A method for detecting the degree of irreversible electroporation using scanning electrochemical microscopy (SECM)

Abstract

The present application relates to a method for detecting the degree of irreversible electroporation by scanning electrochemical microscopy (SECM), which realizes rapid and efficient detection of the degree of cell electroporation by measuring the membrane permeability (Pm) of cells after stimulation by a pulsed electric field. The method first measures the approach curve of the SECM probe to the cells, then converts the approach curve into the membrane permeability of the cells to complete the measurement of the degree of electroporation, and measures the survival rate curve of the cells by the existing detection method, establishes the relationship between the Pm value and the cell survival rate, verifies the detection of irreversible electroporation by the membrane permeability of the cells, and finds that the law of the change of the membrane permeability with the pulsed voltage is almost the same as the law of the change of the cell survival rate with the pulsed voltage, so the method for measuring the degree of cell electroporation by SECM is effective and reliable.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting irreversible electroporation by scanning electrochemical microscopy (SECM) by measuring the membrane permeability (Pm) of cells to achieve rapid and efficient detection of the degree of electroporation. More specifically, the present application relates to measuring the membrane permeability of cells in biological tissues after applying irreversible electroporation pulse ablation to determine the degree of electroporation caused by different pulse conditions on different cells. BACKGROUND

[0002] Malignant tumors affect the normal metabolic function of the human body, seriously impairing the normal function of various organs, and are diseases that endanger human health and life. The 2020 global cancer incidence and mortality estimates data released by the International Agency for Research on Cancer shows that there were 1930 million new cancer cases worldwide in 2020, and nearly 100 million people died of cancer. Cancer treatment has been a field that global scientists and even the entire society have been striving to break through. Despite the large amount of time and effort invested, the road to tackling cancer is still fraught with difficulties, so finding safe, effective and specific cancer treatment methods is still the focus.

[0003] Pulsed electric field (PEF) ablation as a physical minimally invasive ablation method has the advantage of non-thermal selectivity compared to physical thermal therapy, can avoid the thermal sink effect, and produces very little heat that does not cause thermal damage to surrounding structures, and can be applied to the treatment of tumors near important organ structures such as blood vessels. It is achieved by exposing cells to extremely short and super-strong electric fields. After the cancer cells are stimulated by electricity, nanoscale micropores appear on the cell membrane, increasing the membrane permeability, i.e. electroporation of cells. According to whether the micropores can self-close, the pulsed electric field ablation technology is divided into reversible electroporation and irreversible electroporation ablation. Irreversible electroporation uses more powerful pulse conditions, so that the micropores cannot self-repair, the integrity of the cell membrane is destroyed, and cell apoptosis is induced.

[0004] However, after cells are stimulated by a pulsed electric field, different cells form different degrees of electroporation under different pulse parameters, which is an important feature of this method that can specifically treat different tumors. In order to achieve effective, stable and safe killing effects, it is necessary to screen different tumor cells and explore the degree of electroporation under different conditions in advance. Current electroporation detection methods are mainly completed by tracking detection of added substances, detection of cell effluent, and monitoring of changes in physical properties. However, considering the available equipment and experimental conditions used, they still have their own shortcomings, such as the need for cell washing, the use of substances with certain cytotoxicity, long detection time, low accuracy and a series of problems.

