A kind of diaphragm clamp high resistance seal method driven by air pressure-voltage jointly

Abstract

The application discloses a kind of membrane patch clamp high resistance sealing methods driven by air pressure-voltage, belong to the micro-operation technical field of cell level, comprising the following steps: S1, establish the stress model of suction cell membrane under the action of holding air pressure in the process of membrane patch clamp high resistance sealing;S2, establish the stress model of suction cell membrane under the action of applied voltage in the process of membrane patch clamp high resistance sealing;S3, based on the first two steps work, establish the dynamics model of membrane patch clamp high resistance sealing process driven by air pressure-voltage;S4, design adaptive sliding mode controller to track control the sealing resistance of high resistance sealing process;S5, establish the automatic membrane patch clamp high resistance sealing process driven by air pressure-voltage.The application utilizes holding air pressure and voltage to drive membrane patch clamp high resistance sealing process, and establishes adaptive sliding film controller to track control the sealing resistance of high resistance sealing process, finally improves the efficiency of high resistance sealing, and high resistance sealing success rate is improved by 55%, and sealing resistance is improved by 52%.

Description

Technical Field

[0001] The present invention belongs to the field of cell-level micromanipulation technology, and particularly relates to a gas pressure-voltage jointly driven patch clamp high-resistance sealing method. Background Art

[0002] Patch clamp technology can measure the picoampere level (10 -12 A) Weak current is detected, and the key step in using patch clamp technology to measure ion channel signals, which is known as the "gold standard" for ion channel research in brain science and neuroscience, is to use microtubule electrodes to absorb part of the cell membrane into the microtubule, and form a high-resistance seal (high-resistance seal) in the GΩ level between the absorbed cell membrane and the inner wall of the microtubule.

[0003] Because high-resistance seals can effectively shield electromagnetic noise from the environment, the success rate and quality of high-resistance seals are crucial to the efficiency of patch clamping. In current patch clamping procedures, operators often use a mouth pipette or medical syringe to apply a small amount of negative pressure to draw the cell membrane into the microtubule to promote the formation of a high-resistance seal.

[0004] However, due to the complex mechanism of high-resistance sealing, which is influenced by multiple factors, the results of high-resistance sealing using only negative pressure are highly accidental, making it difficult to guarantee both the success rate and quality of the seal. Applying voltage can generate a certain electroosmotic force between the cell membrane and the inner wall of the microtubule, assisting in the formation of the high-resistance seal. However, how to coordinate pressure and voltage to ensure the stability of the high-resistance seal formation process and improve the success rate and quality of the high-resistance seal remains to be studied.

[0005] Therefore, developing a gas pressure-voltage jointly driven patch clamp high-resistance sealing method is of great significance to improving the success rate and quality of high-resistance sealing and improving the efficiency of patch clamp operation. Summary of the Invention

[0006] In order to improve the success rate and quality of current high-resistance sealing and ultimately enhance the efficiency of patch clamp operation, the present invention proposes a patch clamp high-resistance sealing method driven by both air pressure and voltage. By modeling the sealing process under dual drive of air pressure and voltage, an adaptive sliding film controller is designed to compensate for changes in system parameters, thereby achieving smooth control of the sealing process and ensuring the success rate and quality of high-resistance sealing.

[0007] The present invention adopts the following technical solutions to solve the above problems:

[0008] A gas pressure-voltage jointly driven patch clamp high-resistance sealing method, the method comprising the following steps:

[0009] S1: By modeling the force acting on the cell membrane sucked into the microtube under the action of the holding air pressure, the driving force exerted by the holding air pressure on the cell membrane during the high-resistance sealing process is calculated;

[0010] S2: By modeling the force acting on the microtubules sucked into the cell membrane under the action of voltage, the magnitude of the driving force exerted by the voltage on the microtubules sucked into the cell membrane during the high-resistance sealing process is calculated;

[0011] S3: Combining the air pressure-driven and voltage-driven models established above, a dynamic model of the absorption of seeds into the cell membrane during the high-resistance sealing process driven by air pressure and voltage is established;

[0012] S4: Design an adaptive sliding film controller to control the sealing resistance value in the high-resistance sealing process to follow the set curve to complete high-resistance sealing;

[0013] S5: Establish an automatic patch clamp high-resistance sealing process driven by both air pressure and voltage to improve the efficiency of high-resistance sealing.

