A method for dynamic quantitative evaluation of myocardial cell membrane changes after electroporation

Abstract

The present application relates to sensing detection technology, and aims to provide a method for dynamically and quantitatively evaluating the change of myocardial cell membrane after electroporation. The method comprises the following steps: planting purified animal myocardial cells in a culture cavity of a cell electrophysiological sensor device for culture; after the myocardial cells appear spontaneous rhythmic beating, using an electroporation control and signal recording integrated device to perform an electroporation operation, applying a transient pulse electric signal to the cell electrophysiological sensor device to induce the cell membrane to open its nanopore, and recording the change of the cell membrane electrophysiological signal during the process; and evaluating the change of the cell membrane according to the amplitude change of the electrophysiological signal. The present application statistically analyzes the recorded cell action potential, and quantitatively describes the dynamic change process of the cell membrane. Unlike the conventional static processing such as image recognition, the present application can quantitatively describe the whole dynamic evolution process of the nanopore on the cell membrane after electroporation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sensing detection, in particular to a method for dynamically and quantitatively evaluating changes of myocardial cell membranes after electroporation. BACKGROUND

[0002] It is of great significance to study the changes of cell membranes after electroporation for drug research. The changes of cell membranes after electroporation can guide the research on the delivery of drug molecules between cell membranes, and by studying the changes of cell membranes, the most suitable time for cell delivery can be selected to achieve efficient delivery and accurately evaluate the effect of delivered drug molecules, which is beneficial to the development and screening of drug treatment methods.

[0003] In the past few decades, many researchers have tried to describe the mechanism of cell membrane changes by various methods, including optical methods and modeling methods. The optical method refers to observing the expression of fluorescent substances in cells by a microscope to determine the changes of cell membranes after electroporation. However, the changes of cell membranes observed by this method are static and discontinuous, and lack dynamic and quantitative information. The modeling method refers to the continuous medium model based on electroporation, which has been continuously developed under the influence of many factors such as conductive energy, curvature, surface tension change, and nonlinear elasticity. However, these continuous medium models are based on several ideal assumptions, including that the shape of the pore is cylindrical or annular, which is unrealistic at the molecular level. With the significant progress in computing power, molecular dynamics models have been established for molecular-level exploration. In the molecular dynamics model, a simulated lipid bilayer is exposed to an electric pulse to simulate the formation and disappearance of induced transmembrane voltage. However, the simulation results are difficult to completely match the experimental results. Therefore, researchers have introduced other mechanisms such as electric field-mediated lipid peroxidation, membrane proteins, and cytoskeleton networks to explain. Although researchers have conducted experiments by building systems to explore the membrane changes caused by electroporation through multi-scale modeling, there is still a lack of a large amount of quantitative experimental data to compare with the modeling results. Moreover, model verification is difficult to apply to the cell membrane perforation and resealing process.

[0004] Therefore, it is of great significance to propose a method for dynamically and quantitatively evaluating the changes of myocardial cell membranes after electroporation to explore the mechanism of cell membrane electroporation. SUMMARY

[0005] The technical problem solved by the present application is to overcome the shortcomings in the prior art and provide a method for dynamically and quantitatively evaluating the changes of myocardial cell membranes after electroporation.

[0006] To solve the technical problem, the solution of the present application is:

[0007] The application provides a method for dynamically and quantitatively evaluating changes of myocardial cell membranes after electroporation, which is realized based on a cell electrophysiological sensing monitoring system, and the monitoring system comprises a cell electrophysiological sensing device and an electroporation control and signal recording integrated device; wherein the cell electrophysiological sensing device comprises, from top to bottom, a hollow glass culture cavity, a nano microelectrode array chip and a PCB adapter, the nano microelectrode array chip is fixed at the center of the surface of the PCB adapter, and the culture cavity is fixed on the microelectrode array chip; the upper surface of the PCB adapter is connected with the wires on the nano microelectrode array chip through silver paste, and the lower surface is welded with pins; part of the pins of the PCB adapter are connected to the electroporation control and signal recording integrated device through a wire harness, and the latter is connected to a computer and a power supply through a cable; the electroporation control and signal recording integrated device comprises an electric pulse control module, an electric signal conditioning module and a signal acquisition module, which are respectively used for pulse electric signal regulation, filter amplification processing and electrophysiological signal reception.

