A content extraction method based on cell membrane capacitance change

Abstract

The application provides a content extraction method based on cell membrane capacitance change, and belongs to the field of cell-level micromanipulation technology, and comprises the following steps: S1, a change relationship between cell membrane capacitance and cell membrane surface area is established; S2, a change relationship between cell membrane surface area and volume is approximately established; S3, high-resistance sealing and membrane rupture of cells are completed, constant voltage cell content extraction operation is performed, and cell membrane capacitance values in the extraction process are obtained in real time through capacitance fitting; S4, content extraction amount is estimated; S5, actual cytoplasm extraction amount is calculated based on a rotary L-shaped electrode, the relationship model established in S2 is corrected, and finally, quantitative extraction of cell contents is realized. The application utilizes an electrical model of cell membrane capacitance, realizes real-time feedback of cell content extraction amount, realizes quantitative control of content extraction, can realize a cytoplasm extraction success rate of 62.5% in the case of a whole cell patch clamp structure, and the average cytoplasm extraction amount control error is less than 6% of the total cell volume.

Description

Technical Field

[0001] This invention belongs to the field of cell-level micromanipulation technology, and specifically relates to a method for extracting contents based on changes in cell membrane capacitance. Background Technology

[0002] Cellular content extraction refers to the process of extracting the contents of target cells from the cell body for analysis. It is an effective method for obtaining cellular transcriptome information and correlating gene expression and phenotype at the single-cell level. During cellular content extraction, excessive extraction can cause cells to rupture due to the extraction force, introducing impurities that interfere with subsequent purification steps and data analysis, severely impacting the accuracy and reliability of the final results. Conversely, insufficient extraction yields too few usable cellular components, potentially failing to cover all intracellular information and limiting a comprehensive and in-depth understanding of cellular composition. Therefore, it is crucial to determine an appropriate extraction level during cellular content extraction to ensure sufficient contents for compositional analysis while preventing the extraction of extracellular impurities.

[0003] Currently, there are several ways to determine the amount of cell contents extracted: (1) using visual feedback to determine the degree of extraction by observing changes in cell morphology; (2) combining electrical signals to observe whether there is a large current leakage in the cells to determine whether to continue extraction. When using the above common methods to enucleate cells, there are the following defects: a) Visual feedback works well for suspended cells or cells with clear outlines, but it is not effective for cells with unclear morphology and outlines in tissues; b) Visual feedback can only be used in conjunction with a high-power microscope; c) The judgment of the degree of current leakage is more subjective, the judgment standard is not easy to control, and it is difficult to determine whether the extracted cell contents have been extracted when the amount of cell contents to be extracted is small.

[0004] Due to the small size of cells, microneedle extraction is currently the primary method for obtaining cellular contents. The specific method involves the operator using a patch-clamp glass microneedle under a microscope to perform whole-cell patch-clamp manipulation on the target cell. This includes sealing the microneedle against the cell membrane and then breaking the membrane to create an opening for extraction. However, this extraction method has several problems in practical application: After the microneedle enters the biological tissue and forms a whole-cell structure, the cluttered background makes it difficult to observe the flow of cytoplasm during extraction. While fluorescent staining can make the cytoplasm visible, the procedure is cumbersome, and the microneedle position cannot be calculated under a fluorescent field, making microneedle movement control difficult and affecting the extraction of cellular contents.

[0005] Existing research indicates that when using microneedles to extract contents from adherent cells, the length of the extracted contents can be observed using a high-magnification microscope. A geometric model can be established using the microneedle's inner diameter and the extraction length to calculate the extracted volume, thus providing feedback on the extracted cell contents. However, the formula for calculating the extraction volume is complex, and direct observation of the microneedle's inner diameter requires a high-magnification microscope, placing high demands on the equipment. Therefore, developing a suitable method for quantitative extraction of cell contents that is easy to operate and can provide real-time feedback on the extracted volume is essential. Summary of the Invention

[0006] To achieve real-time monitoring of the amount of cell contents extracted and thus quantitative extraction, this invention proposes a method for quantitative extraction of cell contents based on changes in cell membrane capacitance. This method monitors the degree of change in cell membrane capacitance during extraction, estimates the reduction in cell membrane area, and then estimates the degree of extraction of cell contents, ultimately achieving quantitative extraction of cell contents.