[0014] Furthermore, in S1, the portion of the microtube in contact with the cell membrane is limited to the tube mouth, and this portion has been heated and polished. It can be assumed that the inner diameter of the portion of the microtube in contact with the inhaled cell membrane does not change, that is, the microtube is considered to be a hollow cylinder.

[0015] Furthermore, in S1, the cell membrane sucked into the cell membrane is divided into two parts: the wall-adherent part and the free part. The force exerted by the holding pressure on the cell membrane is divided into the suction force on the free part and the Poiseuille flow force on the wall-adherent part, which are calculated separately.

[0016] Furthermore, in S2, during the process of seal formation, there is a gap between the cell membrane and the inner wall of the glass microelectrode. When voltage is applied in the electrode microtube, a potential difference is formed at both ends of the channel, so that the ions in the solution drive the liquid to flow, generating electroosmotic flow. By controlling the voltage to drive the flow of liquid in the gap, electroosmotic force is generated in the attachment area of ​​the cell membrane to promote seal formation.

[0017] Furthermore, in S2, the magnitude of the electroosmotic force in the cell membrane attachment area is calculated based on the Navier-Stokes equation, which is a balance formula between the Coulomb force on ions moving in the gap under the electric field and the viscous force generated by the liquid.

[0018] Furthermore, in S3, after the cell membrane enters the microtube electrode, it is moved along the tube wall by the combined action of the suction force generated by the air pressure and the electroosmotic force generated by the voltage. At the same time, it is affected by the two resistances of the cell viscoelastic force and the force between the cell membrane and the tube wall. The driving force and the resistance jointly affect the formation of a high-resistance seal.

[0019] Furthermore, in S3, the cell membrane and microtubule attachment process assumes that the cell membrane and the microtubule electrode are evenly attached, and the resistance and the attachment length satisfy a linear relationship.

[0020] Furthermore, in S4, the adaptive sliding mode controller is designed to adapt to the change of model parameters caused by the increase in the weight of the sucked cell membrane during the high-resistance sealing process, and the sealing resistance is controlled to track the set change curve to achieve high-resistance sealing.

[0021] Furthermore, in S5, based on the three-dimensional positioning results of the microtube electrode and the cell, the electrode is automatically guided to approach and contact the target cell, and the microtube air pressure and voltage are adjusted through the adaptive sliding mode controller to adjust the sealing resistance along the planned change curve to reach the set value.

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] 1. The voltage driving the high-resistance sealing process in the present invention is the output voltage of the traditional patch clamp system, and no other special voltage generating equipment is required. Therefore, it does not increase the complexity of the system, making the inventive method have good versatility in traditional patch clamp operating systems, facilitating the promotion and application of the inventive method.

[0024] 2. By designing an adaptive synovial controller, the method of the present invention can adaptively adjust the control parameters of the pressure-voltage co-driven model to the parameter changes, without having to remodel or calibrate the parameters of each cell or each type of system. Therefore, this method is adaptive to different cell types.

[0025] 3. Compared with the traditional pure air pressure driving method, the high resistance sealing success rate of the method of the present invention is increased by 55%, and the sealing resistance value is increased by 52%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention, the following will briefly introduce the drawings required for use in the description of the specific embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0027] Figure 1 This is a flow chart of the air pressure-voltage jointly driven patch clamp high-resistance sealing method described in the present invention.

[0028] Figure 2 It is a schematic diagram of the force applied to the cell membrane during the air pressure-driven high-resistance sealing process of the present invention.

[0029] Figure 3 This is a schematic diagram of the force applied to the cell membrane during the voltage-driven high-resistance sealing process of the present invention.

[0030] Figure 4 This is a block diagram of the air pressure-voltage jointly driven high-resistance sealing process control system based on the adaptive sliding film controller of the present invention.

[0031] Figure 5 This is a flow chart of the air pressure-voltage jointly driven high-resistance sealing method of the present invention.