[0008] The method for dynamically and qualitatively evaluating changes of myocardial cell membranes after electroporation comprises the following steps:

[0009] (1) After the cell electrophysiological sensing device is sterilized with 75% alcohol and ultraviolet irradiation, the surface of the device is coated with a 10 μg / mL fibronectin solution, and the device is placed in a 37℃, 5.0% (v / v) CO2 incubator for 4 hours to promote cell adhesion;

[0010] (2) The purified animal myocardial cells are planted in the culture cavity, and the culture is continued in the incubator;

[0011] (3) After the myocardial cells appear spontaneous rhythmic beating, the electroporation control and signal recording integrated device is used to perform electroporation operation, and a transient pulse electric signal is applied to the cell electrophysiological sensing device to induce the cell membrane to open its nanopore, and the change of the cell membrane electrophysiological signal during the process is recorded;

[0012] (4) According to the amplitude change of the electrophysiological signal, the change of the cell membrane is evaluated:

[0013] Before electroporation, the action potential outside the cell membrane is recorded due to the barrier effect of the cell membrane, which reflects the state of the cell membrane after the stable cell-electrode interface is established;

[0014] After electroporation, due to the appearance of nanopores on the cell membrane, the signal recorded at this time is the intracellular action potential; the signal rapidly rises to the maximum amplitude in a short time, then gradually decays to a relatively low level, and finally returns to the initial extracellular potential; the amplitude change process of the signal corresponds to the entire evolution process of the cell membrane from the initial perforation, expansion to partial recovery, stability and finally resealing; among them, in the rapid amplitude rising stage, the cell membrane forms nanopores and the number, size and distribution range reach the maximum at the peak; in the amplitude gradually decaying stage, the nanopores of the cell membrane gradually recover and shrink; when the amplitude tends to be stable, the nanopores of the cell membrane have been resealed.

[0015] As a preferred scheme of the present application, in step (4), the amplitude of the electrophysiological signal is used to further calculate the amplitude change rate, energy integral and energy integral change rate; wherein the amplitude change rate refers to the change of the amplitude of the cell action potential with respect to time, the energy integral refers to the sum of squares of the amplitude of the cell action potential, and the energy integral change rate refers to the change of the energy integral with respect to time; the evaluation is performed in the following manner:

[0016] In the rapid amplitude rising stage: when the energy integral change rate exceeds 20 mV 2 / s, it indicates that nanopores begin to form on the cell membrane; when the signal amplitude exceeds 500 μV, it indicates that effective nanopores have formed on the cell membrane; when the energy integral change rate gradually decreases to close to 0, the nanopores reach the most stable state;

[0017] In the amplitude gradually decaying stage, when the energy integral change rate is lower than 100 mV 2 / s, it indicates that the nanopores on the cell membrane begin to heal; when the change rate is lower than 20 mV 2 / s, it indicates that the nanopores are basically complete, and at this time the amplitude is lower than 100 μV; when the change rate changes from negative to close to 0, it indicates that the nanopores on the cell membrane have completely recovered.

[0018] As a preferred scheme of the present application, when performing electroporation, the sampling rate of the action potential is controlled to be 15-20 kHz, the amplitude of the electroporation pulse is 2-6 V, the pulse width is 0.1-10 ms, and the number of pulses is 1-1000. This ensures effective electroporation of the cardiomyocytes and recording of the electrophysiological signal.

[0019] As a preferred scheme of the present application, the extracellular action potential before electroporation is recorded for 10 s-10 min, and the time for recording the potential change after electroporation is 1-60 min; the amplitude of the electroporation pulse is 3-4 V, the pulse width is 0.2-4 ms, and the number of pulses is 1-20. This ensures that the electroporation does not damage the cell activity, and at the same time, sufficient data is obtained for dynamic quantitative evaluation of the cell membrane change.

[0020] As a preferred scheme of the present application, the number of channels in the nanomicroelectrode array chip is 16-64, the metal layer is 5-20 nm Ti and 50-100 nm Au, the insulating layer is 2-5 um thick SU8, and the electrode diameter is 10-30 um; the diameter of the culture cavity is 0.6-1.5 cm.

[0021] As a preferred scheme of the present application, when the cardiomyocytes are planted in the culture cavity, the cardiomyocytes are uniformly covered on the surface of the nanomicroelectrode array chip and can fuse into a single layer.