[0007] To solve the above problems, the present invention adopts the following technical solution:

[0008] S1: Establish the relationship between cell membrane capacitance and cell membrane surface area: During the extraction process, assuming that the dielectric constant of the cell membrane and the phospholipid bilayer spacing of the cells to be extracted remain unchanged, the relationship between the cell membrane capacitance and cell surface area is established using the cell membrane capacitance calculation formula.

[0009] S2: Approximately establish the relationship between cell membrane surface area and volume: Based on the approximate cell morphology, establish a model of the relationship between cell volume and cell membrane surface area, and calculate the change in cell volume, i.e. the amount of cell contents extracted, from the change in the cell membrane surface area.

[0010] S3: Cell contents extraction: Complete the high-resistance sealing and membrane perforation of the cells, perform constant-pressure cell contents extraction, and obtain the cell membrane capacitance value in real time during the extraction process through capacitance fitting.

[0011] S4: Estimate the amount of contents extracted: Based on the relationship model between cell membrane capacitance and cell membrane surface area established in S1 and cell membrane surface area and cell volume established in S2, estimate the amount of cell membrane area reduction based on the degree of cell membrane capacitance reduction, and then estimate the amount of cell volume reduction to obtain the amount of contents extracted.

[0012] S5: Achieve quantitative extraction: Calculate the actual amount of cytoplasm extracted based on the rotating L-shaped electrode, correct the relationship model between cell membrane surface area and cell volume established in S2, and achieve quantitative extraction of cell contents.

[0013] Furthermore, in S1, the formula for calculating the cell membrane capacitance is:

[0014]

[0015] in, This is the membrane capacitance value. The dielectric constant of the cell membrane, The cell membrane surface area, Let be the spacing between the phospholipid bilayers of the cell membrane. From the formula, we can see that, with the cell membrane dielectric constant and the phospholipid bilayer spacing remaining constant, the cell membrane capacitance is linearly proportional to the membrane surface area.

[0016] Furthermore, in S2, if the cell is considered a sphere, then the formula for calculating the cell membrane surface area is:

[0017]

[0018] The formula for calculating cell volume is:

[0019]

[0020] in The cell membrane surface area, Cell volume Let be the cell radius. From equations (2) and (3), the relationship between the cell membrane surface area and the cell volume can be derived as follows:

[0021]

[0022] As can be seen from equation (4), the surface area of ​​a spherical cell membrane increases with the cell volume. The change in cell membrane capacitance can be used to deduce the change in cell membrane surface area, and further estimate the change in cell volume, i.e., the amount of cell contents extracted. The approximate correspondence between cell membrane capacitance and cell volume is as follows:

[0023]

[0024] Furthermore, in S3, the constant-pressure extraction of cell contents and the acquisition of cell membrane capacitance are based on robotic whole-cell patch-clamp operation. An L-shaped electrode with its tip bent at approximately 30° is used to perform the constant-pressure extraction of cell contents. At the same time, the cell membrane capacitance value is acquired in real time during the extraction process through capacitance fitting, thereby obtaining the degree of change in membrane capacitance during the extraction of cell contents.

[0025] Furthermore, in S3, the extraction of the cell contents involves the following specific steps:

[0026] S301: Place the sample to be extracted under the microscope field of view, select cells of appropriate size and clear edges, and based on the robotic whole-cell patch clamp operation process, control the L-shaped electrode microneedle with the tip bent at about 30° to gradually approach the target cell until a clear indentation is formed.

[0027] S302: Apply a small negative pressure to the cell to seal the tip of the microneedle with the cell membrane. After the seal is stable, slowly apply negative pressure to the cell to break the membrane and make the microneedle and the cell interior connected, in preparation for the extraction of contents.

[0028] S303: Once the cell state is stable, continuously apply a constant negative pressure to the cell to extract the cell contents until the extracted amount is sufficient or the cell can no longer withstand the negative pressure extraction, at which point the extraction can be stopped.

[0029] Furthermore, in S3, the constant-pressure extraction of cell contents and the acquisition of cell membrane capacitance are specifically performed after forming a high-resistance seal and breaking the cell membrane to form a whole-cell record. A constant negative pressure is used to extract the cell contents. As the extraction of cell contents proceeds, the absolute value of the measured cell membrane capacitance will gradually decrease. The volume of the contents in the cell decreases continuously during the extraction process, and the cell will shrink as it is extracted, resulting in a decrease in the surface area of ​​the cell membrane. Since the entire extraction process is short, it is assumed that the dielectric constant of the cell membrane and the spacing of the phospholipid bilayer of the cell membrane remain constant during this short period of time. As can be seen from equation (1), the value of the membrane capacitance also decreases. Therefore, the degree of extraction of cell contents can be reflected by the change in the membrane capacitance value.