[0005] Therefore, it is urgent to construct an effective electroporation detection method to evaluate the degree of electroporation and establish the relationship between the degree of electroporation and pulse conditions. As a non-invasive electrochemical measurement method, scanning electrochemical microscopy (SECM) combines probe microscopy and ultramicroelectrode electrochemistry, can draw surface reactivity, and evaluate surface reaction kinetics and surface chemical imaging, and is particularly prominent in the field of biomedicine. Among them, some experts have successfully measured the change of cell membrane permeability of cells exposed to metal ions by using this method. Zhang M.N. et al. used SECM to measure cisplatin-induced human bladder cancer cells (T24). In the depth scanning mode, SECM can quantify cell morphology and membrane permeability at the same time. The results show that the addition of cisplatin to the external environment of T24 cells can immediately cause changes in cell membrane permeability within 5 minutes. Filice F.P. et al. used depth scanning imaging of SECM to monitor the membrane permeability of T24 cells under the stimulation of different concentrations of Cr(VI). The results show that low concentration of Cr(VI) has little effect on membrane permeability within a short incubation time. With the increase of time, the membrane permeability of the cells increases significantly, and with the increase of concentration, the change of membrane permeability is accelerated. The irreversible electroporation technology changes the cell membrane permeability by means of electric pulse to achieve tumor treatment. However, there is no good detection method at present. It is of great significance to develop a rapid and efficient irreversible electroporation detection method for membrane permeability by using SECM to detect the membrane permeability of cells after irreversible electroporation and establishing the relationship between the degree of irreversible electroporation and the efficiency of irreversible electroporation. SUMMARY

[0006] The application aims to provide a method for detecting the degree of irreversible electroporation by using scanning electrochemical microscopy (SECM), which realizes rapid and efficient detection of the degree of cell electroporation by measuring the membrane permeability (Pm) of cells after pulse electric field stimulation. The method first measures the approach curve of the SECM probe to the cells, then converts the approach curve into the membrane permeability of the cells to complete the measurement of the degree of electroporation, and measures the survival rate curve of the cells by using the existing detection method, establishes the relationship between the Pm value and the cell survival rate, and verifies the detection of irreversible electroporation by the membrane permeability of the cells. The law of change of the membrane permeability of the cells with the pulse voltage is almost the same as the law of change of the cell survival rate with the pulse voltage, so the method for measuring the degree of cell electroporation by using SECM is effective and reliable.

[0007] The technical scheme of the application is as follows:

[0008] A method for detecting irreversible electroporation by using scanning electrochemical microscopy (SECM) can specifically measure the membrane permeability of cells and rapidly and efficiently measure the degree of electroporation of cells after pulse electric field stimulation, and specifically comprises the following steps:

[0009] After the cell culture reaches the logarithmic growth phase density, the corresponding pulse parameters (pulse width, pulse number, voltage amplitude and pulse frequency) are set on the high-voltage pulse generator, and then the irreversible electroporation ablation is performed on the cells by using a metal electrode, and after the ablation is completed, the culture dish is not replaced, and the prepared corresponding cell culture solution is directly replaced with an equal amount of redox medium, and then the scanning electrochemical microscope is used to probe the cell to determine the approach curve of the SECM probe to the cell, and then the cell probe approach curve is corresponded to the simulated standard probe approach curve, and the cell membrane permeability (Pm) value is converted to complete the determination of the degree of electroporation.

[0010] According to the method for detecting irreversible electroporation, further, the Pm value can be related to the cell survival rate by using other electroporation detection means to establish a standard library to further verify and judge the degree of irreversible electroporation.

[0011] According to the method for detecting irreversible electroporation, further, the probe electrode used by the scanning electrochemical microscope is a supermicroelectrode (diameter less than 25 μm), which can be a platinum electrode, a gold electrode, etc.

[0012] According to the method for detecting irreversible electroporation, further, the mode adopted by the scanning electrochemical microscope is a negative feedback working mode, and a redox medium is added in the solution environment of the cell to generate a Faraday current, and the degree of membrane permeability is determined by observing the current change.

[0013] According to the method for detecting irreversible electroporation, further, the redox medium used needs to meet the requirements of not being able to normally penetrate the cell membrane and not producing toxic side effects on the cells, and can be a series of compounds such as ferrocene methanol and ferrocene ethanol.

[0014] According to the method for detecting irreversible electroporation, further, the measured cells need to meet the requirements of adherent growth on the culture dish, and can be any normal cells or tumor cells.