[0022] Principle of the application:

[0023] The present application evaluates the membrane changes of the cardiomyocytes after electroporation by culturing the purified animal cardiomyocytes on a sensing and regulating device. The electroporation is performed after the cardiomyocytes appear spontaneous rhythmic beating, and the device is connected to an integrated device of electroporation control and signal recording. At this time, due to the barrier of the cell membrane, the platform records the extracellular action potential, reflecting the state of the cell membrane after the establishment of a stable cell-electrode interface. Further, by applying a transient electroporation pulse to the system, nanochannels appear on the cell membrane, at which time the signal recorded by the platform is converted into an intracellular action potential, and rapidly rises to a maximum amplitude in a short time, then the signal gradually decays to a lower level, and finally returns to the extracellular potential. Statistical analysis of the electrophysiological signals recorded by the platform before and after electroporation can quantitatively and dynamically evaluate the evolution of the cell membrane after electroporation, including the initial perforation, swelling, partial recovery, stability and gradual resealing of the cell membrane.

[0024] The continuous electrophysiological recording after electroporation can be divided into a short-amplitude rising stage and a long-amplitude falling stage. First, the amplitude increases with time, reaches a maximum value, and the change rate gradually decreases, reflecting the perforation process of the cell membrane after electroporation. With the passage of time, the number of nanochannels formed on the cell membrane is larger, the size is larger, and the range is wider, thereby inducing the amplitude evolution and its change rate in this stage. The change rate decreases in the later stage, indicating that the cell membrane with nanochannels has stability. The amplitude gradually decreases after reaching the maximum value, indicating that the resealing of the cell membrane is slow. More specifically, the amplitude change rate reaches a negative peak and then gradually increases, which characterizes the recovery of the cell membrane. Finally, the intracellular signal returns to the extracellular signal, and the change rate fluctuates around zero, which means the complete resealing of the cell membrane.

[0025] The application innovatively proposes to quantitatively evaluate the cell membrane change process by using energy integral and its change rate. The square sum of the amplitude of the electrophysiological signal is used to reflect the energy of the signal, and the energy of the signal after the maximum amplitude and its change rate dynamically reflect the recovery of the cell membrane. The evolution of the signal energy can be divided into two stages. In the first stage, the signal amplitude in the cell is large at the initial stage of electroporation, the energy increases rapidly, and the energy change rate decreases sharply, indicating that the cell membrane is gradually resealed. In the second stage, the energy and its change rate change slowly, indicating that the amplitude of the electrophysiological signal is low.

[0026] Compared with the prior art, the application has the beneficial effects that:

[0027] 1. The application can record the electrophysiological signal of the myocardial cell while applying electroporation, and realize accurate and high-throughput recording of the extracellular and intracellular action potential of the myocardial cell.

[0028] 2. After the application of transient electroporation on the myocardial cell, statistical analysis is performed on the recorded cell action potential to quantitatively describe the dynamic change process of the cell membrane. This method is significantly different from the conventional image recognition and static processing, and breaks through the conventional thinking mode of the person skilled in the art.

[0029] 3. The application quantitatively describes the entire dynamic process of the nanometer hole on the cell membrane from the beginning of formation, to the stable state, to the beginning of healing, and to the complete healing after electroporation by using the amplitude and its change of the action potential, the energy integral and its change rate. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a microscope graph of a nanometer microelectrode array chip;

[0031] Figure 2 It is a schematic diagram of a cell electrophysiological sensing and control device;

[0032] Figure 3 It is a microscope graph of myocardial cells cultured on a chip;

[0033] Figure 4 It is a signal graph corresponding to the change of the cell membrane before and after electroporation;

[0034] Figure 5 It is an amplitude and its change rate and energy integral and its change rate graph corresponding to the change of the electroporation cell membrane. DETAILED DESCRIPTION

[0035] The structure of the cell electrophysiological sensing device, and the preparation and use method thereof, are based on the previous work of the applicant, and can refer to the disclosure of "nanometer microelectrode array device" in the published patent documents "a sensing and detection method and system for coupling of myocardial cell excitation and contraction" (CN115078466A) and "a detection system and method for intracellular delivery and electrical sensing integration of myocardial cells" (CN115236049A). The electroporation control and signal recording integrated device can be realized according to the conventional scheme, and the application does not have special requirements.

[0036] The implementation modes of the application will be described in detail below with reference to the drawings.