[0032] Figure 6 This is a diagram of the high-resistance sealing and signal recording experimental results described in the present invention. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1

[0035] like Figure 1 As shown in the flowchart of the present invention, a gas pressure-voltage driven patch clamp high-resistance sealing method is provided, comprising the following steps:

[0036] S1: By modeling the force acting on the cell membrane sucked into the microtube under the action of the holding air pressure, the driving force of the holding air pressure on the cell membrane during the high-resistance sealing process is calculated.

[0037] The part of the microtubule that contacts the cell membrane is limited to the tube mouth, and this part has been heated and polished. It can be assumed that the inner diameter of the part of the microtubule that contacts the absorbed cell membrane does not change, that is, the microtubule is considered to be a hollow cylinder, and the absorbed cell membrane is divided into two parts: the wall-attached part and the free part. The force of the suction and holding pressure on the cell membrane is divided into the suction force on the free part and the Poiseuille flow force on the wall-attached part, which are calculated separately.

[0038] S2: By modeling the force acting on the microtubules sucked into the cell membrane under the action of voltage, the magnitude of the driving force of the voltage on the microtubules sucked into the cell membrane during the high-resistance sealing process is calculated.

[0039] During the seal formation process, there is a gap between the cell membrane and the inner wall of the glass microelectrode. When voltage is applied in the electrode microtube, a potential difference is formed at both ends of the channel, causing the ions in the solution to drive the liquid to flow, generating electroosmotic flow. By controlling the voltage to drive the flow of liquid in the gap, electroosmotic force is generated in the attachment area of ​​the cell membrane to promote seal formation.

[0040] The magnitude of the electroosmotic force in the cell membrane attachment area is calculated based on the Navier-Stokes equation, which is the balance between the Coulomb force on the ions moving in the gap under the electric field and the viscous force generated by the liquid.

[0041] S3: Based on the above established air pressure driving and voltage driving models, a kinetic model of the cell membrane being sucked into the cell membrane during the high-resistance sealing process driven by air pressure and voltage is established.

[0042] After the cell membrane enters the micro-tube electrode, it is driven by the suction force generated by air pressure and the electro-osmotic force generated by voltage to move along the tube wall, while being affected by the cell viscoelastic force and the interaction force between the cell membrane and the tube wall, and the driving force and the resistance jointly affect the formation of high-resistance sealing.

[0043] The process of cell membrane adhering to the micro-tube is assumed that the cell membrane and the micro-tube electrode adhere uniformly, and the resistance and the adhering length satisfy a linear relationship

[0044] S4: Design an adaptive sliding mode controller to control the sealing resistance value in the high-resistance sealing process to follow the set curve, and complete the high-resistance sealing.

[0045] By designing an adaptive sliding mode controller to adapt to the change of model parameters caused by the increase of the weight of the cell membrane being sucked in during the high-resistance sealing process, the sealing resistance value is controlled to track the set change curve to achieve high-resistance sealing.

[0046] S5: Establish an automatic patch clamp high-resistance sealing process driven by air pressure and voltage to improve the efficiency of high-resistance sealing.

[0047] According to the three-dimensional positioning results of the micro-tube electrode and the cell, the electrode is automatically guided to approach and contact the target cell, and the adaptive sliding mode controller is used to adjust the micro-tube air pressure and voltage to adjust the sealing resistance value along the planned change curve to reach the set value.

[0048] Embodiment 2

[0049] As shown in Figure 2-Figure 6 , as the best embodiment of the present application, in this embodiment, the target cell of the patch clamp operation is a mouse brain slice visual cortex pyramidal neuron, which specifically includes the following steps:

[0050] S0: Preparation of mouse brain slice neural cells

[0051] (1) Select 4-6 week old C57BL / 6N female mice, after anesthesia, decapitate them, open the skull to remove the whole brain, and quickly immerse the whole brain in 0-4 degrees Celsius artificial cerebrospinal fluid saturated with oxygen mixed gas (95% O2+5% CO2) for 1 minute;

[0052] (2) After removal, cut the cerebellum flat and fix it in the bath of the vibration sectioning machine with 502 glue;

[0053] (3) Use the vibration sectioning machine to prepare brain slices with a thickness of 300 microns, select 2-3 brain slices containing the target brain area according to the mouse brain slice atlas, and place them in oxygen-saturated 33 degrees Celsius artificial cerebrospinal fluid for incubation for 30 minutes.

