A robotic quantitative perforation and membrane breaking method

Abstract

This invention provides a robotic quantitative perforation membrane disruption method, belonging to the field of micromanipulation, comprising the following steps: S1, establishing a perforation patch-clamp cellular circuit model based on cell electrophysiological characteristics; S2, deriving the relationship between the measured resistance and the number of micropores during perforation using the above model; S3, deriving the relationship between the transmembrane current under zero-state response and the measured resistance by applying periodic step voltage signals to the cells; S4, based on this modeling result, combining a robotic perforation patch-clamp system, using the peak value of the transmembrane current under zero-state response to input a step voltage for real-time feedback on the number of micropores, thereby achieving closed-loop control of the release of perforated material and realizing robotic quantitative perforation membrane disruption. This invention overcomes the problem of difficulty in quantifying the perforation degree of current perforation patch-clamp methods by deriving a perforation patch-clamp cellular circuit model, achieving a quantitative perforation membrane disruption success rate of 90%, and an average perforation speed nearly twice that of ordinary perforation membrane disruption methods.

Description

Technical Field

[0001] This invention relates to the field of micromanipulation, and more specifically to a robotic quantitative perforation and membrane breaking method. Background Technology

[0002] Ion channel activity is fundamental to various cellular physiological activities. The patch-clamp technique uses a negative feedback circuit to maintain the membrane potential at a commanded level. It utilizes a high-resistance seal (over 1000 Ω) between the microelectrode tip and the cell membrane surface, creating electrochemical insulation between the small region of the cell membrane (the patch) at the electrode tip opening and the surrounding tissue. Based on this, ion channel currents on the patch (single-channel recording) and the cell (whole-cell recording) are monitored and recorded. Analysis of these channel currents reveals the cell's electrical properties, physiological functions, and the effects of various interventions. In 1976, Neher, Sakmann, and others, building upon voltage clamp technology, elevated traditional electrophysiological methods to the molecular level, enabling the study of individual proteins and marking a new milestone in understanding the structure-function relationship of ion channels. Traditional patch-clamp techniques employ negative pressure aspiration or electroporation to create a whole-cell model. During this process, the diffusion and exchange between the electrode fluid and the intracellular fluid often causes time-dependent rundown of some channel currents regulated by intracellular substances. There are four conventional patch-clamp recording methods: cell-attached patches, inside-out patches, outside-out patches, and whole-cell recording models. In 1988, Horn et al. improved upon traditional whole-cell recording by establishing the perforated patch-clamp technique: utilizing the property of certain antibiotics to form permeable pores in biological membranes, these antibiotics are filled into the electrode fluid, and a whole-cell model spontaneously forms after high-resistance sealing.

[0003] Perforated patch-clamp technology uses a perforating material to drill sub-nanometer-scale pores in the cell membrane of a microtubular electrode. This allows specific ions to enter and exit the cell membrane without damaging its overall structure, achieving electrical perforation and enabling the detection of whole-cell electrophysiological signals. Compared to traditional methods using pneumatic pulses to rupture the membrane, perforation causes less stress on the high-resistivity seal between the cell membrane and the microtubular electrode and does not result in the loss of cellular contents. Therefore, it has been widely applied in neuroscience and brain science in recent years.

[0004] However, unlike pneumatic pulse perforation, which can achieve membrane rupture in a single step, the perforation degree, i.e., the number of perforations, gradually increases in the perforation method. The lack of real-time feedback on the number of perforations makes quantitative perforation very difficult, which greatly limits the widespread application of perforated patch-clamp technology. Therefore, establishing a real-time perforation degree detection method is of great significance for ultimately achieving quantitative perforation and promoting the widespread application of perforated patch-clamp technology. Summary of the Invention

[0005] To overcome the difficulty in quantifying the perforation degree of current patch clamp perforation methods, this invention proposes a robotic quantitative perforation and membrane breaking method, which realizes real-time feedback of the number of perforations, completes closed-loop control of perforation and membrane breaking, and achieves a high success rate of perforation and membrane breaking.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] A robotic quantitative perforation and membrane rupture method, the method comprising the following steps:

[0008] S1: Establishing a perforated patch-clamp cell circuit model: Based on the electrophysiological characteristics of cells, each part of the cell is equivalent to a resistor and a capacitor to establish a perforated patch-clamp cell circuit model.