[0037] As shown in the drawings, Figures 1-4 The cell electrophysiological sensing and monitoring system in the application includes a cell electrophysiological sensing device and an electroporation control and signal recording integrated device. The cell electrophysiological sensing device includes a hollow glass culture cavity, a nanometer microelectrode array chip and a PCB adapter arranged in sequence from top to bottom. The nanometer microelectrode array chip is fixed at the center of the surface of the PCB adapter, and the culture cavity is fixed on the microelectrode array chip. The upper surface of the PCB adapter is connected to the wires on the nanometer microelectrode array chip through silver paste, and the pins are welded on the lower surface. Part of the pins of the PCB adapter are connected to the electroporation control and signal recording integrated device through a wire harness, and the latter is connected to a computer and a power supply through a cable. The electroporation control and signal recording integrated device includes an electric pulse control module, an electric signal conditioning module and a signal acquisition module, which are respectively used for pulse electric signal regulation, filter amplification processing and electrophysiological signal reception.

[0038] Optionally, in the nanometer microelectrode array chip of the cell electrophysiological sensing device, the number of channels is 16-64, the metal layer is 5-20 nm of Ti and 50-100 nm of Au, the insulating layer is 2-5 μm thick SU8, and the electrode diameter is 10-30 μm. The diameter of the culture cavity is 0.6-1.5 cm.

[0039] Based on the above system, the method for dynamically and quantitatively evaluating the change of myocardial cell membrane after electroporation in the application includes the following steps:

[0040] (1) After the cell electrophysiological sensing device is sterilized with 75% alcohol and ultraviolet irradiation, the device surface is coated with a 10 μg / mL fibronectin solution, and placed in a 37℃, 5.0% (v / v) CO2 incubator for 4 hours to promote cell adhesion. Ensure that the myocardial cells are evenly covered on the surface of the nanometer microelectrode array chip and can fuse into a single layer.

[0041] (2) The purified animal myocardial cells are planted in the culture cavity, and continue to be cultured in the incubator.

[0042] (3) After the myocardial cell appears spontaneous rhythmic beating, the electric perforation operation is performed by using the electric perforation control and signal recording integrated device, and the transient pulse electric signal is applied to the cell electric physiological sensing device to induce the cell membrane to open its nanopore, and the change of the cell membrane electric physiological signal during the process is recorded;

[0043] (4) According to the amplitude change of the electric physiological signal, the cell membrane change is evaluated:

[0044] Before the electric perforation, the action potential outside the cell membrane is recorded due to the barrier effect of the cell membrane, reflecting the cell membrane state after the stable cell-electrode interface is established;

[0045] After the electric perforation, the action potential inside the cell is recorded due to the appearance of the nanopore on the cell membrane; the signal rapidly rises to the maximum amplitude in a short time, and then gradually decays to a relatively low level, and finally returns to the initial extracellular potential; the amplitude change process of the signal corresponds to the whole evolution process of the cell membrane from the initial perforation, expansion to partial recovery, stability and finally resealing after the electric perforation; during the rapid amplitude rising stage, the nanopore is formed on the cell membrane and the number, size and distribution range reach the maximum at the peak value; during the amplitude gradually decaying stage, the nanopore on the cell membrane gradually recovers and shrinks; when the amplitude tends to be stable, the nanopore on the cell membrane has been resealed.

[0046] Further, the energy integral and energy integral change rate can be calculated by using the amplitude of the electric physiological signal, and the evaluation can be performed in the following manner: during the rapid amplitude rising stage: when the energy integral change rate exceeds 20 mV 2 / s, it is indicated that the nanopore begins to form on the cell membrane; when the signal amplitude exceeds 500 μV, it is indicated that the effective nanopore has been formed on the cell membrane; when the energy integral change rate gradually decreases to close to 0, the nanopore reaches the most stable state; during the amplitude gradually decaying stage, when the energy integral change rate is lower than 100 mV 2 / s, it is indicated that the nanopore on the cell membrane begins to heal; when the change rate is lower than 20 mV 2 / s, it is indicated that the nanopore is basically complete, and the amplitude is lower than 100 μV; when the change rate changes from the negative value to close to 0, it is indicated that the nanopore on the cell membrane has been completely recovered.

[0047] During the electric perforation operation, the extracellular action potential before the electric perforation is recorded for 10 s to 10 min, and the electric potential change after the electric perforation is recorded for 1 to 60 min; the sampling rate of the action potential is controlled to be 15 to 20 kHz, ensuring the effective electric perforation of the myocardial cell and the recording of the electric physiological signal.