[0015] According to the method for detecting irreversible electroporation, further, the corresponding standard probe approach curve needs to be simulated by using simulation software, the approach curve obtained by the experiment is corresponded to the standard curve, so as to obtain the membrane permeability under different pulse voltage electroporation, the degree of electroporation is represented by the membrane permeability, and the relationship between different pulse parameters and the degree of electroporation is established.

[0016] According to the method for detecting irreversible electroporation, further, the scanning electrochemical microscope works and the cells in the irreversible electroporation ablation are adhered to the culture dish, and the applied pulse electric field needs to meet the requirements of stably covering the bottom of the culture dish and ensuring that the electric field intensity in the area approached by the probe each time is consistent.

[0017] According to the other detection means for detecting irreversible electroporation in the application, further, the related detection methods such as FESEM observation of cell membrane pore forming rule and MTT method for measuring cell survival rate can be used as reference evaluation technology.

[0018] According to the method for detecting irreversible electroporation in the application, further, the cell membrane permeability can be specifically determined after the cell is stimulated by the pulse electric field, and the degree of electroporation can be determined quickly and efficiently. The irreversible electroporation technology is used for tumor treatment by changing the cell membrane permeability. The cell membrane permeability is determined by the negative feedback working mode of the scanning electrochemical microscope. The sample preparation of the SECM is simple, and does not need cell fixation and any dyeing or marking steps. The probe does not need to contact the cell in the operation process, and will not damage the cell. The detection time is short, and the approach curve under the corresponding pulse parameters can be measured in 5 minutes. The method can be applied to the detection of the degree of electroporation under various conditions, such as changing the pulse number, pulse frequency, pulse width, pulse voltage, and using different waveforms and different pulse combinations for electric field ablation. As long as the cell perforation can be formed, the method can be used for detection.

[0019] In some embodiments of the application, the electrode used for the pulse electric field ablation can be a self-made plane electrode sheet, which is placed stably above the bottom of the culture dish when used, and includes at least one electrode for power supply and at least one electrode for grounding. The electrode can withstand 3000-4000V when powered, and the pulse width is 0.01-100μs, and the pulse voltage is 1kHZ-1MHZ.

[0020] In some embodiments of the application, the COMSOL simulation software is used to draw the standard approach curve under different Pm, and each approach curve corresponds to a unique Pm value. Then, the approach curve obtained from the experiment is compared with the standard curve to obtain the membrane permeability under the electric perforation of different pulse voltages. The degree of electric perforation is represented by the membrane permeability, and the relationship between different pulse parameters and the degree of electric perforation is established.

[0021] In some embodiments of the application, the survival rate curve of the cell can be determined by adding exogenous MTT dye, the relationship between the Pm value and the cell survival rate is established, and the detection of the irreversible electroporation of the cell membrane permeability is verified.

[0022] In some embodiments of the application, the feasibility and effectiveness of the method can be further determined by observing the cell membrane perforation under different voltages in the field emission scanning electron microscope. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1is a flow chart of the irreversible electroporation detection method according to the present application;

[0024] Figure 2 is a standard approximation curve drawing diagram simulated by COMSOL;

[0025] Figure 3 is a probe approximation curve diagram of SECM for different cells after electroporation under different pulse voltages;

[0026] Figure 4 is a cell survival rate-pulse voltage curve diagram of MTT for different cells;

[0027] Figure 5 is a comparison diagram of cell membrane permeability measured by SECM and cell survival rate measured by MTT under the same pulse parameters;

[0028] Figure 6 is a magnification diagram of FESEM for different cells under different pulse voltages. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present application will be described herein below with reference to the drawings. It should be noted that the relative arrangement, numerical values of the components and steps set forth in these embodiments do not limit the scope of the present application unless otherwise specifically stated.

[0030] The following description of at least one example implementation is merely illustrative in nature and is in no way intended to limit the scope of the application, its application, or uses.