[0009] In the perforated patch-clamp cell circuit model, the cell membrane, composed of a phospholipid bilayer, is electrically equivalent to a capacitor, and the microtubule electrode resistance R... E , cytoplasmic resistance R C and pore resistance R P With sealing resistor R S Together they constitute the measuring resistance, the cytoplasmic resistance R C and pore resistance R P Connected in series, and with sealing resistor R S After being connected in parallel, it is then connected to the electrode resistance R. E Series;

[0010] The capacitance C of the capacitor formed by the phospholipid bilayer M It is negatively correlated with the phospholipid bilayer thickness d, and positively correlated with the dielectric constant ε and the cell membrane cross-sectional area S, which can be expressed as:

[0011]

[0012] Neglecting the effect of micropores on the cross-sectional area S of the cell membrane, the thickness d and dielectric constant ε of the phospholipid bilayer remain unchanged. According to equation (1), the capacitance C of the capacitor formed by the phospholipid bilayer can be determined. M The size remains constant during the perforation process;

[0013] The microtube electrode used in patch clamp operations acts as a resistor, forming the microtube electrode resistance R. E The cytoplasm inside the cell is a conductor, forming the cytoplasmic resistance R. C During the perforation process, the cell membrane surface drawn in by the microtube electrode forms micropores under the action of the perforating material, and these micropores constitute the pore resistance R. P After the high-resistance seal is formed, a small portion of the cell membrane that is absorbed has an extremely high resistance before the membrane ruptures, and the current will then flow through the seal resistance R. S cytoplasmic resistance R C and pore resistance R P It is in series and connected to the sealing resistor R. S After being connected in parallel, it is then connected to the electrode resistance R. E Series connection can be represented as:

[0014] R M =R E +(R C +R P ) || R S (2)

[0015] The sealing resistor R S It is in the gigaohm range, much larger than R. E R C and R P The sum of these values, where the sealing resistance can be ignored during the process, means that the measured resistance R... M It can be transformed into the following forms:

[0016] R M =R E +R C +R P (3);

[0017] S2: Derive the relationship between the number of micropores and the measured resistance;

[0018] During the perforation process, as micropores gradually form, current will uniformly pass through each micropore into the cell interior. It can be inferred that the pore resistances of each micropore are in parallel, as shown by R. Pi This represents the pore resistance of each of the micropores, where n represents the number of micropores formed. According to the formula for parallel resistance, the total pore resistance can be expressed as:

[0019]

[0020] Treating the pore resistance of each micropore as an almost identical constant, the total pore resistance R P The calculation formula can be expressed as:

[0021]

[0022] S3: Derive the zero-state response of transmembrane current under step voltage input: Based on the zero-state response theory of RC circuit, derive the relationship between the transmembrane current and the measuring resistance under step voltage input.

[0023] S4: Design of a robotic quantitative perforation membrane breaking process: Using the above modeling results and combined with a robotic perforation membrane patch clamp system, the peak value of the transmembrane current is used as feedback for the number of perforations to achieve closed-loop control of the release of perforated material, thereby realizing robotic quantitative perforation membrane breaking.

[0024] Furthermore, the resistance R of each micropore Pi The micropores are connected in parallel and are considered to be of the same size. The number of micropores n and the total pore resistance R are... P The relationship between the two is inverse: the more micropores there are, the lower the total pore resistance.