[0048] When performing electroporation, the electroporation pulse amplitude is 2-6V, the pulse width is 0.1-10ms, and the pulse number is 1-1000. More preferably, the electroporation pulse amplitude is 3-4V, the pulse width is 0.2-4ms, and the pulse number is 1-20; to ensure that electroporation does not damage cell activity, while obtaining sufficient data for dynamic quantitative evaluation of cell membrane changes.

[0049] More detailed specific embodiments:

[0050] 1. A cell electrophysiological sensing and regulation device is prepared and assembled using standard microfabrication techniques.

[0051] The nanomicroelectrode array chip in the cell electrophysiological sensing device is manufactured on a 10x10cm 2 glass substrate with a thickness of 0.5mm. After ultrasonic cleaning in acetone and ethanol for 10 minutes, 2.5μm thick RZJ-390PG-30 photoresist is spin-coated, 120℃ baked for 2min, then exposed to ultraviolet light by a photoetching machine, and developed for 35s. Then 10nm thick titanium layer and 100nm thick gold layer are sputtered onto the substrate, and the excess photoresist is stripped with acetone. Finally, 2μm thick SU-8 2002 is spin-coated on the conductive layer, 95℃ baked for 1min, aligned by a photoetching machine, and exposed to ultraviolet light, then developed in propylene glycol methyl ether acetate for 1min, isopropanol for 1min, and 150℃ baked for 11h. After the chip array is prepared, it is cut into 2x2cm 2 units, and each unit is fixed on a customized printed circuit board and connected by conductive silver paste. Finally, the glass culture cavity is fixed at the center of the chip, and the pins (or pin array) are soldered to the lower surface of the printed circuit board, completing the preparation and assembly of the cell electrophysiological sensing and regulation device.

[0052] The example nanomicroelectrode array chip is shown in Figure 1 , with 16-64 channels and electrode diameters of 10-30μm. The example cell electrophysiological sensing and regulation device is shown in Figure 2 , with a cell culture cavity diameter of 0.6-1.5cm.

[0053] 2. Primary neonatal rat cardiomyocytes are cultured on the cell electrophysiological sensing and regulation device.

[0054] The cell electrophysiological sensor regulating device is sterilized by 75% ethanol and ultraviolet irradiation for 2 hours, then coated with 10 μg / mL fibronectin solution, and stored in a cell incubator at 37°C and 5.0% (v / v) CO2 for 4 hours to promote cell adhesion. Fresh heart tissue of newborn rats is obtained, and washed with ice-cold culture solution to remove blood cells. Then cut into small pieces at 37°C in Hanks balanced salt solution, and add 0.07% trypsin and 0.05% collagenase type II for digestion for 10-12 times, and update the digestion enzyme every 8 minutes. After digestion, the mixed cells are blown down from the loose tissue, centrifuged for 5 minutes to purify the cardiomyocytes, resuspended in the culture medium, filtered through a 70 μm cell sieve, and then subjected to differential adhesion for 45 minutes twice. Finally, the purified cardiomyocytes are seeded on the cell electrophysiological sensor regulating device at a density of 3×10 5 cells / cm 2 . The device is placed in a culture incubator at 37°C and 5.0% (v / v) CO2, and the culture medium is replaced every 48 hours.

[0055] As shown in Figure 3 , the cardiomyocytes cultured on the nano-microelectrode array chip have good morphology and fuse into a monolayer, uniformly covering the sensing electrodes on the chip.

[0056] 3. Evaluation of cell membrane changes after electroporation based on the cell electrophysiological sensor regulating platform.

[0057] After 3-4 days of culture, the cardiomyocytes exhibit spontaneous rhythmic beating, and the device is connected to the electroporation control and signal recording integrated device. The system sampling rate is 15 kHz, and the band-pass filtering is 1 Hz-7.5 kHz. After recording the extracellular action potential, further transient electroporation pulses are applied by the system, with a pulse of 3V, 200 μs, and 20 times. After applying the electroporation, the signal is converted into an intracellular action potential, which is continuously recorded and further statistically analyzed to quantitatively and dynamically evaluate the evolution of the cell membrane after electroporation.