[0031] Figure 1 is a flow chart of the irreversible electroporation detection method according to the present application

[0032] As Figure 1As shown, the flowchart of the irreversible electroporation detection method of the present application can be composed of four parts: irreversible electroporation part 11, SECM probe approach part 12, simulation software simulation part 13 and combination with other detection methods part 14. Among them, the irreversible electroporation part 11 is the part where the cells experience pulse ablation, the pulse generator 15, after adjusting certain pulse parameters, transmits the pulse electric field to the cells through the pulse electrode 16, the pulse electric field fully covers the cell culture dish 17, and makes the cells in it receive uniform irreversible electroporation; the SECM probe approach part 12 is controlled by the SECM central computer 18 to output signals such as potential distance to the probe electrode device 19, and to make probe approach to the cells, and to transmit the cell signals obtained by the probe back to the computer 18 for data processing; the simulation software simulation part 13 is a part simulated by the host software 110 throughout, after setting the computer parameters, simulating and drawing the standard approach curve; the combination with other detection methods part 14 is to further verify the cell membrane permeability to complete the irreversible electroporation detection, and to determine the cell survival rate curve by the existing detection method, and to establish the relationship between Pm value and cell survival rate.

[0033] Figure 2 is a standard approach curve drawing schematic diagram simulated by COMSOL

[0034] The cell probe approach curve obtained by experiment is a curve graph of the tip current and the distance of the cell, which is difficult to directly express the degree of cell membrane permeability. Therefore, COMSOL simulation software is selected to draw the standard approach curve under different Pm, each approach curve corresponds to a unique Pm value, and then the approach curve obtained by experiment is compared with the standard curve to obtain the membrane permeability under different pulse voltage electroporation, the degree of electroporation is expressed by the membrane permeability, and the relationship between different pulse parameters and the degree of electroporation is established.

[0035] Figure 2 (A) is a geometric construction diagram simulated by software. The spatial dimension of the simulation model is selected as two-dimensional axisymmetric, the physical field is selected as dilute substance transfer in chemical substance transfer, the cell 21 adopts a semi-ellipsoidal shape, the diameter is set to 30.0 μm, the height is 10.0 μm, the probe glass sleeve 22 has a diameter of 32.0 μm. Among them, the platinum electrode 23 has a diameter of 10.0 μm (RG=3.2), and the cell 21, the probe glass sleeve 22 and the platinum electrode 23 are placed in the bulk solution 24, after setting the related definition parameters, the grid is divided and optimized, and finally by changing the distance between the probe and the cell, a plurality of points are obtained, the calculated tip current is set as the ordinate value, the distance between the probe and the cell is set as the abscissa, and a plurality of points are connected into a smooth curve to draw the standard approach curve under the corresponding Pm.

[0036] Figure 2 (B) is the theoretical approximation curve 25 corresponding to different Pm values, and seven standard curves are selected, with Pm being 0.0 x 10-5 m / s, 0.5 x 10-5 m / s, 1.0 x 10-5 m / s, 1.5 x 10-5 m / s, 2.0 x 10-5 m / s, 2.5 x 10-5 m / s and 3.0 x 10-5 m / s, respectively. As can be seen from the figure, as the Pm value increases, the curve moves to the left, indicating that the probe will be closer to the cell surface due to current compensation. It can be found from the experimental control group (0 V) 26 that the approximation curve 26 obtained when the cell is not electroporated can well coincide with the curve 25 with Pm being 0. The approximation curve 26 obtained in the experiment can be found in the standard curve 25, so as to obtain the corresponding Pm value under different electroporation.

[0037] Example 1: Probe approximation experiment of three kinds of cancer cells (4T1, B16 and Hela) and two kinds of normal cells (HC11 and L929) after electroporation under different pulse voltages.