[0030] Furthermore, in S3, since the measured membrane capacitance value fluctuates greatly, a capacitance value is recorded at regular intervals, and the average of the two membrane capacitance values ​​following that value is calculated to fit a membrane capacitance change curve, thereby obtaining a more accurate membrane capacitance value.

[0031] Furthermore, in S4, the degree of reduction in the absolute value of the extracted membrane capacitance relative to the initial absolute value of the membrane capacitance is calculated. The amount of reduction in cell membrane area is estimated using the previously established relationship model, and then the amount of cell contents extracted is estimated, thus obtaining the degree of cell contents extraction.

[0032] Furthermore, in S5, after the extraction is complete, L The electrode microneedle rotates 180° along the axis from the tip to the tail, making the tip of the microneedle parallel to the plane of the brain slice, thus enabling clear visualization of the contents inside the microneedle. The success of the extraction can be verified by the change in capacitance and the amount of contents at the tip, and the amount of extracted contents can be calibrated to achieve quantitative extraction of contents.

[0033] Furthermore, in S5, the specific operation is as follows: the cell body of the nerve cell is regarded as spherical, and the initial volume of the cell before extraction is calculated. After extraction, the ratio of the extracted volume to the initial cell volume can be estimated by the ratio of the needle tip inner diameter, the length of the extracted contents within the needle tip, and the cell diameter, thereby estimating the amount of contents extracted; the relationship between cell volume and membrane capacitance before and after extraction can be obtained from equations (1) and (4):

[0034]

[0035] in, , These represent the cell volume and membrane capacitance value before extraction, respectively. , These represent the cell volume and membrane capacitance value after extraction, respectively. The ratio of cell volume before and after extraction can be calculated from the membrane capacitance value, thus obtaining the amount of cell contents extracted. By comparing the amount of cell contents extracted calculated by the two methods, the extraction amount can be calibrated.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. This invention eliminates the need to stain the cytoplasm when processing cell contents, thus avoiding the difficulties in controlling microneedle movement caused by photobleaching and the dark fluorescence field resulting from staining.

[0038] 2. During the extraction process, the present invention can provide real-time feedback on the amount of cell contents extracted by the change in capacitance, which reduces the impact of differences in cell characteristics and differences in the standards for judging extraction volume by different operators on the results, and can reduce the occurrence of too little or too much cell contents extracted.

[0039] 3. The present invention requires simple experimental equipment and does not require complex devices or expensive instruments to complete the relevant operations; at the same time, its operation steps are simple and clear, and the operation difficulty is low.

[0040] 4. This invention can achieve a 62.5% success rate in extracting contents and an average quantitative extraction error of less than 6% of the total cell volume when a whole-cell patch-clamp structure has been formed. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is a flowchart of the method for quantitative extraction of contents based on changes in cell membrane capacitance as described in this invention;

[0043] Figure 2 This is a schematic diagram of the cell contents extraction steps described in this invention;

[0044] Figure 3 This is the equivalent circuit diagram of the film capacitor described in this invention;

[0045] Figure 4 This is a graph showing the change in membrane capacitance fitted during the cell contents extraction process described in this invention. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] like Figure 1 As shown, this invention discloses a method for extracting cell contents based on changes in cell membrane capacitance. This method eliminates the need for staining the cytoplasm during cell content extraction, avoiding the difficulties caused by photobleaching and the dark fluorescence field that hinder microneedle movement control. Simultaneously, the amount of cell contents extracted can be fed back in real-time by capacitance changes during the extraction process, reducing the impact of differences in cell characteristics and varying extraction standards among different operators. This minimizes the occurrence of under- or over-extraction of cell contents. Furthermore, the method requires simple experimental equipment, eliminating the need for complex devices or expensive instruments. The operation steps are simple and straightforward, with low operational difficulty. The method includes the following steps:

[0048] S1: Assuming that the cell membrane dielectric constant and phospholipid bilayer spacing remain unchanged during the extraction process, the capacitance can be calculated using the following formula. ,in This is the membrane capacitance value. The dielectric constant of the cell membrane, The cell membrane surface area, The distance between the phospholipid bilayers of the cell membrane can be used to obtain the relationship between the cell membrane capacitance and the cell membrane surface area, that is, the cell membrane capacitance is linearly proportional to the membrane surface area.