[0025] Furthermore, in S3, since there are both capacitors and resistors in the circuit, the cell circuit model can be regarded as a first-order RC circuit. Under a step voltage input, the zero-state response is used to obtain the transmembrane current calculation formula.

[0026] Furthermore, in S4, a robotic perforated membrane patch-clamp system is used to control the release of the perforated material, thereby achieving robotic quantitative membrane rupture, including the following steps:

[0027] S41, a perforated patch-clamp cell circuit model based on cell electrophysiological characteristics, modeling the relationship between the number of micropores and total pore resistance, and using the zero-state response theory of RC circuits to derive the calculation formula for transmembrane current under step voltage input, obtaining the relationship between the number of micropores and transmembrane current, which can be used to evaluate the degree of perforation.

[0028] S42, after completing the derivation of the transmembrane current, uses the peak value of the transmembrane current as feedback on the number of perforations, and uses a robotic perforation patch clamp system to actively and controllably release the perforated material and detect the change of the peak value of the transmembrane current in real time.

[0029] S43 adjusts the release rate of the perforating material in real time according to the change of the transmembrane current peak, thereby achieving quantitative perforation and realizing robotic quantitative perforation and membrane breaking.

[0030] Furthermore, in step S42, the robotic perforated patch clamp system can release perforated material precisely at time and in a quantitative manner, and use the change in the peak value of the transmembrane current as real-time feedback on the perforation effect.

[0031] Furthermore, in step S42, the magnitude of the transmembrane current is monitored and output in real time by a patch clamp amplifier to obtain the number of micropores formed in real time.

[0032] Furthermore, once the transmembrane current reaches a set threshold or reaches a stable state, the release of perforating material stops, and the robotic quantitative perforation and membrane breaking is completed.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention utilizes the zero-state transmembrane current peak under step voltage input conditions to achieve real-time feedback of the number of perforations for the first time, laying the foundation for achieving closed-loop quantitative membrane rupture.

[0035] 2. In the robotic quantitative perforation membrane breaking method provided by the present invention, the robotic perforation membrane patch clamp device used can complete the active and controllable release of perforation material at fixed time, fixed location and quantitative amount, adjust the release amount of perforation material, and use the peak value of transmembrane current as feedback to realize closed-loop control of perforation membrane breaking.

[0036] 3. In the robotic quantitative perforation membrane breaking method of the present invention, no additional detection equipment is used to provide feedback on the degree of perforation. The quantitative membrane breaking control can be completed using the currently common membrane clamp equipment. The success rate of perforation membrane breaking reaches 90%, and the perforation speed is nearly twice that of ordinary perforation membrane breaking methods.

[0037] 4. This invention applies the perforated patch-clamp cell circuit model obtained through modeling and derivation to further obtain the relationship between the number of micropores and the total pore resistance, and derives the formula for calculating the transmembrane current under step voltage input conditions, which can be used to evaluate the degree of perforation. Attached Figure Description

[0038] 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.

[0039] Figure 1 This is a flowchart illustrating the implementation of a robotic quantitative perforation and membrane breaking method as described in this embodiment of the invention.

[0040] Figure 2 This is a schematic diagram of a robotic perforated patch-clamp system with an additional catheter in an embodiment of the present invention.

[0041] Figure 3 This is a schematic diagram of the perforated patch-clamp cell circuit model and the equivalent circuit of pore resistance in an embodiment of the present invention.

[0042] Figure 4 This is a flowchart illustrating the robotic quantitative perforation and membrane breaking process in an embodiment of the present invention.

[0043] Figure 5This is a block diagram of the overall control of the robotic quantitative perforation and membrane breaking in an embodiment of the present invention.

[0044] Figure 6 This diagram illustrates the robotic quantitative perforation experiment process in this embodiment of the invention, as well as the peak values ​​of the transmembrane current at the 2nd, 5th, and 12th minutes after the start of perforation.