[0038] Since SECM cannot detect suspended cells in actual operation, it is required to complete irreversible electroporation ablation and PACs determination under the condition that the cells are adherent. Therefore, the cells are cultured in a 35 mm culture dish so that the electroporation and probe approximation process can be directly implemented thereon without the need to change the dish. A planar electrode sheet (two pieces of 2 x 1 cm 2 metallic sheet are clamped on an insulating film sheet as the positive and negative electrodes of the electrode, and a copper wire is welded from the back to serve as a lead wire to ensure the smoothness of the electrode output surface) is self-made to complete the electroporation of the adherent cells. L929 and HC11, two kinds of normal cells, and Hela, B16 and 4T1, three kinds of cancer cells are selected as experimental objects. The applied pulse parameters are as follows: pulse voltage is 0-1200 V, and a set of experiments is set every 200 V (0 V is the control group without any pulse intensity voltage), pulse width is 100 μs, pulse number is 60, and pulse frequency is 1 Hz. Each set of experiments is determined in triplicate.

[0039] Figure 3 is the probe approximation curve graph of different cells after electroporation under different pulse voltages (the dots in the figure), and it is compared with the standard approximation curve to obtain the corresponding Pm value. Figure 3 a is the approximation curve graph of 4T1 cells, as shown in the figure. The approximation curves of the cells under pulse voltages of 200 V, 400 V and 600 V are relatively close, and Pm is 0.9 x 10 -5around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s - 5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s Figure 3 b is the approximating curve of B16 cells, as shown in the figure, the cells at the pulse voltage of 200V, Pm is around 1.0 x 10~5m / s, at 400V, Pm is 1.4 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s Figure 3 c is the approximating curve of Hela cells, as shown in the figure, the cells at the pulse voltages of 200V, 400V and 600V are relatively close, and Pm is around 1.4 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V, 800V and 1000V are relatively close, and Pm is around 2.2 x 10~5m / s Figure 3 d is the approximating curve of HC11 cells, as shown in the figure, the cells at the pulse voltage of 200V, Pm is around 0.4 x 10~5m / s, the approximating curves at the pulse voltages of 400V and 600V are relatively close, and Pm is around 0.8 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s Figure 3 e is the approximating curve of L929 cells, as shown in the figure, the cells at the pulse voltage of 200V, Pm is around 0.9 x 10~5m / s, the approximating curves at the pulse voltages of 400V and 600V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s -5 around 2.2 x 10~5m / s, the approximating curves at the pulse voltages of 600V and 800V are relatively close, and Pm is around 1.2 x 10~5m / s

[0040] From the comparison of the experimental and standard approximation curves of cells in SECM, it can be seen that the irreversible electroporation caused by the pulse voltage to the cell membrane will change the permeability of the cell membrane, and it is closely related to the amplitude of the applied pulse voltage. When the Pm values are similar, it indicates that the degree of irreversible electroporation caused by the applied pulse voltage at this time is similar, and as the voltage gradually increases, the Pm also increases accordingly, indicating that the degree of electroporation is greater at this time. And it is found that different cells have different Pm values when the same pulse voltage is applied, and the distribution of the approximation curve changes with the voltage, indicating that the degree of electroporation of different cells under the same conditions will be different. Therefore, by detecting and recording the Pm values of different cells under different voltages, a standard library for specific cells can be established to judge the degree of electroporation under certain pulse parameters.

[0041] Example 2: MTT staining of three cancer cells (4T1, B16 and Hela) and two normal cells (HC11 and L929) after electroporation under different pulse voltages to measure cell survival rate.

[0042] Currently, dyes are often used to detect cell survival rate in cell experiments to determine the degree of electroporation. Thiazolyl blue (MTT) can be reduced to a blue-violet formazan under the action of succinate dehydrogenase to indirectly reflect the number of living cells, so MTT is selected in this paper to verify the reliability of the method of SECM for detecting the degree of electroporation. L929 and HC11 normal cells and Hela, B16 and 4T1 cancer cells were selected as experimental objects, and the applied pulse parameters were: pulse voltage 0-1200V, with a set of experiments every 200V (0V as the control group, without any pulse intensity voltage), pulse width 100μs, pulse number 60, pulse frequency 1Hz, and each group of experiments was measured in triplicate.

