Patch clamp high-resistance sealing method driven by air pressure and voltage

Abstract

The invention discloses an air pressure-voltage co-driven patch clamp high-resistance sealing method, and belongs to the technical field of cell-level micromanipulation, and the method comprises the following steps: S1, building a stress model of a suction cell membrane under the action of suction holding air pressure in a patch clamp high-resistance sealing process; s2, establishing a stress model of a suction cell membrane under the action of external voltage in the high-resistance sealing process of the patch clamp; s3, establishing a dynamic model of the patch clamp high-resistance sealing process under the common driving of air pressure and voltage based on the work of the previous two steps; s4, designing a self-adaptive sliding mode controller to perform tracking control on the sealing resistance value in the high-resistance sealing process; and S5, establishing an air pressure-voltage co-driven automatic patch clamp high-resistance sealing process. The high-resistance sealing process of the patch clamp is jointly driven by the holding air pressure and the voltage, and the self-adaptive sliding film controller is established to track and control the sealing resistance value in the high-resistance sealing process, so that the high-resistance sealing efficiency is finally improved, the high-resistance sealing success rate is improved by 55%, and the sealing resistance value is improved by 52%.

Description

Technical Field

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

[0002] The patch clamp technique can monitor the picoampere level (10 -12 A) Weak current is used for detection, and the key step of using patch clamp technology to measure ion channel signals requires the use of microtube electrodes to absorb part of the cell membrane into the microtube, and to form a high-resistance seal (high-resistance seal) of the GΩ level between the absorbed cell membrane and the inner wall of the microtube.

[0003] Since high-resistance sealing can effectively shield electromagnetic noise in the environment, the success rate and quality of high-resistance sealing are the key factors affecting the efficiency of patch clamp operation. In current patch clamp operation, operators often apply a small amount of negative pressure through a mouth pipette or a medical syringe to suck the cell membrane into the microtube to promote the formation of high-resistance sealing.

[0004] However, due to the complex formation mechanism of high-resistance sealing and the influence of many factors, the result of high-resistance sealing by negative pressure alone is extremely accidental, and the success rate and quality of sealing are difficult to guarantee. By applying voltage, a certain electroosmotic force can be generated between the cell membrane and the inner wall of the microtubule to assist the formation of the high-resistance sealing process. However, how to coordinate air pressure and voltage to ensure the stability of the high-resistance sealing process and improve the success rate and quality of high-resistance sealing still needs 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 improve the efficiency of patch clamp operation, the present invention proposes a patch clamp high-resistance sealing method driven by air pressure and voltage. By modeling the sealing process under the 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 co-driven patch clamp high-resistance sealing method, the method comprising the following steps:

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

[0010] S2: By modeling the force on the microtubules sucked into the cell membrane under the action of voltage, the magnitude of the driving force of the voltage on the sucked-in 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 the high resistance sealing;

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

[0014] Furthermore, 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. It can be considered 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.

[0015] Furthermore, in S1, the cell membrane sucked into the cell membrane is divided into two parts: a wall-attached part and a free part, and the force exerted by the suction and holding pressure on the cell membrane is divided into two parts: the suction force on the free part and the Poiseuille flow force on the wall-attached 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 ions in the solution 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.

[0017] Furthermore, in S2, the magnitude of the electroosmotic force in the attachment area of ​​the cell membrane is calculated according to 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 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 resistances: 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, according to 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 by 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, so the complexity of the system will not be increased, making the inventive method have good versatility in the traditional patch clamp operating system, which is convenient for the promotion and application of the inventive method.

[0024] 2. The method of the present invention can adaptively adjust the control parameters of the air pressure-voltage co-driven model to the parameter changes by designing an adaptive synovial membrane controller, without remodeling or calibrating the parameters for each cell or each type of system. Therefore, the 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 drawings required for use in the description of the specific embodiments will be briefly introduced below. 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 paying creative work.

[0027] Figure 1 The present invention discloses a flow chart of a gas pressure-voltage jointly driven patch clamp high resistance sealing method.

[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 It 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 It is a block diagram of a control system for a high-resistance sealing process driven by air pressure and voltage jointly based on an adaptive sliding film controller according to the present invention.

[0031] Figure 5 It is a flow chart of the gas 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 co-driven patch clamp high-resistance sealing method is provided, comprising the following steps:

[0036] S1: The force modeling of the cell membrane sucked into the microtube under the action of the holding gas pressure is used to calculate the driving force of the holding gas pressure on the cell membrane sucked into the microtube during the high-resistance sealing process.

[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 sucked cell membrane does not change, that is, the microtubule is considered to be a hollow cylinder, and the sucked 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 sucked-in cell membrane during the high-resistance sealing process is calculated.