[0045] Figure 7 This is a curve showing the change of transmembrane current over time during the perforation process in an embodiment of the present 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] This invention proposes a robotic quantitative perforation membrane rupture method. First, a perforation patch-clamp cell circuit model is obtained by modeling based on the electrical properties of cells. Then, the relationship between the measured resistance and the number of micropores is derived and calculated using this model. Furthermore, under a step voltage input, the relationship between the measured resistance and the transmembrane current is obtained by combining the zero-state response of the RC circuit model. Finally, combined with a robotic perforation patch-clamp system, the perforation degree, i.e., the number of perforations, is fed back in real time through the transmembrane current, enabling closed-loop control of the perforation membrane rupture process, and ultimately completing the robotic quantitative perforation membrane rupture.

[0048] like Figure 1 As shown, this invention discloses a robotic quantitative perforation and membrane rupture method, which includes the following steps:

[0049] S1: Establishing a perforated patch-clamp cell circuit model: Based on the electrophysiological characteristics of cells, each part of the cell is equivalent to a resistor and a capacitor to establish a perforated patch-clamp cell circuit model.

[0050] The cell membrane, composed of a phospholipid bilayer, is electrically equivalent to a capacitor, and the resistance R of the microtubule electrode is... E , cytoplasmic resistance R C and pore resistance R P With sealing resistor R S Together they constitute the measured resistance, cytoplasmic resistance R. C and pore resistance R P Connected in series, and with sealing resistor R S After being connected in parallel, it is then connected to the electrode resistance R. E Series connection.

[0051] S2: Derive the relationship between the number of micropores and the measured resistance.

[0052] The formula for calculating parallel resistance is used to derive the number of micropores n and the total pore resistance R. P The relationship between the resistance R of each micropore PiThe micropores are connected in parallel and are considered to be of the same size. The number of micropores n and the total pore resistance R are... P The relationship between the two is inverse: the more micropores there are, the lower the total pore resistance.

[0053] S3: Deriving the zero-state response of transmembrane current under step voltage input: Based on the zero-state response theory of RC circuits, the relationship between the transmembrane current and the measuring resistance under step voltage input is derived.

[0054] Since both capacitors and resistors exist in the circuit, the cell circuit model can be regarded as a first-order RC circuit. Under a step voltage input, the zero-state response is used to obtain the formula for calculating the transmembrane current.

[0055] S4: Design a robotic quantitative perforation membrane breaking process: Using the above modeling results and combined with a robotic perforation membrane patch clamp system, the peak value of the transmembrane current is used as feedback for the number of perforations to achieve closed-loop control of the release of perforated material, thereby realizing robotic quantitative perforation membrane breaking.

[0056] The release of perforated material is controlled using a robotic perforated patch-clamp system, thereby achieving robotic quantitative membrane rupture. This process includes the following steps:

[0057] S41, a perforated patch-clamp cell circuit model based on cell electrophysiological characteristics, modeling the relationship between the number of micropores and total pore resistance, and using the zero-state response theory of RC circuits to derive the calculation formula for transmembrane current under step voltage input, obtains the relationship between the number of micropores and transmembrane current, and uses it to evaluate the degree of perforation.

[0058] S42, after completing the derivation of the transmembrane current, uses the peak value of the transmembrane current as feedback on the number of perforations, and uses a robotic perforation patch clamp system to actively and controllably release the perforated material and detect the changes in the peak value of the transmembrane current in real time.

[0059] The robotic perforating patch-clamp system can release perforating material precisely at time and in quantitative amounts. It uses the change in the peak value of the transmembrane current as real-time feedback on the perforation effect. When the transmembrane current (number of micropores) reaches the set threshold or reaches a stable state, the release of perforating material stops, and the robotic quantitative perforation is completed. The patch-clamp amplifier monitors the magnitude of the transmembrane current in real time and outputs it to obtain the number of micropores formed in real time.