[0043] Figure 4 is the MTT method for determining the cell survival rate-pulse voltage curve of different cells. As can be seen from the figure, with the increase of pulse voltage, the cell survival rate is gradually decreasing, indicating that the degree of electroporation is gradually deepening, and the five curves all have a slow decline and a rapid decline region. The slow decline region indicates that the cell death rate is similar in this pulse voltage range, and the degree of electroporation is similar; while the rapid decline region indicates that the cell death rate has a mutation in this pulse voltage range, and the degree of electroporation is very different. And it is also found that the cell survival rate of different cells is different under the same pulse voltage, that is, the degree of electroporation of different cells under the same pulse conditions is different, which indicates that the killing of electroporation on cells is selective, not indiscriminate.

[0044] Figure 5is a comparison chart of cell membrane permeability measured by SECM and cell survival rate measured by MTT method under the same pulse parameters. The MTT method is used to verify the reliability of the method of SECM for detecting the degree of electroporation. It can be seen from Figure 5 (A) that the Pm of 4T1 cells at 200V, 400V and 600V is similar, and the Pm at 800V, 1000V and 1200V is also similar, and under the same pulse voltage, the cell survival rate also has the same rule, and with the increase of voltage, the Pm and the cell survival rate show the same change rule; from Figure 5 (B) it can be known that the Pm of B16 cells at 600V, 800V and 1000V is similar, and under the same pulse voltage, the cell survival rate also has the same rule, and with the increase of voltage, the Pm and the cell survival rate show the same change rule; from Figure 5 (C) it can be known that the Pm of Hela cells at 200V, 400V and 600V is similar, and the Pm at 1000V and 1200V is also similar, and under the same pulse voltage, the cell survival rate also has some of the same rule, and with the increase of voltage, the Pm and the cell survival rate show the same change rule; from Figure 5 (D) it can be known that the Pm of HC11 cells at 400V and 600V is similar, and the Pm at 800V and 1000V is also similar, and under the same pulse voltage, the cell survival rate also has some of the same rule, and with the increase of voltage, the Pm and the cell survival rate show the same change rule; from Figure 5 (E) it can be known that the Pm of L929 cells at 400V and 600V is similar, and the Pm at 800V, 1000V and 1200V is also similar, and under the same pulse voltage, the cell survival rate also has some of the same rule, and with the increase of voltage, the Pm and the cell survival rate show the same change rule.

[0045] SECM is used to determine the degree of electroporation by measuring the permeability of cell membrane, and MTT method is used to determine the degree of electroporation by measuring the survival rate of cells, and the relationship between Pm value and cell survival rate is established, and it is found that the rule of cell membrane permeability changing with pulse voltage is almost the same as the rule of cell survival rate changing with pulse voltage, so the method of using SECM to determine the degree of electroporation of cells is effective and reliable.

[0046] Example 3: FESEM observation of three kinds of cells (B16, Hela and L929) after electroporation under different pulse voltages.

[0047] The experiment continues to explore the pore-forming rules on the cell membrane under the influence of the pulsed electric field, such as the pore size, the number of pores and the distribution, etc. Hela, B16 and L929 cells are selected as the experimental objects, and the pulsed parameters are as follows: Hela cells: the pulsed voltage is 0V, 100V, 300V, 600V, 800V, 1000V (0V is the control group, and no pulse intensity voltage is given); B16 cells: the pulsed voltage is 0V, 100V, 300V, 500V, 900V; L929 cells: the pulsed voltage is 0V, 100V, 400V, 700V, 1000V, and other conditions are consistent, the pulse width is 100μs, the pulse number is 60, and the pulse frequency is 1Hz.