[0039] 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, 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.

[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: Combining the air pressure-driven and voltage-driven models established above, a dynamic model of the absorption of species 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 microtube electrode, it moves along the tube wall under 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 two resistances: 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.

[0043] The cell membrane and microtubule attachment process assumes that the cell membrane and the microtubule electrode are evenly attached, so the resistance and the attachment length satisfy the linear relationship

[0044] 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 the high resistance sealing;

[0045] An 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.

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

[0047] According to 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 to the set value along the planned change curve.

[0048] Example 2

[0049] like Figure 2-Figure 6 As shown in the figure, as the best embodiment of the present invention, in this implementation, the target cells of the patch clamp operation are pyramidal neurons in the visual cortex of mouse brain slices, and the specific steps include:

[0050] S0: Preparation of Mouse Brain Slices with Neurons

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

[0052] (2) After removal, the cerebellum was cut flat and fixed in the bath of a vibrating microtome with 502 glue;

[0053] (3) Use a vibrating slicer to prepare brain slices with a thickness of 300 μm. Select 2-3 brain slices containing the target brain area according to the mouse brain slice atlas and place them in oxygen-saturated artificial cerebrospinal fluid at 33°C for incubation for 30 minutes.

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

[0055] like Figure 2 As shown in the figure, 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 inhaled cell membrane is only in the size of square micrometers, and contains a small number of cytoskeleton and proteins, so their 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 spherical surface 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 wall 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 tube wall T f2 , where T f1 The adhesion force between the cell membrane and microtubules is balanced, 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 an ordinary 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), we have F S < <F P Therefore, F S It was ignored in the subsequent analysis.

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

[0067] like Figure 3 As shown, during the process of forming the seal, there is a gap between the cell membrane and the inner wall of the glass microelectrode. When a 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, forming an electroosmotic flow. The present invention controls the voltage to drive the liquid flow in the gap, and generates 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 of the velocity flowing along the x-axis, ρ e represents the volume charge density, E x =-φ p / L represents the length L The potential difference across the gap is

[0071]

[0072] We can get:

[0073]

[0074] where φ is the distribution of the 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 equation (10) we 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 joint drive of air pressure and voltage. The driving force it receives is the suction force and electroosmotic force generated by the air pressure. 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 it receives 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 a 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 control box Figure 4 As shown. Define the Lyapunov function as:

[0103]

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

[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 process based on gas pressure-voltage co-driven

[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 three-dimensional positions of the two. After that, the system automatically adjusts the air pressure and voltage, tracks the designed sealing resistance change curve, and completes the automated high-resistance sealing process. According to the sealing resistance feedback, the cell membrane is broken 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, and for comparison, another 20 neurons were subjected to high-resistance sealing using the traditional negative pressure suction 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 method 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 performs current restraint and voltage clamping on the nerve cells after high-resistance sealing and membrane breaking ( Figure 6 (a)) and successfully recorded the action potential of the neuron. Figure 6 (b) and the current generated by ion channel opening ( Figure 6 (c)).

[0111] In addition, it should be noted that the shapes and names of the parts and components of the specific embodiments described in this specification may be different. All equivalent or simple changes made based on the structure, features and principles of the patent concept of the present invention are included in the protection scope of the patent of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A gas pressure-voltage driven patch clamp high resistance sealing method, characterized in that: The method comprises the following steps: S1: By modeling the force on the cell membrane sucked into the microtube under the action of the holding gas pressure, the driving force of the holding gas pressure on the cell membrane sucked into the microtube during the high-resistance sealing process is calculated; S2: By modeling the force on the microtubules sucked into the cell membrane under the action of voltage, the magnitude of the driving force of the voltage on the sucked-in 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 film controller to control the sealing resistance value in the high resistance sealing process to follow the set curve to complete the high resistance sealing; S5: Establish an automatic patch clamp high-resistance sealing process driven by air pressure and voltage to improve the high-resistance sealing efficiency.

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

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

4. The gas pressure-voltage co-driven patch clamp high-resistance sealing method according to claim 1, characterized in that: 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 ions in the solution 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 gas pressure-voltage co-driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S2, the magnitude of the electroosmotic force of the cell membrane attachment region is calculated according to 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 gas pressure-voltage co-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 resistances: 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.

7. The gas pressure-voltage co-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 gas pressure-voltage co-driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S4, the adaptive sliding mode controller is designed to adapt to the change of model parameters caused by the increase of 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.

9. The gas pressure-voltage co-driven patch clamp high-resistance sealing method according to claim 1, characterized in that: In S5, according to 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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