[0049] S2: Based on the approximate morphology of cells, establish a model relating cell volume and cell membrane surface area, treating the cell as a sphere. The formula for cell membrane surface area is: The cell volume formula is: Therefore, the relationship between cell membrane surface area and cell volume is as follows: The approximate correspondence between cell membrane capacitance and cell volume is as follows: This allows us to calculate the change in cell volume, i.e. the amount of cell contents extracted, from the change in cell membrane surface area.

[0050] S3: Based on a robotic whole-cell patch-clamp procedure, high-resistance sealing and cell membrane rupture are achieved using a tip bent at approximately 30°. L The electrode performs constant-voltage cell content extraction, and simultaneously acquires the cell membrane capacitance value in real time during the extraction process through capacitance fitting, thereby obtaining the degree of change in membrane capacitance during cell content extraction. Based on the cell membrane capacitance model, the change in cell volume caused by content extraction leads to a change in cell membrane surface area, which in turn leads to a change in membrane capacitance. The change in membrane capacitance reflects the change in cell content volume caused by extraction, and indirectly reflects the change in cell membrane surface area, which can further estimate the degree of reduction in cell content.

[0051] As can be seen from equation (1), the cell membrane capacitance may also be affected by the cell membrane dielectric constant and the spacing between the phospholipid bilayers. Since the entire extraction process is relatively rapid, it is assumed that the cell membrane dielectric constant and the spacing between the cell membrane phospholipid bilayers remain constant during this short period of time. The membrane capacitance is only affected by the membrane surface area, which simplifies the complexity of the problem. As the volume of the contents in the cell decreases continuously during the extraction process, the cell will shrink as it is extracted, resulting in a reduction in the surface area of ​​the cell membrane. It can be calculated from the formula that the value of the membrane capacitance also decreases. The change in membrane capacitance can indirectly reflect the change in the volume of the cell contents. Therefore, the degree of extraction of cell contents can be reflected by the change in the membrane capacitance value.

[0052] S4. By establishing a model relating cell membrane capacitance to cell membrane surface area and cell volume, the amount of cell membrane area reduction is estimated based on the degree of cell membrane capacitance reduction, and then the amount of cell volume reduction is estimated to obtain the amount of contents extracted.

[0053] S5, based on rotation L The electrode type calculates the actual amount of cytoplasm extracted, revising the previously established model of the relationship between cell membrane area and cell volume, based on the definition of cell membrane capacitance. And the relationship between cell membrane surface area and volume The relationship between cell volume and membrane capacitance before and after extraction can be obtained: ,in, , These represent the cell volume and membrane capacitance value before extraction, respectively. , These represent the extracted cell volume and membrane capacitance value, respectively, enabling quantitative extraction of cell contents.

[0054] The operation steps in this embodiment are as follows:

[0055] In this embodiment, the cells used are cerebral cortical cells from 4-5 week old female mice. The mouse brain was taken and sliced, and the brain slices were placed under the microscope. Suitable cells were selected under the microscope, and whole-cell recording was performed using a robotic patch clamp. After the cell membrane was broken by a microneedle, negative pressure was applied to extract the cell contents.

[0056] In step S1, the cell membrane capacitance is defined as follows:

[0057]

[0058] in This is the membrane capacitance value. The dielectric constant of the cell membrane, The cell membrane surface area, Let be the spacing between the phospholipid bilayers of the cell membrane. Analyzing the formula, we can conclude that, under the condition that the dielectric constant of the cell membrane and the spacing between the phospholipid bilayers remain unchanged during the extraction process, the cell membrane capacitance is linearly proportional to the cell membrane surface area.

[0059] like Figure 2 As shown, in step S2, the principle of establishing an electrical model for cell membrane capacitance is as follows:

[0060] The cell membrane is mainly composed of a phospholipid bilayer. From an electrical perspective, this bilayer can be considered as a parallel-plate capacitor, with the upper and lower layers corresponding to the two plates of the capacitor, and the area of ​​the plates is equal to the surface area of ​​the cell membrane. The cell interior is negatively charged relative to the cell exterior; negative charges are mainly distributed inside the cell membrane, while positive charges are mainly distributed on the outer side.