[0060] S43 adjusts the release rate of the perforating material in real time according to the change of the transmembrane current peak, thereby achieving quantitative perforation and realizing robotic quantitative perforation and membrane breaking.

[0061] The operation steps in this embodiment are as follows:

[0062] S0: Preparations before perforation and membrane breaking.

[0063] S01: Prepare several mouse brain slices containing pyramidal neurons from the visual cortex.

[0064] Brain slices were prepared from 6-7 week old C57BL6 / J mice. After decapitation, the intact brain was quickly removed and placed in an artificial cerebrospinal fluid (aCSF) mixture of ice and water containing a mixture of 95% O2 and 5% CO2. After five minutes, the brain was removed and sliced. Several morphologically sound brain slices were placed in a small beaker containing aCSF with a continuous flow of the mixed gas, and incubated in a 35°C water bath for 33 minutes. After 33 minutes, the brain slices were removed and placed in a brain slice bath. The bath was perfused with recording solution and the mixed gas was continuously introduced. The brain slice cell preparation was complete.

[0065] S02: Prepare the perforation material solution.

[0066] Because nystatin and other perforating materials have low water solubility, a mixture of nystatin and sodium fluorescein was chosen to improve solubility. The stock solution contained 5 mg of nystatin and 20 mg of sodium fluorescein, dissolved in 1 ml of methanol. Before use, 80 μL of the stock solution was added to a dry test tube and mixed with water to obtain the perforation solution of the required concentration, with 400 μg / ml of nystatin and 1.6 mg / ml of sodium fluorescein. Through experiments, we found that excessively high concentrations of perforating materials could lead to dissolution problems and blockage of microtube openings. Conversely, excessively low concentrations would result in a slow perforation process. The experiments ultimately determined that the optimal concentration of the perforating material was 50 μg / ml.

[0067] S03: Prepare a quantitative perforation and membrane breaking device.

[0068] like Figure 2 As shown, this embodiment employs an improved robotic perforated membrane patch clamp device. This device introduces an independent conduit for releasing the perforating material. However, because the introduction of a conduit alters the original pressure balance of the clamp, the external drive device needs to suppress accidental release as needed. This ensures the timely, precise, and quantitative release of the perforating material, providing a hardware foundation for quantitative perforation and membrane breaking.

[0069] S1: Establishing a perforated patch-clamp cell circuit model: Based on the electrophysiological characteristics of cells, each part of the cell is equivalent to a resistor and a capacitor to establish a perforated patch-clamp cell circuit model.

[0070] First, based on the analysis of cellular electrophysiological characteristics, the cell membrane is composed of a phospholipid bilayer. The interior of the phospholipid bilayer is filled with a dielectric, while the outer and inner sides are filled with extracellular fluid and intracellular fluid, respectively. We consider this a conductive solution. Therefore, the cell membrane composed of a phospholipid bilayer is electrically equivalent to a capacitor, with the phospholipid bilayer acting as the two plates. These plates are connected to the conductive extracellular fluid and intracellular fluid, respectively, and the space between the plates is filled with a dielectric. According to the capacitance formula, the capacitance C of the capacitor formed by the phospholipid bilayer is... M It is negatively correlated with the phospholipid bilayer thickness d, and positively correlated with the dielectric constant ε and the cell membrane cross-sectional area S, which can be expressed as:

[0071]

[0072] Secondly, since the micropores formed during the perforation process are only sub-nanometer in size, their influence on the cell membrane cross-sectional area S is negligible. Therefore, the thickness d and dielectric constant ε of the phospholipid bilayer remain unchanged. Based on equation (1), the capacitance C of the capacitor formed by the phospholipid bilayer can be determined. M The size remains constant during the perforation process.