[0048] Figure 6 Figure 1 is a schematic diagram of FESEM observation of different cells under different pulsed voltages, which is used to observe the pore-forming rules on the cell membrane. The image is magnified 20000 times, and the nano-sized structure can be seen. Among them, B16 cells are selected at the pulsed voltages of 100V, 300V, 600V, 800V and 1000V for electroporation, and the images are magnified 20000 times. Figure 6 (A) It can be known from the figure that at 0V and 100V, the cell surface is very regular and no holes are found. At 300V, small holes are found in the local part, and there are not many holes. At 500V, more and more holes are found, but they are still small. At 900V, not only many holes are found, but also large holes are found. Figure 6 (B) It can be known from the figure that at 0V and 100V, almost no holes are found on the cell surface. At 300V, small holes are found, and the hole distribution is not much. At 600V, the small hole distribution is found to be more. At 800V and 1000V, the small holes are found to gradually become large holes. Figure 6 (C) It can be known from the figure that at 0V and 100V, the cell surface has a large number of "antennae" but no holes are found. At 400V, small holes are found in the local part. At 700V, the small hole distribution is found to be more. At 1000V, the holes are found to be further more and larger.

[0049] Under the stimulation of the pulsed voltage, holes appear on the cell membrane, and as the applied voltage increases, the number of holes increases, and the distribution is more dense. When the voltage increases to a certain extent, the holes will cover the surface, and the holes will change from small holes to large holes.

[0050] The above merely describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the concept of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM), characterized by, The degree of electroporation of cells after pulse electric field stimulation is determined by membrane permeability of cells specifically, and comprises the following steps: After the cells are cultured to a certain cell density, the cells are subjected to irreversible electroporation ablation by a high-voltage pulse generator, and after the ablation is completed, the cell culture solution is directly replaced with an oxidation-reduction medium without changing the culture dish, and then the cells are subjected to probe approach by a scanning electrochemical microscope, an approach curve of the SECM probe to the cells is determined, and then the probe approach curve of the cells is matched with a standard probe approach curve simulated, and the membrane permeability Pm value is converted to complete the determination of the degree of electroporation. The approach curve is a curve of the tip current and the distance of the cell.

2. The method of claim 1, wherein the degree of irreversible electroporation is detected using a scanning electrochemical microscope (SECM). The Pm value and the survival rate of the cells after electroporation are related by the SECM probe means, a standard library is constructed, and the degree of irreversible electroporation is further verified and judged.

3. The method of claim 1, wherein the degree of irreversible electroporation is detected using a scanning electrochemical microscope (SECM). The probe electrode used by the scanning electrochemical microscope is a supermicro electrode.

4. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 3, wherein The supermicro electrode is a platinum electrode or a gold electrode.

5. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 1, wherein, The mode adopted by the scanning electrochemical microscope is a negative feedback working mode, an oxidation-reduction medium is added in the solution environment of the cells to generate a Faraday current, and the degree of membrane permeability is determined by observing the current change.

6. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 1, wherein, The oxidation-reduction medium needs to meet the requirements of not normally penetrating the cell membrane and not producing toxic side effects on the cells.

7. The method of claim 6, wherein the degree of irreversible electroporation is detected by scanning electrochemical microscopy (SECM). The oxidation-reduction medium is a ferrocene methanol or a ferrocene ethanol series compound.

8. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 1, wherein, The cells to be determined need to meet the requirement of adherent growth on the culture dish.

9. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 8, wherein, The cells are different normal cells or tumor cells.

10. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 1, wherein, The corresponding standard probe approach curve needs to be simulated by simulation software, the approach curve obtained by the experiment is matched with the standard curve, the membrane permeability under different pulse voltage electroporation is obtained, the degree of electroporation is represented by the membrane permeability, and the relationship between different pulse parameters and the degree of electroporation is established.

11. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 1, wherein, When the scanning electrochemical microscope works, the cells are adherent in the culture dish during the irreversible electroporation ablation, and the applied pulse electric field needs to meet the requirements of stably covering the bottom of the culture dish and ensuring that the electric field intensity in the area approached by the probe each time is consistent.

12. The method for detecting the degree of irreversible electroporation using a scanning electrochemical microscope (SECM) according to claim 2, wherein, The cell membrane pore formation rule is observed by FESEM, and the cell survival rate is measured by MTT method as a reference evaluation technology.

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