[0054] S1: Modeling of cell membrane forces driven by air pressure

[0055] like Figure 2 As shown, considering that the part of the microtubule that contacts the cell membrane is limited to the tube mouth and this part has been heated and polished, it can be assumed that the inner diameter of the part of the microtubule that contacts the inhaled cell membrane does not change, that is, the microtubule is a hollow cylinder. At the same time, the surface of the cell membrane that is inhaled is only in the size of square micrometers, and contains a small number of cytoskeleton and proteins, so its role is ignored in the mechanical modeling process. The inhaled cell membrane is subdivided into a free part at the top and a wall-attached part in contact with the tube wall. According to the observation results under the high-definition camera, the shape of the cell membrane of the free part is simplified to a radius of R f The surface tension T of the partial sphere under the action of the holding pressure ΔP f Satisfies the following formula

[0056] T f =ΔPR f / twenty one)

[0057] At the junction of the free part and the adherent part, T f can be further decomposed into a component perpendicular to the inner wall of the microtubule, T f1 and the component parallel to the wall, T f2 , where T f1 is balanced by the adhesion force between the cell membrane and microtubules, while T f2 It plays the role of pulling the sucked cell membrane to crawl along the tube wall. According to the geometric relationship,

[0058] T f2 = T f cosβ=ΔP*R f / 2*R p / R f =ΔP*R p / twenty two)

[0059] Where β is the contact angle between the cell membrane and the inner wall of the microtubule, R p is the inner diameter of the microtubule. The pulling force that pulls the cell membrane to crawl is

[0060]

[0061] According to the literature, the attachment between the inner wall of the microtubule and the hydrophilic cell membrane can be approximated as a common black membrane structure containing a water layer of about 10nm. When negative pressure acts on it, a parabolic Poiseuille flow with a low Reynolds number is generated. The laminar flow velocity along the Y direction can be calculated as

[0062]

[0063] Where η is the kinematic viscosity coefficient of the liquid, L is the length of the attached cell membrane, and h is the thickness of the liquid layer. According to Newton's law of viscosity, the force exerted by the fluid on the membrane surface is

[0064]

[0065] Where S is the area of ​​the cell membrane, τ p It is the shear force of the liquid flow in the Y direction applied to the unit area of ​​the membrane surface. Since h (about 10nm) is much smaller than R P (1-2μm), according to equations (3) and (5), F S < <F P Therefore, F S It is ignored in the subsequent analysis.

[0066] S2: Modeling of cell membrane forces under voltage drive

[0067] like Figure 3 As shown, during the seal formation process, a gap exists between the cell membrane and the inner wall of the glass microelectrode. When a voltage is applied within the electrode microtube, a potential difference is formed across the channel, causing ions in the solution to drive the liquid flow, forming electroosmotic flow. The present invention controls the voltage to drive the flow of liquid across the gap, generating an electroosmotic force in the cell membrane attachment area to promote the seal.

[0068] The voltage inside and outside the microelectrode is φ p According to the Navier-Stokes equation, the Coulomb force on the ions moving in the gap under the electric field is balanced by the viscous force generated by the liquid, that is,

[0069]

[0070] where v x (z) represents the flow velocity of the fluid, which is the laminar flow with respect to the z distribution along the x-axis. e represents the volume charge density, E x =-φ p / L represents the length L The potential difference across the gap. According to Poisson's equation

[0071]

[0072] We can get:

[0073]

[0074] where φ is the distribution of the electric potential in the gap with respect to z. Integrating this equation twice yields:

[0075]

[0076] ηu x (y)-ε0ε r E x ψ(y)=k0+k1y (10)

[0077] According to the boundary conditions, the fluid velocity on the cell membrane and microtube electrode surface is 0, that is, u x (0) = u x (h) = 0, substituting into formula (10) we can get Substituting into equation (9), according to Newton's law of internal friction, we can obtain the force τ exerted by electroosmotic flow on the cell membrane per unit area in the attachment area: e The voltage φ applied to the electrode microtube p The relationship is

[0078]

[0079] The surface potential of the microtube electrode φ(0), the surface potential of the cell membrane φ(h), and the potential distribution of the sealing gap φ′(z) are all determined by the cell state, the composition and pH value of the electrode solution, and the voltage φ applied to the microtube electrode. p Not relevant.