[0073] Finally, the microtube electrode used in patch-clamp operations itself acts as a resistor, forming the microtube electrode resistance R. E Because the cytoplasm inside the cell is a conductor, it forms the cytoplasmic resistance R. C During the perforation process, as the cell membrane surface drawn in by the microtube electrode forms micropores under the action of the perforating material, these micropores constitute the pore resistance R. P After the high-resistance seal is formed, because the resistance of a small portion of the cell membrane that is absorbed is extremely high before the membrane ruptures, the current will instead flow through the seal resistance R. S At this point, the current is called leakage current. Cellular resistance R C and pore resistance R P It is in series and connected to the sealing resistor R. S After being connected in parallel, it is then connected to the electrode resistance R. E Series connection can be represented as:

[0074] R M =R E +(R C +R P ) || R S (2)

[0075] According to actual measurements, due to the sealing resistance R S It is in the gigaohm range, much larger than R. E R C and R PThe sum of GΩ vs MΩ is given, so the sealing resistance can be ignored during the process. Therefore, the measured resistance R... M It can be transformed into the following forms:

[0076] R M =R E +R C +R P (3)

[0077] S2: Deriving the relationship between the number of micropores and the measured resistance: Since the resistance of each pore is connected in parallel, the relationship between the number of micropores and the measured resistance is derived from the formula for calculating the parallel resistance.

[0078] During the perforation process, as micropores gradually form, current will uniformly pass through each micropore into the cell interior. Therefore, it can be inferred that the pore resistances of each micropore are in parallel, represented by R. Pi This represents the pore resistance of each micropore, where n represents the number of micropores formed. According to the formula for parallel resistance, the total pore resistance can be expressed as:

[0079]

[0080] If the pore resistance of each micropore is considered to be almost the same constant, then the total pore resistance R P The calculation formula can be expressed as:

[0081]

[0082] like Figure 3 As shown, based on the above analysis and derivation, a perforated patch-clamp cell circuit model based on cell electrophysiological characteristics was obtained.

[0083] S3: Deriving the zero-state response of transmembrane current under step voltage input: Based on the zero-state response theory of RC circuits, the relationship between the transmembrane current and the measuring resistance under step voltage input is derived.

[0084] Based on the above perforated patch-clamp cell circuit model, the circuit contains both capacitors and resistors, forming a first-order RC circuit. When a voltage of magnitude U is applied to the cell... S The periodic step voltage can be used to derive the transmembrane current i based on the zero-state response of the RC circuit. M Definition:

[0085]

[0086] Where t is the time for the perforated material to be released, and τ is the time constant.

[0087] In voltage clamp mode, the time required for the transient value of the film capacitor to decay to 37% of its original value during the charging process can be expressed as:

[0088] τ=R M C M (7)

[0089] Combining the above formula for calculating transmembrane current and the above cell circuit model, the formula for calculating transmembrane current is derived as follows:

[0090]

[0091] When time t=0 is substituted, the peak value of the transmembrane current i can be obtained. M (0):

[0092]

[0093] Due to the magnitude of the step voltage U S Microtube electrode resistance R E and cytoplasmic resistance R C Both are constants, and the peak transmembrane current i M (0) is positively correlated with the number of holes n, therefore, during the perforation process, i M (0) can be used as a feedback indicator of the degree of perforation.

[0094] S4: Design of a robotic quantitative perforation membrane breaking process: Using the above modeling results and combined with a robotic perforation membrane patch clamp system, the peak value of the transmembrane current is used as feedback for the number of perforations to achieve closed-loop control of the release of perforated material, thereby realizing robotic quantitative perforation membrane breaking.

[0095] like Figure 4 , Figure 5 As shown, the robotic quantitative perforation process is as follows:

[0096] First, the electrodes and cells are automatically positioned, such as... Figure 6 As shown, negative pressure is applied to the electrode contact with the nerve cell to form a high-resistance seal, while simultaneously inhibiting accidental release. After the high-resistance seal is formed, the perforating material is released, and the change in the peak value of the transmembrane current is monitored in real time. The progress of micropore formation is determined based on the change in the peak value of the transmembrane current. When the peak value of the transmembrane current reaches a stable state, it indicates that the quantitative perforation process is complete, and electrical signal recording can then be performed.

