering the periodic motion of the mass point on the cell membrane patch when the nanotube tip hits it, the cell membrane patch moves periodically in the vertical direction ( Figure 3 b,i), which can be simulated using an ideal spring oscillator model ( Figure 3 b, ii). Here, the spring stiffness coefficient represents the bending modulus of the membrane. Figure 3b, Figure iii, and Figure iv are the corresponding time domain and frequency domain respectively. Figure 3 c shows the periodic motion of the diaphragm in the horizontal direction. By superimposing the two sine functions obtained from the spring oscillator model, we find that the superimposed function fits the original It trajectory well ( Figure 3 d), which confirms the validity of the analytical model.

[0058] Based on the kinematic equation of the cell membrane, the corresponding force conditions and mechanical parameters were further studied through mathematical analysis. Assuming that the impact force F generated by the electroosmotic flow on the cell membrane is proportional to the distance x from the nanotube tip to the cell membrane, it can be deduced that:

[0059] kx (1)

[0060] Where k is the stiffness coefficient. According to Newton's second law:

[0061]

[0062] Combining equation (1) and equation (2):

[0063]

[0064] make Then (3) can be simplified as:

[0065]

[0066] So the equation of motion of the particle is:

[0067]

[0068] And because:

[0069] ω=2πf (6)

[0070] Then we can get:

[0071] k / m=4π 2 f 2 (7)

[0072] Where A is the amplitude, ω is the angular frequency, φ is the phase, and f is the frequency. The motion and mechanical parameters of the diaphragm are as follows Figure 3 e. The elastic parameter k / m can be calculated from the motion equations in the vertical and horizontal directions, where k is the stiffness coefficient and m is the mass. The k / m values ​​in the vertical and horizontal directions are 9.86×10 4 and 3.94×10 5 These results suggest that the cell membrane has mechanical strength in both vertical and horizontal directions, which may be due to the support of the cytoskeleton and the fluidity of phospholipids.

[0073] according to Figure 3 As a result, we expected that the dissolution of the cytoskeleton would significantly reduce the mechanical strength of the cell membrane in the vertical direction. To verify this, we attempted to measure the changes in the mechanical properties of the cell membrane under external stimuli.

[0074] like Figure 4 As shown in a, the cytoskeleton, a system of fibers or fibres that serve as the main structural backbone, is present in the cytoplasm to support the cell membrane. To alter the strength of the cytoskeleton, cells were treated with two different reagents, Nocodazole and Taxol. Nocodazole can disrupt microtubules by binding to β-tubulin, while Taxol can promote microtubule polymerization and inhibit depolymerization. We expected that when the cytoskeleton is disturbed in living cells, the mechanical strength of the cell membrane would change accordingly. According to the established PEOM strategy, the current signals were recorded during the treatment with various reagents ( Figure 4 b) By extracting the current peaks before and after drug treatment, a scatter plot of current block and peak duration is obtained as shown in Figure 4 c, d. As can be seen from the figure, after the addition of nocodazole, the signal duration increased significantly. In contrast, paclitaxel treatment led to an increase in the intensity of current blockade. Nonparametric tests of current blockade and duration ( Figure 4 e) The significant differences in the mechanical properties of the cell membrane under three different conditions (control, nocodazole treatment and paclitaxel treatment) are highlighted. These changes can be attributed to the interference of the drugs on the cytoskeleton. Nocodazole can disrupt the cytoskeleton and reduce membrane stiffness, thereby weakening the cell membrane's feedback to the electroosmotic flow induced by the nanoprobe, resulting in a decrease in membrane displacement. In contrast, paclitaxel showed the opposite effect by increasing the strength of the cytoskeleton.

[0075] To further explore the changes in the entire oscillatory system, we analyzed 10 representative waveforms corresponding to cells under three different conditions (control, nocodazole, and paclitaxel treatment). Figure 3 In the frequency domain, we observed two characteristic frequencies, indicating two different membrane vibration modes. By statistically analyzing the amplitudes of these two characteristic frequencies, we found that there were significant differences between the three cases ( Figure 4 f). Next, the linear discriminant analysis (LDA) machine learning method was used to classify the three current signals. The results show that at a 95% confidence level, LDA can well divide the signals into three clusters. ( Figure 4 ). These results indicate that PEOM can be effectively used to analyze mechanical changes in cell membranes after drug treatment.