[0080] S3: Dynamic model of high-resistance sealing process driven by gas pressure and voltage

[0081] After the cell membrane enters the microtube electrode, it moves backward under the combined drive of air pressure and voltage. The driving force it receives is the suction force generated by the air pressure and the electroosmotic force. According to the cell viscoelastic model, the resistance it receives is the viscoelastic force of the cell and the force between the cell membrane and the tube wall. The combined force is:

[0082]

[0083] where μ a is the adhesion coefficient between the cell membrane and the tube wall, k e is the elastic coefficient of the cell, c a is the viscosity coefficient of the cell.

[0084] The equation of motion for the cell sucked into the microelectrode is:

[0085]

[0086] The measured value of the system is resistance. Assuming that the cell membrane and the microtube electrode are evenly attached, the relationship between resistance and attachment length is:

[0087]

[0088]

[0089] Rewrite the dynamic model of the system by equation (13):

[0090]

[0091] Where x=L, u1=ΔP, u2=-ψ p , Under experimental conditions, c and d are both positive, d(t) represents a bounded external perturbation and |f(t)|≤δ≤cδ1+dδ2.

[0092] S4: Design of Adaptive Sliding Mode Controller

[0093] like Figure 4 As shown, during the cell sealing process, the actual parameters of the system are difficult to measure, and as the sealing proceeds, the mass m of the controlled object will continue to increase, causing the above parameters to change continuously. Therefore, the present invention designs an adaptive sliding mode controller to adapt to the changes in parameters and control the sealing to be formed at the desired speed.

[0094] The tracking error of the system is defined as

[0095] e=xx d (17)

[0096] where x d is the desired sealing trajectory. Set the sliding surface to

[0097]

[0098] Defining uncertain parameters Design the controller as:

[0099]

[0100] Where tanh(·) represents the hyperbolic tangent function, k1 and k2 are control gains, represents the estimated values ​​of these parameters, and the update law of the designed estimated values ​​is:

[0101]

[0102] System controller enclosure Figure 4 As shown. Define the Lyapunov function as:

[0103]

[0104] Taking the derivative of this function with respect to time, we can obtain:

[0105]

[0106] This proves that the tracking error will converge to 0 and the system is asymptotically stable.

[0107] S5: Robotic high-resistance sealing and patch clamping procedures based on pneumatic-voltage co-actuated

[0108] Based on the aforementioned key technologies, the present invention uses conventional image processing algorithms to perform three-dimensional positioning of microtube electrodes and cells, and automatically guides the microtubes to contact the target cell surface based on the obtained three-dimensional positions of the two. The system then automatically adjusts the air pressure and voltage, tracks the designed sealing resistance change curve, and completes the automated high-resistance sealing process. Based on the sealing resistance feedback, the cell membrane is ruptured and the signal is recorded by applying a negative pressure pulse. Figure 5 shown.

[0109] like Figure 6 As shown, the results of the high-resistance sealing and patch clamp experiments in this embodiment are as follows: the present invention conducted an automated high-resistance sealing experiment on 20 pyramidal neurons in the visual cortex of mouse brain slices. For comparison, another 20 neurons were subjected to high-resistance sealing using the traditional negative pressure holding method, of which 11 cells completed high-resistance sealing, with a success rate of 55% (11 / 20) and an average sealing resistance of 2.03±0.62GΩ (n=11); in comparison, 17 cells successfully completed high-resistance sealing using the method of the present invention, with a success rate of 85% and an average sealing resistance of 3.09±0.58GΩ (n=17).