[0097] Figure 7 The graph shows the peak value of the transmembrane current changing over time. The peak value of the transmembrane current tends to stabilize about 13 minutes after the perforation begins.

[0098] To verify the effectiveness of the quantitative perforation method of this invention, we selected multiple mouse brain slices containing pyramidal cells from the visual cortex and conducted experiments using the method described in the embodiments. The experiments showed that the quantitative perforation success rate of the method in this invention reached 90% (9 / 10), with only one cell failing due to high-resistance seal detachment during the perforation process. Comparing the operation speed of the method described in this invention with that of traditional patch-clamp perforation, the traditional perforation operation takes approximately 23.2 minutes, while the perforation operation time of the method described in this invention is approximately 13.9 minutes, making the operation speed of this method approximately twice that of traditional perforation.

[0099] This invention applies the modeled and derived perforated patch-clamp cell circuit model to further obtain the relationship between the number of micropores and the total pore resistance, and derives the formula for calculating the transmembrane current under a step voltage input condition to evaluate the degree of perforation. A robotic perforated patch-clamp system is used for timed and quantitative release of the perforating material, with the peak value change of the transmembrane current serving as real-time feedback on the perforation effect. When the transmembrane current (number of micropores) reaches a set threshold or reaches a stable state, the release of the perforating material stops, and the robotic quantitative perforation and membrane breaking is completed.

[0100] The present invention has been described in detail above through embodiments, but the content is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A robotic quantitative perforation and membrane breaking method, characterized in that: The method includes the following steps: S1: Establishing a perforated patch-clamp cell circuit model: Based on the electrophysiological characteristics of cells, each part of the cell is equivalent to a resistor and a capacitor to establish a perforated patch-clamp cell circuit model. In the perforated patch-clamp cell circuit model, the cell membrane, composed of a phospholipid bilayer, is electrically equivalent to a capacitor, and the microtubule electrode resistance... R E cytoplasmic resistance R C and pore resistance R P With sealing resistor R S Together they constitute the measured resistance, the cytoplasmic resistance R C and pore resistance R P Connected in series, and with the sealing resistor R S After being connected in parallel, it is then connected to the electrode resistance. R E Series; The capacitance of the capacitor formed by the phospholipid bilayer C M With phospholipid bilayer thickness d It is negatively correlated with the dielectric constant. ε and cell membrane cross-sectional area S Positive correlation, represented as: ； Neglecting the effect of micropores on the cross-sectional area of ​​the cell membrane S Changes in phospholipid bilayer thickness d Dielectric constant ε All remain unchanged. Based on equation (1), determine the capacitance of the capacitor formed by the phospholipid bilayer. C M The size remains constant during the perforation process; The microtube electrode used in patch clamp operations acts as a resistor, forming the microtube electrode resistor. R E The cytoplasm inside the cell acts as a conductor, forming cytoplasmic resistance. R C During the perforation process, the cell membrane surface drawn in by the microtube electrode forms micropores under the action of the perforating material, and these micropores constitute the pore resistance. R P After the high-resistance seal is formed, a small portion of the cell membrane that is absorbed has an extremely high resistance before the membrane ruptures, and the current will then flow through the seal resistance. R S cytoplasmic resistance R C and pore resistance R P In series, and with the sealing resistor R S After being connected in parallel, it is then connected to the electrode resistance. R E Series connection, represented as: ； The sealing resistor R S It is in the gigaohm range, much larger than R E , R C and R P The sum of these values ​​is used, therefore the sealing resistance is ignored during the process. R S Then measure the resistance R M It can be transformed into the following form: ； S2: Derive the relationship between the number of micropores and the measured resistance; During the perforation process, as micropores gradually form, current will uniformly pass through each micropore into the cell interior. It is inferred that the pore resistances of each micropore are in a parallel state. R Pi This represents the pore resistance of each of the micropores. n Representing the number of micropores already formed, the total pore resistance is expressed as follows, according to the parallel resistance calculation formula: ； Treating the pore resistance of each micropore as an almost identical constant, the total pore resistance... R P The calculation formula is expressed as follows: ； S3: Derive the zero-state response of transmembrane current under step voltage input: Based on the zero-state response theory of RC circuit, derive the relationship between the transmembrane current and the measuring resistance under step voltage input. Based on the perforated patch-clamp cell circuit model established in S1 and S2, the circuit contains both capacitors and resistors, forming a first-order RC circuit, which applies a voltage of magnitude [value missing] to the cell. U S Based on the periodic step voltage and the zero-state response of the RC circuit, the transmembrane current is derived. i M Definition: ； in: t The time for the perforated material to be released is τ, where τ is a time constant. In voltage clamp mode, the time required for the transient value of the film capacitor to decay during the charging process is expressed as: ； Combining the above formula for calculating transmembrane current and the above cell circuit model, the formula for calculating transmembrane current is derived as follows: ； Substituting time t=0, the peak value of the transmembrane current is obtained. i M (0): ； Due to the magnitude of the step voltage U S Microtube electrode resistance R E and cytoplasmic resistance R C Both are constants, peak transmembrane current i M (0) and number of holes n They are positively correlated, therefore during the perforation process, i M (0) As a feedback indicator of the degree of perforation; S4: Design of a robotic quantitative perforation membrane breaking process: Using the above modeling results and combined with a robotic perforation membrane patch clamp system, the peak value of the transmembrane current is used as feedback for the number of perforations to achieve closed-loop control of the release of perforated material, thereby realizing robotic quantitative perforation membrane breaking.