[0076] On this basis, the next step is to detect the periodic current signals at different points on the membrane of a single living cell to explore the uneven distribution of membrane mechanical properties. Figure 5 As shown in a, after the nano-micromanipulator moves precisely, the tip of the nanotube scans along the outer edge of the cell and then gradually moves toward the center, selecting 10 different sampling points ( Figure 5 b) The cell images, It traces and spectrum corresponding to these 10 sampling points are shown in Figure 2. Figure 5 As shown in c.

[0077] We note that there is a clear correlation between the vibration modes and the membrane curvature. Figure 5 The corresponding parameters shown in d show that the k / m value measured at the point with larger curvature at the outer edge of the cell is higher than the k / m value measured at the point with smaller curvature. There is a large amount of cytoplasm in the cell body, and its k / m value is higher than that of the extracellular part. For overall observation, we will Figure 5 The time domain and frequency domain signals at different sampling points in c are converted into pixel intensity values ​​to generate the corresponding imaging images ( Figure 5 e,f), which helps to have an overview of the distribution of mechanical properties. In summary, the mechanical properties of cell membranes show obvious heterogeneity, indicating the mechanical polarity of cell membranes. These different distributions may be due to the fact that the mechanical properties of the membrane are affected by the cell shape.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow, characterized in that: The method comprises constructing a single cell sensing platform, inserting an Ag / AgCl electrode at the tip of a nanotube, wherein the tip size of the nanotube is 80-100 nm, and under the stimulation of an external electric field of 50 mV, the tip of the nanotube generates a picoliter of electroosmotic fluid, triggering periodic cell membrane vibration, and linking the distance between the tip of the nanotube and the membrane surface with the collected current signal. The tiny displacement of the membrane is reflected as an observable change in the current waveform, and the frequency f of the current signal is obtained by fast Fourier transform, and substituted into the cell membrane elastic parameter k / m=4π 2 f 2 The calculation formula for the mechanical properties of cell membrane is obtained.

2. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow according to claim 1, characterized in that: The tip size of the nanotubes was 100 nm.

3. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow according to claim 1, characterized in that: When the nanotube tip moves from the outer surface of the cell membrane to the inner surface of the cell membrane, under a continuous external potential of 50 mV, the current trajectory shows different stages: before the nanotube tip touches the cell membrane, the current does not change. After the nanotube tip moves downward, the current suddenly decreases, indicating that the nanotube tip is in contact with the cell membrane. Stable and regular periodic oscillations are observed from the current time trajectory. As the contact between the nanotube tip and the cell membrane deepens, the waveform of the oscillation signal changes in the middle of the waveform. When the nanotube tip penetrates the cell membrane, the regular periodic current signal disappears.

4. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow according to claim 1, characterized in that: The current wave can be decomposed into two sine waves of different frequencies. The vertical and horizontal directions have different elastic parameters k / m. The cell membrane has mechanical strength in both vertical and horizontal directions.

5. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow according to claim 1, characterized in that: Place the tip of the nanopipette near the edge of a selected cell to record picoliter-scale electroosmotic measurement signals, move the tip to collect signals at multiple locations around the cell membrane, move the tip to the center of the cell to collect vibration information from different areas of the cell surface, and obtain measurement data on the mechanical polarity of the cell membrane.

6. A method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow according to claim 5, characterized in that: The mechanical properties of the cell membrane are heterogeneous. At the outer edge of the cell, the elastic parameter k / m value measured at the point with larger curvature is higher than the k / m value measured at the point with smaller curvature. The k / m value of the cytoplasm in the cell body is higher than that of the extracellular part.

7. Application of the method for measuring cell membrane mechanical properties based on nanotube electroosmotic flow as described in claim 1 in screening pathological cells and analyzing mechanical changes of cell membrane after drug treatment.

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