[0110] Compared with the traditional air pressure-based sealing method, the high-resistance sealing success rate of the present invention is increased by 55%, and the sealing resistance is increased by 52%. In order to verify the activity of the cells after the operation, the present invention conducted current restraint and voltage clamping on the nerve cells after high-resistance sealing and membrane rupture ( Figure 6 (a)), the action potential of the neuron was successfully recorded. Figure 6 (b) and the current generated by ion channel opening ( Figure 6 (c)).

[0111] Furthermore, it should be noted that the specific embodiments described in this specification may vary in the shapes and names of their components. Any equivalent or simple variations based on the structure, features, and principles of the present invention are included within the scope of protection of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, and such modifications and additions shall fall within the scope of protection of the present invention.

Claims

1. A gas pressure-voltage driven patch clamp high-resistance sealing method, characterized by: The method comprises the following steps: S1: By modeling the force acting on the cell membrane sucked into the microtube under the action of the holding air pressure, the driving force exerted by the holding air pressure on the cell membrane during the high-resistance sealing process is calculated; S2: By modeling the force acting on the microtubules sucked into the cell membrane under the action of voltage, the magnitude of the driving force exerted by the voltage on the microtubules sucked into the cell membrane during the high-resistance sealing process is calculated; S3: Combining the air pressure-driven and voltage-driven models established above, a dynamic model of the absorption of seeds into the cell membrane during the high-resistance sealing process driven by air pressure and voltage is established; S4: Design an adaptive sliding mode controller to control the sealing resistance value in the high resistance sealing process to follow the set curve to complete the high resistance sealing. By designing the adaptive sliding mode controller to adapt to the change of model parameters caused by the increase in the weight of the inhaled cell membrane during the high resistance sealing process, the sealing resistance value is controlled to track the set change curve to achieve high resistance sealing. The tracking error of the system is defined as ； in For the desired sealing trajectory, set the sliding surface to ； Defining uncertain parameters , the designed controller is: ； in represents the hyperbolic tangent function, To control the gain, represents the estimated values ​​of these parameters; S5: Establish an automatic patch clamp high-resistance sealing process driven by both air pressure and voltage to improve the efficiency of high-resistance sealing.

2. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S1, the part of the microtube that contacts the cell membrane is limited to the tube mouth, and this part has been heated and polished, so it is believed that the inner diameter of the part of the microtube that contacts the inhaled cell membrane does not change, that is, the microtube is considered to be a hollow cylinder.

3. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S1, the cell membrane sucked in is divided into two parts: the wall-adherent part and the free part. The force exerted by the holding pressure on the cell membrane is divided into the suction force on the free part and the Poiseuille flow force on the wall-adherent part, which are calculated separately.

4. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S2, during the seal formation process, there is a gap between the cell membrane and the inner wall of the glass microelectrode. When voltage is applied in the electrode microtube, a potential difference is formed at both ends of the channel, causing the ions in the solution to drive the liquid to flow, generating electroosmotic flow. By controlling the voltage to drive the liquid flow in the gap, electroosmotic force is generated in the attachment area of ​​the cell membrane to promote seal formation.

5. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S2, the magnitude of the electroosmotic force in the cell membrane attachment area is calculated based on the Navier-Stokes equation, which is a balance formula between the Coulomb force on ions moving in the gap under the electric field and the viscous force generated by the liquid.

6. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S3, after the cell membrane enters the microtube electrode, it moves along the tube wall under the combined action of two driving forces: the suction force generated by the air pressure and the electroosmotic force generated by the voltage. At the same time, it is affected by two resistance forces: the viscoelastic force of the cell and the force between the cell membrane and the tube wall. The driving force and the resistance jointly affect the formation of a high-resistance seal.

7. The air pressure-voltage driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S3, the cell membrane and microtubule attachment process assumes that the cell membrane and the microtubule electrode are evenly attached, and the resistance and the attachment length satisfy a linear relationship.

8. The air pressure-voltage co-driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S5, based on the three-dimensional positioning results of the microtube electrode and the cell, the electrode is automatically guided to approach and contact the target cell, and the microtube air pressure and voltage are adjusted through the adaptive sliding mode controller to adjust the sealing resistance along the planned change curve to reach the set value.

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