2. The robotic quantitative perforation and membrane breaking method according to claim 1, characterized in that: Resistance of each micropore R Pi The micropores are connected in parallel and are considered to be of the same size. The number of micropores n and the total pore resistance R P The relationship between the two is inverse: the more micropores there are, the lower the total pore resistance.

3. The robotic quantitative perforation and membrane breaking method according to claim 1, characterized in that: In S4, a robotic perforated membrane patch-clamp system is used to control the release of the perforated material, thereby achieving robotic quantitative membrane rupture, including the following steps: S41: Based on the electrophysiological characteristics of cells, a perforated patch-clamp cell circuit model was developed, and the relationship between the number of micropores and the total pore resistance was modeled. The calculation formula for transmembrane current under step voltage input was derived using the zero-state response theory of RC circuits. The relationship between the number of micropores and transmembrane current was obtained to evaluate the degree of perforation. S42: After completing the derivation of the transmembrane current, the peak value of the transmembrane current is used as feedback on the number of perforations. A robotic perforation patch clamp system is used to actively and controllably release the perforated material and detect the changes in the peak value of the transmembrane current in real time. S43: Based on the changes in the peak value of the transmembrane current, the release rate of the perforating material is adjusted in real time to achieve quantitative perforation and realize robotic quantitative perforation and membrane breaking.

4. The robotic quantitative perforation and membrane breaking method according to claim 3, characterized in that: In step S42, the robotic perforating patch clamp system releases perforating material precisely at timed and quantitative intervals, and uses the change in the peak value of the transmembrane current as real-time feedback on the perforation effect.

5. The robotic quantitative perforation and membrane breaking method according to claim 3, characterized in that: In step S42, the transmembrane current is monitored and output in real time by a patch clamp amplifier to obtain the number of micropores formed in real time.

6. The robotic quantitative perforation and membrane breaking method according to claim 4, characterized in that: When the transmembrane current reaches a set threshold or reaches a stable state, the release of perforating material stops, and the robotic quantitative perforation and membrane breaking is completed.

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