[0061] Specifically, by continuously acquiring membrane capacitance values, the degree of change in membrane capacitance during cell content extraction can be obtained. Based on the cell membrane capacitance model, the reduction in cell volume caused by content extraction leads to cell membrane shrinkage, resulting in a change in cell membrane surface area, which in turn leads to a change in membrane capacitance. Therefore, the change in membrane capacitance can indirectly reflect the degree of reduction in cell content. It is worth noting that since the entire extraction process is very rapid and lasts for a very short time, it is assumed that the dielectric constant of the cell membrane and the interlayer spacing of the phospholipid bilayer remain constant during this short period of time, thus simplifying the problem. That is, the measured value of membrane capacitance is only affected by the cell membrane surface area. The change in cell membrane capacitance can be reflected by equation (1). As can be seen from the definition, cell membrane capacitance may also be affected by the dielectric constant of the cell membrane and the size of the interlayer spacing of the phospholipid bilayer. Since the entire extraction process is relatively rapid, it is assumed that the dielectric constant of the cell membrane and the interlayer spacing of the phospholipid bilayer remain constant during this short period of time, and the size of the membrane capacitance is only affected by the membrane surface area, thus simplifying the complexity of the problem. As the cell contents decrease in volume during extraction, the cell shrinks, leading to a reduction in the cell membrane surface area. The membrane capacitance also decreases, as can be calculated from the formula. Changes in membrane capacitance indirectly reflect changes in the volume of cell contents; therefore, the degree of cell contents extraction can be reflected by changes in membrane capacitance value, and the greater the extraction, the greater the absolute change in membrane capacitance. To more accurately estimate the amount of cell contents extracted, a model of the relationship between cell volume and cell membrane surface area is established. If the cell is considered a sphere, the formula for calculating the cell membrane surface area is:

[0062]

[0063] The formula for calculating cell volume is:

[0064]

[0065] in The cell membrane surface area, Cell volume Let be the cell radius. From equations (2) and (3), the relationship between the cell membrane surface area and the cell volume can be derived as follows:

[0066]

[0067] As can be seen from equation (4), the surface area of ​​a spherical cell membrane increases with the cell volume. The change in cell membrane capacitance can be used to deduce the change in cell membrane surface area, and further estimate the change in cell volume, i.e., the amount of cell contents extracted. The approximate correspondence between cell membrane capacitance and cell volume is as follows:

[0068]

[0069] like Figure 3 As shown, the specific method for extracting cell contents in step S3 is as follows:

[0070] S301: Place a slice of mouse brain under a microscope, select appropriately sized and clearly defined cerebral cortex cells, and, based on a robotic whole-cell patch-clamp procedure, gradually approach the target cells with an L-shaped electrode microneedle that is bent at about 30° until a clear indentation is formed.

[0071] S302: Apply a small negative pressure to the cell to seal the microneedle tip with the cell membrane. Once the seal is stable, i.e. the seal resistance reaches the GΩ level and the absolute current value is less than 10pA, slowly apply negative pressure to the cell to perform the membrane rupture operation, so that the microneedle and the cell interior are connected, preparing for the extraction of contents.

[0072] S303: Once the cell state is stable, continuously apply a constant amount of negative pressure to the cell to extract the cell contents until the extracted amount is sufficient or the cell can no longer withstand the negative pressure extraction, at which point the extraction can be stopped.

[0073] Specifically, in this embodiment, 4-5 week old female mice were used, and their brains were harvested and sliced ​​into 300-micrometer-thick slices. The sliced ​​brains were incubated in artificial cerebrospinal fluid at a constant temperature of 37°C for approximately 40 minutes. Then, the cortical region was located and positioned under a microscope at 4x magnification. Target cells were selected under a microscope at 40x magnification, such as... Figure 3 (a) As shown in the white dashed area, cells with a teardrop shape and relatively clear outline are generally selected, while cells that are obviously swollen or shrunken are not. The curved microneedle tip used has the following shape: Figure 3 As shown in (b). After identifying the target cells, the microneedles are gradually inserted into the brain slice tissue and approach the cell membrane surface. During this process, close monitoring of the interaction between the microneedles and the target cells is necessary until a clear indentation appears on the surface of the target cells, as shown in (b). Figure 3 (c) shows that the microneedle has established initial contact with the cell membrane. A slight negative pressure is applied to the cell through the microneedle, and then immediately stopped, allowing the microneedle to seal against the cell membrane. After sealing, a relatively stable high-resistance seal is considered to be formed when the resistance reaches the GΩ level and the absolute current value is less than 10pA. At this point, a continuous slight negative pressure is applied to the cell to break the cell membrane, creating a gap on the cell surface that connects to the microneedle. After the membrane rupture operation, wait for a period of time until the cell state stabilizes, and then continue applying negative pressure to extract the cell contents. After extraction, rotate the microneedle 180° around the axis from the tip to the tail, making the microneedle tip parallel to the brain slice plane. At this point, the contents inside the microneedle tip are clearly visible, as shown in the image. Figure 3 (d) shows the part indicated by the white dashed line.