[0058] After the cell electrophysiological sensor device is connected to the device, the electrophysiological signal is amplified and filtered by the signal conditioning module, further transmitted to the signal acquisition module, and then transmitted to the computer and displayed, stored, and processed using the software program. The software program can be implemented in a conventional manner according to actual needs, and the present application does not make special requirements for the specific implementation of the program. The software program can also be used to apply different conditions of electroporation to the cell electrophysiological sensor device through the electric pulse control module and the signal acquisition module.

[0059] For the recorded data, computer software can be used to process for evaluating the electroporation membrane change process. The routine software functions can include: extracting and formatting the raw data record, multi-channel data display, de-noising, feature point extraction and result calculation. For example, first, the signal is de-noised by wavelet decomposition and selective reconstruction based on the "db06" wavelet base, and the effective signal component is retained. Second, the feature points of the filtered signal are defined, and the characteristic parameters (amplitude and firing rate) of the signal changing with time are extracted. The peak points are determined by setting the minimum distance between adjacent peak values to avoid false peak detection. The minimum distance set depends on the firing period of the signal and is shorter than the firing period of the signal. In this example, a value of 0.25-0.3 s is set according to the actual firing rate of the recorded signal. For intracellular electrophysiological signals, the starting point of the signal is determined by a dynamic threshold. For extracellular electrophysiological signals, the valley point is determined by searching for the minimum value in a 20 ms data window after the peak point of the same signal. According to the extracted features, the amplitude, slope, energy and spectrum can be calculated subsequently. The amplitude of the action potential is defined as the change of the signal from the starting point to the peak value, and the rate is defined as the rate of change of the amplitude. The energy is defined as the square of the amplitude, and the time-varying spectrum is calculated using continuous wavelet transform based on the "db6" wavelet base.

[0060] The continuous electrophysiological recording after electroporation can be divided into a short amplitude rising phase and a long amplitude falling phase. First, the amplitude increases with time, reaches a maximum value, and the rate of change gradually decreases, reflecting the pore formation process of the cell membrane after electroporation. With the passage of time, the number of nanopores formed on the cell membrane is larger, the size is larger, and the range is wider, thereby inducing the evolution of the amplitude and its rate of change in this stage. The change rate decreases in the later stage, indicating that the cell membrane with nanopores has stability. The amplitude gradually decreases after reaching the maximum value, indicating that the resealing of the cell membrane is slow. More specifically, the amplitude change rate reaches a negative peak value, and then gradually increases, characterizing the recovery of the cell membrane. Finally, the intracellular signal returns to the extracellular signal, and the change rate fluctuates around zero, which means the complete resealing of the cell membrane.

[0061] The square sum of the amplitude of the electrophysiological signal is used to reflect the energy of the signal. The energy of the signal after the maximum amplitude and its rate of change dynamically reflect the recovery of the cell membrane. The evolution of the signal energy can be divided into two stages. In the first stage, the intracellular signal amplitude is large at the initial stage of electroporation, the energy increases rapidly, and the energy change rate decreases sharply, indicating that the cell membrane is gradually resealed. In the second stage, the energy and its change rate change slowly, indicating that the amplitude of the electrophysiological signal is low.

[0062] Figure 4The signal corresponding to the change of cell membrane before and after electroporation is shown. The initially recorded extracellular action potential reflects the state of the cell membrane after the establishment of a stable cell-electrode interface. After the application of electroporation, the cell membrane opens the nanopore, and then gradually swells, at which time the recorded signal is converted into an intracellular action potential, and rapidly rises to a maximum amplitude in a short time. As the cell membrane continues to recover and reseal, the recorded signal gradually decays to a lower level, and finally returns to the extracellular potential.

[0063] Figure 5 The evolution of the amplitude, amplitude rate of change, energy integral and energy integral rate of change of the recorded signal after electroporation is shown, which quantitatively reflects the evolution of the cell membrane after electroporation.

[0064] The left graph shows the change of action potential amplitude and its rate of change over time after electroporation. As can be seen from the graph, the amplitude gradually decreases after reaching a maximum value, indicating that the cell membrane is slowly resealed. More specifically, the amplitude rate of change reaches a negative peak and then gradually increases, indicating the recovery of the cell membrane. Finally, the intracellular signal returns to the extracellular signal, and the rate of change fluctuates around zero, which means the complete resealing of the cell membrane. The right graph shows the change of energy integral and its rate of change over time after electroporation. As can be seen from the graph, the evolution of signal energy can be divided into two stages. In the first stage, the intracellular signal amplitude is large at the beginning of electroporation, the energy increases rapidly, and the energy rate of change decreases sharply, indicating that the cell membrane is gradually resealed. In the second stage, the energy and its rate of change change slowly, indicating that the amplitude of the electrophysiological signal is low. Therefore, it can be confirmed that this method can quantitatively evaluate the change of cell membrane after electroporation in real time by analyzing the cell action potential.