[0074] The amount of contents was calibrated by selecting an experiment where the extracted contents accumulated at the tip of the microneedle. First, the estimated amount of extracted contents was calculated based on the relationship between the membrane capacitance change curve over extraction time and cell volume. Then, the extraction amount was calibrated based on the volume of contents inside the needle tip after extraction. Thus, the success of content extraction can be verified by the change in capacitance value and the amount of contents at the tip, and the extraction amount can be calibrated, achieving quantitative content extraction. Specifically, the cell body of a nerve cell is considered spherical, and the initial cell volume before extraction can be calculated. After extraction, the ratio of the extracted content volume to the initial cell volume can be estimated by the ratio of the needle tip diameter, the length of the extracted contents within the needle tip, and the cell diameter, thereby estimating the amount of extracted contents. This is based on the definition of cell membrane capacitance. And the relationship between cell membrane surface area and volume The relationship between cell volume and membrane capacitance before and after extraction can be obtained:

[0075]

[0076] in, , These represent the cell volume and membrane capacitance value before extraction, respectively. , These represent the cell volume and membrane capacitance value after extraction, respectively. Therefore, the ratio of cell volume before and after extraction can be calculated from the membrane capacitance values, thus yielding the amount of cell contents extracted. Comparing the cell contents extraction amounts calculated by the two methods completes the calibration of the extraction amount.

[0077] Different experimental results were selected for calibration, and the results are shown in Table 1:

[0078] Table 1

[0079]

[0080] In the first experiment, the diameter of the cells before extraction was [missing information]. D The length of the contents extracted from the microneedle was 2.308 mm. D The contents within the microneedle can be considered as a frustum of a cone. The estimated diameter of the microneedle tip is 0.308D, and the diameter of the lower base of the contents is 0.385. D The volume of a frustum can be calculated using the formula... Calculate the volume of cell contents within the needle tip. L Let the height of the frustum be _____. R and r ... Treating the cells as spherical, the initial volume of the cells before extraction was calculated to be approximately 0.524. Comparing the calculated volume of extracted contents with the initial cell volume, we can see that approximately 42% of the contents were extracted in this instance. The membrane capacitance before extraction was 48.75 pF, and the cell membrane capacitance after extraction was 31.07 pF. Based on the membrane capacitance values ​​before and after extraction, as well as the relationship between volume and membrane capacitance, we can conclude that the ratio of cell volume before and after extraction is approximately 0.51, meaning that approximately 49% of the contents were extracted.

[0081] In the second experiment, the diameter of the cells before extraction was... D The length of the contents extracted from the microneedle was 0.971 mm. D The contents within the microneedle can be considered as a frustum of a cone. The estimated diameter of the microneedle tip is 0.319D, and the diameter of the lower base of the contents is 0.399. D Using the formula for the volume of a frustum of a cone, the volume of the contents extracted in this experiment can be calculated to be approximately 0.099. Treating the cells as spherical, the initial volume of the cells before extraction was calculated to be approximately 0.524. Comparing the calculated volume of extracted contents with the initial cell volume, we can conclude that approximately 19% of the contents were extracted in this instance. The membrane capacitance before extraction was 22.78 pF, and the cell membrane capacitance after extraction was 18.53 pF. Based on the membrane capacitance values ​​before and after extraction, as well as the relationship between volume and membrane capacitance, we can conclude that the ratio of cell volume before and after extraction is approximately 0.73, meaning that approximately 27% of the contents were extracted.

[0082] In the third experiment, the diameter of the cells before extraction was... D The length of the contents extracted from the microneedle was 1.25 mm. D The contents within the microneedle can be considered as a frustum of a cone. The estimated diameter of the microneedle tip is 0.125D, and the diameter of the lower base of the contents is 0.275. D Using the formula for the volume of a frustum of a cone, the volume of the contents extracted in this experiment can be calculated to be approximately 0.041. Treating the cells as spherical, the initial volume of the cells before extraction was calculated to be approximately 0.524. Comparing the calculated extracted volume with the initial cell volume, approximately 7.82% of the contents were extracted. The membrane capacitance before extraction was 19.88 pF, and after extraction stopped, it was 18.13 pF. Based on the membrane capacitance values ​​before and after extraction, and the relationship between volume and membrane capacitance, the ratio of cell volume before and after extraction is approximately 0.87, meaning approximately 13% of the contents were extracted. The average error of the three extractions was less than 6% of the total cell volume.