Claims

1. A method of dynamically quantifying changes in myocardial cell membranes after electroporation, comprising: The method is realized based on a cell electrophysiological sensing monitoring system, which comprises a cell electrophysiological sensing device and an electroporation control and signal recording integrated device; the cell electrophysiological sensing device comprises, from top to bottom, a hollow glass culture cavity, a nano microelectrode array chip and a PCB adapter, the nano microelectrode array chip is fixed at the center of the surface of the PCB adapter, and the culture cavity is fixed on the microelectrode array chip; the upper surface of the PCB adapter is connected with the wires on the nano microelectrode array chip through silver paste, and the lower surface is welded with pins; part of the pins are connected to the electroporation control and signal recording integrated device through a wire harness, and the latter is connected to a computer and a power supply through a cable; the electroporation control and signal recording integrated device comprises an electric pulse control module, an electric signal conditioning module and a signal acquisition module, which are respectively used for pulse electric signal regulation, filter amplification processing and electrophysiological signal reception; The method comprises the following steps: (1) after the cell electrophysiological sensing device is sterilized with alcohol and irradiated with ultraviolet rays, the surface of the device is coated with a fibronectin solution, and the device is placed in a culture box to promote cell adhesion; (2) the purified animal myocardial cells are planted in the culture cavity, and the culture is continued in the culture box; (3) after the myocardial cells appear spontaneous rhythmic beating, the electroporation control and signal recording integrated device is used to perform electroporation operation, and a transient pulse electric signal is applied to the cell electrophysiological sensing device to induce the cell membrane to open its nanopore, and the change of the cell membrane electrophysiological signal during the process is recorded; (4) according to the amplitude change of the electrophysiological signal, the change of the cell membrane is evaluated: the amplitude change rate, the energy integral and the energy integral change rate are calculated by using the amplitude of the electrophysiological signal; wherein the amplitude change rate refers to the change of the amplitude of the cell action potential with respect to time, the energy integral refers to the sum of squares of the amplitude of the cell action potential, and the energy integral change rate refers to the change of the energy integral with respect to time; During the phase of rapid amplitude increase: the rate of change of energy integral exceeds 20mV. 2 When the signal amplitude reaches 500 μV, it indicates that nanopores have begun to form on the cell membrane; when the signal amplitude exceeds 500 μV, it indicates that effective nanopores have formed on the cell membrane; when the energy integral change rate gradually decreases to zero, the nanopores reach their most stable state. During the phase of gradual amplitude decay, the rate of change of energy integral is less than 100 mV. 2 When the rate of change is / s, it indicates that the nanopores on the cell membrane have begun to heal; when the rate of change is less than 20mV 2 When the amplitude is / s, it indicates that the nanopores are basically healed, and the amplitude is less than 100 μV; when the rate of change fluctuates near zero, it indicates that the nanopores on the cell membrane have been fully restored.

2. The method of claim 1, wherein, when performing electroporation, the sampling rate of the action potential is controlled to be 15-20 kHz, the amplitude of the electroporation pulse is 2-6 V, the pulse width is 0.1-10 ms, and the number of pulses is 1-1000.

3. The method of claim 1, wherein, The extracellular action potential before electroporation is recorded for 10 s-10 min, and the time for recording the potential change after electroporation is 1-60 min; the amplitude of the pulse applied during electroporation is 3-4 V, the pulse width is 0.2-4 ms, and the number of pulses is 1-20.

4. The method of claim 1, wherein, In the nano microelectrode array chip, the number of channels is 16-64, the metal layer is 5-20 nm of Ti and 50-100 nm of Au, the insulating layer is 2-5 μm thick SU8, and the electrode diameter is 10-30 μm; the diameter of the culture cavity is 0.6-1.5 cm.

5. The method of claim 1, wherein, When the myocardial cells are planted in the culture cavity, it is ensured that the myocardial cells uniformly cover the surface of the nano microelectrode array chip and can fuse into a single layer.

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