[0083] Comparing the extraction volume obtained from the capacitance value with the actual calculated extraction volume, it was found that the two are relatively close in value, but the extraction volume obtained from the capacitance value is larger. This reflects, to some extent, that the rate of decrease in cell membrane surface area during extraction is greater than the rate of decrease in volume. This may be because nerve cells are not perfect spheres; they have relatively long dendrites and axons containing less content. During extraction, the contents of the dendrites and axons may be extracted more quickly, causing this part of the cell membrane to fold or break, thus failing to be included in the effective area for membrane capacitance calculation, increasing the rate of decrease in cell membrane surface area. Therefore, the previously established model of the relationship between cell membrane surface area and volume still needs to be revised to minimize errors. Thus, quantitative extraction of cell contents can be achieved by measuring changes in cell membrane capacitance value.

[0084] like Figure 4 As shown, in step S3, a fitted curve is plotted to show the change in cell membrane capacitance with extraction time during a constant pressure extraction process. Since the membrane capacitance changes drastically, the capacitance value is sampled every five seconds, and the two capacitance values ​​adjacent to the sampling point are averaged. From the fitted curve, it is found that the cell membrane capacitance changed significantly before and after the extraction of cell contents. The cell contents are extracted using a pressure of -2000 Pa.

[0085] Before extraction begins, the cell membrane capacitance remains at a relatively stable level, reflecting the charge distribution and capacitance characteristics of the cell membrane in its resting state. However, after extraction begins, as intracellular substances are continuously removed, the cell volume decreases, leading to a reduction in the cell membrane area and thus a gradual decrease in the absolute value of the membrane capacitance. Table 2 presents the results of eight content extraction experiments. In some experiments, the whole-cell patch-clamp structure was disrupted at the initial stage of extraction, resulting in minimal changes in membrane capacitance after extraction. Under the premise of a fully formed whole-cell patch-clamp structure, the absolute value of the cell membrane capacitance significantly decreased with extraction in five out of the eight experiments, resulting in an extraction success rate of 62.5%.

[0086] Table 2

[0087]

[0088] The present invention has been described in detail above through embodiments. The present invention can achieve a 62.5% success rate in content extraction and an average quantitative extraction error of less than 6% of the total cell volume when a whole-cell patch-clamp structure has been formed. However, the descriptions are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for extracting contents based on changes in cell membrane capacitance, characterized in that: The method includes the following steps: S1: Establish the relationship between cell membrane capacitance and cell membrane surface area: During the extraction process, assuming that the dielectric constant of the cell membrane and the phospholipid bilayer spacing of the cells to be extracted remain unchanged, the relationship between the cell membrane capacitance and cell surface area is established using the cell membrane capacitance calculation formula. S2: Approximately establish the relationship between cell membrane surface area and volume: Based on the approximate cell morphology, establish a model of the relationship between cell volume and cell membrane surface area, and calculate the change in cell volume, i.e. the amount of cell contents extracted, from the change in the cell membrane surface area. S3: Cell contents extraction: Complete the high-resistance sealing and membrane perforation of the cells, perform constant-pressure cell contents extraction, and obtain the cell membrane capacitance value in real time during the extraction process through capacitance fitting. S4: Estimate the amount of contents extracted: Based on the relationship model between cell membrane capacitance and cell membrane surface area established in S1 and cell membrane surface area and cell volume established in S2, estimate the amount of cell membrane area reduction based on the degree of cell membrane capacitance reduction, and then estimate the amount of cell volume reduction to obtain the amount of contents extracted. S5: Achieve quantitative extraction: Calculate the actual amount of cytoplasm extracted based on the rotating L-shaped electrode, correct the relationship model between cell membrane surface area and cell volume established in S2, and achieve quantitative extraction of cell contents.

2. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S1, the formula for calculating the cell membrane capacitance is: C m =εA / d (1) where, C m Let ε be the membrane capacitance, A be the cell membrane dielectric constant, d be the cell membrane surface area, and d be the spacing between the phospholipid bilayers of the cell membrane. According to the formula, when the cell membrane dielectric constant and the phospholipid bilayer spacing remain unchanged, the cell membrane capacitance is linearly proportional to the membrane surface area.

3. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S2, if the cell is considered as a sphere, then the formula for calculating the cell membrane surface area is: A = 4πR 2 (2) The formula for calculating cell volume is: V = 4 / 3 * πR 3 (3) Where A is the cell membrane surface area, V is the cell volume, and R is the cell radius. From equations (2) and (3), the relationship between cell membrane surface area and cell volume can be derived as follows: As can be seen from equation (4), the surface area of ​​the spherical cell membrane changes with the 2 / 3 power of the cell volume. Therefore, the change in cell membrane surface area can be deduced from the change in cell membrane capacitance, and the change in cell volume can be further estimated, i.e., the amount of cell contents extracted. The approximate correspondence between cell membrane capacitance and cell volume is as follows:

4. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S3, the constant-pressure extraction of cell contents and the acquisition of cell membrane capacitance are based on robotic whole-cell patch-clamp operation. The constant-pressure extraction of cell contents is performed using an L-shaped electrode with its tip bent at about 30°. At the same time, the cell membrane capacitance value is acquired in real time during the extraction process through capacitance fitting, so as to obtain the degree of change of membrane capacitance when extracting cell contents.

5. The method for extracting contents based on changes in cell membrane capacitance according to claim 4, characterized in that: In S3, the extraction of the cell contents involves the following specific steps: S301: Place the sample to be extracted under the microscope field of view, select cells of appropriate size and clear edges, and based on the robotic whole-cell patch clamp operation process, control the L-shaped electrode microneedle with the tip bent at about 30° to gradually approach the target cell until a clear indentation is formed. S302: Apply a small negative pressure to the cell to seal the tip of the microneedle with the cell membrane. After the seal is stable, slowly apply negative pressure to the cell to break the membrane and make the microneedle and the cell interior connected, in preparation for the extraction of contents. S303: Once the cell state is stable, continuously apply a constant negative pressure to the cell to extract the cell contents until the extracted amount is sufficient or the cell can no longer withstand the negative pressure extraction, at which point the extraction can be stopped.

6. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S3, the constant-pressure extraction of cell contents and the acquisition of cell membrane capacitance are specifically performed after forming a high-resistance seal and breaking the cell membrane to form a whole-cell record. Then, a constant negative pressure is used to extract the cell contents. As the extraction of cell contents proceeds, the absolute value of the measured cell membrane capacitance will gradually decrease.

7. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S3, since the measured membrane capacitance value fluctuates greatly, a capacitance value is recorded at regular intervals, and the average of the two membrane capacitance values ​​after that value is calculated to fit a membrane capacitance change curve, thereby obtaining a more accurate membrane capacitance value.

8. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S4, the degree of reduction in the absolute value of the extracted membrane capacitance relative to the initial absolute value of the membrane capacitance is calculated. The amount of reduction in cell membrane area is estimated by the previously established relationship model, and then the amount of cell contents extracted is estimated, thus obtaining the degree of cell contents extraction.

9. The method for extracting contents based on changes in cell membrane capacitance according to claim 1, characterized in that: In S5, after extraction is completed, the L-shaped electrode microneedle is rotated 180° along the axis from the tip to the tail, so that the tip of the microneedle is parallel to the brain slice plane, thus making the contents inside the microneedle visible. The success of the extraction can be verified by the change in capacitance and the amount of contents at the tip, and the amount of contents extracted can be calibrated to achieve quantitative extraction of contents.

10. The method for extracting contents based on changes in cell membrane capacitance according to claim 9, characterized in that: In S5, the specific operation is as follows: the cell body of the nerve cell is regarded as spherical, and the initial volume of the cell before extraction is calculated. After extraction, the ratio of the extracted volume to the initial cell volume can be estimated by the ratio of the inner diameter of the needle tip, the length of the extracted contents inside the needle tip, and the cell diameter, thereby estimating the amount of contents extracted; the relationship between cell volume and membrane capacitance before and after extraction can be obtained from equations (1) and (4): V2 / V1=(C2 / C1) 3 / 2 (6) Where V1 and C1 represent the cell volume and membrane capacitance value before extraction, respectively, and V2 and C2 represent the cell volume and membrane capacitance value after extraction, respectively. The ratio of cell volume before and after extraction can be calculated from the membrane capacitance values ​​before and after extraction, and thus the amount of cell contents extracted can be obtained. By comparing the amount of cell contents extracted calculated by the two methods, the extraction amount can be calibrated.

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